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. 2015 Dec 3:6:10127.
doi: 10.1038/ncomms10127.

The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs

Benedikt M Beckmann  1 , Rastislav Horos  1 , Bernd Fischer  1 , Alfredo Castello  1 , Katrin Eichelbaum  1 , Anne-Marie Alleaume  1 , Thomas Schwarzl  1 , Tomaž Curk  1 , Sophia Foehr  1 , Wolfgang Huber  1 , Jeroen Krijgsveld  1 , Matthias W Hentze  1
Affiliations

The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs

Benedikt M Beckmann et al. Nat Commun. .

Abstract

RNA-binding proteins (RBPs) exert a broad range of biological functions. To explore the scope of RBPs across eukaryotic evolution, we determined the in vivo RBP repertoire of the yeast Saccharomyces cerevisiae and identified 678 RBPs from yeast and additionally 729 RBPs from human hepatocytic HuH-7 cells. Combined analyses of these and recently published data sets define the core RBP repertoire conserved from yeast to man. Conserved RBPs harbour defined repetitive motifs within disordered regions, which display striking evolutionary expansion. Only 60% of yeast and 73% of the human RBPs have functions assigned to RNA biology or structural motifs known to convey RNA binding, and many intensively studied proteins surprisingly emerge as RBPs (termed 'enigmRBPs'), including almost all glycolytic enzymes, pointing to emerging connections between gene regulation and metabolism. Analyses of the mitochondrial hydroxysteroid dehydrogenase (HSD17B10) uncover the RNA-binding specificity of an enigmRBP.

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Figures

Figure 1
Figure 1. mRNA interactome capture in yeast and HuH-7 cells.
(a) Schematic representation of the mRNA interactome capture protocol in yeast and HuH-7 cells using PAR crosslinking (PAR-CL) or conventional crosslinking (cCL). Quality controls for both yeast and HuH-7 include silver staining (middle upper and lower panel) and western blotting (right upper and lower panel) (M, protein marker, KHD1, KH-domain containing protein1 fused to eGFP, PUB1, polyU binding protein 1, TUB1, tubulin 1, H4, histone 4, PTBP1, polypyrimidine tract binding protein 1, CSDE1, cold shock domain containing protein E1). (b) Volcano plot showing the distribution of proteins according to their enrichment in crosslinked (CL) over non-CL samples. Proteins shown in red (FDR 0.01) represent the mRNA interactome. (c) Overlap of mRNA interactome proteins in yeast and HuH-7. (d) Validation of the yeast mRNA interactome using western blotting of input samples and eluate after interactome capture with specific antibodies (ADH1, alcohol dehydrogenase 1, PUB1) or against TAP-tagged proteins (PGK1, phosphoglycerate kinase 1, TDH1, triose phosphate dehydrogenase, TRX2, thioredoxine 2, SHE2, Swi5p-dependent HO Expression 2). (e) Validation of HuH-7 mRNA interactome RBPs using polynucleotide kinase-mediated 32P labelling after IP with an anti-eGFP antibody (negative control eGFP and MRPP3, mitochondrial RNAseP protein 3; positive control MOV10, moloney leukaemia virus 10; and novel RBPs, HSD17B10, hydroxysteroid dehydrogenase 17B 10, c11orf68, chromosome 11 open reading frame 68, CRKL, v-crk avian sarcoma virus CT10 oncogene homologue-like, LLPH, long-term synaptic facilitation homolog, PGK1, phosphoglycerate kinase 1). (f) Comparison of mRNA interactome RBPs from HuH-7 with those from HeLa and HEK293 cells. (g) Comparison of the yeast mRNA interactome RBPs to a published study using cCL.
Figure 2
Figure 2. Evolutionary expansion of RBP short peptide motifs.
(a) Overlap of yeast and human mRNA interactome orthologs. (b) Definition of InParanoid ortholog groups of mRNA interactome RBPs. (c) Tripeptide motifs showing expansion in number between human RBPs compared with their orthologous yeast RBPs (FDR 0.2). (d) Average copy number of motifs from (c), calculated for yeast, C. elegans, D. melanogaster, D. rerio and human orthologous RBPs of the core mRNA interactome and for non-RNA-binding orthologous proteins (non-RNA binders). (e) Examples of orthologous RBPs with increased copy number of motifs from c. Protein sequences are not drawn to scale; the relative position of domains and motifs within proteins is retained.
Figure 3
Figure 3. Yeast and human mRNA interactomes harbour hundreds of enigmRBPs.
(a) Yeast and human mRNA interactome protein annotations according to domains and functional characteristics. (b) Subset of classic metabolic enzymes within mRNA interactomes. (c) Proportion of classic metabolic enzymes within the conserved core mRNA interactome. (d) GO biochemical pathway enrichment of the enzyme-RBPs from yeast and HuH-7. (e) Simplified scheme of central carbon metabolism in yeast and human. Blue lines represent enzymes, filled green circles indicate activity of enzyme as RBPs, empty circles indicate lack of evidence for RNA binding.
Figure 4
Figure 4. HSD17B10 iCLIP.
(a,b) Raw crosslink sites reads from for HSD17B10 wt and eGFP plotted on heavy and light mt strand, respectively. (c) Enrichment plot for differential crosslink sites on mt RNAs for HSD17B10 wt compared with eGFP. (d) Average fold enrichments for the HSD17B10/eGFP of the crosslink sites plotted on generic tRNA bound by HSD17B10 (15/22), normalized by the region length. (e) Enrichment plot for differential crosslink clusters on mt RNAs for HSD17B10 wt compared with the R130C disease variant.
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