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Originally published In Press as doi:10.1074/mcp.M600384-MCP200 on February 26, 2007.
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Molecular & Cellular Proteomics 6:1049-1058, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

The Myotonic Dystrophy Type 2 Protein ZNF9 Is Part of an ITAF Complex That Promotes Cap-independent Translation*,S

Vincent R. Gerbasi{ddagger} and Andrew J. Link§

From the Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The 5'-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational analysis of the ODC IRES has led to the identification of sequences necessary for cap-independent translation of the ODC mRNA. To discover novel IRES trans-acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements. Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex. Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy.

Most eukaryotic translation initiation involves the interaction of the 43 S preinitiation complex (comprised of the 40 S ribosomal subunit plus initiation factors, Met-tRNAi, and GTP) with the 7-methylguanosine cap complex at the 5'-end of the mRNA (1). During such cap-dependent translation initiation, the 43 S preinitiation complex is recruited to the 5'-cap structure and scans the 5'-untranslated region (UTR).1 Recognition of the AUG start codon is followed by joining of the 40 S-Met-tRNAi complex to the 60 S large ribosomal subunit to form the 80 S initiation complex. In contrast, translation mediated by an internal ribosomal entry site (IRES) does not require the 5'-cap structure. Instead translation initiates at internal sites in the mRNA (2–4).

IRESs are cis-acting RNA sequences found in the 5'-region of a subset of eukaryotic mRNAs. Originally discovered in viral mRNAs, IRESs were later found in cellular transcripts (5). It is postulated that 3–5% of all human mRNAs are translated in a cap-independent manner (6, 7). Some translational initiation factors used in cap-dependent initiation also stimulate translation of some, but not all, IRES-containing transcripts. There are examples of IRES-containing mRNAs that appear to recruit ribosomes directly, independently of the classical initiation factors (8–10). Each IRES may use a unique mechanism of translational initiation.

Evidence from studies of both viral and cellular IRESs has led to several hypotheses as to how and why IRESs initiate translation of their cognate mRNAs. First, IRESs may be an efficient alternative to cap-dependent translation initiation, an observation supported by multiple studies (6, 7, 11, 12). Second, specific RNA-binding proteins may be dedicated to facilitating IRES-mediated translation initiation. This model has gained favor through the discovery that viral and cellular IRESs share a common set of RNA-binding proteins that stimulate their translation (13–18). Finally there is evidence that a subset of IRESs can recruit ribosomes directly through a complex RNA secondary structure (9, 19).

The sequences and structures of viral and cellular IRESs vary widely although they often contain pyrimidine-rich sequences. The conserved pyrimidine tract is found proximal to the start codon (1). Mutagenesis of pyrimidines proximal to the start codon or in other locations throughout an IRES reduces activity and results in the disruption of specific protein-RNA interactions (12, 20). Thus, these pyrimidine-rich sequences in IRESs are necessary for complete IRES activity and recruitment of trans-acting factors.

The IRES trans-acting factor PTBP binds pyrimidine-rich sequences in viral and cellular IRESs (18, 20, 21). It functions with UNR, another ITAF, to enhance the activity of viral and cellular IRESs (17, 18). In addition, several other RNA-binding proteins have been shown to bind and enhance the activity of IRESs (22). However, the high sequence variability between IRESs suggests that there is a wide array of ITAFs, many of which are unknown.

Pancreatic tumor cells alternatively splice the 5'-UTR of ODC to generate an IRES that is translated in a cap-independent and cell cycle-dependent manner (12, 23, 24). Pyrimidine tracts in the ODC 5'-UTR that are necessary for IRES activity have been identified by site-directed mutagenesis (12). Mutations that disrupt either RNA secondary structure or interactions with trans-acting factors compromise IRES activity. As such, we chose the ODC IRES as the target in our search for novel proteins that modulate cellular IRES activity.

To identify potential proteins that associate with the ODC IRES, we utilized a proteomics approach (25). Using RNA affinity capture and mass spectrometry, we found that two nucleic acid-binding proteins, PCBP2 and ZNF9, associate with the wild-type ODC IRES. Mutations in the IRES sequence that compromise ODC IRES function reduced binding to these two proteins. PCBP2 is a known IRES-binding protein that enhances cap-independent translation (13–16, 26). The function of ZNF9 is unknown, although its non-coding region is mutated in patients with type 2 myotonic dystrophy (27). Our results suggest that one function of ZNF9 is to enhance cap-independent translation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—
3'-Biotinylated RNAs used for affinity capture reactions were purchased from Dharmacon Inc. The wild-type ODC RNA sequence was 5'-UUUCUGUCUUAUUGUUUC-3' (12). The mutant ODC RNA sequence was 5'-AAACUGUCUUAUUGAAAC-3' (12). RAJI human B-cell lymphocytes were grown in RPMI 1640 medium, 10% FCS, 1% penicillin-streptomycin at 37 °C and 5% CO2. Human 293T cells were grown in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and 1% penicillin-streptomycin at 37 °C and 5% CO2. Antibodies raised against PCBP2 were kindly provided by Raul Andino. {alpha}-V5 antibodies were purchased from Invitrogen. {alpha}-Protein kinase C and {alpha}-actin antibodies were purchased from Santa Cruz Biotechnology Inc. To clone the ZNF9 cDNA, total RNA was isolated from human RAJI B-cells. cDNA was generated using Superscript II (Invitrogen) primed with oligo(dT). The ZNF9 cDNA was amplified using the primers 5'-GGCAAGGACCCTCAAAATAAAC-3' (forward) and 5'-TGTAGCCTCAATTGTGCATTC-3' (reverse). The 620-bp RT-PCR product was cloned in-frame into the pcDNA3.1/V5-His-TOPO plasmid to create the plasmid pcDNA-ZNF9-V5 expressing a ZNF9-V5 fusion. The plasmid pcDNA3.1/V5-His-TOPO/lacZ expressing a lacZ-V5 fusion was obtained from Invitrogen.

