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Proteomics Uncovers Novel Components of an Interactive Protein Network Supporting RNA Export in Trypanosomes

Open AccessPublished:January 25, 2022DOI:https://doi.org/10.1016/j.mcpro.2022.100208

      Highlights

      • Gene expression in trypanosomes is mediated by noncanonical mechanisms.
      • Trypanosome mRNA nuclear export system comprises unique proteins to kinetoplastids.
      • The present work highlights an amalgam of kinetoplastid-specific and conserved components.
      • Our data support a highly coupled mRNA maturation pathway.

      Abstract

      In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation.

      Graphical Abstract

      Keywords

      Abbreviations:

      EJC (exon junction complex), INT (integrator complex), mRNP (messenger ribonucleoprotein), NMD (nonsense-mediated mRNA decay), NPC (nuclear pore complex), NPGs (nuclear peripheral granules), TREX (TRanscription-EXport), UTR (mRNA untranslated region)
      Trypanosoma cruzi is the causative agent of Chagas disease, a significant infection of humans and animals in Latin America where 10 million people are estimated to be infected (
      WHO
      ). Climate change and human migration have both increased the range of the disease and risk for much of the Americas and Europe; recent global events will likely inflate this further, as well as compromise the ability of healthcare organizations to deliver therapeutics (
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      ). Trypanosomes have multiple distinct RNA granules, and their composition is divergent from animals and fungi. For example, during translational repression, some populations of RNA granule are compositionally stable (
      • Krüger T.
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      • Strome S.
      SCD6 induces ribonucleoprotein granule formation in trypanosomes in a translation- independent manner, regulated by its Lsm and RGG domains.
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      ,
      • Ferreira J.
      • Ferrarini M.G.
      • Nardelli S.C.
      • Goldenberg S.
      • Ávila A.R.
      • Holetz F.B.
      Trypanosoma cruzi XRNA granules colocalise with distinct mRNP granules at the nuclear periphery.
      ). RNA granules at the nuclear periphery may regulate mRNA fate by preventing misprocessed mRNAs from reaching the translation machinery (
      • Kramer S.
      • Marnef A.
      • Standart N.
      • Carrington M.
      Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate.
      ,
      • Goos C.
      • Dejung M.
      • Wehman A.M.
      • M-natus E.
      • Schmidt J.
      • Sunter J.
      • Engstler M.
      • Butter F.
      • Kramer S.
      Trypanosomes can initiate nuclear export co-transcriptionally.
      ). However, it remains unclear how these complexes interact with the nuclear components involved in RNA export.
      In animals and fungi, mRNA export is initiated by cotranscriptional association of nascent mRNAs with the THO complex. THO is a conserved multimeric complex required for mRNP biogenesis consisting of at least four components: Tho2, Hpr1, Mft1, and Thp2 (
      • Jimeno S.
      • Rondón A.G.
      • Luna R.
      • Aguilera A.
      The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability.
      ). THO also associates with two mRNA export factors, UAP56 and ALY (Sub2 and Yra1 in budding yeast), to form the TREX complex that couples transcription with mRNA nuclear export (
      • Strässer K.
      • Masuda S.
      • Mason P.
      • Pfannstiel J.
      • Oppizzi M.
      • Rodriguez-Navarro S.
      • Rondón A.G.
      • Aguilera A.
      • Struhl K.
      • Reed R.
      • Hurt E.
      TREX is a conserved complex coupling transcription with messenger RNA export.
      ). In animals, recruitment of TREX to mRNA occurs by a splicing-coupled mechanism rather than a direct transcription-coupled mechanism as in yeast (
      • Chi B.
      • Wang Q.
      • Wu G.
      • Tan M.
      • Wang L.
      • Shi M.
      • Chang X.
      • Cheng H.
      Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA.
      ,
      • Masuda S.
      • Das R.
      • Cheng H.
      • Hurt E.
      • Dorman N.
      • Reed R.
      Recruitment of the human TREX complex to mRNA during splicing.
      ). A second complex, TREX-2, links transcription and the nuclear pore complex (NPC) by facilitating transfer of mature mRNPs from the nuclear interior to the NPC (
      • Jani D.
      • Lutz S.
      • Hurt E.
      • Laskey R.A.
      • Stewart M.
      • Wickramasinghe V.O.
      Functional and structural characterization of the mammalian TREX-2 complex that links transcription with nuclear messenger RNA export.
      ). In yeast, TREX-2, composed of Sac3, Thp1, Sem1, Sus1 and Cdc3, regulates a surprisingly diverse number of chromatin-associated processes (
      • Jani D.
      • Valkov E.
      • Stewart M.
      Structural basis for binding the TREX2 complex to nuclear pores, GAL1 localisation and mRNA export.
      ,
      • Schubert T.
      • Köhler A.
      Mediator and TREX-2: Emerging links between transcription initiation and mRNA export.
      ).
      The transport receptor, TAP (Mex67 in yeast), is recruited by TREX or TREX-2 via the UAP56/Sub2 subunits (
      • Wickramasinghe V.O.
      • Laskey R.A.
      Control of mammalian gene expression by selective mRNA export.
      ), to interact with the NPC and export mRNA to the cytoplasm. TAP/Mex67 share a modular domain organization that includes the aminoterminal RNA recognition motif (RRM) followed by Nuclear Transport Factor 2-like (NTF2L) and the ubiquitin associated (UBA) (
      • Katahira J.
      • Dimitrova L.
      • Imai Y.
      • Hurt E.
      NTF2-like domain of Tap plays a critical role in cargo mRNA recognition and export.
      ). The NTF2-like superfamily is a large group of related proteins that share a common fold, first observed in the structure of the rat NTF2 protein (
      • Bullock T.L.
      • Clarkson W.D.
      • Kent H.M.
      • Stewart M.
      The 1.6 Å resolution crystal structure of nuclear transport factor 2 (NTF2).
      ,
      • Eberhardt R.Y.
      • Chang Y.
      • Bateman A.
      • Murzin A.G.
      • Axelrod H.L.
      • Hwang W.C.
      • Aravind L.
      Filling out the structural map of the NTF2-like superfamily.
      ).
      Alternatively, UAP56/Aly can also be recruited by the exon-junction complex (EJC), which is composed of eIF4AIII, MLN51 (also known as BTZ/Barentsz), Y14 and Mago (or Magoh/Mago-nashi) in mammalian cells. The EJC is loaded onto immature mRNA molecules during cis-splicing at a conserved position upstream of the exon–exon junction (
      • Hir H. Le
      • Izaurralde E.
      • Maquat L.E.
      • Moore M.J.
      The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions.
      ) to form a stable mRNA export complex and is only removed from mRNP complexes when translation is initiated in the cytoplasm; hence, the EJC accompanies an mRNA molecule throughout nuclear export (
      • Gehring N.H.
      • Lamprinaki S.
      • Kulozik A.E.
      • Hentze M.W.
      Disassembly of exon junction complexes by PYM.
      ). The EJC also acts as a platform for transient binding of many proteins that define the fate of individual mRNAs via specific maturation pathways (
      • Tange T.Ø.
      • Shibuya T.
      • Jurica M.S.
      • Moore M.J.
      Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core.
      ,
      • Andreou A.Z.
      • Klostermeier D.
      The DEAD-box helicase eIF4A paradigm or the odd one out?.
      ). For example, the EJC can bind to the mRNA export factors REF/Aly and TAP/p15 to enhance the efficiency of transport of spliced mRNAs into the cytoplasm (
      • Hir H. Le
      • Gatfield D.
      • Izaurralde E.
      • Moore M.J.
      The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay.
      ). EJC complex components have diversified in individual lineages, and not all components are retained universally, for example, MLN51 is not conserved in some trypanosomes (
      • Bercovich N.
      • Levin M.J.
      • Clayton C.
      • Vazquez M.P.
      Identification of core components of the exon junction complex in trypanosomes.
      ,
      • Bannerman B.P.
      • Kramer S.
      • Dorrell R.G.
      • Carrington M.
      Multispecies reconstructions uncover widespread conservation, and lineage-specific elaborations in eukaryotic mRNA metabolism.
      ). Some EJC components have been identified in trypanosomes, but the direct association with the mRNA export machinery remains to be demonstrated.
      Although many components of the RNA export machinery are known from animals, fungi, plants, and other organisms, in silico identification of components in trypanosomatid genomes is unreliable due to high divergence and few proteins appear conserved or recognizable (
      • Serpeloni M.
      • Vidal N.M.
      • Goldenberg S.
      • Ávila A.R.
      • Hoffmann F.G.
      Comparative genomics of proteins involved in RNA nucleocytoplasmic export.
      ). In fact, several nuclear complexes and nuclear-pore-associated proteins that play major roles in mRNA export in opisthokonts are lacking in trypanosomes, and a recent comparative review described the main differences in nuclear mRNA maturation and mRNA export (
      • Kramer S.
      Nuclear mRNA maturation and mRNA export control: From trypanosomes to opisthokonts.
      ). While UAP56/Sub2 (
      • Serpeloni M.
      • Moraes C.B.
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      • Motta M.C.M.
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      • Kessler R.L.
      • Inoue A.H.
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      • Fragoso S.P.
      • Goldenberg S.
      • Freitas-Junior L.H.
      • Ávila A.R.
      An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.
      ) and TAP/Mex67 (
      • Schwede A.
      • Manful T.
      • Jha B.A.
      • Helbig C.
      • Bercovich N.
      • Stewart M.
      • Clayton C.
      The role of deadenylation in the degradation of unstable mRNAs in trypanosomes.
      ,
      • Kramer S.
      • Kimblin N.C.
      • Carrington M.
      Genome-wide in silico screen for CCCH-type zinc finger proteins of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major.
      ,
      • Dostalova A.
      • Käser S.
      • Cristodero M.
      • Schimanski B.
      The nuclear mRNA export receptor Mex67-Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif.
      ) are essential for mRNA transport in trypanosomes, little else has been identified with significant roles (
      • Schwede A.
      • Manful T.
      • Jha B.A.
      • Helbig C.
      • Bercovich N.
      • Stewart M.
      • Clayton C.
      The role of deadenylation in the degradation of unstable mRNAs in trypanosomes.
      ,
      • Dostalova A.
      • Käser S.
      • Cristodero M.
      • Schimanski B.
      The nuclear mRNA export receptor Mex67-Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif.
      ,
      • Neumann N.
      • Lundin D.
      • Poole A.M.
      Comparative genomic evidence for a complete nuclear pore complex in the last eukaryotic common ancestor.
      ,
      • Bühlmann M.
      • Walrad P.
      • Rico E.
      • Ivens A.
      • Capewell P.
      • Naguleswaran A.
      • Roditi I.
      • Matthews K.R.
      NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway.
      ). In Trypanosoma brucei, the general mRNA export factors Mex67 and Mtr2 interact with members of the 60S ribosomal subunit (
      • Rink C.
      • Williams N.
      Unique interactions of the nuclear export receptors TbMex67 and TbMtr2 with components of the 5S ribonuclear particle in Trypanosoma brucei.
      ) and play distinct roles in tRNA export (
      • Hegedűsová E.
      • Kulkarni S.
      • Burgman B.
      • Alfonzo J.D.
      • Paris Z.
      The general mRNA exporters Mex67 and Mtr2 play distinct roles in nuclear export of tRNAs in Trypanosoma brucei.
      ), confirming the presence of unique structures and potentially parasite-specific interactions for trypanosome RNA export. Further, Mex67 associates with Ran in trypanosomes, which is distinct from animals and fungi. Trypanosomes can initiate mRNA nuclear export cotranscriptionally, and initiation of export does not depend on completion of trans-splicing, suggesting that trypanosomes regulate completion as opposed to initiation of export. This is supported by the divergence of the cytoplasmic face composition of the NPC in trypanosomes (
      • Obado S.O.
      • Brillantes M.
      • Uryu K.
      • Zhang W.
      • Ketaren N.E.
      • Chait B.T.
      • Field M.C.
      • Rout M.P.
      Interactome mapping reveals the evolutionary history of the nuclear pore complex.
      ) together with cytoplasmic nuclear peripheral granules (NPGs), but the components of both the NPC and export machinery responsible await identification (
      • Goos C.
      • Dejung M.
      • Wehman A.M.
      • M-natus E.
      • Schmidt J.
      • Sunter J.
      • Engstler M.
      • Butter F.
      • Kramer S.
      Trypanosomes can initiate nuclear export co-transcriptionally.
      ).
      Progress in characterizing the trypanosome NPC, kinetochores, and nuclear lamina has been achieved mainly through proteomics, essentially the only available approach due to high primary structure divergence (
      • Obado S.O.
      • Brillantes M.
      • Uryu K.
      • Zhang W.
      • Ketaren N.E.
      • Chait B.T.
      • Field M.C.
      • Rout M.P.
      Interactome mapping reveals the evolutionary history of the nuclear pore complex.
      ,
      • DuBois K.N.
      • Alsford S.
      • Holden J.M.
      • Buisson J.
      • Swiderski M.
      • Bart J.M.
      • Ratushny A.V.
      • Wan Y.
      • Bastin P.
      • Barry J.D.
      • Navarro M.
      • Horn D.
      • Aitchison J.D.
      • Rout M.P.
      • Field M.C.
      NUP-1 is a large coiled-coil nucleoskeletal protein in trypanosomes with lamin-like functions.
      ,
      • Akiyoshi B.
      • Gull K.
      Discovery of unconventional kinetochores in kinetoplastids.
      ,
      • D’Archivio S.
      • Wickstead B.
      Trypanosome outer kinetochore proteins suggest conservation of chromosome segregation machinery across eukaryotes.
      ). Here we have also explored proteomics with cryomilling and immunoisolation to identify proteins associated with validated components of the mRNA export pathway in T. cruzi, TcSub2, and TceIF4AIII. TcSub2 is a TREX complex protein involved in mRNA export (
      • Serpeloni M.
      • Moraes C.B.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Ramos A.S.P.
      • Kessler R.L.
      • Inoue A.H.
      • DaRocha W.D.
      • Yamada-Ogatta S.F.
      • Fragoso S.P.
      • Goldenberg S.
      • Freitas-Junior L.H.
      • Ávila A.R.
      An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.
      ), while TceIF4AIII is an RNA helicase that shuttles between the nucleus and cytoplasm via an Mex67-dependent route (
      • Inoue A.H.
      • Serpeloni M.
      • Hiraiwa P.M.
      • Yamada-Ogatta S.F.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Vidal N.M.
      • Goldenberg S.
      • Avila A.R.
      Identification of a novel nucleocytoplasmic shuttling RNA helicase of trypanosomes.
      ). We identify not only conserved components but also proteins specific to trypanosomatids. Among them, we identified a kinetoplastid-specific NTF2-like protein that interacts with Mex67, crucial for mRNA export. These observations indicate considerable divergence in the composition and function of trypanosome mRNA export factors.

