Abstract
RNA-binding proteins (RBPs) are essential for post-transcriptional regulation of gene expression. Recent high-throughput screens have dramatically increased the number of experimentally identified RBPs; however, comprehensive identification of RBPs within living organisms is elusive. Here we describe the repertoire of 765 and 594 proteins that reproducibly interact with polyadenylated mRNAs in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively. Furthermore, we report the differential association of mRNA-binding proteins (mRPBs) upon induction of apoptosis in C. elegans L4-stage larvae. Strikingly, most proteins composing mRBPomes, including components of early metabolic pathways and the proteasome, are evolutionarily conserved between yeast and C. elegans. We speculate, on the basis of our evidence that glycolytic enzymes bind distinct glycolytic mRNAs, that enzyme-mRNA interactions relate to an ancient mechanism for post-transcriptional coordination of metabolic pathways that perhaps was established during the transition from the early 'RNA world' to the 'protein world'.
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Acknowledgements
We are grateful to K. Heesom (Proteomics Facility, University of Bristol) for performing the MS analysis; M. Hengartner and D. Subasic (Institute of Molecular Life Sciences, University of Zurich) for support and C. elegans strains; S. Leidel (Max-Planck-Institute for Molecular Biomedicine, Münster) for GFP and TAP-tagged S. cerevisiae strains; M. Seedorf (Center for Molecular Biology, University of Heidelberg) and R. Ciosk (Friedrich Miescher Institute for Biomedical Research) for anti-Scp160 and anti–GLD-1 antibodies, respectively; D. Pérez-Mendoza and D. Subasic for reading of the manuscript; and members of the Gerber laboratory and the Sinergia project for discussions. This study was funded by a 'Sinergia' grant (CSRII3-141942 (A.P.G.)) from the Swiss National Science Foundation and (in part) by the Biotechnology and Biological Sciences Research Council (BB/K009303/1 (A.P.G.)).
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A.M.M.-G. and A.P.G. conceived and designed the experiments. A.M.M.-G. performed laboratory experiments. E.E.L. performed bioinformatics analyses. All authors analyzed data, discussed the results, and wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 RNA integrity after UV-cross-linking of cells at 254 nm.
One μg of total RNA was electrophoresed on a 1% agarose gel and visualized with Red-safe. Total RNA from non-irradiated cells was used as a reference. Total RNA after RNase ONE digestion was analyzed to confirm effective RNA degradation. Total RNA isolation was routinely performed prior to isolation of poly(A) mRNAs from (a) yeast and (b) nematodes.
Supplementary Figure 2 The mRBPome overlaps significantly with previously identified sets of RBPs.
(a) Venn diagrams showing overlap of the yeast mRBPome and data from Mitchell et al. (Mitchell, S.F et al., Nat Struct Mol Biol. 20, 127-33, 2013); Scherrer et al. (Scherrer, T. et al., PLoS One. 5, e15499, 2010); Tsvetanova et al. (Tsvetanova, N.G. et al., PLoS One. 5, e12671, 2010) and Hogan et al. (Hogan, D.J. et al., PLoS Biology. 6, 2297-2313, 2008); and (b) of the C. elegans mRBPome and data from Tamburino et al. (Tamburino, A.M. et al., G3 (Bethesda). 3, 297-304, 2013). P-values relate to the significance of overlap (hypergeometric test).
Supplementary Figure 3 Comparing the expression of mRBPs to non-mRBPs in yeast.
(a) Boxplot depicting the expression dynamics of genes coding for proteins of the yeast mRBPome (red) and non-mRBPs (green) in the entire genome. Filled boxes extend from the first to the third quartile and whiskers extend to minimum and maximum values. Data was retrieved for protein abundance (Ghaemmaghami, S. et al., Nature. 425, 737-41, 2003), protein half-life (Belle, A. et al., Proc Natl Acad Sci U S A. 103, 13004-9, 2006), protein noise (Newman, J.R. et al., Nature. 441, 840-6, 2006), mRNA copy number (Miura, F. et al., BMC Genomics. 9, 574, 2008), mRNA half-life (Shalem, O. et al., Mol Syst Biol. 4, 223, 2008) and ribosome occupancy (Arava, Y. et al., Proc Natl Acad Sci U S A. 100, 3889-94, 2003). Asterisks refer to P-values determined in a Mann-Whitney, two-tailed test comparing the distribution of mRBPs with non-mRBPs; *** P < 0.001. (b) Intracellular distribution of proteins comprising the yeast mRBPome and the yeast proteome reported by Breker et al. containing 5,330 proteins (Breker M. et al., Nucleic Acids Res. 42, D726-30, 2014). Asterisks refer to the significant overrepresentation of indicated cellular compartments in the mRBPome compared to the reference proteome. *P < 0.05, ***P < 0.1% at 5% FDR, hypergeometric test.
