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• W2480300184 abstract "•Experimental generation of an atlas of RNA-binding sites (RBS) in human cells•RBS overlap with enzymatic cores and protein-protein interaction sites•About half of the total RBS map to disordered protein regions•RBS are enriched for phosphorylation, acetylation, and methylation sites Mammalian cells harbor more than a thousand RNA-binding proteins (RBPs), with half of these employing unknown modes of RNA binding. We developed RBDmap to determine the RNA-binding sites of native RBPs on a proteome-wide scale. We identified 1,174 binding sites within 529 HeLa cell RBPs, discovering numerous RNA-binding domains (RBDs). Catalytic centers or protein-protein interaction domains are in close relationship with RNA-binding sites, invoking possible effector roles of RNA in the control of protein function. Nearly half of the RNA-binding sites map to intrinsically disordered regions, uncovering unstructured domains as prevalent partners in protein-RNA interactions. RNA-binding sites represent hot spots for defined posttranslational modifications such as lysine acetylation and tyrosine phosphorylation, suggesting metabolic and signal-dependent regulation of RBP function. RBDs display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles. RBDmap thus yields profound insights into native protein-RNA interactions in living cells. Mammalian cells harbor more than a thousand RNA-binding proteins (RBPs), with half of these employing unknown modes of RNA binding. We developed RBDmap to determine the RNA-binding sites of native RBPs on a proteome-wide scale. We identified 1,174 binding sites within 529 HeLa cell RBPs, discovering numerous RNA-binding domains (RBDs). Catalytic centers or protein-protein interaction domains are in close relationship with RNA-binding sites, invoking possible effector roles of RNA in the control of protein function. Nearly half of the RNA-binding sites map to intrinsically disordered regions, uncovering unstructured domains as prevalent partners in protein-RNA interactions. RNA-binding sites represent hot spots for defined posttranslational modifications such as lysine acetylation and tyrosine phosphorylation, suggesting metabolic and signal-dependent regulation of RBP function. RBDs display a high degree of evolutionary conservation and incidence of Mendelian mutations, suggestive of important functional roles. RBDmap thus yields profound insights into native protein-RNA interactions in living cells. RNA metabolism relies on the dynamic interplay of RNAs with RNA-binding proteins (RBPs) forming ribonucleoprotein complexes, which control RNA fate from synthesis to decay (Glisovic et al., 2008Glisovic T. Bachorik J.L. Yong J. Dreyfuss G. RNA-binding proteins and post-transcriptional gene regulation.FEBS Lett. 2008; 582: 1977-1986Crossref PubMed Scopus (923) Google Scholar). Due to their central role in cell biology, it is unsurprising that mutations in RBPs underlie numerous hereditary diseases (Castello et al., 2013aCastello 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, Lukong et al., 2008Lukong K.E. Chang K.W. Khandjian E.W. Richard S. RNA-binding proteins in human genetic disease.Trends Genet. 2008; 24: 416-425Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). Many RBPs are modular, built from a limited pool of RNA-binding domains (RBDs), including the RNA recognition motif (RRM) and other canonical RBDs (Lunde et al., 2007Lunde B.M. Moore C. Varani G. RNA-binding proteins: modular design for efficient function.Nat. Rev. Mol. Cell Biol. 2007; 8: 479-490Crossref PubMed Scopus (867) Google Scholar). These domains have been characterized biochemically and structurally, furthering our understanding of protein-RNA interactions. The identification of unorthodox RBPs lacking canonical RBDs expands the scope of physiologically important protein-RNA interactions (e.g., Jia et al., 2008Jia J. Arif A. Ray P.S. Fox P.L. WHEP domains direct noncanonical function of glutamyl-Prolyl tRNA synthetase in translational control of gene expression.Mol. Cell. 2008; 29: 679-690Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). System-wide approaches to identify RBPs have recently been developed, including immobilization of RNA probes (Butter et al., 2009Butter F. Scheibe M. Mörl M. Mann M. Unbiased RNA-protein interaction screen by quantitative proteomics.Proc. Natl. Acad. Sci. USA. 2009; 106: 10626-10631Crossref PubMed Scopus (104) Google Scholar) or proteins (Scherrer et al., 2010Scherrer T. Mittal N. Janga S.C. Gerber A.P. A screen for RNA-binding proteins in yeast indicates dual functions for many enzymes.PLoS ONE. 2010; 5: e15499Crossref PubMed Scopus (108) Google Scholar, Tsvetanova et al., 2010Tsvetanova N.G. Klass D.M. Salzman J. Brown P.O. Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae.PLoS One. 2010; 5: e12671Crossref PubMed Scopus (131) Google Scholar), followed by in vitro selection of their interaction partners. These experiments identified numerous proteins previously unknown to bind RNA. While informative, in vitro protein-RNA interactions may arise non-physiologically from the electrostatic properties of RNA. To address this limitation, in vivo UV crosslinking has been used to covalently stabilize native protein-RNA interactions occurring in living cells. After cell lysis, proteins covalently bound to polyadenylated [poly(A)] RNAs are isolated by oligo(dT) selection and identified by quantitative mass spectrometry (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, 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). This approach (named RNA interactome capture) identified over a thousand RBPs in HeLa and HEK293 cells, hundreds of which were previously unknown to bind RNA. Subsequently, similar data sets were obtained from mouse embryonic stem cells, Saccharomyces cerevisiae, and Caenorhabditis elegans (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, 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, 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), confirming earlier findings and further uncovering the repertoire of RBPs. Several of the unorthodox RBPs identified in these studies have been characterized for their physiological roles in RNA biology. These include metabolic enzymes (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), regulators of alternative splicing (Papasaikas et al., 2015Papasaikas P. Tejedor J.R. Vigevani L. Valcárcel J. Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery.Mol. Cell. 2015; 57: 7-22Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, Tejedor et al., 2015Tejedor J.R. Papasaikas P. Valcárcel J. Genome-wide identification of Fas/CD95 alternative splicing regulators reveals links with iron homeostasis.Mol. Cell. 2015; 57: 23-38Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), the E3 ubiquitin ligase TRIM25 (Choudhury et al., 2014Choudhury N.R. Nowak J.S. Zuo J. Rappsilber J. Spoel S.H. Michlewski G. Trim25 is an RNA-specific activator of Lin28a/TuT4-mediated uridylation.Cell Rep. 2014; 9: 1265-1272Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), or the FAST kinase domain-containing protein 2 (FASTKD2) (Popow et al., 2015Popow J. Alleaume A.M. Curk T. Schwarzl T. Sauer S. Hentze M.W. FASTKD2 is an RNA-binding protein required for mitochondrial RNA processing and translation.RNA. 2015; 21: 1873-1884Crossref PubMed Scopus (61) Google Scholar). However, the RNA-binding regions of these unorthodox RBPs remain largely unknown. To identify the interaction sites of such proteins with RNA, UV crosslinking followed by extensive RNase treatment has been used to detect the peptide mass shift induced by the crosslinked RNA remnant via mass spectrometry (Schmidt et al., 2012Schmidt C. Kramer K. Urlaub H. Investigation of protein-RNA interactions by mass spectrometry--techniques and applications.J. Proteomics. 2012; 75: 3478-3494Crossref PubMed Scopus (36) Google Scholar). While conceptually simple, the mass heterogeneity of the nucleotide remnant has rendered this approach challenging in practice. Some RBDs have been characterized in vitro using this approach (reviewed in Schmidt et al., 2012Schmidt C. Kramer K. Urlaub H. Investigation of protein-RNA interactions by mass spectrometry--techniques and applications.J. Proteomics. 2012; 75: 3478-3494Crossref PubMed Scopus (36) Google Scholar), and a sophisticated algorithm allowed assignment of 257 binding sites from 124 proteins in yeast (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). While informative, this data set is strongly enriched for interactions mediated by RRMs, because the challenging identification of peptides with aberrant mass spectra requires both abundance and high crosslinking efficiency for detection. Nonetheless, 10% of the identified interaction sites mapped to non-canonical RBDs, supporting the existence of unanticipated modes of RNA binding. Here, we develop and exploit RBDmap as a method for the in vivo identification of RBDs on a proteome-wide scale. We identified 1,174 high-confidence RNA-binding sites in 529 RBPs from HeLa cells, generating an unprecedented atlas of RNA-binding architectures in vivo. To define how RBPs bind to RNA in living cells, we extended RNA interactome capture (Castello et al., 2013bCastello A. Horos R. Strein C. Fischer B. Eichelbaum K. Steinmetz L.M. Krijgsveld J. Hentze M.W. System-wide identification of RNA-binding proteins by interactome capture.Nat. Protoc. 