RNA Affinity Chromatography—
To generate the RNA affinity chromatography resin, 100 µl of streptavidin-agarose beads (Pierce) were incubated with 30 nmol of wild-type or mutant 3'-biotinylated RNA in binding buffer (12 mM HEPES in diethyl pyrocarbonate-treated water, pH 8.0, 15 mM KCl, 15 mM dithiothreitol, 5 mM MgCl2, 10% glycerol with one mini-CompleteTM protease inhibitor tablet (Roche Applied Science)/50 ml of binding buffer) at 4 °C for 30 min with gentle mixing. Following incubation of biotinylated RNA with the streptavidin beads, the chromatography resin was washed with binding buffer (200x bead volumes) to remove excess biotinylated RNA. To generate cell extracts, 109 cells were suspended in 3 ml of binding buffer and lysed in 2-ml tubes with 0.5-mm glass beads using a bead beater (Biospec, Inc). The supernatant was removed from the glass beads into sterile microcentrifuge tubes and centrifuged at 20,000 x g for 15 min. The cleared supernatant was incubated with the agarose-coupled RNA affinity resin for 30 min at 4 °C with gentle mixing. After incubation, the chromatography resin was washed with binding buffer (200x bead volumes) to remove nonspecific proteins. Proteins were eluted from the RNA affinity resin with 1-ml washes of increasing salt concentration (150, 250, 350, and 600 mM NaCl) in binding buffer. Each salt wash was then subjected to a 10% TCA precipitation. Protein pellets were suspended in 100 mM ammonium bicarbonate prior to SDS-PAGE, trypsinization, and mass spectrometry analysis.

Experiments utilizing a competitive, non-biotinylated RNA were performed essentially as described above with the exception that a wild-type RNA affinity column was incubated with cell extract and then washed twice with 30 nmol of non-biotinylated ODC RNA suspended in 1 ml of binding buffer. Proteins that remained associated with the resin were eluted with four separate salt washes (150, 250, 350, and 600 mM NaCl) and prepared for mass spectrometry analysis as described below.

Mass Spectrometry Analysis of RNA-binding Proteins—
Eluted proteins from the RNA affinity capture experiments were reduced, alkylated, and trypsinized in 100 mM ammonium bicarbonate as described previously (28). The tryptic peptide fragments were desalted using a C18 reverse-phase salt trap (Michrom) and were subjected to reverse-phase microcapillary LC-ESI-MS/MS. A fritless, microcapillary column (100-µm inner diameter) was packed with 10 cm of 5-µm C18 reverse-phase material (Synergi 4u Hydro RP80a, Phenomenex). The trypsin-digested peptides were loaded onto the reverse-phase column equilibrated in buffer A (0.1% formic acid, 5% acetonitrile). The column was placed in line with an LTQ linear ion trap mass spectrometer (Thermo Electron, Inc.). Peptides were eluted using a 60-min linear gradient from 0 to 60% buffer B (0.1% formic acid, 80% acetonitrile) at a flow rate of 0.3 µl/min. During the gradient, the eluted ions were analyzed by one full precursor MS scan (400–2000 m/z) followed by five MS/MS scans of the five most abundant ions detected in the precursor MS scan while operating under dynamic exclusion. The program extractms2 was used to generate the ASCII peak list and identify +1 or multiply charged precursor ions from the native mass spectrometry data file.2 Tandem spectra were searched with no protease specificity using SEQUEST-PVM (29) against the RefSeq human protein database (released May 2005) containing 28,818 entries. For multiply charged precursor ions (z ≥ +2), an independent search was performed on both the +2 and +3 mass of the parent ion. Data were processed and organized using the BIGCAT software analysis suite (30). A weighted scoring matrix was used to select the most likely charge state of multiply charged precursor ions (25, 30). From the database search, fully tryptic peptide sequences with Sequest cross-correlation scores ≥1.5 for +1 ions, ≥2 for +2 ions, and ≥2 for +3 ions were considered significant and used to create the list of identified proteins. For the proteins ZNF9 and PCBP2 pursued in this study, the annotated MS/MS spectra identifying the two proteins are available in Supplemental Fig. 3. A complete listing of all identified peptides and proteins along with the relevant scoring metrics is available in Supplemental Tables 1 and 2. To estimate the relative abundance of a protein from the mass spectrometry data, a protein abundance factor (PAF) was calculated for each identified protein (31, 32). To calculate PAF values, the total number of non-redundant spectra that correlated significantly with each cognate protein was normalized to the molecular weight of the protein (x104) (31, 32).

Mass Spectrometry Analysis of Trypsinized Proteins from SDS-PAGE Gels—
Protein bands corresponding to the predicted molecular weights of PCBP2 and ZNF9 were excised from silver-stained gels and sliced into 1-mm cubes. The gel pieces were dehydrated in acetonitrile and then rehydrated in 100 mM ammonium bicarbonate. After rehydration, the samples were brought to a 1:1 equal volume of ammonium bicarbonate and acetonitrile. Gel pieces were lyophilized to dryness. The samples were suspended in digestion buffer (50 mM ammonium bicarbonate, 0.5 mM CaCl2, and 0.0125 µg/µl trypsin). The gel pieces remained in digestion buffer for 45 min on ice. Following incubation, 20 µl of additional digestion buffer without trypsin was added, and the gel pieces were incubated for 18 h at 37 °C. Trypsinized peptides were extracted three times from the gel pieces with 50-µl washes of 25 mM ammonium bicarbonate and acetonitrile (1:1). The pooled supernatants were frozen and lyophilized. The dried peptides were resuspended in 10 µl of 0.1% formic acid and subjected to the LC-ESI-MS/MS analysis described above.