Supplementary Figure 4 Apoptosis induction in C. elegans and MS analysis.
(a) Induction of germline apoptosis with 5 mm ENU. RT-PCR was performed on total RNA isolated from synchronized animals at L4 stage as well as L4 stage larvae treated with 5 mM ENU, using egl-1 and mpk-1 specific primers. Products were visualized on an agarose gel. Increased egl-1 mRNA level are a marker for germline apoptosis, mpk-1 mRNA levels are not expected to change and served as a negative control. (b) Pairwise comparisons of C. elegans mRBPome samples. Scatterplots comparing the processed (background subtraction followed by total area normalization and addition of the arbitrary value 0.000001, see Methods) protein peak areas between all C. elegans samples in this study, generated using R (Team, R.C. R Foundation for Statistical Computing, Vienna, Austria, 2012). For visualization purposes the peak areas have been transformed via log2 (processed_peak_area) +20, such that any proteins measured as having no abundance in a particular sample has a transformed value of 0 (as the log2 (pseudo value of 0.000001) + 20 = 0). The samples being directly compared within a plot can be identified by the ‘label’ boxes along the diagonal, where the sample plotted on the y-axis is identified by the label box to the left of the scatterplot and the sample on the x-axis is identified by the label box beneath the scatterplot. Proteins that do not form part of the C. elegans mRBPome (i.e. not identifiable by two peptides) are indicated in black and proteins of the C. elegans mRBPome are in red. The respective Pearson correlation coefficient, calculated using the processed (non-transformed) peak areas, of a comparison is indicated to the bottom right of each scatterplot. (c) Venn diagram showing the occurrence of the 594 proteins identified with at least 2 peptides at less than 1% FDR within mixed-stage, L4-, and L4-staged animals treated with 5 mM ENU.
Supplementary Figure 5 Association of Pfk2 and Eno1 with mRNAs is not ribosome dependent.
Reverse transcription (RT)-PCR with gene specific primers to detect mRNAs in affinity isolates of indicated TAP-tagged proteins (Shg1, Pfk2 and Eno1) in the presence or absence of 1 mM puromycin (Puro). Shg1-TAP was used as a positive control to assess the efficiency of puromycin treatment to relieve co-translational association of SET1 mRNA with the SET1C complex, as shown previously (Halbach, A. et al., EMBO J. 28, 2959-70, 2009). Untagged cells (BY4741) served as a negative control (Ctrl). The input refers to total RNA from non-UV crosslinked cells. Products were visualized on an agarose gel.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5 and Supplementary Notes 1 and 2 (PDF 1078 kb)
Supplementary Data Set 1
Uncropped gels and blots (PDF 817 kb)
Supplementary Table 1
MS data for six S. cerevisiae samples defining the mRBPome (XLS 606 kb)
Supplementary Table 2
Analysis of the S. cerevisiae mRBPome (XLS 1982 kb)
Supplementary Table 3
MS data for C. elegans samples defining the mRBPome (XLS 1121 kb)
Supplementary Table 4
Analysis of the C. elegans mRBPome (XLS 2020 kb)
Supplementary Table 5
Conservation between S. cerevisae and C. elegans mRBPomes (XLS 710 kb)
Supplementary Table 6
Oligonucleotide sequences (XLS 38 kb)
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Matia-González, A., Laing, E. & Gerber, A. Conserved mRNA-binding proteomes in eukaryotic organisms. Nat Struct Mol Biol 22, 1027–1033 (2015). https://doi.org/10.1038/nsmb.3128
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DOI: https://doi.org/10.1038/nsmb.3128
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