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• W2487752912 abstract "•mRNA interactome capture and RBDmap reveal the cardiomyocyte RNA-binding proteome•1,148 RBPs are identified, 393 of which are thus far unique to cardiomyocytes•Many cardiac RBPs have links to heart disease and mitochondrial metabolism•Contacts of metabolic enzymes with RNA frequently involve Rossmann fold domains RNA functions through the dynamic formation of complexes with RNA-binding proteins (RBPs) in all clades of life. We determined the RBP repertoire of beating cardiomyocytic HL-1 cells by jointly employing two in vivo proteomic methods, mRNA interactome capture and RBDmap. Together, these yielded 1,148 RBPs, 391 of which are shared with all other available mammalian RBP repertoires, while 393 are thus far unique to cardiomyocytes. RBDmap further identified 568 regions of RNA contact within 368 RBPs. The cardiomyocyte mRNA interactome composition reflects their unique biology. Proteins with roles in cardiovascular physiology or disease, mitochondrial function, and intermediary metabolism are all highly represented. Notably, we identified 73 metabolic enzymes as RBPs. RNA-enzyme contacts frequently involve Rossmann fold domains with examples in evidence of both, mutual exclusivity of, or compatibility between RNA binding and enzymatic function. Our findings raise the prospect of previously hidden RNA-mediated regulatory interactions among cardiomyocyte gene expression, physiology, and metabolism. RNA functions through the dynamic formation of complexes with RNA-binding proteins (RBPs) in all clades of life. We determined the RBP repertoire of beating cardiomyocytic HL-1 cells by jointly employing two in vivo proteomic methods, mRNA interactome capture and RBDmap. Together, these yielded 1,148 RBPs, 391 of which are shared with all other available mammalian RBP repertoires, while 393 are thus far unique to cardiomyocytes. RBDmap further identified 568 regions of RNA contact within 368 RBPs. The cardiomyocyte mRNA interactome composition reflects their unique biology. Proteins with roles in cardiovascular physiology or disease, mitochondrial function, and intermediary metabolism are all highly represented. Notably, we identified 73 metabolic enzymes as RBPs. RNA-enzyme contacts frequently involve Rossmann fold domains with examples in evidence of both, mutual exclusivity of, or compatibility between RNA binding and enzymatic function. Our findings raise the prospect of previously hidden RNA-mediated regulatory interactions among cardiomyocyte gene expression, physiology, and metabolism. RNA-binding proteins (RBPs) are critical interaction partners for all cellular RNAs. RNAs recruit RBPs to form dynamic ribonucleoprotein particles (RNPs), and it is these assemblies that execute RNA function (Castello et al., 2013Castello A. Fischer B. Hentze M.W. Preiss T. RNA-binding proteins in Mendelian disease.Trends Genet. 2013; 29: 318-327Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, Chen and Shyu, 2014Chen C.Y. Shyu A.B. Emerging mechanisms of mRNP remodeling regulation.Wiley Interdiscip. Rev. RNA. 2014; 5: 713-722Crossref PubMed Scopus (14) Google Scholar, Singh et al., 2015Singh G. Pratt G. Yeo G.W. Moore M.J. The clothes make the mRNA: past and present trends in mRNP fashion.Annu. Rev. Biochem. 2015; 84: 325-354Crossref PubMed Scopus (237) Google Scholar). Multiple sequencing-based RBP footprinting studies (König et al., 2012König J. Zarnack K. Luscombe N.M. Ule J. Protein-RNA interactions: new genomic technologies and perspectives.Nat. Rev. Genet. 2012; 13: 77-83Crossref PubMed Scopus (314) Google Scholar) have now attested to the long-held view that RBPs interact with RNA in an intricate and highly combinatorial manner (Keene, 2007Keene J.D. RNA regulons: coordination of post-transcriptional events.Nat. Rev. Genet. 2007; 8: 533-543Crossref PubMed Scopus (1008) Google Scholar). The identification of proteins that co-purify with polyadenylated RNA from native cellular contexts by mass spectrometry, dubbed mRNA interactome capture, has yielded the first comprehensive views of active RBPs in eukaryotic cells. Specifically, mRNA interactome capture from human cervical cancer (HeLa) (Castello et al., 2012Castello A. Fischer B. Eichelbaum K. Horos R. Beckmann B.M. Strein C. Davey N.E. Humphreys D.T. Preiss T. Steinmetz L.M. et al.Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.Cell. 2012; 149: 1393-1406Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar) and embryonic kidney (HEK293) cells (Baltz et al., 2012Baltz A.G. Munschauer M. Schwanhäusser B. Vasile A. Murakawa Y. Schueler M. Youngs N. Penfold-Brown D. Drew K. Milek M. et al.The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.Mol. Cell. 2012; 46: 674-690Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar), murine embryonic stem cells (ESCs) (Kwon et al., 2013Kwon S.C. Yi H. Eichelbaum K. Föhr S. Fischer B. You K.T. Castello A. Krijgsveld J. Hentze M.W. Kim V.N. The RNA-binding protein repertoire of embryonic stem cells.Nat. Struct. Mol. Biol. 2013; 20: 1122-1130Crossref PubMed Scopus (332) Google Scholar), S. cerevisiae (Beckmann et al., 2015Beckmann B.M. Horos R. Fischer B. Castello A. Eichelbaum K. Alleaume A.M. Schwarzl T. Curk T. Foehr S. Huber W. et al.The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs.Nat. Commun. 2015; 6: 10127Crossref PubMed Scopus (285) Google Scholar, Matia-González et al., 2015Matia-González A.M. Laing E.E. Gerber A.P. Conserved mRNA-binding proteomes in eukaryotic organisms.Nat. Struct. Mol. Biol. 2015; 22: 1027-1033Crossref PubMed Scopus (111) Google Scholar, Mitchell et al., 2013Mitchell S.F. Jain S. She M. Parker R. Global analysis of yeast mRNPs.Nat. Struct. Mol. Biol. 2013; 20: 127-133Crossref PubMed Scopus (252) Google Scholar), C. elegans (Matia-González et al., 2015Matia-González A.M. Laing E.E. Gerber A.P. Conserved mRNA-binding proteomes in eukaryotic organisms.Nat. Struct. Mol. Biol. 2015; 22: 1027-1033Crossref PubMed Scopus (111) Google Scholar), as well as human hepatoma cells (HuH-7) (Beckmann et al., 2015Beckmann B.M. Horos R. Fischer B. Castello A. Eichelbaum K. Alleaume A.M. Schwarzl T. Curk T. Foehr S. Huber W. et al.The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs.Nat. Commun. 2015; 6: 10127Crossref PubMed Scopus (285) Google Scholar) together identified over 1,000 RBPs, many of which had no prior RNA-related annotation. Despite this already considerable expansion of mRNP componentry, more distinct cellular contexts need to be studied to define context-specific RBP repertoires. The identification of so many RBPs indicates hitherto-unknown connections between seemingly disparate cellular processes and unexpected “moonlighting” activities that proteins carry out in a highly compartmentalized cellular environment (Copley, 2012Copley S.D. Moonlighting is mainstream: paradigm adjustment required.BioEssays. 2012; 34: 578-588Crossref PubMed Scopus (152) Google Scholar). One such area deserving of further exploration is suggested by the reported ability of several metabolic enzymes to interact with RNA (Castello et al., 2015Castello A. Hentze M.W. Preiss T. Metabolic enzymes enjoying new partnerships as RNA-binding proteins.Trends Endocrinol. Metab. 2015; 26: 746-757Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, Cieśla, 2006Cieśla J. Metabolic enzymes that bind RNA: yet another level of cellular regulatory network?.Acta Biochim. Pol. 2006; 53: 11-32Crossref PubMed Scopus (86) Google Scholar, Hentze, 1994Hentze M.W. Enzymes as RNA-binding proteins: a role for (di)nucleotide-binding domains?.Trends Biochem. Sci. 1994; 19: 101-103Abstract Full Text PDF PubMed Scopus (123) Google Scholar, Hentze and Preiss, 2010Hentze M.W. Preiss T. The REM phase of gene regulation.Trends Biochem. Sci. 2010; 35: 423-426Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Enzymes could moonlight to regulate mRNA utilization in response to co-factor or metabolite levels, as documented for the cytosolic aconitase (ACO1)/iron regulatory protein (IRP1) paradigm (Muckenthaler et al., 2008Muckenthaler M.U. Galy B. Hentze M.W. Systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network.Annu. Rev. Nutr. 2008; 28: 197-213Crossref PubMed Scopus (480) Google Scholar). Conversely, RNA could affect enzyme activity, and collectively these interactions could form regulatory RNA-enzyme-metabolite (REM) networks (Hentze and Preiss, 2010Hentze M.W. Preiss T. The REM phase of gene regulation.Trends Biochem. Sci. 2010; 35: 423-426Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Here, we report the adaptation and parallel application of mRNA interactome capture (Castello et al., 2012Castello A. Fischer B. Eichelbaum K. Horos R. Beckmann B.M. Strein C. Davey N.E. Humphreys D.T. Preiss T. Steinmetz L.M. et al.Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.Cell. 2012; 149: 1393-1406Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar) and RBDmap (Castello et al., 2016Castello A. Fischer B. Frese C.K. Horos R. Alleaume A.-M. Föhr S. Curk T. Krijgsveld J. Hentze M.W. Comprehensive identification of RNA-binding domains in human cells.Mol Cell. 2016; 63https://doi.org/10.1016/j.molcel.2016.06.029Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) to beating murine HL-1 cardiomyocytes (Claycomb et al., 1998Claycomb W.C. Lanson Jr., N.A. Stallworth B.S. Egeland D.B. Delcarpio J.B. Bahinski A. Izzo Jr., N.J. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.Proc. Natl. Acad. Sci. USA. 1998; 95: 2979-2984Crossref PubMed Scopus (1235) Google Scholar). We chose cardiomyocytes because of their unique metabolic requirements and importance to human disease (Rosca et al., 2013Rosca M.G. Tandler B. Hoppel C.L. Mitochondria in cardiac hypertrophy and heart failure.J. Mol. Cell. Cardiol. 2013; 55: 31-41Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), gaining insight especially into the scope of mitochondrial RBPs. Both proteomic methods require large numbers of cells, and thus we employed the murine HL-1 cardiomyocyte cell line as readily renewable source material. HL-1 cells are widely used as a model as they retain many characteristics of adult cardiomyocytes (Claycomb et al., 1998Claycomb W.C. Lanson Jr., N.A. Stallworth B.S. Egeland D.B. Delcarpio J.B. Bahinski A. Izzo Jr., N.J. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.Proc. Natl. Acad. Sci. USA. 1998; 95: 2979-2984Crossref PubMed Scopus (1235) Google Scholar), although compared to native myocardium their energy metabolism is less organized (e.g., Eimre et al., 2008Eimre M. Paju K. Pelloux S. Beraud N. Roosimaa M. Kadaja L. Gruno M. Peet N. Orlova E. Remmelkoor R. et al.Distinct organization of energy metabolism in HL-1 cardiac cell line and cardiomyocytes.Biochim. Biophys. Acta. 2008; 1777: 514-524Crossref PubMed Scopus (41) Google Scholar). Confluent and spontaneously beating murine HL-1 cells were irradiated with UV light (150 mJ/cm2 at 254 nm) to induce covalent crosslinks (CLs) between proteins and RNA within native complexes. Under these conditions, crosslink formation will typically be sub-stoichiometric and selectively occur at “zero” distance protein-RNA interactions (Castello et al., 2012Castello A. Fischer B. Eichelbaum K. Horos R. Beckmann B.M. Strein C. Davey N.E. Humphreys D.T. Preiss T. Steinmetz L.M. et al.Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.Cell. 2012; 149: 1393-1406Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar, Castello et al., 2016Castello A. Fischer B. Frese C.K. Horos R. Alleaume A.-M. Föhr S. Curk T. Krijgsveld J. Hentze M.W. Comprehensive identification of RNA-binding domains in human cells.Mol Cell. 2016; 63https://doi.org/10.1016/j.molcel.2016.06.029Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). After denaturing cell lysis, RNA-protein complexes were captured on oligo(dT) beads and washed with high-salt/anionic detergent buffer to remove non-crosslinked proteins. Bound material was then processed for “mRNA interactome capture” to determine the scope of cardiomyocyte RBPs or for “RBDmap”, which maps protein regions contacting RNA, as schematized in Figure 1A. Three independent biological replicates were processed for each approach, as well as parallel non-crosslinked controls (noCL). Aliquots taken after the first round of capture demonstrated selective purification of polyadenylated RNAs (cytosolic and mitochondrial; Figure 1B), known RBPs such as ELAVL1 and PTBP1 (Figures 1C and S1A), and a distinct subset of cellular proteins as the mRNA interactome (Figures 1D and S1B). For mRNA interactome capture (Castello et al., 2012Castello A. Fischer B. Eichelbaum K. Horos R. Beckmann B.M. Strein C. Davey N.E. Humphreys D.T. Preiss T. Steinmetz L.M. et al.Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.Cell. 2012; 149: 1393-1406Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar), RBPs were liberated by RNase treatment, digested with Trypsin/Lys-C protease mix, and analyzed by quantitative mass spectrometry. This identified 963 high-confidence RBPs (false discovery rate [FDR], 1%; see Figure S1D for reproducibility between replicates). Mass spectrometry was also performed on whole-cell lysate (WCL) samples, which identified 4,749 proteins (Table S1). The RBDmap approach is described and validated in detail elsewhere (Castello et al., 2016Castello A. Fischer B. Frese C.K. Horos R. Alleaume A.-M. Föhr S. Curk T. Krijgsveld J. Hentze M.W. Comprehensive identification of RNA-binding domains in human cells.Mol Cell. 2016; 63https://doi.org/10.1016/j.molcel.2016.06.029Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). In applying RBDmap here, complexes eluted after a first round of capture were digested with Lys-C only, before a second round of oligo(dT) purification (Figure S1C). This separated protein fragments into a “released” and an “RNA bound” pool, which were both treated with RNase and Trypsin. All tryptic fragments within the released pool, termed Rpeps, can in principle be detected by mass spectrometry (Figure 1A). By contrast, tryptic digestion of the “RNA-bound” pool will yield two types of peptides. One type (termed Xpep, depicted in green, Figure 1A) will still carry a remnant, or remnants, of crosslinked RNA. Due to this heterogeneous mass shift, Xpeps are difficult to be detected by mass spectrometry (see Castello et al., 2016Castello A. Fischer B. Frese C.K. Horos R. Alleaume A.-M. Föhr S. Curk T. Krijgsveld J. Hentze M.W. Comprehensive identification of RNA-binding domains in human cells.Mol Cell. 2016; 63https://doi.org/10.1016/j.molcel.2016.06.029Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, Kramer et al., 2014Kramer K. Sachsenberg T. Beckmann B.M. Qamar S. Boon K.L. Hentze M.W. Kohlbacher O. Urlaub H. Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins.Nat. Methods. 2014; 11: 1064-1070Crossref PubMed Scopus (159) Google Scholar). The other type (termed neighboring peptide or Npep, depicted in red, Figure 1A) is of predictable mass and thus readily detectable. Extension from the Npep boundaries to adjacent Lys-C sites can nevertheless still predict the Xpep coordinates, although sub-stoichiometric crosslinking and protease cleavage will add some complexity to the assessment of individual examples. Note that proteolytic processing efficiency is close to 100% (Figure S1C). Therefore, matching Npep and Xpep(s) together constitute the original RNA-bound Lys-C fragment (termed RBDpep, Figure 1A). Proteins derived from 368 genes exhibited RBDpeps that were enriched in the RNA bound over the released pool at an FDR of 1% (see Figure S1E for reproducibility between replicates). Proteins that lack a Trypsin cleavage site within their RBDpeps will not be “visible” by RBDmap but may still be detected by mRNA interactome capture. Conversely, the additional proteolytic step and enrichment by a second oligo(dT) capture round in RBDmap will reduce sample complexity and experimental noise, thus improving detection for another subset of proteins (Castello et al., 2016Castello A. Fischer B. Frese C.K. Horos R. Alleaume A.