2013; 8: 491-500Crossref PubMed Scopus (137) Google Scholar) by addition of an analytical protease digestion step followed by a second round of oligo(dT) capture and mass spectrometry (Figure 1A). First, UV light is applied to cell monolayers to covalently stabilize native protein-RNA interactions taking place at “zero” distance (Pashev et al., 1991Pashev I.G. Dimitrov S.I. Angelov D. Crosslinking proteins to nucleic acids by ultraviolet laser irradiation.Trends Biochem. Sci. 1991; 16: 323-326Abstract Full Text PDF PubMed Scopus (89) Google Scholar). While UV exposure using dosages exceeding those used here can potentially promote protein-protein crosslinking (Davidenko et al., 2016Davidenko N. Bax D.V. Schuster C.F. Farndale R.W. Hamaia S.W. Best S.M. Cameron R.E. Optimisation of UV irradiation as a binding site conserving method for crosslinking collagen-based scaffolds.J. Mater. Sci. Mater. Med. 2016; 27: 14Crossref PubMed Scopus (62) Google Scholar, Suchanek et al., 2005Suchanek M. Radzikowska A. Thiele C. Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells.Nat. Methods. 2005; 2: 261-267Crossref PubMed Scopus (366) Google Scholar), we could not detect such crosslinks under our conditions, evidenced by the lack of UV-dependent, high molecular weight complexes in RNase-treated samples (Figures S1A and S4A; Strein et al., 2014Strein C. Alleaume A.M. Rothbauer U. Hentze M.W. Castello A. A versatile assay for RNA-binding proteins in living cells.RNA. 2014; 20: 721-731Crossref PubMed Scopus (28) Google Scholar). Proteins crosslinked to poly(A) RNA are isolated using oligo(dT) magnetic beads and purified by stringent washes that include 500 mM LiCl and chaotropic detergents (0.5% LiDS), efficiently removing non-covalent binders (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., 2013bCastello A. Horos R. Strein C. Fischer B. Eichelbaum K. Steinmetz L.M. Krijgsveld J. Hentze M.W. System-wide identification of RNA-binding proteins by interactome capture.Nat. Protoc. 2013; 8: 491-500Crossref PubMed Scopus (137) Google Scholar). After elution, RBPs are proteolytically digested by either LysC or ArgC. These proteases were selected as best suited for RBDmap by an in silico simulation of their predicted cleavage patterns of known HeLa 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) and their compatibility with subsequent tryptic digestion (Figure S1B). Analysis by mass spectrometry (MS) of LysC- and ArgC-treated samples revealed an excellent match with the in silico predictions, as reflected by the low number of missed cleavages (Figures 1B and 1C). The extensive proteolysis of HeLa RBPs is achieved without compromising RNA integrity (Figures 1D and S1C–S1E). The average peptide length after LysC and ArgC treatment is ∼17 amino acids, which defines the resolution of RBDmap (Figure 1C). Note that the extensive protease treatment disrupts protein integrity, and thus protein-protein complexes that might have withstood the experimental conditions will be released into the supernatant. We collected an input sample aliquot after UV irradiation, oligo(dT) selection, and protease digestion, which in principle should reflect the RNA interactome (Figure 1A). When compared to a non-irradiated specificity control, the resulting high-confidence RBPs overlap 82% with the previously published human RNA interactomes (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). This high concordance shows that LysC and ArgC treatments are fully compatible with the RNA interactome capture protocol. The remaining two thirds of the LysC or ArgC-treated samples were subjected to a second round of oligo(dT) purification leading to two peptide pools (Figure 1A): (1) peptides released from the RNA into the supernatant, and (2) peptides remaining covalently bound to the RNA, representing the RNA-binding sites of the respective RBPs. Importantly, subsequent tryptic digestion of the RNA-bound LysC/ArgC fragments yields two classes of peptides: the portion that still remains crosslinked to the RNA (X-link) and its neighboring peptides (N-link) (Figure 1A). While the directly crosslinked peptides (X-link) are difficult to identify due to the heterogeneous mass shift induced by the residual nucleotides (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, Schmidt et al., 2012Schmidt C. Kramer K. Urlaub H. Investigation of protein-RNA interactions by mass spectrometry--techniques and applications.J. Proteomics. 