      Experimental Procedures

      Parasite Culture

      T. cruzi Dm28c epimastigotes were maintained in axenic culture in liver infusion tryptose (LIT) medium at 28 °C (
      • Contreras V.T.
      • Salles J.M.
      • Thomas N.
      • Morel C.M.
      • Goldenberg S.
      In vitro differentiation of Trypanosoma cruzi under chemically defined conditions.
      ). Procyclic forms of T. brucei Lister 427 and RNA interference T. brucei 29-13 line were maintained in SDM-79 medium supplemented with 10% fetal bovine serum at 28 °C (
      • Brun R.
      • Schonenberger M.
      Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication.
      ). For T. brucei 29-13, medium was also supplemented with G418 (15 μg/ml) and hygromycin (50 μg/ml).

      Affinity Purification of Protein Complexes

      Anti-GFP nanobodies were expressed and purified (
      • Fridy P.C.
      • Li Y.
      • Keegan S.
      • Thompson M.K.
      • Nudelman I.
      • Scheid J.F.
      • Oeffinger M.
      • Nussenzweig M.C.
      • Fenyö D.
      • Chait B.T.
      • Rout M.P.
      A robust pipeline for rapid production of versatile nanobody repertoires.
      ). Antibodies (Sigma) or anti-GFP nanobodies were coupled to Dynabeads M-270 epoxy (Life technologies) (
      • Obado S.O.
      • Field M.C.
      • Chait B.T.
      • Rout M.P.
      High-efficiency isolation of nuclear envelope protein complexes from trypanosomes.
      ). T. cruzi cell extracts were prepared by cryomilling with a Planetary Ball PM100 (RETSCH, UK) and affinity purification of GFP-tagged proteins (
      • Obado S.O.
      • Field M.C.
      • Chait B.T.
      • Rout M.P.
      High-efficiency isolation of nuclear envelope protein complexes from trypanosomes.
      ). Pullout of TcSub2::GFP was achieved using a buffer containing 20 mM HEPES pH 7.4, 10 μM CaCl2, 1 mM MgCl2, 10 mM sodium citrate, 0.1% CHAPS (w/v), and protease inhibitors (COMPLETE Mini Protease inhibitor cocktail tablet, Roche); for TceIF4AIII, TcMago, TcNTF2L, and TcMex67 a buffer with 20 mM HEPES pH 7.4, 10 μM CaCl2, 1 mM MgCl2, 50 mM sodium citrate, 0.1% Triton X-100 (v/v), and protease inhibitors (COMPLETE Mini Protease inhibitor cocktail tablet, Roche); and for TcFOP and TcAPI5, a buffer with 20 mM HEPES pH 7.4, 10 μM CaCl2, 1 mM MgCl2, 50 mM sodium citrate, 0.1% Triton X-100 (v/v), and 10% glycerol (v/v). Aliquots of purified complexes were separated on 4 to 12% NuPAGE Novex Bis-Tris precast gels (Life technologies) and stained with SilverQuest Silver Staining (Life technologies).
      For TcFOP, TcAPI5, TcNTF2L, and TcHYP, the complex was analyzed by mass spectrometry. T. cruzi cell extracts were prepared using 5 × 108 parasites lysed in 1 ml of citrate buffer (50 mM sodium citrate, 20 mM Hepes pH 7.4, 1 mM MgCl2, 1 μM CaCl2, 0,5% Triton X-100, protease inhibitor (COMPLETE Mini Protease inhibitor cocktail tablet, Roche) by sonication. Pullout of TcAPI5::GFP, TcFOP::GFP, TcNTF2L::GFP, and TcHYP::GFP was achieved by affinity purification of GFP-tagged proteins (
      • Obado S.O.
      • Field M.C.
      • Chait B.T.
      • Rout M.P.
      High-efficiency isolation of nuclear envelope protein complexes from trypanosomes.
      ). For TcFOP and TcAPI5 pullout, 10% glycerol was added to citrate buffer. The complexes were eluted using 15 μl of elution buffer (2% SDS, 20 mM Tris-HCl pH 8.0) at 72 °C for 20 min. Aliquots of purified complexes were separated on 13% SDS-PAGE and stained with Silver Staining protocol.

      Proteomics: Experimental Design and Statistical Rationale

      Initially, TcSub2 and TceIF4AIII complexes were affinity purified and, for those analyses, three different technical replicates were performed for each TcSub2::GFP or TceIF4AIII::GFP complex. In each experiment, two samples were compared: TcSub2::GFP or TceIF4AIII::GFP pullout with a wild-type pullout (control). Next, samples were processed and identified by LC-MS/MS as previously described. Proteins were compared between TcSub2 and TceIF4AIII complex with the control samples, and only proteins identified in at least two replicates and with emPAI index >0.1 were considered for analyses.
      For TcMex67, TcAPI5, TcFOP, TcNTF2L, and TcHYP complexes, different replicate numbers were processed or reprocessed in different experiments to increase the reliability and the coverage. Three biological replicates were used for TcMex67 analyses. For TcAPI5, seven replicates were used: three biological replicates, but technical triplicates were performed for two biological replicates. For FOP, TcNTF2L, and TcHYP, five technical replicates were used for each protein: three biological replicates, but technical replicates were performed for one biological replicates. In each replicate, two samples were compared: target pullout and wild-type pullout (control). The TcMex67, TcAPI5, TcFOP, TcNTF2L, and TcHYP data analysis was performed based on the averages of all replicates and comparing the protein intensities between the target and control samples. The fold change ≥2 was considered to select the proteins.

      Mass-Spectrometry-Based Proteomics

      For TcSub2 and TceIF4AIII samples, mass spectrometry was performed at the FingerPrints Proteomics Facility at the University of Dundee (Scotland, UK). Samples were purified and desalted by electrophoresis into 4 to 12% NuPAGE Novex Bis-Tris precast gel (Life technologies), silver-stained, excised, and dehydrated in a SpeedVac (Thermo Scientific). The dehydrated gel was digested with trypsin (Modified Sequencing Grade, Roche) for 18 h at 30 °C. Tryptic peptides were extracted from the gel with 95% Acetonitrile/5% Formic acid (v/v) and captured with an Acclaim PepMap 100 system (C18, 100 μm × 2 cm) and fractionated on a C18 Easy-Spray PepMap RSLC (75 μm × 50 cm) (Thermo Scientific) columns with an Ultimate 3000 RS LCnano system (Thermo Scientific) coupled to an LTQ OrbiTrap Velos Pro (Thermo Scientific). For identification of proteins, the Mascot Search Daemon (Version 2.4.1) (Matrix Science) (http://www.matrixscience.com/) was used with a custom T. cruzi protein sequence library of 54,610 sequences from five different strains (CL Brener Esmeraldo-like, CL Brener non-Esmeraldo-like, Sylvio, Dm28c, and Marinkellei, downloaded on February 4, 2015 from Uniprot). This strategy increased coverage and hence the likelihood of identification of peptides. MS tolerance allowed up to two missed and/or nonspecific tryptic cleavages, and peptide mass tolerance and fragment mass tolerance were set as ±10 ppm and ±0.6 Da, respectively. Carbamidomethylation (C) was set as a fixed modification, and Acetyl (N-term), Dioxidation (M), Glu->pyro-Glu (N-term E), Oxidation (M) were included as variable modifications. FDR was calculated using a reverse translated proteome. This target-decoy strategy uses along with the target database a decoy database (randomized database, reverse target database used in maxquant). The number of matches to this decoy database equals the number of random matches (false positives) obtained in the target database and is used to calculate local or global FDRs. The relative abundance of proteins was estimated using the exponentially modified protein abundance index (emPAI score), determined in a peptide mixture based on the coverage of peptides from the identified protein (
      • Ishihama Y.
      • Oda Y.
      • Tabata T.
      • Sato T.
      • Nagasu T.
      • Rappsilber J.
      • Mann M.
      Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.
      ) with a 1% FDR and significance p value ≤0.05.
      TcMex67, TcAPI5, TcFOP, TcNTF2L, and TcHYP samples were processed at the mass spectrometry facility at the Carlos Chagas Institute/FIOCRUZ-PR (RPT02H PDTIS/Instituto Carlos Chagas—Fiocruz Paraná, Brazil). Samples were purified by electrophoresis into SDS–polyacrylamide gels; lanes were excised and dehydrated with 100% ethanol and dried in a vacuum centrifuge. After reduction with 10 mM DTT, 50 mM ammonium bicarbonate (ABC), samples were alkylated with 50 mM iodacetamide, 50 mM ABC. Gels were washed with 50 mM ABC and dehydrated with 100% ethanol, and this step was repeated. Then, gels were incubated with 12.5 ng/ml trypsin (Promega, V5113), 50 mM ABC at 37 °C for 18 h. After trypsinization, trifluoroacetic acid (TFA) was added to a final concentration of 0.5%. Peptides were extracted from the gel matrix through incubation twice with 30% MeCN, 3% TFA, and twice with 100% MeCN. The extracted peptide solution was concentrated in a vacuum centrifuge to remove MeCN and desalted with homemade C18 spin columns and analyzed by LC-MS/MS in a Thermo Scientific Easy-nLC 1000 system coupled to a LTQ Orbitrap XL ETD. Peptide separation was carried out in a 30 cm (75 μm inner diameter) fused silica in-house packed column with reverse-phase ReproSil-Pur C18-AQ 1.9 μm resin (Dr Maisch GmbH). Chromatography runs were performed with a flow rate of 250 nl/min from 5 to 40% MeCN in 0.1% formic acid, 5% DMSO in a 120 min gradient. The mass spectrometer operated in a data-dependent mode to automatically switch between MS and MS/MS (MS2) acquisition. Survey full-scan MS spectra (at 300–2000 m/z range) were acquired in the Orbitrap analyzer with resolution of 60,000 at m/z 400 (after accumulation to a target value of 1,000,000 in the C-trap). The 12 most intense ions were sequentially isolated and fragmented in the linear ion trap using collision-induced dissociation at a target value of 30,000. The “lock mass” option was enabled in all full scans to improve mass accuracy of precursor ions (
      • Olsen J.V.
      • de Godoy L.M.F.
      • Li G.
      • Macek B.
      • Mortensen P.
      • Pesch R.
      • Makarov A.
      • Lange O.
      • Horning S.
      • Mann M.
      Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap.
      ). Peaklist picking, protein identification, quantification, and validation were done using the MaxQuant platform (version 1.5.5.1) (
      • Cox J.
      • Mann M.
      MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
      ), which includes the algorithm Andromeda (
      • Cox J.
      • Neuhauser N.
      • Michalski A.
      • Scheltema R.A.
      • Olsen J.V.
      • Mann M.
      Andromeda: A peptide search engine integrated into the MaxQuant environment.
      ) for database searching. Default parameters of the software were used for all analysis steps, unless stated otherwise. Proteins were searched against a “decoy database” prepared by reversing the sequence of each entry of the T. cruzi CL Brener protein sequence database (containing 19,242 protein sequences, downloaded on August 10, 2016 from Uniprot) and appending them to the forward sequences. This database was complemented with frequently observed contaminants (porcine trypsin, Achromobacter lyticus lysyl endopeptidase, and human keratins) and their reversed sequences. Search parameters specified an MS tolerance of 20 ppm, an MS/MS tolerance of 0.5 Da, and full trypsin specificity, allowing for up to two missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine and N-terminal acetylation (protein) were allowed as variable modifications.
      For validation of the identifications, a minimum of seven amino acids for peptide length and two peptides per protein was required. In addition, an FDR threshold of 1% was applied at both peptide and protein levels. When transforming peptide identifications into protein identifications, similar protein sequences (e.g., isoforms) present in the database that could not be distinguished by the experimentally detected peptides were grouped and referred to as protein groups. Protein quantification was performed using a label-free approach, where peptides eluting from each LC run are detected as three-dimensional features—retention time versus signal intensity (extracted ion chromatogram, XIC) versus mass/charge—aligned and compared across runs, as previously described (
      • Luber C.A.
      • Cox J.
      • Lauterbach H.
      • Fancke B.
      • Selbach M.
      • Tschopp J.
      • Akira S.
      • Wiegand M.
      • Hochrein H.
      • O’Keeffe M.
      • Mann M.
      Quantitative proteomics reveals subset-specific viral recognition in dendritic cells.
      ).
      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (https://www.ebi.ac.uk/pride/) partner repository with the dataset identifier PXD028072.