Sucrose Gradient Analysis of ZNF9—
Sucrose gradient analysis was performed as described previously (33).

Reporter Assays and RNA Interference—
To analyze the activity of the bicistronic reporter (12), 293T cells were transfected with either 10 µg of the bicistronic reporter plus 10 µg of pcDNA-ZNF9-V5 or 10 µg of the bicistronic reporter plus 10 µg of pcDNA3.1/V5-His-TOPO/lacZ. Each transfection mixture contained 60 µl of Lipofectamine 2000 (Invitrogen). All reporter assays were performed in triplicate. All transfections were performed in Opti-MEM minimal medium as recommended by the manufacturer (Invitrogen). After 4 h of transfection, the Opti-MEM medium was replaced with full medium containing serum. Cells were harvested 48 h after transfection and assayed for luciferase and chloramphenicol acetyltransferase (CAT) activity (34). Reporter assays using RNA interference were similar to the assays described above. On day 1, 293T cells were simultaneously transfected with 10 µg of the bicistronic reporter and 400 nM siRNAs specific for either lamin A/C (35), ZNF9 (5'-GCUAUUCUUGUGGAGAAUU-3'), PCBP2 (5'-GCAUUCCACAAUCCAUCAUUU-3'), or a combination of ZNF9 and PCBP2 siRNAs using 60 µl of LipofectAMINE 2000. All siRNAs used in this study were purchased from Dharmacon Inc. siRNAs for PCBP2 targeted both isoforms detected in our mass spectrometry analysis. On day 2, 293T cells were retransfected with a 400 nM concentration of the same siRNAs except that 60 µl of Oligofectamine/transfection was used instead of the LipofectAMINE 2000. Twenty-four hours after the second transfection, the cells were harvested and assayed for luciferase and CAT activity. Experiments were performed in triplicate. A Student's two-tailed t-test was performed to test the statistical significance of the difference between the experimental and control results.

Electrophoretic Mobility Shift Assays—
Electrophoretic mobility shift assays (EMSAs) were performed essentially as described previously (36). We used oligoribonucleotides corresponding to the wild-type ODC IRES RNA sequence (5'-UUUCUGUCUUAUUGUUUC-3') and the mutant sequence (5'AAACUGUCUUAUUGAAAC-3'). Briefly 50 pmol of an oligonucleotide was end-labeled with 50 pmol of {gamma}-ATP using T4 polynucleotide kinase. Two picomoles of the radiolabeled oligonucleotide was incubated with 10 µg of human cell extract in the presence of binding buffer (20 mM HEPES, pH 7.9, 2 mM MgCl2, 10% glycerol, 50 mM KCl, 1 mM EDTA) at room temperature for 30 min. Supershifting experiments used either {alpha}-PCBP2 or control antibodies at 1 µg of polyclonal antibody/EMSA reaction. Following formation of ribonucleoprotein (RNP) complexes, the EMSA reactions were electrophoresed on 6% native polyacrylamide gels at 145 V for 3 h. The gels were dried and exposed to autoradiographic film to visualize RNP complexes.

Immunoprecipitation-RT-PCR—
293T cells were transfected with 5 µg of the bicistronic ODC IRES reporter or 5 µg of the reporter plus 5 µg of pcDNA-ZNF9-V5 using Lipofectamine 2000 (Invitrogen). Following transfection, cell extracts were prepared and immunoprecipitated with either 10 µg of {alpha}-V5 antibody, 10 µg of {alpha}-PCBP2 antibody, or 10 µg of preimmune serum as a negative control in the presence of 50 µl of protein G beads (Pierce). Immunoprecipitations (IPs) were performed at 4 °C for 30 min prior to washing with 100x bead volumes of 12 mM HEPES, pH 8.0, 15 mM KCl, 0.25 mM dithiothreitol, 5 mM MgCl2, 0.1 mM PMSF, 40 units of RNasin, 10% glycerol. The IPs were subjected to RNA extraction and RT-PCR of mRNA. RNA extraction and cDNA synthesis were performed as described previously (37). The sequences of the primers used to amplify the ODC IRES cDNA product were 5'-TGAGGCATTTCAGTCAGTTG-3' (forward) and 5'-GGATGAGCATTCATCAGGC-3' (reverse). The sequences of the primers used to amplify the GAPDH product were 5'-GGAGAAGGGTGTTAAGGTGG-3' (forward) and 5'-GATCTCATGGTTGTCCACG-3' (reverse). The sequences of the primers used to amplify the RPL32 product were 5'-CCTTGTGAAGCCCAAGATC-3' (forward) and 5'-AATGTTGGGCATCAAGATCTG-3' (reverse). PCR was performed as described previously (37). Samples were taken after 24 PCR cycles, electrophoresed on 6% native polyacrylamide gels, and stained with ethidium bromide.