-M. Föhr S. Curk T. Krijgsveld J. Hentze M.W. Comprehensive identification of RNA-binding domains in human cells.Mol Cell. 2016; 63https://doi.org/10.1016/j.molcel.2016.06.029Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Consequently, proteins identified by RBDmap substantially (65%), but not completely, overlap with those of the mRNA interactome approach (183 RBPs are shared with the HL-1 mRNA interactome Figure 1A; an additional 51 are shared with the following mRNA interactomes: HeLa, HEK293, HuH-7). Taken together, both approaches define a “superset” of 1,148 cardiomyocyte RBPs (Tables S1, S2, and S3). Compared to the WCL proteome, cardiomyocyte RBPs were enriched for RNA-related functions (56% have RNA-related annotation, Figure 1E; see Figure S1F for the most enriched/depleted Gene Ontology [GO] terms), as expected. A similar GO term enrichment was seen with either the mRNA interactome or RBDmap set individually (data not shown). KEGG pathway enrichment analysis confirms this but interestingly also indicates overrepresentation of several pathways of intermediary metabolism among RBPs without prior RNA-related annotation (Figure 1G; see below). Similarly, proteins with classic or non-classic RNA-binding domains (RBD) are also overrepresented (Figure 1F). Many known RBD types are enriched among the cardiomyocyte RBPs (Figures S1G and S1H). For instance, we captured most of the RRM-containing proteins expressed in HL-1 cells (139 out of 168; Figure S1G). Similar trends were seen for other RBDs, including DEAD box helicase, KH, PWI, and PUF domains as well as many of the expected zinc finger domain subtypes (Figures S1I and S1J). We further identified all eight proteins with the MIF4G fold expressed in HL-1 cells as RBPs (i.e., EIF4G1-3, UPF2, CWC22, CTIF, NOM1, NCBP1). These proteins relate to the nexus of mRNA splicing, translation initiation, and nonsense-mediated decay. Enrichment of the Nol1_Nop2_Fmu domain was driven by capture of four members of the NSUN family of RNA:m5C (5-methylcytosine) methyltransferases (NSUN1, -2, -4, -5). Nucleoside modifications in polyadenylated RNA have received much attention lately and indeed, 29 proteins with annotations related to “RNA modification” were detected as cardiomyocyte RBPs (Table S3), prominently covering the enzymology of m5C, m6A (N6-methyladenosine), and pseudouridine modifications, as well as adenosine to inosine editing, all shown to occur in mRNA (Jaffrey, 2014Jaffrey S.R. An expanding universe of mRNA modifications.Nat. Struct. Mol. Biol. 2014; 21: 945-946Crossref PubMed Scopus (28) Google Scholar, Sibbritt et al., 2013Sibbritt T. Patel H.R. Preiss T. Mapping and significance of the mRNA methylome.Wiley Interdiscip. Rev. RNA. 2013; 4: 397-422Crossref PubMed Scopus (71) Google Scholar). Beyond that, we find componentry involved in several additional RNA modifications, including m5U (5-methyluridine), m66A (N6,N6-dimethyladenosine), and D (dihydrouridine), suggesting that the diversity of RNA modifications in polyadenylated RNAs may be richer than currently documented. All seven mammalian pentatricopeptide repeat (PPR)-containing proteins were identified here. PPR proteins function in mitochondrial RNA metabolism (Lightowlers and Chrzanowska-Lightowlers, 2013Lightowlers R.N. Chrzanowska-Lightowlers Z.M. Human pentatricopeptide proteins: only a few and what do they do?.RNA Biol. 2013; 10: 1433-1438Crossref PubMed Scopus (41) Google Scholar) and recognize RNA in a modular manner (Filipovska and Rackham, 2013Filipovska A. Rackham O. Pentatricopeptide repeats: modular blocks for building RNA-binding proteins.RNA Biol. 2013; 10: 1426-1432Crossref PubMed Scopus (33) Google Scholar). Several other protein domains lacking reported RNA-binding activity were also well represented in our datasets (Figure S1K), possibly reflecting direct involvement in RNA binding in some cases. Histones were notable (mostly H1 and H2A variants), possibly relating to the known association of RNA with chromatin (Mondal et al., 2010Mondal T. Rasmussen M. Pandey G.K. Isaksson A. Kanduri C. Characterization of the RNA content of chromatin.Genome Res. 2010; 20: 899-907Crossref PubMed Scopus (148) Google Scholar). Consistent with observations in the HeLa cell mRNA interactome (Castello et al., 2012Castello A. Fischer B. Eichelbaum K. Horos R. Beckmann B.M. Strein C. Davey N.E. Humphreys D.T. Preiss T. Steinmetz L.M. et al.Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.Cell. 2012; 149: 1393-1406Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar), we detected RNA binding by 11 peptidyl-prolyl isomerases (PPIases, Table S3). Several of these are known for their involvement in nuclear gene expression (i.e., chromatin structure, gene transcription, mRNA splicing, and export) and some have recognized RBDs (Schiene-Fischer, 2015Schiene-Fischer C. Multidomain Peptidyl Prolyl cis/trans Isomerases.Biochim. Biophys. Acta. 2015; 1850: 2005-2016Crossref PubMed Scopus (58) Google Scholar); however, for others these findings suggest hitherto-unknown roles for RNA in PPIase biology and pathology (see below). Cardiomyocyte RBPs also display the biophysical and sequence features expected of bona fide RBPs. They range from low to high abundance, with some tendency toward higher abundance compared to the WCL proteome (Figure S2A), reflecting similar observations reported elsewhere (Castello et al., 2016Castello A. Fischer B. Frese C.K. Horos R. Alleaume A.-M. Föhr S. Curk T. Krijgsveld J. Hentze M.W. Comprehensive identification of RNA-binding domains in human cells.Mol Cell. 2016; 63https://doi.org/10.1016/j.molcel.2016.06.029Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, Matia-González et al., 2015Matia-González A.M. Laing E.E. Gerber A.P. Conserved mRNA-binding proteomes in eukaryotic organisms.Nat. Struct. Mol. Biol. 2015; 22: 1027-1033Crossref PubMed Scopus (111) Google Scholar). Cardiomyocyte RBPs have no length bias, but they are shifted toward a more alkaline isoelectric point and lower hydrophobicity (Figures S2B–S2D), as reported for other mRNA interactomes (Castello et al., 2012Castello A. Fischer B. Eichelbaum K. Horos R. Beckmann B.M. Strein C. Davey N.E. Humphreys D.T. Preiss T. Steinmetz L.M. et al.Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.Cell. 2012; 149: 1393-1406Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar, Kwon et al., 2013Kwon S.C. Yi H. Eichelbaum K. Föhr S. Fischer B. You K.T. Castello A. Krijgsveld J. Hentze M.W. Kim V.N. The RNA-binding protein repertoire of embryonic stem cells.Nat. Struct. Mol. Biol. 2013; 20: 1122-1130Crossref PubMed Scopus (332) Google Scholar). They further display an elevated content of intrinsically disordered regions and are enriched for the amino acids arginine (R), lysine (K), tyrosine (Y), and glycine (G) (Figures S2E and S2F). Low-complexity and repetitive regions, known to favor formation of RNA-protein granules (Kato et al., 2012Kato M. Han T.W. Xie S. Shi K. Du X. Wu L.C. Mirzaei H. Goldsmith E.J. Longgood J. Pei J. et al.Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.Cell. 2012; 149: 753-767Abstract Full Text Full Text PDF PubMed Scopus (1301) Google Scholar) and to frequently occur in RBPs (Castello et al., 2012Castello A. Fischer B. Eichelbaum K. Horos R. Beckmann B.M. Strein C. Davey N.E. Humphreys D.T. Preiss T. Steinmetz L.M. et al.Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.Cell. 2012; 149: 1393-1406Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar), are also overrepresented (Figures S2G and S2H). All of these features also apply to the subset of 688 cardiomyocyte RBPs lacking previously known RBDs. Amino acid patterns that center around the enriched residues R, K, Y, S, and G include RS repeats often found in splicing factors and resemble RGG and YGG boxes or poly(K) stretches, each previously implicated in RNA binding (Figure S2I) (Castello et al., 2012Castello A. Fischer B. Eichelbaum K. Horos R. Beckmann B.M. Strein C. Davey N.E. Humphreys D.T. Preiss T. Steinmetz L.M. et al.Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.Cell. 2012; 149: 1393-1406Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar). Overall, the RNA-binding proteome of cardiomyocytes shows substantial overlap with other mRNA interactomes: 429 RBPs are shared with the murine ESC set (Kwon et al., 2013Kwon S.C. Yi H. Eichelbaum K. Föhr S. Fischer B. You K.T. Castello A. Krijgsveld J. Hentze M.W. Kim V.N. The RNA-binding protein repertoire of embryonic stem cells.Nat. Struct. Mol. Biol. 2013; 20: 1122-1130Crossref PubMed Scopus (332) Google Scholar), and 717 are in common with three available human mRNA interactomes from HEK293, HeLa, and HuH-7 cells (Baltz et al., 2012Baltz A.G. Munschauer M. Schwanhäusser B. Vasile A. Murakawa Y. Schueler M. Youngs N. Penfold-Brown D. Drew K. Milek M. et al.The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.Mol. Cell. 2012; 46: 674-690Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar, Beckmann et al., 2015Beckmann B.M. Horos R. Fischer B. Castello A. Eichelbaum K. Alleaume A.M. Schwarzl T. Curk T. Foehr S. Huber W. et al.The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs.Nat. Commun. 