      Bioinformatic Analyses

      For TceIF4AIII phylogenetic analysis, database searches were done using BLASTp (
      • Altschul S.F.
      • Madden T.L.
      • Schäffer A.A.
      • Zhang J.
      • Zhang Z.
      • Miller W.
      • Lipman D.J.
      Gapped BLAST and PSI-BLAST: A new generation of protein database search programs.
      ) and ortholog proteins identified by reciprocal best hit (RBH). Protein sequences from representative species were obtained from the NCBI database and included proteomes from Saccharomyces cerevisiae (fungi), Homo sapiens (metazoa), Dictyostelium discoideum (amoebozoa), Arabidopsis thaliana (Viridiplantae), Plasmodium falciparum (Chromalveolata), Toxoplasma gondii (Chromalveolata), T. cruzi (Excavata), T. brucei (Excavata), and Leishmania major (Excavata). For calculating the similarity and identity among amino acid sequences (supplemental Table S1, sheet2), the Needle program from the EMBOSS package was used (
      • Rice P.
      • Longden I.
      • Bleasby A.
      EMBOSS: The European Molecular Biology Open Software Suite.
      ), which allows a global alignment between two protein sequences. Phylogenetic analysis was performed using maximum likelihood (ML) by FastTree 2.1 using WAG model (
      • Price M.N.
      • Dehal P.S.
      • Arkin A.P.
      FastTree 2 - approximately maximum-likelihood trees for large alignments.
      ). The phylogenetic tree was drawn using FigTree v1.4 program (http://tree.bio.ed.ac.uk/software/figtree/). TcHYP ortholog proteins were identified by RBH criteria and synteny evaluation. A total of 108 positions with more reliable alignment obtained by PSI-coffee (
      • Chang J.
      • Tommaso P. Di
      • Taly J.
      • Notredame C.
      Accurate multiple sequence alignment of transmembrane proteins with PSI-Coffee.
      ) composed the final dataset. The tree was rooted using the Bodo saltans sequence. An evolutionary tree of TcHYP (TcCLB.506435.150) orthologs was inferred by using MrBayes 3.2.6 (
      • Ronquist F.
      • Teslenko M.
      • Van Der Mark P.
      • Ayres D.L.
      • Darling A.
      • Höhna S.
      • Larget B.
      • Liu L.
      • Suchard M.A.
      • Huelsenbeck J.P.
      Mrbayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space.
      ). Markov Chain Monte Carlo (MCMC) was run for 1,000,000 generations, sampling trees every 1000 generations. An ML tree (JTT +F + G + I) was also inferred using MegaX (
      • Kumar S.
      • Stecher G.
      • Li M.
      • Knyaz C.
      • Tamura K.
      Mega X: Molecular evolutionary genetics analysis across computing platforms.
      ) with 1000 bootstrap replicates. For TcAPI5, TcFOP, and TcNTF2L, T. cruzi CL Brener Non-Esmeraldo-like sequences were obtained from TriTrypDB version 33 and used as queries for BLASTp against a database containing 121 eukaryotic predicted proteomes (supplemental Table S2) with an e-value threshold of 0.1. All hits identified were subject to reverse BLASTp using an e-value threshold of 0.1. Orthology was predicted based upon by RBH criteria being the original T. cruzi query sequence. Additional hits were identified using sequences obtained from B. saltans and Dictyostelium purpureum. Predicted orthologous sequences were aligned using MUSCLE (
      • Edgar R.C.
      Muscle: Multiple sequence alignment with high accuracy and high throughput.
      ), trimmed manually, and both ML and BA phylogenetic trees were constructed using PhyML (
      • Guindon S.
      • Dufayard J.F.
      • Lefort V.
      • Anisimova M.
      • Hordijk W.
      • Gascuel O.
      New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0.
      ) and MrBayes (
      • Ronquist F.
      • Teslenko M.
      • Van Der Mark P.
      • Ayres D.L.
      • Darling A.
      • Höhna S.
      • Larget B.
      • Liu L.
      • Suchard M.A.
      • Huelsenbeck J.P.
      Mrbayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space.
      ), respectively. PhyML 3.0 was used with default settings, and a bootstrap of 1000 and MrBayes 3.2.6 was used with an MCMC generation of 800,000, Γ shape rate variation with four discreet categories and a sampling frequency of 1000 with the first quarter as burn-in. MrBayes trees were run using the CIPRES Science Gateway portal (
      • Miller M.A.
      • Pfeiffer W.
      • Schwartz T.
      Creating the CIPRES Science Gateway for inference of large phylogenetic trees.
      ). Proteins were identified by accession number in the TriTryp database (
      • Aslett M.
      • Aurrecoechea C.
      • Berriman M.
      • Brestelli J.
      • Brunk B.P.
      • Carrington M.
      • Depledge D.P.
      • Fischer S.
      • Gajria B.
      • Gao X.
      • Gardner M.J.
      • Gingle A.
      • Grant G.
      • Harb O.S.
      • Heiges M.
      • et al.
      TriTrypDB: A functional genomic resource for the trypanosomatidae.
      ). Protein domains were searched using InterProScan (
      • Jones P.
      • Binns D.
      • Chang H.Y.
      • Fraser M.
      • Li W.
      • McAnulla C.
      • McWilliam H.
      • Maslen J.
      • Mitchell A.
      • Nuka G.
      • Pesseat S.
      • Quinn A.F.
      • Sangrador-Vegas A.
      • Scheremetjew M.
      • Yong S.Y.
      • et al.
      InterProScan 5: Genome-scale protein function classification.
      ) or HMMscan from HMMER (
      • Eddy S.R.
      Profile hidden Markov models.
      ) looking for hidden Markov models (HMM) available in Pfam database 27.0 version (
      • Finn R.D.
      • Bateman A.
      • Clements J.
      • Coggill P.
      • Eberhardt R.Y.
      • Eddy S.R.
      • Heger A.
      • Hetherington K.
      • Holm L.
      • Mistry J.
      • Sonnhammer E.L.L.
      • Tate J.
      • Punta M.
      Pfam: The protein families database.
      ).
      Interactome visualization was performed using Cytoscape (available in https://cytoscape.org) from selected data from Tables 1 and 2. A static representation was generated from Cytoscape and processed using Affinity Designer (available in https://affinity.serif.com/en-gb/).
      Table 1Lists of filtered proteins identified in TceIF4AIII and TcSub2 isolates
      eIF4III isolation onlySub2 isolation onlyBoth Sub2 and eIF4III isolation
      Designation
      Protein name according to conserved and specific domain identified using INTERPRO, HMMER, PFAM, or ortholog in NCBI nr database. Bold represents kinetoplastid-specific granules, and italic represents nuclear peripheral granules.
      Location, annotation
      Location based on TrypTag, mass spectrometry, or direct experimental evidence. Restricted to either T. cruzi or T. brucei evidence, except in case of highly conserved ortholog, where evidence from animal or fungal cells is accepted.
      AccessionDesignationLocation, annotationAccessionDesignationLocation, annotationAccession
      NTF2L
      mRNA, underline trans-splicing factor.
      Cytoplasmic, RNA binding, N-terminal NTF2 domainTcCLB.511367.220BTZL
      mRNA, underline trans-splicing factor.
      Nuclear, RRM superfamilyTcCLB.508879.80Sub2
      mRNA, underline trans-splicing factor.
      Nuclear, ATP-dependent RNA helicase SUB2TcCLB.508319.40
      PABP2
      mRNA, underline trans-splicing factor.
      Cytoplasmic (granules), polyadenylate-binding proteinTcCLB.508461.140Y14
      mRNA, underline trans-splicing factor.
      Nuclear, RNA-binding protein Y14/8ATcCLB.508213.40eIF4AIII
      mRNA, underline trans-splicing factor.
      Nuclear/Nucleolar ATP-dependent RNA helicase FAL1TcCLB.506587.40
      Ce1
      rRNA/nucleolar.
      Nucleolar, RNA 3′-terminal phosphate cyclaseTcCLB.510761.30TSR1
      mRNA, underline trans-splicing factor.
      Nuclear, splicing factor TSR1, SR typeTcCLB.503715.10API5
      mRNA, underline trans-splicing factor.
      Nuclear, mRNA binding, ARM repeatsTcCLB.511807.280
      Lark-like
      mRNA, underline trans-splicing factor.
      Cytoplasmic, dual RRM RNA-binding proteinTcCLB.511727.270TSR1IP
      mRNA, underline trans-splicing factor.
      Nuclear, splicing factor ptsr1 interacting protein, SR typeTcCLB.504105.160FOP
      mRNA, underline trans-splicing factor.
      Nuclear, Zn finger CCCHTcCLB.509033.80
      U3 snRAP6
      sn/snoRNA.
      Nucleolar, U3 small nucleolar RNA-associated protein 6TcCLB.510769.30U2AF26
      mRNA, underline trans-splicing factor.
      Nuclear, U2 splicing auxiliary factorTcCLB.503577.20Mago
      mRNA, underline trans-splicing factor.
      Nuclear, Mago nashiTcCLB.506945.200
      Dhh1
      mRNA, underline trans-splicing factor.
      Cytoplasmic, ATP-dependent DEAD/H RNA helicaseTcCLB.506959.30RRM1
      mRNA, underline trans-splicing factor.
      Nuclear, RNA-binding protein SR typeTcCLB.511621.50PRP19
      mRNA, underline trans-splicing factor.
      Nuclear, PRP19 domain, WD40 repeatsTcCLB.509103.10
      ZC3H41
      mRNA, underline trans-splicing factor.
      Cytoplasmic, Zn-finger and RNA helicase, ZC3H41TcCLB.508355.330RBSR2/SR34
      mRNA, underline trans-splicing factor.
      Nuclear, spicing factor, SR typeTcCLB.503919.30Hel67/DDX3/

      DBP1
      Translation.
      Nuclear/cytoplasmic, ATP-dependent RNA helicase Hel67/DDX3TcCLB.506213.120
      ZC3H39
      mRNA, underline trans-splicing factor.
      Cytoplasmic (granules), RING and RNA-binding proteinTcCLB.506211.70RBP33
      mRNA, underline trans-splicing factor.
      Nuclear, RNA-binding protein 33, spicing factorTcCLB.508569.90DRBD2
      mRNA, underline trans-splicing factor.
      Nuclear, Double RNA-binding domain protein 2TcCLB.510755.120
      NOP5
      sn/snoRNA.
      Nucleolar, pre-snoRNA splicing protein, NOP domainTcCLB.508277.230DRBD18
      mRNA, underline trans-splicing factor.
      Nuclear, double RBD, extensive interactionsTcCLB.511727.190DRBD3
      mRNA, underline trans-splicing factor.
      Nuclear/cytoplasmic, double RNA-binding domain protein 3TcCLB.506649.80
      NOP56
      sn/snoRNA.
      Nucleolar, pre-snoRNA splicing protein, NOP domainTcCLB.506189.10ZC3H40
      mRNA, underline trans-splicing factor.
      Cytoplasmic, mRNA binding and stabilizing, Zn finger typeTcCLB.506211.60ALBA1
      Chromatin.
      ALBA1TcCLB.504001.10
      NOP136/

      BMS1
      rRNA/nucleolar.
      Nucleolar, ribosomal biogenesisTcCLB.510899.59UBP-2
      mRNA, underline trans-splicing factor.
      U-rich RNA-binding protein, mRNA destabilizerTcCLB.507093.229ALBA2
      Chromatin.
      ALBA2TcCLB.504001.20
      Tc38
      mRNA, underline trans-splicing factor.
      Nucleolar/cytoplasmic, RNA-bindingTcCLB.503833.50Hel64/