Co-immunoprecipitation Analysis of PCBP2 and ZNF9-V5—
2.5 x 108 293T cells were transfected with plasmid pcDNA-ZNF9-V5 or no plasmid (negative control) using LipofectAMINE 2000 (Invitrogen). Cells from either the ZNF9 or control transfections were lysed by bead beating with 0.5-mm glass beads in 1 ml of lysis buffer (12 mM HEPES in diethyl pyrocarbonate-treated water, pH 8.0, 15 mM KCl, 15 mM dithiothreitol, 5 mM MgCl2, 10% glycerol, plus one Complete EDTA-free protease inhibitor tablet (Roche Applied Science)/50 ml). The supernatant from the cell extract was then removed from the glass beads into sterile microcentrifuge tubes and centrifuged at 20,000 x g for 15 min. Supernatants were transferred to new microcentrifuge tubes and incubated with 10 µg of {alpha}-PCBP2 antibody and 50 µl of protein G beads. The immunoprecipitations were then incubated with gentle agitation overnight at 4 °C. The precipitations were then washed with 100x column volumes of lysis buffer. The beads were then suspended in 50 µl of 2x Laemmli buffer, boiled for 2 min, subjected to SDS-PAGE, immunoblotted, and probed with {alpha}-V5 antibodies. Ten microliters of each extract was assayed for actin levels by immunoblotting to quantitate protein levels prior to immunoprecipitation of the samples.

RT-PCR of Cells Transfected with siRNAs—
293T cells were transfected with siRNAs using protocols described previously (35). Individual samples of 293T cells were transfected with siRNAs specific to either lamin A/C, PCBP2, or ZNF9. Following transfection, RNA was extracted using TRI Reagent (Molecular Research Center, Inc.) as described by the manufacturer. cDNA products coding for GAPDH, PCBP2, or ZNF9 were amplified by RT-PCR as described previously (37) and analyzed at cycle 24 by native gel electrophoresis and ethidium bromide staining. The sequences of the primers used to amplify the PCBP2 product were 5'-GCCTGCAGTTTTTGGCTTTC-3' (forward) and 5'-TCACAAAGAAAAAGCTCCAGT-3' (reverse). The sequences of the primers used to amplify the ZNF9 product were 5'-GGCAAGGACCCTCAAAATAAAC-3' (forward) and 5'-TGTAGCCTCAATTGTGCATTC-3' (reverse). The primers used to amplify the GAPDH product were 5'-GGAGAAGGGTGTTAAGGTGG-3' (forward) and 5'-GATCTCATGGTTGTCCACG-3' (reverse).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Sequence of the ODC IRES Is Important for Formation of RNPs and Stimulation of Translation—
Mutation of a pyrimidine (PY) tract in the 5'-UTR of the ODC mRNA has been shown to inhibit IRES activity (12). We refer to this 18-base sequence in the ODC 5'-UTR as the IRES regulatory element (RE). To analyze the effects on secondary structure caused by these mutations in the RE, we compared the wild-type and PY-mutated ODC IRES structures using an RNA folding algorithm (38, 39). Although minor structural differences were predicted, the pyrimidine tract mutations are not expected to result in a complete restructuring of the ODC IRES (Supplemental Fig. 1). Because mutations in the pyrimidine tract are predicted to have only a minor effect on the structure of the ODC IRES RNA, we hypothesized that these mutations might directly disrupt protein-RNA interactions. To test this hypothesis, we performed EMSAs with human cell extracts and radiolabeled RNAs containing the wild-type or PY mutant RE (12). Interestingly we found that the wild-type but not the PY mutant RE formed RNP complexes in an EMSA (Fig. 1). We conclude that an RNA-binding protein or a group of RNA-binding proteins associates with the ODC IRES RE and that this association requires the wild-type pyrimidine tract.


Figure 1
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  		FIG. 1. Wild-type, not mutant, form of the ODC IRES forms RNPs in vitro. Oligonucleotides containing sequence from either wild-type (WT) ODC IRES RNA or mutant (Mut) ODC IRES RNA was end-labeled with {gamma}-ATP, incubated with extract from either RAJI lymphocytic B-cells or 293T cells, and electrophoresed on native polyacrylamide gels in an EMSA. Arrows point to RNP complex(es) that formed between the radiolabeled oligonucleotides and proteins in the RAJI and 293T cell lysates. The mutant ODC IRES has been shown to have repressed translational activity compared with the wild-type IRES (12).

 
The ODC IRES Recruits Specific RNA-binding Proteins—
Because this ODC PY mutation reduced translation mediated by the IRES (12) and reduced the ability of the RE to recruit RNA-binding proteins, we hypothesized that these RNA-binding proteins represent ITAFs that bind to and stimulate the ODC IRES. Using RNA affinity capture combined with LC-MS/MS mass spectrometry, we designed a proteomics screen to identify proteins from human cells that bind to the wild-type IRES RE but not the mutant (Fig. 2).


Figure 2
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  		FIG. 2. Schematic of proteomics screen for proteins associated with ODC wild-type (active) and a mutant IRES (repressed) element. Two separate RNA affinity chromatography samples were generated. In the top sample, the wild-type form of the ODC IRES RNA was used to affinity capture human RNA-binding proteins. In the bottom sample, a mutated version of the ODC IRES RNA was used in a control reaction. After isolation of proteins from the wild-type and mutant IRES affinity capture reactions, proteins were trypsinized, desalted, and analyzed by microcapillary LC-MS/MS mass spectrometry. Acquired tandem mass spectra were searched against the human proteome using the Sequest algorithm (50). Identified proteins bound to the wild-type and the mutant RNA affinity resin were then processed and compared using the BIGCAT software suite (30).