2015; 6: 10127Crossref PubMed Scopus (285) Google Scholar, Castello et al., 2012Castello A. Fischer B. Eichelbaum K. Horos R. Beckmann B.M. Strein C. Davey N.E. Humphreys D.T. Preiss T. Steinmetz L.M. et al.Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.Cell. 2012; 149: 1393-1406Abstract Full Text Full Text PDF PubMed Scopus (1353) Google Scholar). 391 “core” RBPs emerge as “in common” between all reported mammalian mRNA interactomes, while 393 RBPs are thus far unique to the cardiomyocyte RBP set (Figure S2J). As expected, the core RBPs are particularly enriched for RNA-related GOMF annotations and recognized RBDs. The “unique” cardiomyocyte RBPs have lower proportions of these attributes and instead feature elevated proportions of proteins with mitochondrial localization, links to cardiovascular disease and development, genetic disease, and metabolic enzyme function (Figure S2K). This likely reflects the unique physiology of cardiomyocytes, e.g., their heavy reliance on mitochondrial metabolism, and their importance to human disease, aspects that we explore further below. RBDmap identified 568 RBDpeps at 1% FDR, representing RNA-binding regions of 368 cardiomyocyte RBPs. The distances between Lys-C and Trypsin cleavage sites in individual proteins define the resolution achievable by RBDmap by determining the lengths of individual RBDpeps, Npeps and Xpeps (range/median: 7–161/20 amino acids (aa) for RBDpeps, 7–30/11 aa for Npeps, 1–148/11 aa for Xpeps) (Figure S3A). Limitations of RBDmap are that some protein-RNA contacts can be absent from the data due to (1) absence of suitable crosslink geometry; (2) lack of trypsin cleavage site(s) within RBDpeps; and (3) peptide detection biases of mass spectrometry. Moreover, Xpep can only give the boundaries within which the actual RNA-protein crosslink site is situated but cannot locate it further (see Castello et al., 2016Castello A. Fischer B. Frese C.K. Horos R. Alleaume A.-M. Föhr S. Curk T. Krijgsveld J. Hentze M.W. Comprehensive identification of RNA-binding domains in human cells.Mol Cell. 2016; 63https://doi.org/10.1016/j.molcel.2016.06.029Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar for a comprehensive evaluation). That said, RBDpeps and released peptides exhibit the expected divergent properties. Positively charged (R, K), aromatic (F, W, Y, H), and tiny amino acids (A, G, S, T, C) are enriched among RBDpeps, whereas negatively charged (D, E) and aliphatic amino acids (I, L, V) are more prevalent in released peptides (Figure 2A). A majority (347, 61.1%) of RBDpeps map to disordered regions (Figure 2B), and this subset retains the same amino acid bias (Figure 2A), evoking a broader role of disordered regions in RNA binding. These observations match those with RBDmap of HeLa cells (Castello et al., 2016Castello A. Fischer B. Frese C.K. Horos R. Alleaume A.-M. Föhr S. Curk T. Krijgsveld J. Hentze M.W. Comprehensive identification of RNA-binding domains in human cells.Mol Cell. 2016; 63https://doi.org/10.1016/j.molcel.2016.06.029Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). To assess the consistency of RBDmap assignments, we identified proteins with homologs present in both the HL-1 and HeLa datasets (Figure S3A) and tested for concordance of RNA-binding site identifications. Approximately two-thirds of the associated mouse RBDpeps (161 of 237) had an equivalent counterpart in the human dataset (Figure S3B), a high proportion given the use of different cell lines from different organisms as well as the relative inefficiency and spatial limitations of UV crosslinking (Castello et al., 2012Castello A. Fischer B. Eichelbaum K. Horos R. Beckmann B.M. Strein C. Davey N.E. Humphreys D.T. Preiss T. Steinmetz L.M. et al.Insights int" @default.
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