      DBP2B
      mRNA, underline trans-splicing factor.
      Nuclear/nucleolar, ATP-dependent RNA helicase DDX3X domainTcCLB.508973.50ALBA3
      Chromatin.
      ALBA3TcCLB.510877.30
      Mle
      mRNA, underline trans-splicing factor.
      Cytoplasmic, dsRNA unwindling Mle RNA helicaseTcCLB.505123.4Nucleolar RNA helicase II
      rRNA/nucleolar.
      Nucleolar, DEAD box helicase, GUCT domainTcCLB.508205.20
      eEF1a
      Translation.
      Cytoplasmic, elongation factor 1aTcCLB.511369.10SCD6
      Translation.
      Cytoplasmic, LSm domain, Trailer hitch proteinTcCLB.507093.300
      Mex67B
      Translation.
      Cytoplasmic/nuclear Mex67BTcCLB.506127.50Histone 3
      Chromatin.
      Nuclear, histone H3TcCLB.505931.50
      HypotheticalUnknown, low complexity regionsTcCLB.506435.150
      a Protein name according to conserved and specific domain identified using INTERPRO, HMMER, PFAM, or ortholog in NCBI nr database. Bold represents kinetoplastid-specific granules, and italic represents nuclear peripheral granules.
      b Location based on TrypTag, mass spectrometry, or direct experimental evidence. Restricted to either T. cruzi or T. brucei evidence, except in case of highly conserved ortholog, where evidence from animal or fungal cells is accepted.
      c mRNA, underline trans-splicing factor.
      d rRNA/nucleolar.
      e sn/snoRNA.
      f Translation.
      g Chromatin.
      Table 2Lists of filtered proteins identified in TcHyp, TcAPI5, TcFOP, and TcNTF2L isolates
      TcCLB.506435.150 isolationAPI5 isolationFOP isolationNTF2L isolation
      Designation
      Protein name according to conserved and specific domain identified using INTERPRO, HMMER, PFAM, or ortholog in NCBI nr database. Bold represents kinetoplastid-specific granules, and italic represents nuclear peripheral granules.
      Location, annotation
      Location based on TrypTag, mass spectrometry, or direct experimental evidence. Restricted to either T. cruzi or T. brucei evidence, except in case of highly conserved ortholog, where evidence from animal or fungal cells is accepted.
      AccessionDesignation
      Protein name according to conserved and specific domain identified using INTERPRO, HMMER, PFAM, or ortholog in NCBI nr database. Bold represents kinetoplastid-specific granules, and italic represents nuclear peripheral granules.
      Location, annotation
      Location based on TrypTag, mass spectrometry, or direct experimental evidence. Restricted to either T. cruzi or T. brucei evidence, except in case of highly conserved ortholog, where evidence from animal or fungal cells is accepted.
      AccessionDesignation
      Protein name according to conserved and specific domain identified using INTERPRO, HMMER, PFAM, or ortholog in NCBI nr database. Bold represents kinetoplastid-specific granules, and italic represents nuclear peripheral granules.
      Location, annotation
      Location based on TrypTag, mass spectrometry, or direct experimental evidence. Restricted to either T. cruzi or T. brucei evidence, except in case of highly conserved ortholog, where evidence from animal or fungal cells is accepted.
      AcessionDesignation
      Protein name according to conserved and specific domain identified using INTERPRO, HMMER, PFAM, or ortholog in NCBI nr database. Bold represents kinetoplastid-specific granules, and italic represents nuclear peripheral granules.
      Location, annotation
      Location based on TrypTag, mass spectrometry, or direct experimental evidence. Restricted to either T. cruzi or T. brucei evidence, except in case of highly conserved ortholog, where evidence from animal or fungal cells is accepted.
      Acession
      Hyp
      mRNA, underline trans-splicing factor.
      Unknown, low complexity regionsTcCLB.506435.150API5
      mRNA, underline trans-splicing factor.
      Nuclear, mRNA binding, ARM repeatsTcCLB.511807.280FOP
      mRNA, underline trans-splicing factor.
      Nuclear, Zn finger CCCHTcCLB.509033.80NTF2L
      mRNA, underline trans-splicing factor.
      Cytoplasmic, RNA binding, N-terminal NTF2 domainTcCLB.511367.220
      Mex67B
      mRNA, underline trans-splicing factor.
      Cytoplasmic/nuclear Mex67BTcCLB.506127.50FOP
      mRNA, underline trans-splicing factor.
      Nuclear, Zn finger CCCHTcCLB.509033.80Importin - beta
      mRNA, underline trans-splicing factor.
      importin beta-1 subunit, putative
      mRNA, underline trans-splicing factor.
      TcCLB.504105.150PABP2
      mRNA, underline trans-splicing factor.
      Cytoplasmic (granules), polyadenylate-binding proteinTcCLB.508461.140
      S27a
      rRNA/nucleolar.
      ubiquitin/ribosomal protein S27a, putativeTcCLB.510409.39Mex67B
      mRNA, underline trans-splicing factor.
      Cytoplasmic/nuclear Mex67BTcCLB.506127.50eiF4AIII
      mRNA, underline trans-splicing factor.
      Nuclear/Nucleolar ATP-dependent RNA helicase FAL1TcCLB.506587.40DRBD2
      mRNA, underline trans-splicing factor.
      Nuclear, Double RNA-binding domain protein 2TcCLB.510755.120;
      PABP1
      Translation.
      polyadenylate-binding protein 1, putativeTcCLB.506885.70eiF4AIII
      mRNA, underline trans-splicing factor.
      Nuclear/Nucleolar ATP-dependent RNA helicase FAL1TcCLB.506587.40Mago
      mRNA, underline trans-splicing factor.
      Nuclear, Mago nashiTcCLB.506945.200Tc38
      mRNA, underline trans-splicing factor.
      Nucleolar/cytoplasmic, RNA-bindingTcCLB.503833.50
      DED1
      mRNA, underline trans-splicing factor.
      ATP-dependent RNA helicase, putativeTcCLB.510661.90DRBD4
      mRNA, underline trans-splicing factor.
      polypyrimidine tract-binding protein, putativeTcCLB.511727.160NAP
      Chromatin.
      nucleosome assembly protein (NAP), putativeTcCLB.509003.10ALBA4
      Translation.
      DNA/RNA-binding protein Alba 4TcCLB.510877.40
      SUB2
      mRNA, underline trans-splicing factor.
      Nuclear, ATP-dependent RNA helicase SUB2TcCLB.508319.40Hyp
      mRNA, underline trans-splicing factor.
      Unknown, low complexity regionsTcCLB.506435.150Importin alfa
      mRNA, underline trans-splicing factor.
      Importin subunit alphaTcCLB.509057.20Hel67/DDX3/DBP1
      Translation.
      Nuclear/cytoplasmic, ATP-dependent RNA helicase Hel67/DDX3TcCLB.511285.120
      hnRNPH
      mRNA, underline trans-splicing factor.
      heterogeneous nuclear ribonucleoprotein H/F, putativeTcCLB.511109.130Mago
      mRNA, underline trans-splicing factor.
      Nuclear, Mago nashiTcCLB.506945.200DRBD4
      mRNA, underline trans-splicing factor.
      polypyrimidine tract-binding protein, putativeTcCLB.511727.160ZC3H41
      mRNA, underline trans-splicing factor.
      Cytoplasmic, Zn-finger and RNA helicase, ZC3H41TcCLB.508355.330
      ZC3H40
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.508895.60Fibrillarin
      rRNA/nucleolar.
      nucleolar RNA binding protein, putativeTcCLB.508277.230hnRNPH
      mRNA, underline trans-splicing factor.
      Heterogeneous nuclear ribonucleoprotein H/F, putativeTcCLB.511109.130DRBD4
      mRNA, underline trans-splicing factor.
      polypyrimidine tract-binding protein, putativeTcCLB.511727.160
      eiF4AIII
      mRNA, underline trans-splicing factor.
      Nuclear/Nucleolar ATP-dependent RNA helicase FAL1TcCLB.506587.40DRBD3
      mRNA, underline trans-splicing factor.
      Nuclear/cytoplasmic, double RNA-binding domain protein 3TcCLB.508349.39NRBD
      mRNA, underline trans-splicing factor.
      nuclear RNA binding domainTcCLB.511727.290RBP
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.509317.60
      RanBP1
      mRNA, underline trans-splicing factor.
      Ran-binding protein 1, putativeTcCLB.507099.30SMD3
      mRNA, underline trans-splicing factor.
      small nuclear ribonucleoprotein sm d3TcCLB.508257.150Dhh1
      mRNA, underline trans-splicing factor.
      Cytoplasmic, ATP-dependent DEAD/H RNA helicaseTcCLB.506959.30eEF1a
      Translation.
      Cytoplasmic, elongation factor 1aTcCLB.511369.10;
      DBP2A
      mRNA, underline trans-splicing factor.
      ATP-dependent RNA helicase DBP2A,TcCLB.510187.290UBP-2
      mRNA, underline trans-splicing factor.
      U-rich RNA-binding protein, mRNA destabilizerTcCLB.507093.229Hel67/DDX3/DBP1
      Translation.
      Nuclear/cytoplasmic, ATP-dependent RNA helicase Hel67/DDX3TcCLB.506213.120Nonsense Reg
      mRNA, underline trans-splicing factor.
      regulator of nonsense transcripts 1TcCLB.511317.30
      MTR2
      mRNA, underline trans-splicing factor.
      mRNA transport regulator MTR2, putativeTcCLB.508173.180SUB2
      mRNA, underline trans-splicing factor.
      Nuclear, ATP-dependent RNA helicase SUB2TcCLB.508319.40NOP5
      rRNA/nucleolar.
      Nucleolar, pre-snoRNA splicing protein, NOP domainTcCLB.508277.230ZC3H39
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.508895.50
      RiboHII
      mRNA, underline trans-splicing factor.
      ribonuclease HII, putativeTcCLB.510287.60SCD6
      Translation.
      Cytoplasmic, LSm domain, Trailer hitch proteinTcCLB.507093.300ZC3H40
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.506211.60ZC3H40
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.508895.60
      eIF5A
      Translation.
      eukaryotic translation initiation factor 5ATcCLB.506925.130NTF2L
      mRNA, underline trans-splicing factor.
      Cytoplasmic, RNA binding, N-terminal NTF2 domainTcCLB.511367.220ZC3H39
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.508895.50PABP1
      Translation.
      polyadenylate-binding protein 1, putativeTcCLB.506885.70
      snoRBP
      rRNA/nucleolar.
      nucleolar RNA-binding protein, putativeTcCLB.507649.80ZC3H40
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.508895.60DRBD3
      mRNA, underline trans-splicing factor.
      Nuclear/cytoplasmic, double RNA-binding domain protein 3TcCLB.506649.80Hel67/DDX3/DBP1
      Translation.
      Nuclear/cytoplasmic, ATP-dependent RNA helicase Hel67/DDX3TcCLB.506213.120
      Fibrillarin
      rRNA/nucleolar.
      Nucleolar RNA binding protein, putativeTcCLB.508277.230Dhh1
      mRNA, underline trans-splicing factor.
      ATP-dependent DEAD/H RNA helicase, putativeTcCLB.506959.30SCD6
      Translation.
      Cytoplasmic, LSm domain, Trailer hitch proteinTcCLB.507093.300PUM
      mRNA, underline trans-splicing factor.
      pumilio protein, putativeTcCLB.507049.199
      FOP
      mRNA, underline trans-splicing factor.
      Nuclear, Zn finger CCCHTcCLB.509033.80PABP2
      mRNA, underline trans-splicing factor.
      Cytoplasmic (granules), polyadenylate-binding proteinTcCLB.508461.140RBP42
      mRNA, underline trans-splicing factor.
      RNA-binding protein 42 (RNA-binding motif protein 42), putativeTcCLB.509167.140Ran
      mRNA, underline trans-splicing factor.
      GTP-binding nuclear protein rtb2, putativeTcCLB.509455.80
      NOP56
      sn/snoRNA.
      Nucleolar, ribosomal biogenesisTcCLB.506189.10PRP19
      mRNA, underline trans-splicing factor.
      Nuclear, PRP19 domain, WD40 repeatsTcCLB.509103.10Hyp
      mRNA, underline trans-splicing factor.
      Unknown, low complexity regionsTcCLB.506435.150PUF6
      mRNA, underline trans-splicing factor.
      pumilio/PUF RNA binding protein 6, putativeTcCLB.510125.10
      RNA Hel
      mRNA, underline trans-splicing factor.
      ATP-dependent DEAD/H RNA helicase, putativeTcCLB.507641.120Histone H3
      Chromatin.
      Nuclear, histone H3TcCLB.505931.50snoRBP
      rRNA/nucleolar.
      Nucleolar RNA-binding protein, putativeTcCLB.507649.80snoRBP
      rRNA/nucleolar.
      nucleolar RNA-binding protein, putativeTcCLB.507649.80
      DRBD2
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.510755.120RBP2
      mRNA, underline trans-splicing factor.
      DNA-directed RNA polymerase III subunit, putativeTcCLB.505997.210ZFP2
      mRNA, underline trans-splicing factor.
      zinc finger protein 2, putativeTcCLB.503989.10
      eEF1-beta
      Translation.
      translation elongation factor 1-beta, putativeTcCLB.509733.100RNA Hel II
      mRNA, underline trans-splicing factor.
      nucleolar RNA helicase II, putativeTcCLB.506123.40CAP - methyltransferase
      Translation.
      mRNA cap guanine-N7 methyltransferase, putativeTcCLB.508799.80
      snoRBP
      rRNA/nucleolar.
      nucleolar RNA-binding protein, putativeTcCLB.510859.17RBP42
      mRNA, underline trans-splicing factor.
      RNA-binding protein 42 (RNA-binding motif protein 42),TcCLB.509167.140RBP31
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.510007.30
      NTF2L
      mRNA, underline trans-splicing factor.
      Cytoplasmic, RNA binding, N-terminal NTF2 domainTcCLB.511367.220DBP2A
      mRNA, underline trans-splicing factor.
      ATP-dependent RNA helicase DBP2A, putativeTcCLB.510187.290u2af26
      mRNA, underline trans-splicing factor.
      U2 splicing auxiliary factor, putativeTcCLB.510943.60;
      PABP2
      mRNA, underline trans-splicing factor.
      Cytoplasmic (granules), polyadenylate-binding proteinTcCLB.508461.140SmD2
      mRNA, underline trans-splicing factor.
      small nuclear ribonucleoprotein SmD2, putativeTcCLB.511189.80UBP-2
      mRNA, underline trans-splicing factor.
      U-rich RNA-binding protein, mRNA destabilizerTcCLB.507093.229;
      eIF4A1
      Translation.
      Eukaryotic initiation factor 4A-1TcCLB.511585.190DRBD2
      mRNA, underline trans-splicing factor.
      Nuclear, Double RNA-binding domain protein 2TcCLB.510755.120Histone H2B
      Chromatin.
      histone H2B, putativeTcCLB.511635.20
      eEF1-gamma
      Translation.
      elongation factor 1-gamma (EF-1-gamma, pseudogene), putativeTcCLB.506459.290ALBA3
      Chromatin.
      ALBA3TcCLB.510877.30RNA Hel II
      mRNA, underline trans-splicing factor.
      nucleolar RNA helicase II, putativeTcCLB.506123.40
      DRBD2
      mRNA, underline trans-splicing factor.
      Nuclear, Double RNA-binding domain protein 2TcCLB.510755.120NTF2L
      mRNA, underline trans-splicing factor.
      Cytoplasmic, RNA binding, N-terminal NTF2 domainTcCLB.511367.220PUF2
      mRNA, underline trans-splicing factor.
      pumilio/PUF RNA binding protein 2, putativeTcCLB.511261.120
      Ran
      mRNA, underline trans-splicing factor.
      GTP-binding nuclear protein rtb2, putativeTcCLB.509455.80SUB2
      mRNA, underline trans-splicing factor.
      Nuclear, ATP-dependent RNA helicase SUB2TcCLB.508319.40eiF4AIII
      mRNA, underline trans-splicing factor.
      Nuclear/Nucleolar ATP-dependent RNA helicase FAL1TcCLB.506587.40
      RRM1
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.511621.50PABP2
      mRNA, underline trans-splicing factor.
      Cytoplasmic (granules), polyadenylate-binding proteinTcCLB.508461.140RRM1
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.511621.50
      Hel67/DDX3/DBP1
      Translation.
      ATP-dependent RNA helicase HEL67TcCLB.511285.120NOP5
      sn/snoRNA.
      Nucleolar, ribosomal biogenesisTcCLB.506189.10DED1
      mRNA, underline trans-splicing factor.
      ATP-dependent RNA helicase, putativeTcCLB.510661.90
      eEF1-alfa
      Translation.
      elongation factor 1-alpha, putativeTcCLB.510119.20RRM1
      mRNA, underline trans-splicing factor.
      RNA-binding protein, putativeTcCLB.511621.50Histone H2A
      Chromatin.
      histone H2A, putative (fragment)TcCLB.508321.11
      Histone H4
      Chromatin.
      histone H4, putativeTcCLB.508203.29API5
      mRNA, underline trans-splicing factor.
      Nuclear, mRNA binding, ARM repeatsTcCLB.511807.280PBP1
      Translation.
      PAB1-binding proteinTcCLB.511409.10
      Histone H3
      Chromatin.
      Nuclear, histone H3TcCLB.505931.50kZFP2
      Chromatin.
      poly-zinc finger protein 2, putativeTcCLB.509731.10
      Histone H2B
      Chromatin.
      histone H2B, putativeTcCLB.511635.20
      SCD6
      Translation.
      Cytoplasmic, LSm domain, Trailer hitch proteinTcCLB.507093.300
      DUF 1126
      mRNA, underline trans-splicing factor.
      Repeat of unknown function (DUF1126)/EF-hand domain pair, putativeTcCLB.510797.30
      NUP59
      mRNA, underline trans-splicing factor.
      Nucleoporin NUP59TcCLB.506301.30
      a Protein name according to conserved and specific domain identified using INTERPRO, HMMER, PFAM, or ortholog in NCBI nr database. Bold represents kinetoplastid-specific granules, and italic represents nuclear peripheral granules.
      b Location based on TrypTag, mass spectrometry, or direct experimental evidence. Restricted to either T. cruzi or T. brucei evidence, except in case of highly conserved ortholog, where evidence from animal or fungal cells is accepted.
      c mRNA, underline trans-splicing factor.
      d rRNA/nucleolar.
      e Translation.
      f Chromatin.
      g sn/snoRNA.

      Polyclonal Antibody Production

      The open reading frames of TcAPI5, TcFOP, TcNTF2L, and TcMago were amplified by PCR using specific oligonucleotide primers as shown in supplemental Table S3. T. cruzi Dm28c genomic DNA was used as template. The PCR products were cloned into the pDONR221 vector from Gateway technology (Invitrogen) and further recombination into the pDEST17 vector (Invitrogen) to produce his-tagged recombinant protein, according to the manufacturer’s protocol. Expression of recombinant protein was induced in Escherichia coli BL21 (DE3) by addition of 1 mM IPTG and incubation for 3 h at 37 °C. His-tagged protein was purified by affinity chromatography on Ni-NTA resin (Qiagen) under denaturing conditions and used to inoculate mice to produce polyclonal antibodies, according to Inoue et al. (2014) (
      • Inoue A.H.
      • Serpeloni M.
      • Hiraiwa P.M.
      • Yamada-Ogatta S.F.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Vidal N.M.
      • Goldenberg S.
      • Avila A.R.
      Identification of a novel nucleocytoplasmic shuttling RNA helicase of trypanosomes.
      ). Animals were immunized by intraperitoneal injection with approximately 50 μg of the his6-tagged protein in Freund's complete adjuvant (Sigma) for the first inoculation. For the three booster injections, we used 20 μg of the recombinant protein in Alu-Gel (Serva) administered at 2-week intervals. Antiserum was obtained 5 days after the last booster injection and by blood collection via cardiac puncture. The protocol was approved by Ethics Commission on Animal Use (CEUA) from FICRUZ, protocol 47/12-3, license LW15/13; project title: "Characterization of trypanosome proteins."