 
Affinity columns containing either the wild-type or mutant IRES RE RNA were used to capture proteins, and the proteins were then eluted with increasing concentrations of salt. We fractionated and detected the eluted proteins using SDS-PAGE and silver staining (Fig. 3A). The gel revealed several proteins that were present in both the wild-type and mutant eluates. In contrast, the mutant IRES RE failed to form any RNP complexes in our EMSA analysis. We speculate that many of the proteins present in both the wild-type and mutant affinity eluates bound to the streptavidin resin nonspecifically. Additionally the buffering environment was more stringent in the EMSA analysis compared with the affinity purification. Thus the EMSA analysis revealed only high affinity interactions. Of particular interest were several additional proteins that were eluted from the wild-type affinity column but were absent from the mutant column. We pursued these candidate proteins that bind to the wild-type IRES RE but not the mutant.


Figure 3
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  		FIG. 3. Proteomics identification of proteins associated with the ODC IRES RNA. A, proteins bound to either the wild-type (WT) IRES or mutant (M) IRES RNA affinity capture reactions were eluted with 150, 250, 350, and 600 mM NaCl. Five micrograms of total protein from each salt fractionation was subjected to SDS-PAGE and detected by silver staining. The arrows point to gel bands that were in-gel digested and shown to contain spectra corresponding to PCBP2 and ZNF9, respectively (Supplemental Fig. 4). B, the heat map shows the 10 most abundant proteins associated with the wild-type RNA affinity column that were not detected from the mutant RNA affinity column. Ten micrograms of protein from each salt fractionation in A was trypsinized, desalted, and analyzed by LC-MS/MS. The acquired tandem mass spectra were searched against a human protein database using the Sequest algorithm. Proteins identified from the wild-type (left side) and mutant (right side) RNA affinity capture reactions were displayed using the BIGCAT Clusterer visualization application (30). Red indicates a protein with a high PAF score (an estimate of the relative abundance of each protein in a given sample), yellow indicates proteins with an intermediate PAF score, and black indicates that the protein was not detected (PAF = 0). All of the peptides and proteins identified in this experiment are listed in Supplemental Table 1 and Supplemental Fig. 2. Mut, mutant. C, the heat map shows that ZNF9 and PCBP2 were competed away from the wild-type affinity column with excess competitor oligonucleotide. Human cellular proteins were bound to two separate RNA affinity columns that contained the wild-type ODC IRES RNA sequence. One of the affinity columns was treated with wash buffer, whereas the second column was treated with 60 nmol (a 2-fold excess to column) of non-biotinylated (competitor) oligonucleotide identical in sequence to the wild-type ODC IRES RNA. Proteins bound to both columns were eluted with 150, 250, 350, and 600 mM NaCl. Each protein fraction was analyzed as described in B to generate the heat maps of ZNF9 and PCBP2. All of the peptides and proteins identified in this experiment are listed in Supplemental Table 2 and Supplemental Fig. 5. comp, competitor.

 
To directly identify proteins that were associated with both the wild-type and mutant IRES RE, we performed LC-MS/MS mass spectrometry on both sets of chromatography elutions (25). The mass spectrometry data were processed and analyzed using our bioinformatic graphical comparative analysis software tools (BIGCAT) (30). The mass spectrometry analysis identified 219 individual human proteins (Supplemental Table 1 and Supplemental Fig. 2).

To estimate the relative abundance of each protein from the mass spectrometry data, we used a normalized label-free method of quantification (31, 32). A PAF was calculated for each identified protein to quantify its relative amount in the samples. PAF values in our samples ranged from 0 to 4.64 (Supplemental Table 1 and Supplemental Fig. 2). A PAF value of 0 indicated that the protein was not identified in the sample.

We were specifically interested in those proteins that eluted preferentially from the wild-type RNA affinity column (Fig. 3B). The proteins ZNF9 and PCBP2 scored the highest PAF values among those proteins that bound to the wild-type IRES RE but not to the mutant. From the LC-MS/MS analysis, multiple, independent peptides (n ≥ 3) were identified for both ZNF9 and PCBP2, strongly supporting these identifications (Supplemental Fig. 3). Although two protein isoforms of PCBP2 are predicted from alternative splicing of the cognate gene (40), our LC-MS/MS analysis could not distinguish between the isoforms (Supplemental Table 1 and Supplemental Fig. 3). Among all the proteins that associated with the wild-type but not the mutant IRES, ZNF9 had the highest PAF value (0.899 in the wild-type samples and 0 in the mutant samples) (Supplemental Table 1 and Supplemental Fig. 2). PCBP2 had PAF values of 0.523 in the wild-type samples and 0 in the mutant samples (Supplemental Table 1 and Supplemental Fig. 2).

The LC-MS/MS identifications of ZNF9 and PCBP2 were supported by the PAGE analysis of the same affinity purifications. Fig. 3A shows silver-stained bands corresponding to the predicted mobilities of PCBP2 and ZNF9 in the proteins eluted from the wild-type IRES RE but not the mutant. In-gel digestion and LC-MS/MS analysis of these gel regions confirmed the bands as PCBP2 and ZNF9, respectively.

We next tested whether the ZNF9 and PCBP2 proteins could be competed away from the ODC IRES RE. A 2-fold molar excess of non-biotinylated wild-type ODC IRES RE RNA released a number of RNA-binding proteins from the wild-type RE affinity column (Supplemental Table 2 and Supplemental Fig. 5). Importantly both ZNF9 and PCBP2 were successfully competed away from the affinity column by the non-biotinylated oligonucleotide (Fig. 3C). Therefore, both ZNF9 and PCBP2 were bound to the ODC wild-type IRES RE RNA.

