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Research: Lars Jønson, Jonas Vikesaa, Anders Krogh, Lars K. Nielsen, Thomas vO. Hansen, Rehannah Borup, Anders H. Johnsen, Jan Christiansen, and Finn C. Nielsen Molecular Composition of IMP1 Ribonucleoprotein Granules Mol Cell Proteomics 2007; 6: 798-811 [Abstract] [Full text] [PDF]

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Response to Denman Letter

Finn C. Nielsen, Lars Jønson and Jan Christiansen   (2 April 2007)

FMRP and IMP1: Evidence of Absence or Absence of Evidence, Noise in the Protein Interactome

Robert B. Denman   (2 April 2007)

Response to Denman Letter 2 April 2007
Finn C. Nielsen University of Copenhagen, Lars Jønson and Jan Christiansen Send letter to journal: Re: Response to Denman Letter FCN{at}rh.dk Finn C. Nielsen, et al.	FMRP and IMP1 are both found in large RNP granules, and we agree that it is unclear if the two factors interact directly. We have no data to support a direct interaction between the molecules, but it is possible that FMRP and IMP1 may become closely associated on common target mRNAs or in a common RNA transit compartment such as P-bodies (1), or even in a subpopulation of RNP granules. Using a trimolecular fluorescence approach, Rackham and Brown (2) showed that FMRP and IMP1 are present on common reporter mRNAs in cytoplasmic granules in COS-7 cells. Based on immunoprecipitation and RNase treatment, the data indicate that IMP1 and FMRP associate independently of RNA, but it is not determined whether the interaction requires a bridging molecule. In fact, the potential of the method to demonstrate ternary complexes accommodating an unknown factor is one of its advantages. FMRP is not necessary for IMP1 granule formation (3), and the distinct spatial and temporal expression of the two proteins should also be borne in mind when discussing the putative physiological connection between the two factors (4-6), So even if they participate in a number of common processes, both factors obviously perform independent functions in the intact organism. 1. Stohr, N., Lederer, M., Reinke, C., Meyer, S., Hatzfeld, M., Singer, R. H., and Huttelmaier, S. (2006) ZBP1 regulates mRNA stability during cellular stress. J Cell Biol 175, 527-34. 2. Rackham, O., and Brown, C. M. (2004) Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs. Embo J 23, 3346-55. 3. Nielsen, F. C., Nielsen, J., Kristensen, M. A., Koch, G., and Christiansen, J. (2002) Cytoplasmic trafficking of IGF-II mRNA-binding protein by conserved KH domains. J Cell Sci 115, 2087-97. 4. Hansen, T. V., Hammer, N. A., Nielsen, J., Madsen, M., Dalbaeck, C., Wewer, U. M., Christiansen, J., and Nielsen, F. C. (2004) Dwarfism and impaired gut development in insulin-like growth factor II mRNA-binding protein 1-deficient mice. Mol Cell Biol 24, 4448-64. 5. Hinds, H. L., Ashley, C. T., Sutcliffe, J. S., Nelson, D. L., Warren, S. T., Housman, D. E., and Schalling, M. (1993) Tissue specific expression of FMR-1 provides evidence for a functional role in fragile X syndrome. Nat Genet 3, 36-43. 6. Nielsen, F. C., Nielsen, J., and Christiansen, J. (2001) A family of IGF-II mRNA binding proteins (IMP) involved in RNA trafficking. Scand J Clin Lab Invest Suppl 234, 93-9. Finn C. Nielsen[1], Lars Jønson[1] and Jan Christiansen[2] Department of Clinical Biochemistry[1] and Institute of Molecular Biology[2], University of Copenhagen.
FMRP and IMP1: Evidence of Absence or Absence of Evidence, Noise in the Protein Interactome 2 April 2007
Robert B. Denman, Head of the Biochemical Molecular Neurobiology Laboratory New York State Institute for Basic Research in Developmental Disabilities Send letter to journal: Re: FMRP and IMP1: Evidence of Absence or Absence of Evidence, Noise in the Protein Interactome rbdenman{at}yahoo.com Robert B. Denman	In, “Molecular Composition of IMP1 RNP Granules”, Jønson et al characterize IMP1-containing granules. Specifically, they show by confocal imaging of FLAG-IMP1-expressing HEK293 cells, which were transiently transfected with YFP-FMRP, or YFP-hStaufen expression vectors, that the IMP1 granules did not co-localize with these proteins. In addition, anti-FLAG immunoprecipitation reactions did not co- immunoprecipitate either FMRP or hStaufen. The results appear clear and compelling because in control transfections with expression vectors encoding IMP3 and HuB, known IMP1 interactors, anti-FLAG co- imunoprecipitated both proteins and HuB co-localized with IMP1 by confocal microscopy. Equally compelling however, are data on the interaction of IMP1 and FMRP provided by Rackham et al (1). In this case the authors studied the interaction using a tri-molecular fluorescence approach (reviewed in (2)). They demonstrated that by co-transfecting COS cells with vectors coding for an MS2 protein fused to the N terminus of the Venus fluorescent protein, the C-terminal portion of the Venus fluorescent protein linked to IMP1, FMRP, hStau or PTB and a MS2-RNA-mRNA reporter that these proteins associate with similar mRNA sequences in vivo. Importantly, they also demonstrated that FMRP and IMP1 associate independently of the presence of the fused MS2-mRNA reporter. Finally, they demonstrated in cells transfected with FMRP fused to the C-terminal portion of the Venus fluorescent protein and FLAG-tagged IMP1 that both FMRP and IMP1 were immunoprecipitated with an anti-GFP antibody targeting the Venus fluorescent protein. Again, a myriad of control reactions were performed suggesting the interaction was specific. Several points are worth mentioning. First, Jønson et al’s proteomic studies of IMP1 granules detected the RNA binding proteins nucleolin and YB-1, the latter being confirmed independently by Western analysis (Table S1 and Figure S1). Both proteins were previously found associated with FMRP in cultured cells and in mouse brain by Ceman et al (3,4). Interestingly, neither this information, nor the studies of Rackham et al were mentioned in the present study. Regardless, none of the studies adequately address whether endogenous IMP1 and FMRP associate, which is the more important question. Finally, it is clear from the present considerations that there is noise within the protein interactome. This noise may be attributed to the inherent dynamics and heterogeneity of mRNPs (5,6), but as importantly, may have much to do with the tools RNP biologists use to study these complexes. Specifically, the antibodies employed to immunoprecipitate and detect RNP components, the conditions under which immunoprecipitation is performed, and the efficiency of immunoprecipitation must be given closer scrutiny. References 1. Rackham, O., and Brown, C. E. (2004) EMBO J 23(16), 3346-3355 2. Denman, R. B. (2006) BioEssays 28(11), 1132-1143 3. Ceman, S., Brown, V., and Warren, S. (1999) Mol. Cell Biol. 19(12), 7925-7932 4. Ceman, S., Nelson, R., and Warren, S. (2000) Biochem Biophys Res Commun 279, 904-908 5. Keene, J., and Tenenbaum, S. (2002) Mol Cell 9, 1161-1167 6. Hieronymus, H., and Silver, P. A. (2004) Genes Dev. 18(23), 2845- 2860