      Immunoblotting

      Proteins were separated by gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Hybond C, Amersham Biosciences). The membrane was blocked with 0.1% Tween 20 and 5% milk in phosphate-buffered saline (PBS). Primary antibodies were diluted in blocking solution at the following concentrations: mouse anti-TceIF4AIII (diluted 1:500) (
      • Inoue A.H.
      • Serpeloni M.
      • Hiraiwa P.M.
      • Yamada-Ogatta S.F.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Vidal N.M.
      • Goldenberg S.
      • Avila A.R.
      Identification of a novel nucleocytoplasmic shuttling RNA helicase of trypanosomes.
      ), rabbit anti-Protein A (Sigma-Aldrich, diluted 1:40,000), rabbit anti-TcSub2 (diluted 1:1000) (
      • Serpeloni M.
      • Moraes C.B.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Ramos A.S.P.
      • Kessler R.L.
      • Inoue A.H.
      • DaRocha W.D.
      • Yamada-Ogatta S.F.
      • Fragoso S.P.
      • Goldenberg S.
      • Freitas-Junior L.H.
      • Ávila A.R.
      An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.
      ), mouse anti-TcAPI5 (diluted 1:500), mouse anti-TcFOP (diluted 1:500), mouse anti-TcNTF2L (diluted 1:500), mouse anti-TcMago (diluted 1:500), rabbit anti-GFP (kindly provided by Dr Stenio Fragoso Perdigão, diluted 1:500), mouse anti-GFP (Roche, diluted 1:500), rat anti-HA (Roche, diluted 1:500). Antibodies were incubated with the membrane for 1 h. The membrane was washed three times in 0.1% Tween 20 in PBS. Bound antibodies were detected by fluorescence with IRDye whole IgG secondary antibody (LI-COR). Relative quantification of Western blots was performed using ImageJ software (available in https://imagej.nih.gov/ij/).

      Protein Tagging

      The coding sequences of TceIF4AIII, TcMago, TcAPI5, TcHYP, TcFOP, and TcFOPlΔFOP, a mutated version of TcFOP without the FoP domain, cloned previously into pDONR221 (Invitrogen) were recombined pTcGW (
      • Batista M.
      • Marchini F.K.
      • Celedon P.A.F.
      • Fragoso S.P.
      • Probst C.M.
      • Preti H.
      • Ozaki L.S.
      • Buck G.A.
      • Goldenberg S.
      • Krieger M.A.
      A high-throughput cloning system for reverse genetics in Trypanosoma cruzi.
      ) for N-terminal eGFP or PTP tagging. Similarly, the coding sequence of TcNTF2L was cloned into pTcGW1.1 (
      • Jones P.
      • Binns D.
      • Chang H.Y.
      • Fraser M.
      • Li W.
      • McAnulla C.
      • McWilliam H.
      • Maslen J.
      • Mitchell A.
      • Nuka G.
      • Pesseat S.
      • Quinn A.F.
      • Sangrador-Vegas A.
      • Scheremetjew M.
      • Yong S.Y.
      • et al.
      InterProScan 5: Genome-scale protein function classification.
      ) for C-terminal eGFP/PTP tagging. T. cruzi epimastigotes were transfected with these plasmids, and stable lines were selected by adding 500 μg/ml G418 to the culture medium. Cell lines carrying TcSub2 tagged at N-terminal with GFP and TcMex67 tagged at C-terminal with PTP were described in Serpeloni et al. (2011) (
      • Serpeloni M.
      • Moraes C.B.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Ramos A.S.P.
      • Kessler R.L.
      • Inoue A.H.
      • DaRocha W.D.
      • Yamada-Ogatta S.F.
      • Fragoso S.P.
      • Goldenberg S.
      • Freitas-Junior L.H.
      • Ávila A.R.
      An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.
      ) and Kugeratski et al. (2015) (
      • Kugeratski F.G.
      • Batista M.
      • Inoue A.H.
      • Ramos B.D.
      • Krieger M.A.
      • Marchini F.K.
      pTcGW plasmid vectors 1.1 version: A versatile tool for Trypanosoma cruzi gene characterisation.
      ), respectively. In. T. brucei, cell lines expressing HA-tagged or GFP-tagged TbMago were generated by in situ tagging with the pMOTag4H or pMOTag4G plasmid, as described previously in Oberholzer et al. (2006) (
      • Oberholzer M.
      • Morand S.
      • Kunz S.
      • Seebeck T.
      A vector series for rapid PCR-mediated C-terminal in situ tagging of Trypanosoma brucei genes.
      ). The parasites were analyzed by FACSAria II (BD) in the flow cytometry facility at Carlos Chagas Institute–Fiocruz/PR. GFP-positive parasites were cloned using single cell precision mode into 96-well plate containing 100 μl of LIT medium/well and the selection antibiotic.
      The endogenous and tagged proteins were localized by indirect immunofluorescence assays, as described by Serpeloni et al. (2011) (
      • Serpeloni M.
      • Moraes C.B.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Ramos A.S.P.
      • Kessler R.L.
      • Inoue A.H.
      • DaRocha W.D.
      • Yamada-Ogatta S.F.
      • Fragoso S.P.
      • Goldenberg S.
      • Freitas-Junior L.H.
      • Ávila A.R.
      An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.
      ). The parasites were incubated with specific antibodies for 1 h at 37 °C and then washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG or Alexa Fluor 594-conjugated goat anti-rabbit IgG antibodies (Invitrogen, 1:600 dilution), as appropriate, for 1 h. DNA was stained by incubation with 5 μg/ml DAPI for 15 min. Slides were analyzed by fluorescence microscopy (Nikon 80i), and images were captured with a CoolSnap PROcf (Media Cybernetics) camera and analyzed with Image Pro-Plus v. 4.5.1.22 (Media Cybernetics). Images were also obtained by inverted microscopy (Leica DMI6000B) associated with deconvolution software Leica AF6000 (microscope facility RPT07C PDTIS/Carlos Chagas Institute–Fiocruz Paraná).

      RNA Interference

      The coding sequences of orthologs of TcFOP (TbFOP, Gene ID Tb927.6.1470), TcAPI5 (TbAPI5, Gene ID Tb927.7.2240), TcNTF2L (TbNTF2L, Gene ID Tb927.10.2240), and TcHYP (TbHYP, Gene ID Tb927.04.3060) from T. brucei were analyzed by RNAit (
      • Redmond S.
      • Vadivelu J.
      • Field M.C.
      RNAit: An automated web-based tool for the selection of RNAi targets in Trypanosoma brucei.
      ) to identify specific target nucleotide sequences for RNA interference. The oligonucleotides predicted by RNAit were used to amplify a DNA fragment (supplemental Table S3) for cloning into the p2T7-177 vector (
      • Wickstead B.
      • Ersfeld K.
      • Gull K.
      Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei.
      ). A total of 10 μg insert-containing vector was linearized with NotI and transfected into procyclic forms of T. brucei 29-13. Transfected parasites were selected by the addition of 5 μg/ml phleomycin to the medium, and RNAi was induced by adding 2 μg/ml tetracycline to log phase parasite cultures.

      mRNA in Situ Hybridization

      To analyze the distribution of poly(A)+ RNA in T. brucei, a digoxigenin-conjugated oligo(dT) probe was used as previously protocol described in Inoue et al. (2014) (
      • Inoue A.H.
      • Serpeloni M.
      • Hiraiwa P.M.
      • Yamada-Ogatta S.F.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Vidal N.M.
      • Goldenberg S.
      • Avila A.R.
      Identification of a novel nucleocytoplasmic shuttling RNA helicase of trypanosomes.
      ). Probe binding was detected by indirect immunofluorescence analysis, as described above, with mouse monoclonal anti-digoxigenin antibody (Sigma-Aldrich, 1:300 dilution) and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Invitrogen, 1:600 dilution).

      Quantitative PCR Analysis

      T. brucei total RNA was extracted using TRIzol Reagent (Invitrogen). To remove residual DNA, the DNase treatment was performed with RQ1 (RNA Qualified) RNase-Free DNase (Promega) according to the manufacturer's instruction. RNA quantification was performed with NanoDrop One Spectrophotometer (Thermo Fisher Scientific), and 1 μg of RNA was run on a formaldehyde gel to check the integrity of RNA. To prepare templates for quantitative PCR, cDNA was synthesized from 1 μg of RNA by ImProm-II Reverse Transcription System (Promega), using random primers (Invitrogen. Real-time PCR was performed in triplicate with the SYBERSelect Master Mix (Applied Biosystems) on LightCycle 96 system (Roche).
      qPCR analysis for TbAPI5, TbFOP, TbNTF2L, and TbHYP was normalized using the data obtained by amplification of 7SL RNA. supplemental Table S3 lists the primers used for qPCR, PCR conditions were as follows: preincubation at 95 °C for 10 min, followed by 65 cycles of denaturation at 95 °C for 20 s, annealing at 55 °C for 20 s, and extension at 72 °C for 20 s, one cycle of melting at 95 °C for 60 s, 40 °C for 60 s, 65 °C for 1 s, and 97 °C for 1 s. Expression levels were calculated by Pfaffl’s method (
      • Pfaffl M.W.
      A new mathematical model for relative quantification in real-time RT-PCR.
      ).

      Transcriptome Analysis

      The TbSub2 RNAi lineage, previously obtained in Serpeloni et al. (2011) (
      • Serpeloni M.
      • Moraes C.B.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Ramos A.S.P.
      • Kessler R.L.
      • Inoue A.H.
      • DaRocha W.D.
      • Yamada-Ogatta S.F.
      • Fragoso S.P.
      • Goldenberg S.
      • Freitas-Junior L.H.
      • Ávila A.R.
      An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.
      ), was used to isolate RNA samples. The induction of RNAi was performed in triplicate by adding 2 μg/ml tetracycline to log phase parasite culture. Total RNA from 1 × 109 cells was isolated using RNeasy Mini Kit (Qiagen) at 24- and 48-h postinduction. RNA was treated with DNAse1 on-column using a RNase-Free DNase Set (Qiagen) following the manufacturer protocol. The integrity was determined by nanoelectrophoresis using an Agilent 2100 Bioanalyzer (Agilent) and quantified by spectrophotometry using a NanoDrop One Spectrophotometer (Thermo Scientific). Approximately 5 μg RNA was sent to BGI services (China) for transcriptome sequencing using Illumina HiSeq 2000 technology (Agilent). The transcriptome data have been submitted to SRA at NCBI with the accession number SUB10190938. We used https://www.bioinformatics.babraham.ac.uk/projects/fastqc and MultiQC for quality control, and analysis was done with Snakemake script. We performed differential expression between each pair of conditions, using edgeR (specifically, exactTest), selecting only coding sequences with at least ten counts in at least one condition (mean across replicates). We used sleuth for differential expression of Salmon data, which simulates technical replicates and improves accuracy. There are 3736 transcripts “differentially expressed” across all three conditions (that is changed in at least one condition) out of 8485 total transcripts analyzed.