PCBP2 and ZNF9 Bind the ODC IRES—
We next used independent methods to validate the interactions of ZNF9 and PCBP2 with the ODC IRES RE. We used extracts from a human B-cell line for RNA EMSAs. Again we found that the radiolabeled ODC IRES RE formed RNP complexes. Addition of {alpha}-PCBP2 polyclonal antibodies resulted in disruption of the primary RNP and formation of a slightly larger supershifted RNP (Fig. 4A). Only a fraction of the IRES-protein complexes were supershifted. We speculate that the {alpha}-PCBP2 antibodies mostly disrupt the interaction between the IRES RE and PCBP2. Similar results have been observed in other studies when antibodies were added to EMSA reactions (41). Because the reduced electrophoretic mobility of the supershifted RNP was relatively small, we speculate that antibodies raised against PCBP2 are disrupting other interactions within the RNP. In a control reaction, an {alpha}-protein kinase C antibody did not produce a supershifted RNP (Fig. 4A). These data, together with our proteomics analysis of ODC IRES-binding proteins, support our conclusion that PCBP2 is a bona fide ODC IRES-binding protein.


Figure 4
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  		FIG. 4. Biochemical analysis of interactions with the ODC IRES and binding proteins. A, radiolabeled oligonucleotide probes containing the wild-type ODC IRES RNA sequence were incubated with either diethyl pyrocarbonate-treated water (Probe alone), human RAJI lymphocyte lysate, RAJI lysate and {alpha}-protein kinase C (PKC) antibodies, or RAJI lysate and {alpha}-PCBP2 antibodies. EMSA analysis was performed on all the reactions. Arrows point to the RNPs formed between RAJI lysate proteins and the oligonucleotide probe. Arrows pointing to RNP1 indicate the primary RNP formed between RAJI extracts and wild-type ODC IRES RNA. Arrows pointing to RNP2 indicate a second minor RNP that had a slower electrophoretic mobility (supershift) and specifically formed when {alpha}-PCBP2 antibodies were added to the EMSA reaction. B, 293T cells were transfected with the ODC bicistronic reporter (lanes 1 and 3) or the bicistronic reporter and pcDNA-ZNF9-V5 (lane 2). Cell extracts were immunoprecipitated with either preimmune (lane 1), {alpha}-V5 (lane 2), or {alpha}-PCBP2 (lane 3) antibodies and analyzed for the presence of ODC IRES reporter, GAPDH, and RPL32 mRNAs by RT-PCR. C, 10% of the total extract mRNA in B was analyzed for the presence of ODC IRES reporter, GAPDH, and RPL32 mRNAs by RT-PCR.

 
Because we failed to detect supershifted RNP complexes in EMSA experiments using ZNF9-V5 and {alpha}-V5 antibody (data not shown), we used immunoprecipitations and RT-PCR to validate biochemical interactions between ZNF9 or PCBP2 and the ODC IRES. First ZNF9-V5 was immunoprecipitated from 293T cells expressing a bicistronic reporter mRNA containing the entire ODC IRES (5'-UTR) (24). RNA was extracted from the precipitates, and the samples were analyzed by RT-PCR for the presence of the ODC IRES and mRNAs for GAPDH and RPL32 (controls). GAPDH is a highly abundant mRNA that is translated in a cap-dependent fashion, and the RPL32 mRNA contains a 5'-terminal oligopyrimidine tract. We identified the ODC IRES in the samples immunoprecipitated with an {alpha}-V5 antibody (Fig. 4B). Using the {alpha}-PCBP2 antibodies, we found the ODC mRNA also co-immunoprecipitated with PCBP2 (Fig. 4B). RT-PCR did not detect GAPDH and RPL32 in the precipitated samples (Fig. 4B). Specificity of the interactions was demonstrated by the absence of the ODC IRES RT-PCR product in extracts immunoprecipitated using nonspecific polyclonal antibodies (Fig. 4B). A loading control showed that all samples contained similar levels of the ODC IRES reporter, GAPDH, and RPL32 mRNAs prior to immunoprecipitation (Fig. 4C). In combination, these experiments confirm that ZNF9 and PCBP2 specifically bind the ODC IRES.

ZNF9 Co-purifies with the ITAF PCBP2 and Partially Co-localizes with the Ribosomal Density—
PCBP2 has been shown previously to bind to and regulate the activity of cellular and viral IRESs (14–16, 42). Our results suggested that PCBP2 as well as ZNF9 are part of an ITAF complex that binds to the ODC IRES. We hypothesized that if ZNF9 is a cellular IRES-associated protein and functions in cap-independent translation then ZNF9 should associate with mRNAs that are actively being translated. To test this model, we used sucrose gradient ultracentrifugation to separate particles containing the different ribosomal subunits and performed immunoblot analysis to identify ZNF9. Fractions from cells transfected with pcDNA-ZNF9-V5 were analyzed. We found that the ZNF9 protein partially co-localizes with the ribosome- and polysome-containing portion of the sucrose density gradient (Fig. 5A). These data suggest that ZNF9 associates with both actively translating and ribosome-free mRNAs.


Figure 5
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  		FIG. 5. ZNF9 associates with ribosome-containing portions of a sucrose gradient and co-purifies with the ITAF PCBP2. A, 293T cells were transfected with plasmid pcDNA-ZNF9-V5. Transfected cell extracts were fractionated over 7–47% sucrose gradients as described previously (33). The UV trace from the sucrose gradient shows the position of free RNPs, 80 S monosomes, and polyribosomes (polysomes). Twelve fractions from the sucrose gradient were subjected to SDS-PAGE, immunoblotted, and probed for the V5 epitope (ZNF9-V5). B, 293T cells were either mock-transfected with either no plasmid (lane 1) or transfected with plasmid pcDNA-ZNF9-V5 (lane 2). Extracts from both samples were immunoprecipitated with {alpha}-PCBP2 antibodies. Following immunoprecipitation, both samples were subjected to SDS-PAGE, immunoblotted, and probed with an antibody against the V5 epitope. Immunoreactivity above the 50-kDa marker represents nonspecific background proteins detected in both samples. As a loading control, extracts from both samples were probed for actin levels by immunoblotting prior to immunoprecipitation.

