Conserved mRNA-binding proteomes in eukaryotic organisms

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'.

Figure 1

Identification of mRBPs in S. cerevisiae. (a) Silver stained polyacrylamide gel. Lanes 2-4, input extracts and poly(A) and RNase treated control samples; lanes 6-8, eluates from poly(A) mRNA isolation. Lane 1 depicts the marker (M) with molecular weights (MW) indicated to the left. (b) Immunoblot analysis with Scp160p and Act1p antibodies. Original images are shown in the Supplementary Data Set 1. (c) Heatmap representation of the abundance of 765 proteins comprising the yeast mRBPome. Columns refer to three independent experiments and respective controls, rows represent individual proteins; for visualization purposes, the white-red color bar represents log₁₀ transformed raw (non-normalized) MS peak areas of respective proteins. The Venn diagram represents the overlap of identified proteins across the three experiments. The P-value (hypergeometric test) relates to the significance of overlap (see Methods). (d) Selective samples of significantly shared GO terms from SGD (P < 0.01, FDR < 5%) among proteins of the yeast mRBPome. Bars indicate the fraction of proteins annotated with respective GO term in the yeast mRBPome (765 proteins; black bars) and all GO annotated proteins in SGD (6,607 proteins; grey bars). (e) Selection of domains enriched in the mRBPome. Bars indicate fraction of annotated proteins bearing at least one of the indicated domains (InterPro) in the yeast mRBPome (765 proteins; black bars) and the reference proteome (6,621 proteins in UniProt; grey bars). The number of proteins within each fraction is shown to the right. *P < 0.05, **P < 0.01, ***P < 0.001 at FDR < 5% (hypergeometric test).

Figure 2

Identification of mRBPs in C. elegans. (a) Silver stained polyacrylamide gel of UV-crosslinked samples from mixed-stages nematodes. (b) Immunoblot analysis with GLD-1 and CYC-1 antibodies. Original images of blots can be found in Supplementary Data Set 1. (c) Cluster heatmap representing the abundance of 594 proteins comprising the mRBPome within mixed-stage, L4-, and L4-staged animals treated with 5 mM ENU; and in the corresponding negative control samples. The white-blue scale shows raw (non-normalized) peak areas (log₁₀ transformed) and number of proteins within groups is indicated to the left. (d) Relative changes of 96 proteins in the mRBPomes of synchronized L4-stage animals upon apoptosis (FDR < 5%). Rows indicate three pairwise comparisons within matched samples (a, b, c), columns refer to proteins. Fold changes are indicated with the blue-yellow color bar. (e) Significantly shared GO terms among proteins of the C. elegans mRBPome (FWER adj. P < 0.01). Bar diagrams indicate the fraction of proteins in the respective GO term among the C. elegans mRBPome (594 proteins; black bars) and the 22,817 GO annotated proteins in the UniProt reference proteome (grey bars). (f) Domains enriched in the mRBPome. Bars relate to the fraction of proteins bearing at least one of the domains (InterPro) in the C. elegans mRBPome (594 proteins; black) and 26,165 proteins comprising the UniProt reference proteome (grey). Numbers of proteins is shown to the right. *P < 0.05, **P < 0.01, ***P < 0.001 at FDR < 5% (hypergeometric test).

Figure 3

Conservation of the mRBPome across species. (a) Conservation between S. cerevisiae and C. elegans proteins. Yellow columns refer to the fraction of conserved S. cerevisiae proteins in C. elegans considering the reference proteome (6,621 proteins) and mRBPome (765 proteins). Blue columns refer to the fraction of conserved C. elegans proteins in S. cerevisiae considering the worm reference proteome (26,165 proteins) and mRBPome (594 proteins). Stars demarcate the significant difference between fractions (P < 0.001, hypergeometric test). (b) Venn diagram showing overlap of orthologous proteins of the mRBPome in S. cerevisiae (476 proteins) and C. elegans (330 proteins). The P-value relates to the significance of overlap (hypergeometric test). (c) Schematic view of the glycolytic pathway. Proteins highlighted in yellow are exclusively present in the yeast mRBPome, proteins in blue are exclusively found in C. elegans mRBPome, and proteins shared in both mRBPomes are in green. (d) Schematic view of the proteasome comprising the core particle (20S CP) that can bind to one or two regulatory particles (19S RP).

Figure 4

Validation of mRNA-protein interactions by quantitative dual fluorescence-based mRNA detection assay. Normalized Alexa 594 fluorescence signal was used to monitor the presence of poly(A) mRNAs (left panel, black bars). As a control, the samples were treated with RNase ONE (grey bars). Data was normalized to untagged wild type cells (Ctrl). Error bars, s.e.m. (n = 3 independent immunopurifications). *P ≤ 0.05; **P ≤ 0.01 by paired, two-tailed, Student’s t test. The right panel shows the normalized GFP fluorescence signal of immunopurified proteins. Data underlying the graphical representation can be found in Supplementary Data Set 2.

Figure 5

Glycolytic enzymes selectively interact with glycolytic mRNAs. Agarose gel showing products from reverse transcription (RT)-PCR reactions with gene specific primers to detect glycolytic mRNAs (right) bound to immunopurified GFP or TAP-tagged proteins of indicated glycolytic proteins (top). Input refers to total RNA from crosslinked cells; Ctrl, untagged control cells. Full-sized images of gels can be found in Supplementary Data Set 1.