      Results

      TcSub2 and TceIF4AIII Share Kinetoplastid-Specific Protein Interactions

      To identify components of the T. cruzi mRNA maturation machinery, we used T. cruzi Dm28c ectopically expressing N-terminal GFP fusions of TcSub2 (
      • Serpeloni M.
      • Moraes C.B.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Ramos A.S.P.
      • Kessler R.L.
      • Inoue A.H.
      • DaRocha W.D.
      • Yamada-Ogatta S.F.
      • Fragoso S.P.
      • Goldenberg S.
      • Freitas-Junior L.H.
      • Ávila A.R.
      An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.
      ) and TceIF4AIII (
      • Inoue A.H.
      • Serpeloni M.
      • Hiraiwa P.M.
      • Yamada-Ogatta S.F.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Vidal N.M.
      • Goldenberg S.
      • Avila A.R.
      Identification of a novel nucleocytoplasmic shuttling RNA helicase of trypanosomes.
      ) as bait. We used cryomilling in liquid nitrogen to preserve dynamic and low-affinity protein–protein interactions (
      • Obado S.O.
      • Field M.C.
      • Chait B.T.
      • Rout M.P.
      High-efficiency isolation of nuclear envelope protein complexes from trypanosomes.
      ). Complexes were affinity-captured using anti-GFP nanobodies conjugated to magnetic beads (
      • Fridy P.C.
      • Li Y.
      • Keegan S.
      • Thompson M.K.
      • Nudelman I.
      • Scheid J.F.
      • Oeffinger M.
      • Nussenzweig M.C.
      • Fenyö D.
      • Chait B.T.
      • Rout M.P.
      A robust pipeline for rapid production of versatile nanobody repertoires.
      ). Cell grindates were directly thawed into buffers to determine optimum conditions for preservation and isolation of complexes. Following analysis by SDS-PAGE (Fig. 1A), where we confirmed the enrichment of the tagged-proteins, TcSub2 and TceIF4AIII isolates were analyzed by mass spectrometry and protein identifications filtered according to exponentially modified protein abundance index (emPAI). Proteins from three independent experiments with emPAI >0.1, and identified in at least two of the three replicates, are listed in the supplemental material (supplemental Table S4). In addition to proteins known to be involved with RNA metabolism, many additional proteins were recovered among the significant identifications (Table 1).
      Figure thumbnail gr1
      Fig. 1Identification of proteins copurified with TcSub2 and TceIF4AIII. A, affinity purified TcSub2 (Sub2) and TceIF4AIII complexes were analyzed on 4 to 12% NuPAGE Novex Bis-Tris precast gel (Life technologies) and stained with SilverQuest Silver Stain (Life technologies). As a negative control, cryomilled cell extracts from wild-type (WT) parasites were treated in the same conditions. Arrowheads indicate the bands corresponding to TcSub2 and TceIF4AIII. B, schematic representation of selected proteins identified by mass spectrometry: Trypanosome EJC proteins (TcMago, TcY14 and TcBTZL) and three novel proteins (TcAPI5, TcFOP, and TcNTF2L). The motifs and domains were predicted using INTERPRO, HMMER, and PFAM databases and are shown in colors.
      There was considerable coincidence in the proteins interacting with TcSub2 and TceIF4AIII, together with specific interactions (Table 1). The coincident identifications suggest that TcSub2 and TceIF4AIII associate as a supercomplex, further supported by copurification of TcSub2 and TceIF4AIII. The TcSub2/TceIF4AIII cohort includes orthologs of PRP19-like, Dbp1, Alba1-3, and orthologs of DRBD2 and 3, all of which have defined functions in mRNA metabolism (
      • Mani J.
      • Güttinger A.
      • Schimanski B.
      • Heller M.
      • Acosta-Serrano A.
      • Pescher P.
      • Späth G.
      • Roditi I.
      Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery.
      ,
      • Das A.
      • Bellofatto V.
      • Rosenfeld J.
      • Carrington M.
      • Romero-zaliz R.
      • Estévez A.M.
      High throughput sequencing analysis of Trypanosoma brucei DRBD3/PTB1-bound mRNAs.
      ,
      • Wippel H.H.
      • Malgarin J.S.
      • Inoue A.H.
      • Leprevost F. da V.
      • Carvalho P.C.
      • Goldenberg S.
      • Alves L.R.
      Unveiling the partners of the DRBD2-mRNP complex, an RBP in Trypanosoma cruzi and ortholog to the yeast SR-protein Gbp2.
      ,
      • Pérez-díaz L.
      • Caroline T.
      • Teixeira S.M.
      Involvement of an RNA binding protein containing Alba domain in the stage-specific regulation of beta-amastin expression in Trypanosoma cruzi.
      ,
      • Subota I.
      • Rotureau B.
      • Blisnick T.
      • Ngwabyt S.
      • Durand-Dubief M.
      • Engstler M.
      • Bastin P.
      ALBA proteins are stage regulated during trypanosome development in the tsetse fly and participate in differentiation.
      ). Significantly, components of TREX or TREX-2 were not identified, in accordance with their apparent absence from trypanosomatid genomes (
      • Serpeloni M.
      • Vidal N.M.
      • Goldenberg S.
      • Ávila A.R.
      • Hoffmann F.G.
      Comparative genomics of proteins involved in RNA nucleocytoplasmic export.
      ). However, proteins associated with chromatin and splicing were identified, including TSR1, TSR1IP, U2AF26, and SR34 (
      • Gupta S.K.
      • Chikne V.
      • Eliaz D.
      • Tkacz I.D.
      • Naboishchikov I.
      • Carmi S.
      • Ben-Asher H.W.
      • Michaeli S.
      Two splicing factors carrying serine-arginine Two splicing factors carrying serine-arginine motifs, TSR1 and TSR1IP, regulate splicing, mRNA stability, and rRNA processing in Trypanosoma brucei.
      ,
      • Holden J.M.
      • Koreny L.
      • Obado S.
      • Ratushny A.V.
      • Chen W.
      • Bart J.
      • Navarro M.
      • Chait B.T.
      • Aitchison J.D.
      • Rout M.P.
      • Field M.C.
      Involvement in surface antigen expression by a moonlighting FG-repeat nucleoporin in trypanosomes.
      ,
      • Elvira-Matelot E.
      • Bardou F.
      • Ariel F.
      • Jauvion V.
      • Bouteiller N.
      • Masson L.
      • Cao J.
      • Crespi M.D.
      • Vaucheret H.
      The nuclear ribonucleoprotein SmD1 interplays with splicing, RNA quality control, and posttranscriptional gene silencing in Arabidopsis.
      ,
      • Jiang J.
      • Liu X.
      • Liu C.
      • Liu G.
      • Li S.
      • Wang L.
      Integrating omics and alternative splicing reveals insights into grape response to high temperature 1 [OPEN].
      ), which interact with quality control (QC) components and the NPC.
      In addition, proteins involved in ribosome biogenesis, SDA1 (
      • Buscemi G.
      • Saracino F.
      • Masnada D.
      • Carbone M.L.A.
      The Saccharomyces cerevisiae SDA1 gene is required for actin cytoskeleton organization and cell cycle progression.
      ) and NOP5 (
      • Michaeli S.
      ), as well as RNA granule associators, SCD6 (
      • Krüger T.
      • Hofweber M.
      • Kramer S.
      • Strome S.
      SCD6 induces ribonucleoprotein granule formation in trypanosomes in a translation- independent manner, regulated by its Lsm and RGG domains.
      ,
      • Cristodero M.
      • Schimanski B.
      • Heller M.
      • Roditi I.
      Functional characterization of the trypanosome translational repressor SCD6.
      ) and TcDHH1 (
      • Kramer S.
      • Marnef A.
      • Standart N.
      • Carrington M.
      Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate.
      ,
      • Holetz F.B.
      • Correa A.
      • Avila A.R.
      • Nakamura C.V.
      • Krieger M.A.
      • Goldenberg S.
      Evidence of P-body-like structures in Trypanosoma cruzi.
      ), were also detected, indicating an interplay of transcription/mRNA export with other RNA metabolism pathways. The domain architectures of several hypothetical proteins were predicted and include representatives containing an FOP domain (TcFOP), a dual API5 domain (TcAPI5), and an NTF2-like domain (TcNTF2L), while others were identified as core components of the EJC (Fig. 1B). Phylogenetic reconstructions demonstrated that these proteins are restricted to the trypanosomatids and eubodonids and hence are likely kinetoplastid-specific mRNA export factors (Fig. 2 and supplemental Fig. S1). Among them, only NTF2L has been already described in trypanosomes as a component of mRNA export, both associated to EJC complex (
      • Bercovich N.
      • Levin M.J.
      • Clayton C.
      • Vazquez M.P.
      Identification of core components of the exon junction complex in trypanosomes.
      ) and to nuclear peripherical granules (
      • Goos C.
      • Dejung M.
      • Wehman A.M.
      • M-natus E.
      • Schmidt J.
      • Sunter J.
      • Engstler M.
      • Butter F.
      • Kramer S.
      Trypanosomes can initiate nuclear export co-transcriptionally.
      ).
      Figure thumbnail gr2
      Fig. 2TcNTF2L orthologs were found in trypanosomatids and Eubodonida. A list of TcNTF2L homologs and the organisms searched are shown in . Red arrow head indicates Euglena, which is outside of the Kinetoplastoda lineage but a relative. Colors represent homolog proteins from representative species of distinct eukaryotic groups, which grouped into a distinct clade from TcNTF2L. Near the nodes are Bayesian posterior probabilities and ML bootstrap values. Bar indicates the estimated number of changed sites.
      For TcNTF2L, while the N-terminal NTF2-like domain is broadly conserved, the overall architecture is clearly kinetoplastid-specific. Other proteins correspond to the human Ran GTPase-activating protein-binding protein 1 and 2 (Q13283 and Q9UN86) and contain an RNA recognition motif (RRM) in the C-terminal region absent from the kinetoplastid NTF2L proteins (supplemental Table S2, sheet 3). Phylogenetic reconstructions for NTF2L (Fig. 2) recovered two major clades, one containing only kinetoplastid NTF2L proteins and the other all other sequences, consistent with unique pathways for mRNA export (
      • Ebenezer T.E.
      • Carrington M.
      • Lebert M.
      • Kelly S.
      • Field M.C.
      Euglena gracilis genome and transcriptome: Organelles, nuclear genome assembly strategies and initial features.
      ).
      To provide orthogonal evidence for the identified interactions between TcAPI5, TcFOP, and TcNTF2L with TcSub2 and TceIF4AIII, we raised antibodies to TcAPI5, TcFOP, and TcNTF2L. Western blots confirmed TcAPI5 and TcFOP as present in the TcSub2 isolates, while TcNTF2L was present in TceIF4AIII pullouts, confirming the assignments (supplemental Fig. S2).

      EJC Proteins Interact With the T. cruzi mRNA Export Pathway

      We previously described the T. cruzi ortholog of eIF4AIII (
      • Inoue A.H.
      • Serpeloni M.
      • Hiraiwa P.M.
      • Yamada-Ogatta S.F.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Vidal N.M.
      • Goldenberg S.
      • Avila A.R.
      Identification of a novel nucleocytoplasmic shuttling RNA helicase of trypanosomes.
      ), and phylogenetic tree indicated that TceIF4AIII grouped into the eIF4AIII clade (supplemental Fig. S3). eIF4AIII is a core component of the metazoan EJC and functions in multiple pathways of mRNA metabolism (
      • Tange T.Ø.
      • Shibuya T.
      • Jurica M.S.
      • Moore M.J.
      Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core.
      ), while the S. cerevisiae ortholog, Fal1, is involved in 40S-ribosomal-subunit biogenesis (
      • Kressler D.
      • de la Cruz J.
      • Rojo M.
      • Linder P.
      Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.
      ). Additional proteins related to the mammalian EJC core were identified here; TcMago is represented in the TceIF4AIII/TcSub2 cohort, while TcY14 was identified only in TcSub2 isolations. A protein containing a Barentsz/BTZ domain, named as TcBarentsz-like (TcBTZL), was also present among the TcSub2 interactors.
      To confirm the interaction between TcMago and TcSub2/TceIF4AIII, we created T. cruzi parasites expressing TcMago::GFP. The fusion protein localized mainly in the nucleus (Fig. 3A), and the T. brucei ortholog tagged with HA or GFP displayed a nuclear localization (supplemental Fig. S4), as described in Tryp Tag database (http://tryptag.org). TcMago localizes with TcSub2 in the nucleus (Fig. 3, A and B), and Western blot analysis of TcMago::GFP immunoprecipitations (Fig. 3D) identified both TcSub2 and TceIF4AIII (Fig. 3E), robustly confirming that conserved core EJC proteins interact with components of the trypanosome mRNA export pathway.
      Figure thumbnail gr3
      Fig. 3Localization and protein interactions of TcMago. A, cellular localization of TcSub2 (Sub2) and TcMago tagged with eGFP (Mago) by confocal microscopy using anti-GFP antibodies. Nucleus is circled in white. DAPI = DNA stained with DAPI. MERGE = merged images. N = nucleus. K = kinetoplast. The kinetoplast DNA usually displays stronger DAPI signal than nucleus. B, graphs show the quantification of fluorescence intensity detected across the dotted line in merge images. DAPI in blue, TcMago::GFP in green and TcSub2 in red. C, TcMago::GFP pullout (Mago) was analyzed in silver-stained SDS–polyacrylamide gel. As a negative control, cryomilled cell extracts from wild-type (WT) parasites were treated in the same conditions. D, TcMago::GFP isolates were analyzed by Western blot using the following antibodies: mouse anti-GFP, mouse anti- TceIF4AIII, and rabbit anti-TcSub2 and detection occurred with IRDye whole IgG secondary antibody (LI-COR).

      Kinetoplastid-Specific Proteins Are Associated With mRNA Export Pathways

      We selected four proteins identified here and which are restricted to kinetoplastids for further characterization. TcFOP, TcAPI5, and TcHYP are also localized to the nucleus, like TcSub2 (Fig. 4, Aand B and supplemental Fig. S7A) while TcNTF2L is cytoplasmic, like TceIF4AIII (Fig. 4C). To investigate the physical interactions of these proteins, we created lines expressing TcFOP, TcAPI5, TcNTF2L, and TcHYP tagged with GFP (supplemental Figs. S5 and S7A). We confirmed that TcSub2 is present in immunoprecipitates from both TcFOP::GFP (Fig. 5B) and TcAPI5::GFP, while TceIF4AIII is indeed present in TcNTF2L::GFP pullouts (Fig. 6B). To ensure that the GFP tag was not affecting TcNTF2L location, we used antibodies to TcNTF2L and observed a similar cytoplasmic localization to TcNTF2L::GFP (supplemental Fig. S5E). Altogether, these data also provide robust support for the presence of kinetoplastid-specific proteins TcFOP, TcAPI5, TcNTF2L, and TcHYP in association with conserved components within trypanosomatid mRNA export pathways.
      Figure thumbnail gr4
      Fig. 4Localizations of TcFOP, TcAPI5, and TcNTF2L. Cellular localization of (A) TcSub2 (Sub2) and TcFOP (FOP); (B) TcSub2 and TcAPI5 (API5); (C) TceIF4AIII (eIF4AIII) and TcNTF2L::GFP (NTF2L). DAPI = DNA stained with DAPI. MERGE = merged images. N = nucleus. K = kinetoplast. Bar = 10 μm (A and B) and 7.5 μm (C). At right, graphs show the quantification of fluorescence intensity detected across the dotted line in merge images.
      Figure thumbnail gr5
      Fig. 5Interactions of TcFOP depend on the FOP domain. A, TcFOP (FOP) and mutated TcFOP (FOPΔC) tagged with GFP were pulled out and the copurified proteins were analyzed on silver-stained SDS-polyacrylamide gels. As a negative control, cryomilled cell extracts from wild-type (WT) parasites were treated in the same conditions. B, the interaction with TcSub2 (Sub2) and TceIF4AIII was analyzed by Western blot. Isolates were analyzed by Western blot using the following antibodies: mouse anti-TceIF4AIII, rabbit anti-TcSub2, or mouse anti-TcFOP and detection were carried out with IRDye whole IgG secondary antibody (LI-COR). C, relative quantification by densitometry of TcSub2 signal, normalized against FOP and ΔFOP signal. Data are expressed as means and standard deviation of three independent experiments.
      Figure thumbnail gr6
      Fig. 6Protein interaction of TcAPI5 and TcNTF2L. A, copurified proteins with TcAPI5 (API5) and TcNTF2L (NTF2L) were analyzed on silver-stained SDS–polyacrylamide gels. As a negative control, cryomilled cell extracts from wild-type (WT) parasites were treated in the same conditions. B, TcAPI5 and TcNTF2L isolates were analyzed by Western blot using the following antibodies: mouse anti-TceIF4AIII, rabbit anti-TcSub2, mouse anti-TcAPI5, or mouse anti-TcNTF2L. Detection was carried out with IRDye whole IgG secondary antibody (LI-COR).
      The FOP domain of TcFOP is divergent from canonical domains (supplemental Fig. S6). In metazoa, FOP contains a conserved LDXXLDAYM UAP56-binding motif (UBM) that is duplicated in the C-terminal region (X = any amino acid) (
      • Chang C.-T.
      • Hautbergue G.M.
      • Walsh M.J.
      • Viphakone N.
      • van Dijk T.B.
      • Philipsen S.
      • Wilson S.A.
      Chtop is a component of the dynamic TREX mRNA export complex.
      ,
      • van Dijk T.B.
      • Gillemans N.
      • Stein C.
      • Fanis P.
      • Demmers J.
      • van de Corput M.
      • Essers J.
      • Grosveld F.
      • Bauer U.-M.
      • Philipsen S.
      Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases.
      ), but in the trypanosomatid FOP, the UBMs are separated by an RGG motif. We asked if these are functional for binding TcSub2, the trypanosome ortholog of the binding partner UAP56. Deletion of the C-terminal UBM from TcFOP (FOPΔC) altered interactions with TcSub2 (Fig. 5B), decreasing TcSub2 recovery in pullouts by ∼50%, indicating that despite divergence, the FOP domain does mediate interaction with TcSub2 (Fig. 5C).