 
We also tested whether PCBP2 and ZNF9 co-purify biochemically. By co-immunoprecipitation analysis, we found that PCBP2 and ZNF9 are biochemically associated (Fig. 5B). Because PCBP2 is often associated with larger ITAF assemblies, we hypothesize that ZNF9 might also function as a novel component of ITAF complexes.

The Function of PCBP2 and ZNF9 in ODC IRES Translation—
ITAF proteins that bind to viral and cellular IRESs also enhance IRES activity (13, 15, 17, 18, 20, 26, 42–44). Our data strongly suggested that PCBP2 and ZNF9 function as ITAFs of the ODC IRES. To test this hypothesis, we used a bicistronic reporter driven by the wild-type ODC IRES (12) to determine whether ZNF9 plays a role in translation mediated by the ODC IRES (Fig. 6). We overexpressed ZNF9 in 293T cells using plasmid pcDNA-ZNF9-V5 and found that overexpression of ZNF9 enhanced the activity of the ODC IRES 3-fold in comparison with controls (p value = 0.013) (Fig. 6B).


Figure 6
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  		FIG. 6. ZNF9 and PCBP2 enhance the translation of the ODC IRES. A, experiments utilized this bicistronic reporter construct for experimental results shown in B and C (12). B, 293T cells were co-transfected with a plasmid expressing the bicistronic reporter and either pcDNA3.1/V5-His-TOPO/lacZ or pcDNA-ZNF9-V5 expression plasmids. Following transfection, cells were lysed and assayed separately for luciferase (Luc) and CAT activity. The white bars indicate CAT (cap-dependent) activity for cells that were co-transfected with the bicistronic reporter and either pcDNA3.1/V5-His-TOPO/lacZ (negative control) or pcDNA-ZNF9-V5. The black bars show the detected luciferase activity of cells transfected with the indicated regiment. Values on the y axis indicate the -fold difference in activity between cells transfected with pcDNA3.1/V5-His-TOPO/lacZ and cells transfected with pcDNA-ZNF9-V5. The activity of CAT and luciferase for samples transfected with pcDNA3.1/V5-His-TOPO/lacZ was set to one. Error bars indicate the standard deviation. C, RT-PCR showing the effectiveness of PCBP2 and ZNF9 siRNAs repressing expression of the endogenous mRNA levels. 293T cells were transfected with either lamin A/C, ZNF9, or PCBP2 siRNAs. Following transfection, RNA was isolated from cells with TRI Reagent, and RT-PCR was performed on each individual sample to amplify either GAPDH, ZNF9, or PCBP2 products. D, 293T cells were co-transfected with the bicistronic reporter and lamin A/C, ZNF9, PCBP2, or PCBP2 and ZNF9 siRNAs combined. After co-transfection, cells were lysed and assayed separately for luciferase and CAT activity. IRES activity was calculated as a ratio of luciferase to CAT activity. The graph shows the percentage of ODC IRES activity in each sample relative to samples transfected with lamin A/C control siRNAs. Error bars indicate the standard deviation.

 
In a second set of experiments, we analyzed the function of endogenous ZNF9 and PCBP2 in the translation of the ODC IRES by transfecting cells with siRNAs targeting PCBP2 and ZNF9. In control experiments to test the effectiveness of the siRNAs, we found that siRNAs directed against PCBP2 and ZNF9 were effective at specifically reducing the PCBP2 and ZNF9 transcript levels (Fig. 6C). These siRNAs also reduced expression of the ODC IRES reporter by 30% in comparison with control siRNAs (p value = 0.043 for ZNF9 siRNAs and p value = 0.020 for PCBP2 siRNAs) (Fig. 6D). To determine whether PCBP2 and ZNF9 had an additive effect on ODC IRES activity, we transfected cells with a combination of both PCBP2 and ZNF9 siRNAs. The combination of PCBP2 and ZNF9 siRNAs reduced ODC IRES activity ~75% (p value = 0.001) (Fig. 6D). Collectively these data suggest that PCBP2 and ZNF9 function as enhancers of the ODC IRES. These results provide evidence that ZNF9 functions as a novel component of a cellular ITAF complex.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies of the ODC 5'-UTR have led to the identification of an RNA element that is necessary for IRES activity (12, 23). We performed a proteomics screen for proteins that bind to this regulatory element. We found that two nucleic acid-binding proteins, PCBP2 and ZNF9, bind to this region of the ODC IRES. Additionally we found that both PCBP2 and ZNF9 enhance the activity of the ODC IRES. Our results suggest that PCBP2 and ZNF9 function as IRES trans-acting factors. Importantly these data show that the myotonic dystrophy type 2 protein ZNF9 can function as an IRES trans-acting factor.

Proteins That Associate with the ODC IRES—
The purpose of our proteomics screen was to identify proteins that specifically bind to the wild-type ODC IRES sequence element but not a mutant IRES sequence. Although PCBP2 and ZNF9 specifically bound the active form of the IRES RNA element, our mass spectrometry approach also identified proteins that are associated with both wild-type and mutant IRES sequences (Supplemental Fig. 2). Many of the apparently nonspecific cellular proteins were detected from both affinity columns. These proteins likely represent background binding to the streptavidin beads. Some of the proteins from both the wild-type and mutant RNA affinity columns are previously described RNA-binding proteins that may be involved in ITAF activity. Two of the proteins, UNR and PTBP, have been shown to be ITAFs (17, 18). Therefore, it is possible that additional ITAFs interact with the ODC IRES element independently of the PY tract by binding to the RNA sequence that is shared by both the wild-type and mutant ODC IRES (12). We speculate that a combination of proteins including PCBP2, ZNF9, PTBP, and UNR might function as a holo-ITAF complex to stimulate ODC IRES activity. Similar models of multiprotein ITAF complexes that assemble on IRESs have been described in other studies (17, 18, 44, 45).

PCBP2 and ZNF9 Function as ODC ITAFs—
Our proteomics screen revealed that PCBP2 and ZNF9 bind to the active form of the ODC IRES RE but not an inactive form. Because we were able to detect an interaction of ZNF9 with the ODC IRES by mass spectrometry and IP-RT-PCR approaches but not by EMSA, we speculate that ZNF9 might partially associate with the ODC IRES through protein-protein interactions. Because we observed co-immunoprecipitation between PCBP2 and ZNF9, it is possible that ZNF9 binds the ODC IRES through interactions with PCBP2.

To dissect the function of these two proteins in the translation of the ODC IRES, we utilized both overexpression and RNA interference approaches. Overexpression of ZNF9 in 293T cells enhanced the activity of the ODC IRES. Disruption of endogenous levels of ZNF9 and PCBP2 by RNA interference also supported a role for these two proteins in enhancing the activity of the ODC IRES. Because the dual RNA interference of PCBP2 and ZNF9 had an additive but not a synergistic effect on ODC IRES activity, it is possible that ZNF9 and PCBP2 play redundant roles in ODC IRES translation. In contrast, the observation that PCBP2 and ZNF9 interact biochemically and bind to and enhance the activity of the ODC IRES suggests that these two proteins function as part of a cellular ITAF complex.

Potential Role of ZNF9 as an ITAF in Type 2 Myotonic Dystrophy—
Myotonic dystrophy is the most common adult form of muscular dystrophy. The disease effects ~1 in every 8,000 individuals (46). There are two types of myotonic dystrophy. Patients afflicted with either type 1 or 2 muscular dystrophy express symptoms that include myotonia, muscle weakness, cataracts, testicular atrophy, and defects in cardiac conduction (46). Both type 1 and type 2 myotonic dystrophy present strikingly similar symptoms in adults. However, type 1 myotonic dystrophy presents a congenital form, whereas type 2 does not. Type 1 myotonic dystrophy results from CTG expansions in the 3'-UTR of DMPK, a gene that encodes a serine/threonine kinase (47). Type 2 myotonic dystrophy, is caused by tetranucleotide (CCTG) repeat expansions in the first intron of the ZNF9 gene (27).

It is possible that these mutations in the ZNF9 gene cause changes in translation of mRNAs containing IRESs. However, the favored model of the CCTG expansions in type 2 myotonic dystrophy is that the long CCUG repeats in the ZNF9 RNA sequester essential cellular ribonucleoproteins (48, 49). Sequestration of these RNPs is believed to disrupt an essential cellular process, such as RNA processing, leading to the disease symptoms.

We showed that ZNF9 functions as an ITAF. Because it is possible that ZNF9 activates many unidentified cellular IRESs, disruption of ZNF9 function through genetic mutations should result in an abnormal profile of cellular proteins. Modifying specific cellular protein levels through a gain or loss of ZNF9 function might contribute to the pathogenesis of type 2 myotonic dystrophy. Identification of such changes in protein levels will lead to a better understanding of the targets of ZNF9 action.


   ACKNOWLEDGMENTS
 
We thank Nahum Sonenberg and Raul Andino for sharing reagents. We thank Jill McAfee, Dexter Duncan, and Jennifer Jennings for technical assistance. We also thank Tracey Fleischer and Elizabeth Link for editorial assistance.


  FOOTNOTES
 
Received, October 6, 2006, and in revised form, February 15, 2007.

Published, MCP Papers in Press, February 26, 2007, DOI 10.1074/mcp.M600384-MCP200

1 The abbreviations used are: UTR, untranslated region; IRES, internal ribosomal entry site; ITAF, IRES trans-acting factor; ODC, ornithine decarboxylase; BIGCAT, Bioinformatic Graphical Comparative Analysis Tools; PAF, protein abundance factor; CAT, chloramphenicol acetyltransferase; siRNA, small interfering RNA; EMSA, electrophoretic mobility shift assay; RNP, ribonucleoprotein; IP, immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PY, pyrimidine; RE, regulatory element; PTBP, polypyrimidine tract-binding protein; UNR, upstream of N-ras. Back

2 J. Eng and J. R. Yates III, unpublished software. Back

* This project was supported in part by National Institutes of Health Grant GM64779. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

{ddagger} Supported by National Institutes of Health Training Grant T32 CA009385 and Grant GM64779. Back

§ Supported by National Institutes of Health Grants GM64779, HL68744, ES11993, and CA098131. Also funded in part with federal funds from the NIAID, National Institutes of Health, Department of Health and Human Services, under Contract HHSN266200400079C/N01-AI-40079. To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Vanderbilt University School of Medicine, 1161 21st Ave. S., Nashville, TN 37232-2363. Tel.: 615-343-6823; Fax: 615-343-7392; E-mail: andrew.link{at}vanderbilt.edu


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