      A Superinteractome Connecting Splicing, Translation, and mRNA Export

      Mex67, a major mRNA export factor, has been partially characterized in T. brucei (
      • Schwede A.
      • Manful T.
      • Jha B.A.
      • Helbig C.
      • Bercovich N.
      • Stewart M.
      • Clayton C.
      The role of deadenylation in the degradation of unstable mRNAs in trypanosomes.
      ,
      • Kramer S.
      • Kimblin N.C.
      • Carrington M.
      Genome-wide in silico screen for CCCH-type zinc finger proteins of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major.
      ,
      • Dostalova A.
      • Käser S.
      • Cristodero M.
      • Schimanski B.
      The nuclear mRNA export receptor Mex67-Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif.
      ). Using T. cruzi cells expressing TcMex67 tagged with PTP (
      • Kugeratski F.G.
      • Batista M.
      • Inoue A.H.
      • Ramos B.D.
      • Krieger M.A.
      • Marchini F.K.
      pTcGW plasmid vectors 1.1 version: A versatile tool for Trypanosoma cruzi gene characterisation.
      ) and the same conditions as the TceIF4AIII pullout (Fig. 7A), we confirmed interactions with several proteins including Mtr2 and Ran previously identified in T. brucei (
      • Dostalova A.
      • Käser S.
      • Cristodero M.
      • Schimanski B.
      The nuclear mRNA export receptor Mex67-Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif.
      ,
      • Obado S.O.
      • Brillantes M.
      • Uryu K.
      • Zhang W.
      • Ketaren N.E.
      • Chait B.T.
      • Field M.C.
      • Rout M.P.
      Interactome mapping reveals the evolutionary history of the nuclear pore complex.
      ) (supplemental Table S5). Although TceIF4AIII itself was not detected, several proteins common to the TceIF4AIII and TcSub2 interactomes were found, including PABP2, RNA helicase Hel67, and DRBD2, together with TcFOP and TcNTF2L (Fig. 7B). This further supports the presence of kinetoplastid-specific proteins as core components of the trypanosome mRNA export machinery.
      Figure thumbnail gr7
      Fig. 7Interactions of TcMex67. A, proteins copurified with TcMex67 (Mex67) were analyzed on silver-stained SDS–polyacrylamide gels. As a negative control, cryomilled cell extracts from wild-type (WT) parasites were treated in the same conditions. B, TcMex67 isolates were analyzed using the following antibodies: rabbit anti-Protein A, mouse anti-TcNTF2L, mouse anti-TceIF4AIII, mouse anti-TcFOP and detection were carried out with IRDye whole IgG secondary antibody (LI-COR).
      Analysis of the kinetoplastid-specific protein interactions (supplemental Table S6) confirmed the presence of several conserved components of the processing, export, quality control, and translation systems (Table 2). These include TcSub2, ALBA, DRBD2, NOP, Mago, TceIF4AIII, TceEF1a, and TcPABP2. Most proteins identified with TcFOP, TcAPI5, TcNTF2L, and TcHYP were also identified as TcSub2 and TceIF4AIII interactors. RNA granule proteins were also identified, including TcDhh1, TcSCD6, and TcPABP2, reinforcing connectivity between mRNA export and the cytoplasmic processing machinery. Further, we identified PUMILIO proteins, RNA-binding proteins involved in controlling gene expression (
      • Azizi H.
      • Dumas C.
      • Papadopoulou B.
      The Pumilio-domain protein PUF6 contributes to SIDER2 retroposon-mediated mRNA decay in Leishmania.
      ,
      • Dallagiovanna B.
      • Correa A.
      • Probst C.M.
      • Holetz F.
      • Smircich P.
      • De Aguiar A.M.
      • Mansur F.
      • Vieira C.
      • Mortara R.A.
      • Garat B.
      • Buck G.A.
      • Goldenberg S.
      • Krieger M.A.
      Functional genomic characterization of mRNAs associated with TcPUF6, a Pumilio-like protein from Trypanosoma cruzi.
      ,
      • Simon M.J.
      • Bindu N.
      Functions, mechanisms and regulation of Pumilio/Puf family RNA binding proteins: A comprehensive review.
      ). The intersection of the interacting proteins recovered with the six isolated proteins was processed using a Venn Diagram tool (https://bioinformatics.psb.ugent.be/cgi-bin/liste/Venn/calculate_venn.htpl), and those proteins recovered with three or more protein complexes plotted (Fig. 8). Overall, these data indicate an unexpected high level of connectivity between multiple aspects of mRNA processing and transport.
      Figure thumbnail gr8
      Fig. 8Summary of coincident interacting proteins with three or more isolated proteins. The six isolated proteins are represented in lines and interacting proteins are in columns. Black circles indicate the presence of the protein in the isolated complex.

      Phenotypic Screening by RNAi in T. brucei of New mRNA Export Factors

      We analyze the roles of TcFOP, TcAPI5, and TcNTF2L by silencing their T. brucei orthologs. Quantitative PCR confirmed the efficiency of RNAi against all targets (Fig. 9A). Knockdown of TbAPI5 and TbNTF2L affected proliferation, while knockdown of TbFOP had little proliferative impact (Fig. 9B). None of the proteins appear involved in global export of mRNAs since accumulation of mRNA in the nucleus by polyA FISH was not seen (Fig. 9C). Two different controls (without probe and RNAse treatment) confirmed the specificity of probe signal (supplemental Fig. S8). Additionally, we also analyzed TbHYP knockdown and obtained a similar phenotype to TbAPI5, TbFOP, and TbNTF2L with no obvious inhibition of global mRNA export (supplemental Fig. S7G). Hence, these new proteins may be mediating the processing of a subset of RNA molecules or that their functions are redundant.
      Figure thumbnail gr9
      Fig. 9TbFOP, TbAPI5, and TbNTF2L are nonessential proteins. A, real-time PCR analysis using noninduced (N) and induced parasites (I) for RNAi of TbFOP (FOP), TbAPI5 (API5), and TbNTF2L (NTF2L). The relative mRNA levels were quantified at 72 h postinduction and normalized by detection of 7SL RNA. B, growth curve of cell lines after RNAi induction for TbFOP, TbAPI5 and TbNTF2L. The density of cells in the culture was determined by triplicate counting in a particle counter (Beckman Coulter, Z Series Coulter Counter Cell). Both noninduced and induced cultures were diluted daily to 5 × 105 cells/ml for all RNAi inductions. C, cellular localization of mRNA by fluorescence in situ hybridization (FISH) in noninduced (Con) and induced (Ind) parasites. Digoxigenin-conjugated oligo(dT) was used as probe. DAPI = DNA stained with DAPI. N = nucleus. K = kinetoplast. Bar = 10 μm.

      Sub2 Knockdown Leads to Restricted Impact on the Transcriptome

      TbSub2 is the sole protein analyzed whose absence clearly blocked mRNA export (
      • Serpeloni M.
      • Moraes C.B.
      • Muniz J.R.C.
      • Motta M.C.M.
      • Ramos A.S.P.
      • Kessler R.L.
      • Inoue A.H.
      • DaRocha W.D.
      • Yamada-Ogatta S.F.
      • Fragoso S.P.
      • Goldenberg S.
      • Freitas-Junior L.H.
      • Ávila A.R.
      An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.
      ). The impact of TbSub2 knockdown on the transcriptome was restricted and given a supposed central role in mRNA maturation, surprising. Only seven genes demonstrated highly significant increases in abundance (Fig. 10), of which three correspond to 28S and 16S rRNAs (Fig. 11) and the remaining four are protein-encoding. The top protein hits Tb927.7.6960, Tb927.5.680, and Tb927.4.910, with the fourth (Tb927.9.7290) corresponding to a VSG pseudogene. Tb927.7.6960 is an E2 ubiquitin ligase with a Ubc2 domain, while Tb927.5.680 and Tb927.4.910 are hypothetical proteins, both kinetoplast-restricted; based on localization at TrypTag Tb927.5.680 is likely endolysosomal. Similarly, few genes were found to have decreased associated transcript levels, and there was no obvious functional or other feature uniting the cohort (supplemental Table S7). The possibility that TbSub2 is involved in rRNA processing is intriguing as it is associated with mRNA export and quality control in other organisms, but the presence of several nucleolar proteins in our interactome would be consistent with an expanded role or close associations between proteins of the mRNA and rRNA export pathways.
      Figure thumbnail gr10
      Fig. 10TbSub2 is involved in a small number of RNA processing events. A, Manhattan plots for all RBNAseq datasets mapped against the T. brucei TREU927 genome. Reads are indicated as the y-axis and chromosome numbers as the x-axis. Note that overall, there is little major variation in the overall transcriptional pattern. B, Volcano plot of the same dataset. Solid red line indicates a log fold change of zero, and the dotted red line a significance of 2.0, above which data are considered robust. C, ratio of fold change (linear) against rank order; only seven transcripts demonstrate a greater than fivefold increase. Inset data for Sub2, the knockdown target. D, plots for the seven most increased transcripts, where green dots are rRNA and blue correspond to protein-coding transcripts. Identities of the transcripts are indicated, and data are available in full as . A ratio of 1.0 indicates no change.
      Figure thumbnail gr11
      Fig. 1128S and 16S RNA levels dramatically increase on Sub2 silencing. Screengrabs from IG Viewer showing two regions of the 28S rRNA loci reads mapped to the T. brucei TREU927 genome. Top two systems represent induced samples in each panel, and lower three are unindexed control cells. Genes in bold are rRNA genes, and those in regular font are protein coding genes. Note that the protein coding genes are unchanged in terms of reads mapped. A, region of chromosome 3. B, region of chromosome 2. Annotation taken rom TritrypDB.

      Discussion

      Messenger RNA maturation requires successful completion of multiple processes, including splicing, polyadenylation, 5′-cap modification, and export to the cytoplasm prior to translation. These steps ensure that only mRNA molecules encoding native proteins are translated, preventing accumulation of unfolded proteins and also supporting differential expression (
      • Siegel T.N.
      • Gunasekera K.
      • Cross G.A.
      • Ochsenreiter T.
      Gene expression in Trypanosoma brucei: Lessons from high throughput RNA sequencing studies.
      ,
      • Kramer S.
      Developmental regulation of gene expression in the absence of transcriptional control: The case of kinetoplastids.
      ,
      • Smircich P.
      • Eastman G.
      • Bispo S.
      • Duhagon M.A.
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      • Sotelo-silveira J.R.
      Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi.
      ,
      • Clayton C.E.
      Gene expression in kinetoplastids.
      ). In animals and fungi, TREX/TREX-2, the EJC, and the integrator complex (INT) are known players in mRNA processing (
      • Skaar J.R.
      • Ferris A.L.
      • Wu X.
      • Saraf A.
      • Khanna K.K.
      • Florens L.
      • Washburn M.P.
      • Hughes S.H.
      • Pagano M.
      The Integrator complex controls the termination of transcription at diverse classes of gene targets.
      ,
      • Rienzo M.
      • Casamassimi A.
      Integrator complex and transcription regulation: Recent findings and pathophysiology.
      ). NPC components, both at the nuclear basket and the cytoplasmic face, together with the Mex67/Mtr2 transport receptor and a vast repertoire of ATP-dependent RNA helicases are critically important. Many factors are widely conserved, but distinct features between lineages are clear. In Arabidopsis, several INT complex proteins have divergent architectures compared with their animal orthologs (
      • Liu Y.
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      • Kimberlin A.N.
      • Cahoon E.B.
      • Yu B.
      snRNA 3′ end processing by a CPSF73-containing complex essential for development in Arabidopsis.
      ) and multiple differences between animals and fungi are known (
      • Skaar J.R.
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      • Wu X.
      • Saraf A.
      • Khanna K.K.
      • Florens L.
      • Washburn M.P.
      • Hughes S.H.
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      The Integrator complex controls the termination of transcription at diverse classes of gene targets.
      ,
      • Rienzo M.
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      ,
      • Wu Y.
      • Albrecht T.R.
      • Baillat D.
      • Wagner E.J.
      • Tong L.
      Molecular basis for the interaction between Integrator subunits IntS9 and IntS11 and its functional importance.
      ). Trypanosomes exhibit even greater divergence as orthologs of many mRNA processing proteins are undetectable in silico.
      Established differences in mRNA processing between trypanosomatids and other eukaryotes include cotranscriptional nuclear export and polycistronic transcription, placing additional burdens on posttranscriptional systems. Additionally, trypanosomes have two PABP paralogs with distinct functions (
      • Zoltner M.
      • Krienitz N.
      • Field M.C.
      • Kramer S.
      Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA.
      ), three Mex67 paralogs (
      • Kramer S.
      • Kimblin N.C.
      • Carrington M.
      Genome-wide in silico screen for CCCH-type zinc finger proteins of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major.
      ) and major mRNA processing activity within nuclear periphery granules (NPGs) proximal to the NPC cytoplasmic face (
      • Kramer S.
      • Marnef A.
      • Standart N.
      • Carrington M.
      Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate.
      ,
      • Goos C.
      • Dejung M.
      • Wehman A.M.
      • M-natus E.
      • Schmidt J.
      • Sunter J.
      • Engstler M.
      • Butter F.
      • Kramer S.
      Trypanosomes can initiate nuclear export co-transcriptionally.
      ). Deferment of mRNA QC, at least in part to the cytoplasm, is in agreement with the absence of several proteins involved in animal/yeast mRNA maturation (
      • Goos C.
      • Dejung M.
      • Wehman A.M.
      • M-natus E.
      • Schmidt J.
      • Sunter J.
      • Engstler M.
      • Butter F.
      • Kramer S.
      Trypanosomes can initiate nuclear export co-transcriptionally.
      ,
      • Obado S.O.
      • Brillantes M.
      • Uryu K.
      • Zhang W.
      • Ketaren N.E.
      • Chait B.T.
      • Field M.C.
      • Rout M.P.
      Interactome mapping reveals the evolutionary history of the nuclear pore complex.
      ). Remarkably, using TcSub2 and TceIF4AIII as affinity handles, we uncovered an interactome encompassing chromatin-associated proteins through to NPG components and several Zn2+-finger RNA-binding proteins (Tables 1 and 2, Fig. 12). One-third of identifications were coincident between both TcSub2 and TceIF4AIII, and neither PABP1 nor any PABP1 interactor was found, indicating our data are robust and specific (
      • Zoltner M.
      • Krienitz N.
      • Field M.C.
      • Kramer S.
      Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA.
      ). Identification of components across the entire journey of mRNA processing suggests a highly integrated system.
      Figure thumbnail gr12
      Fig. 12Schematic representation of T. cruzi mRNA export machinery. A, using a cohort of six affinity handles, we identified a large interactome that illustrates multiple interactions as well as the presence of conserved and divergent proteins. Furthermore, these data validate the absence of many components that have failed to be identified in silico, including proteins of the TREX and TREX2 complexes. Cohorts are arranged as identified in the pullouts and colored by predicted function using the scheme as in and . Large boxes represent a single interactome, with the affinity handle indicated. Dotted lines between entries indicate a detected interaction. Many potential interactions and the TcMex67 isolation have been omitted for clarity. Conserved proteins include TcSub2, TceIF4AIII and multiple RNA binding proteins and EJC components. kinetoplastid-specific proteins include TcAPI5, TcFOP and TcNTF2L, and TcFOP and TcAPI5 interact directly with TcSub2. Core components of the EJC are directly associated with the mRNA machinery. TcNTF2L seems to be a shuttling protein that interacts with TceIF4AIII and the export receptor TcMex67. Connectivity data are also available as a cytoscape file for readers who wish to explore further. B, model for interactome and interactions with the NPC and export systems. Conserved proteins, such as TcSub2, TceIF4AIII, RNA binding proteins, components of the EJC, and kinetoplastid-specific proteins, such as TcAPI5, TcFOP, and TcNTF2L are shown. The nuclear proteins TcFOP and TcAPI5 interact directly with TcSub2. Core components of EJC are directly associated to mRNA machinery. TcNTF2L seems to be a shuttling protein that interacts with TceIF4AIII and the export receptor TcMex67.
      There are several functional cohorts within this TcSub2/eIF4AIII interactome, including proteins associated with chromatin, the trans-splicing machinery, and the EJC. Only a single Mex67 paralog (Mex67B) was recovered, suggesting specificity between mRNA export interactors. With TcMex67 as the affinity handle, we identified Ran, Mtr2 and PABP2 as expected, in addition to the kinetoplastid-specific proteins TcFOP and TcNTF2L, themselves also within the TcSub2/eIF4AIII interactome. A further cohort, preferentially identified with TceIF4AIII is likely involved in rRNA processing and/or ribosomal biogenesis and suggests a close connection between mRNA and rRNA pathways. Significantly, neither TcSub2 nor TceIF4AIII retrieved proteins that would constitute a canonical TREX or EJC.
      In metazoa, the EJC is deposited upstream of exon–exon boundaries during cis-splicing (
      • Hir H. Le
      • Izaurralde E.
      • Maquat L.E.
      • Moore M.J.
      The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions.
      ) and contributes to multiple activities including NMD (
      • Brogna S.
      • Wen J.
      Nonsense-mediated mRNA decay (NMD) mechanisms.
      ,
      • Kataoka N.
      • Yong J.
      • Kim V.N.
      • Velazquez F.
      • Perkinson R.A.
      • Wang F.
      • Dreyfuss G.
      Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm.
      ), export (
      • Hir H. Le
      • Gatfield D.
      • Izaurralde E.
      • Moore M.J.
      The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay.
      ,
      • Kataoka N.
      • Yong J.
      • Kim V.N.
      • Velazquez F.
      • Perkinson R.A.
      • Wang F.
      • Dreyfuss G.
      Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm.
      ,
      • Luo M.
      • Zhou Z.
      • Magni K.
      • Christoforides C.
      • Rappsilber J.
      • Mann M.
      • Reed R.
      Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly.
      ,
      • Palacios I.M.
      • Gatfield D.
      • Johnston D.S.
      • Izaurralde E.
      An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay.
      ), and translation (
      • Nott A.
      • Hir H. Le
      • Moore M.J.
      Splicing enhances translation in mammalian cells: An additional function of the exon junction complex.
      ). Trypanosomes have both components of the EJC and NMD-related pathway (
      • Bercovich N.
      • Levin M.J.
      • Clayton C.
      • Vazquez M.P.
      Identification of core components of the exon junction complex in trypanosomes.
      ,
      • Delhi P.
      • Queiroz R.
      • Inchaustegui D.
      • Carrington M.
      • Clayton C.
      Is there a classical nonsense-mediated decay pathway in trypanosomes?.
      ) and associations between the EJC (Mago, eIF4AIII) and Sub2 suggest a platform linking splicing and export (
      • Strässer K.
      • Masuda S.
      • Mason P.
      • Pfannstiel J.
      • Oppizzi M.
      • Rodriguez-Navarro S.
      • Rondón A.G.
      • Aguilera A.
      • Struhl K.
      • Reed R.
      • Hurt E.
      TREX is a conserved complex coupling transcription with messenger RNA export.
      ,
      • Strässer K.
      • Hurt E.
      Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yra1p.
      ). This is further supported by identification of TSR1, TSR1IP, SR34, and additional splicing machinery proteins. Another SR protein, RRM1, is associated with the core spliceosome and modulates chromatin structure (
      • Naguleswaran A.
      • Gunasekera K.
      • Schimanski B.
      • Heller M.
      • Hemphill A.
      • Ochsenreiter T.
      • Roditi I.
      Trypanosoma brucei RRM1 is a nuclear RNA-binding protein and modulator of chromatin structure.
      ). Interestingly, we also observed RBSR2 that is associated with RRM1, RBSR1, and other RNA-binding proteins that were also identified, such as UBP-1, UBP-2, and ALBA3 (
      • Wippel H.H.
      • Malgarin J.S.
      • De Toledo S.
      • Vidal N.M.
      • Marcon B.H.
      • Miot H.T.
      • Marchini F.K.
      • Goldenberg S.
      • Alves L.R.
      The nuclear RNA-binding protein RBSR1 interactome in Trypanosoma cruzi.
      ). Additionally, DRBD18, identified in TcSub2 and TcMex67 isolates, is involved in mRNA export in association with Mex67/Mtr2 by regulating the mobilization of mRNPs through the nuclear pore complex (
      • Mishra A.
      • Kaur J.N.
      • McSkimming D.I.
      • Hegedűsová E.
      • Dubey A.P.
      • Ciganda M.
      • Paris Z.
      • Read L.K.
      Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei.
      ).
      TcFOP, TcAPI5, TcNTF2L, and TcHYP possess lineage-specific features, indicating mechanistic divergence (
      • Rout M.P.
      • Obado S.O.
      • Schenkman S.
      • Field M.C.
      Specialising the parasite nucleus: Pores, lamins, chromatin, and diversity.
      ). TcFOP contains an FOP-like domain initially described in mammalian CHTOP, a TREX component (
      • van Dijk T.B.
      • Gillemans N.
      • Stein C.
      • Fanis P.
      • Demmers J.
      • van de Corput M.
      • Essers J.
      • Grosveld F.
      • Bauer U.-M.
      • Philipsen S.
      Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases.
      ). CHTOP binds Sub2 and activates mRNA delivery to Mex67 (
      • Chang C.-T.
      • Hautbergue G.M.
      • Walsh M.J.
      • Viphakone N.
      • van Dijk T.B.
      • Philipsen S.
      • Wilson S.A.
      Chtop is a component of the dynamic TREX mRNA export complex.
      ). The trypanosome FOP domain is distinct from metazoan FOP but is required for interaction with TcSub2. TcFOP interacts with TcMex67, supporting a role in mRNA export. However, knockdown of TbFOP did not drastically affect mRNA export, although this is also noted for CHTOP protein, where mRNA export is only affected by a dual knockdown of CHTOP and partner ALYREF (
      • Chang C.-T.
      • Hautbergue G.M.
      • Walsh M.J.
      • Viphakone N.
      • van Dijk T.B.
      • Philipsen S.
      • Wilson S.A.
      Chtop is a component of the dynamic TREX mRNA export complex.
      ). Since ALYREF orthologs are absent from trypanosome genomes (
      • Jimeno S.
      • Rondón A.G.
      • Luna R.
      • Aguilera A.
      The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability.
      ) and was not identified here by proteomics, TcFOP may have both divergent architecture and distinct mechanisms in trypanosomes. Knockdown of API5 also did not significantly impair mRNA export, despite interacting with mRNA export factors. Moreover, API5 interacts directly with Sub2 in mammalian cells and depletion results in accumulation of poly(A)+ RNA in the nucleus, which was not found here (
      • Strässer K.
      • Hurt E.
      Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yra1p.
      ). TcNTF2L has a domain common to several transport receptors, including NTF2 (
      • Bayliss R.
      • Leung S.W.
      • Baker R.P.
      • Quimby B.B.
      • Corbett A.H.
      • Stewart M.
      Structural basis for the interaction between NTF2 and nucleoporin FxFG repeats.
      ) and Mex67 (
      • Fribourg S.
      • Braun I.C.
      • Izaurralde E.
      • Conti E.
      Structural basis for the recognition of a nucleoporin FG repeat by the NTF2-like domain of the TAP/p15 mRNA nuclear export factor.
      ). Although TcNTF2L RNAi did not cause clear nuclear mRNA accumulation, this result cannot exclude the involvement of TcNTF2L in mRNA export pathway since the T. brucei ortholog copurifies with NPG proteins (
      • Kramer S.
      • Marnef A.
      • Standart N.
      • Carrington M.
      Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate.
      ,
      • Goos C.
      • Dejung M.
      • Wehman A.M.
      • M-natus E.
      • Schmidt J.
      • Sunter J.
      • Engstler M.
      • Butter F.
      • Kramer S.
      Trypanosomes can initiate nuclear export co-transcriptionally.
      ). Furthermore, TbNTF2L is a chromatin-associated protein (
      • Leandro de Jesus T.C.
      • Calderano S.G.
      • Vitorino F.N. de L.
      • Llanos R.P.
      • Lopes M. de C.
      • Araújo C. B. de
      • Thiemann O.H.
      • Reis M. da S.
      • Elias M.C.
      • da Cunha J.P.C.
      Quantitative proteomic analysis of replicative and nonreplicative forms reveals important insights into chromatin biology of Trypanosoma cruzi.
      ) and copurifies with EJC components (
      • Bercovich N.
      • Levin M.J.
      • Clayton C.
      • Vazquez M.P.
      Identification of core components of the exon junction complex in trypanosomes.
      ). These results and the very mild impact on mRNA abundance from Sub2 silencing may indicate a level of redundancy and/or additional mechanisms remaining to be uncovered.
      Cytoplasmic proteins PABP2, DHH1, and multiple Zn-finger RNA helicases that associate with NPGs were identified (
      • Kramer S.
      • Marnef A.
      • Standart N.
      • Carrington M.
      Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate.
      ). PABP2 regulates bulk mRNA directed for translation (
      • Zoltner M.
      • Krienitz N.
      • Field M.C.
      • Kramer S.
      Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA.
      ) while DHH1 is localized in cytoplasmic granules (
      • Holetz F.B.
      • Alves L.R.
      • Probst C.M.
      • Dallagiovanna B.
      • Marchini F.K.
      • Manque P.
      • Buck G.
      • Krieger M.A.
      • Correa A.
      • Goldenberg S.
      Protein and mRNA content of TcDHH1-containing mRNPs in Trypanosoma cruzi.
      ,
      • Alves L.R.
      • Ávila A.R.
      • Correa A.
      • Holetz F.B.
      • Mansur F.C.B.
      • Manque P.A.
      • De Menezes J.P.B.
      • Buck G.A.
      • Krieger M.A.
      • Goldenberg S.
      Proteomic analysis reveals the dynamic association of proteins with translated mRNAs in Trypanosoma cruzi.
      ,
      • Holetz F.B.
      • Correa A.
      • Avila A.R.
      • Nakamura C.V.
      • Krieger M.A.
      • Goldenberg S.
      Evidence of P-body-like structures in Trypanosoma cruzi.
      ). Although DHH1 removes polysome mRNAs and interacts with decapping and deadenylase complexes in yeast (
      • Coller J.M.
      • Tucker M.
      • Sheth U.
      • Valencia-sanchez M.A.
      • Parker R.
      The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes.
      ), DHH1 acts only as a translational repressor in trypanosomes (
      • Kramer S.
      • Queiroz R.
      • Ellis L.
      • Hoheisel J.D.
      • Clayton C.
      • Carrington M.
      The RNA helicase DHH1 is central to the correct expression of many developmentally regulated mRNAs in trypanosomes.
      ). In yeast, DHH1 is associated with SCD6, a decapping activator that binds eIF4G and inhibits translation initiation (
      • Cristodero M.
      • Schimanski B.
      • Heller M.
      • Roditi I.
      Functional characterization of the trypanosome translational repressor SCD6.
      ,
      • Nissan T.
      • Rajyaguru P.
      • She M.
      • Song H.
      • Parker R.
      Article decapping activators in Saccharomyces cerevisiae act by multiple mechanisms.
      ). Unlike yeast, SCD6 in T. brucei does not form a complex with DHH1 (
      • Cristodero M.
      • Schimanski B.
      • Heller M.
      • Roditi I.
      Functional characterization of the trypanosome translational repressor SCD6.
      ), even though it also represses translation and significantly we identified TcSCD6 with both TcSub2 and TceIF4AIII.
      In conclusion, we report both novel and conserved components of the mRNA export pathway in trypanosomes identified through immunoisolation and proteomics (Fig. 12). Among many interactions identified, we highlight the kinetoplastid-specific proteins TcFOP, TcAPI5, and TcHYP that interact directly with TcSub2, while TcNTF2L, a shuttling protein, interacts with the export receptor TcMex67. TcNTFL2 and TcFOP are kinetoplastid-specific but connect transcription/splicing components with the conserved export factor Mex67 and other components of mRNA export. We illustrated the comparison between major proteins described in metazoa and those identified in T. cruzi (Fig. 13). Overall, we propose that trypanosome mRNA export comprises a core of pan-eukaryotic proteins together with a significant number of proteins unique to kinetoplastids and a further example of the power of proteomics to uncover evolutionary divergent mechanisms.
      Figure thumbnail gr13
      Fig. 13Comparision between S. cerevisae and T. cruzi components of mRNA export coupled to transcription/splicing. The figure contains the major components of S. cerevisiae as described on Kohler and Hurt (2007) (
      • Köhler A.
      • Hurt E.
      Exporting RNA from the nucleus to the cytoplasm.
      ) and Kramer (2021) (
      • Kramer S.
      Nuclear mRNA maturation and mRNA export control: From trypanosomes to opisthokonts.
      ). To simplify, components of Peripheral Nuclear Granules and Nuclear Pore Complex are omitted. In T. cruzi, the specific proteins are in red.

      Data Availability

      All mass spectrometry data have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository (https://www.ebi.ac.uk/pride/) with the dataset identifier PXD028072. The TbSub2 RNAi transcriptome data have been submitted to SRA at NCBI with the accession number SUB10190938.

      Supplemental data

      This article contains supplemental data.

      Conflict of interest

      The authors declare no competing interests.

      Acknowledgments

      We thank the Program for Technological Development in Tools for Health–RPT FIOCRUZ for Technological Platforms of Microscopy, Flow Cytometry and Mass spectrometry, and the FingerPrints proteomics facility at the University of Dundee, supported by the “Wellcome Trust Technology Platform” award [097945/B/11/Z]. We would like to thank Wagner Nagib de Souza Birbeire for assistance in producing Figures 11B and 13 and James Abbott for assistance in producing Figure 11A.

      Funding and additional information

      This work was supported by the Wellcome Trust (204697/Z/16/Z to M. C. F.), the Medical Research Council (MR/N010558/1 to M. C. F.), Coordenação De Aperfeiçoamento De Pessoal De Nível Superior (CAPES to A. R. Á., A. H. I., P. F. D., P. M. H. and M. S.), Fundação Oswaldo Cruz (FIOCRUZ to A. R. Á.), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (421990/2017-1 CNPq to A. R. Á.).

      Author contributions

      A. H. I., M. C. F., and A. R. Á. conceptualization; M. S., P. M. H., N. M. V., E. R. B., R. C. d. P., and A. L. data curation; M. C. F. and A. R. Á. funding acquisition; A. H. I. and P. F. D. investigation; A. H. I. and P. F. D. methodology; A. R. Á. project administration; P. M. H., R. C. d. P., and C. B. resources; C. B. and M. C. F. supervision; M. S., N. M. V., and A. L. validation; M. C. F. and A. R. Á. visualization; A. H. I. and P. F. D. writing—original draft; M. C. F. and A. R. Á. writing—review and editing.

      Supplemental Data

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