2012; 75: 3478-3494Crossref PubMed Scopus (36) Google Scholar), the native peptides adjacent to the crosslinking site (N-link) can be identified by standard MS and peptide search algorithms. The original RNA-bound region of the RBP (i.e., RBDpep; Figure 1A), which includes both the crosslinked peptide (X-link) and its unmodified neighboring peptides (N-link), is then re-derived in silico by extending the MS-identified peptides to the two nearest LysC or ArgC cleavage sites. Analysis of the RNA-bound and released fractions by quantitative proteomics shows high correlation of the resulting peptide intensity ratios between independent biological replicates. These ratios follow a bimodal distribution with one mode representing the released peptides (gray) and the other the RNA-bound ones (red; Figures 1E and S1F). We detected 909 and 471 unique N-link peptides as significantly enriched in the RNA-bound fractions of LysC- or ArgC samples, respectively (1% false discovery rate, FDR) (Figure S1G). Notably, computed RNA-bound/released peptide intensity ratios also correlate between the LysC and ArgC data sets (Figure 1F), supporting the robustness of the workflow. Due to their different specificities, each protease also contributes unique 1% FDR RBDpeps to the complete peptide superset (Figure S1G), covering 529 RBPs that highly overlap with human RNA interactomes (Figure 1G) (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). Proteins within the RBDmap data set range from low to high abundance (Figure S1H), following a similar distribution as the input fraction and the HeLa RNA 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). Thus, RBDmap is not selective for highly abundant proteins. There were 154 additional RBPs that were identified here, helped by the reduction of sample complexity and of experimental noise by the additional proteolytic step and the second oligo(dT) capture. In agreement with this explanation, the relative abundance of corresponding RBDpeps is higher in the RNA-bound fractions than in the “input” samples (Figures 1H and S1I). Thus, RBDmap detects RNA-binding regions within hundreds of RBPs in one approach, even if it does not cover all RBPs identified by RNA interactome capture (Figure 1G). Proteins will be missed by RBDmap when (1) binding to non-polyadenylated RNAs, (2) displaying low crosslinking efficiency, (3) interacting with the phospho-sugar backbone, but not the nucleotide bases, or (4) lacking suitable cleavage sites for trypsin within the LysC and ArgC proteolytic fragments and hence lacking MS-identifiable N-link peptides. Thus, the distribution of arginines (R) and lysines (K) will influence whether a given RBP can be studied by RBDmap, and we used two different proteases to maximize the identification of RBDpeps. About half of the RBPs covered by RBDpeps harbor well-established RBDs and play known functions in RNA biology, reflected by a strong and significant enrichment of RNA-related protein domains and biological processes comparable to the HeLa RNA interactome (Figures 1I and S1J). Note that the reduced RBP coverage of RBDmap compared to RNA interactome capture equally affects both well-established and unorthodox RBPs (Figures 1I and S1J). Interestingly, RNA-bound and released proteolytic fragments display distinct chemical properties. Released peptides are rich in negatively charged and aliphatic residues, which are generally underrepresented in RNA-binding protein surfaces (Figures 2A, 2B , and S2A). Conversely, RBDpeps are significantly enriched in amino acids typically involved in protein-RNA interactions, including positively charged and aromatic residues. These data show that the chemical properties of the RBDpeps resemble those expected of bona fide RNA-binding surfaces. As a notable exception, glycine (G) is enriched in RBDpeps, but depleted from protein-RNA interfaces derived from available structures (Figures 2A and 2B). Flexible glycine tracks can contribute to RNA binding via shape-complementarity interactions as described for RGG boxes (Phan et al., 2011Phan A.T. Kuryavyi V. Darnell J.C. Serganov A. Majumdar A. Ilin S. Raslin T. Polonskaia A. Chen C. Clain D. et al.Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction.Nat. Struct. Mol. Biol. 2011; 18: 796-804Crossref PubMed Scopus (173) Google Scholar). Hence, lack of glycine at binding sites of protein-RNA co-structures reflects the technical limitations of crystallographic studies regarding disordered protein segments. Validating the RBDmap data, classical RBDs such as RRM, KH, cold shock domain (CSD), and Zinc finger CCHC, are strongly enriched in the RNA-bound fraction (Figure 2C). This enrichment can also be appreciated at the level of individual protein maps (Figures 2D and S2B–S2D). To evaluate the capacity of RBDmap to identify bona fide RBDs, we focused on RBPs that harbor at least one classical RBD (as listed in Lunde et al., 2007Lunde B.M. Moore C. Varani G. RNA-binding proteins: modular design for efficient function.Nat. Rev. Mol. Cell Biol. 2007; 8: 479-490Crossref PubMed Scopus (867) Google Scholar). MS-identified peptides from these proteins were classified as “within” or “outside” a classical RBD, according to their position within the proteins’ architecture (Figure 2E). The relative fraction of peptides within versus outside of the RBD was then plotted for each possible RNA-bound/released intensity ratio (Figure 2F). Correct re-identification of classical RBDs would lead to an ascending line (i.e., within/outside ratios should grow in parallel to the RNA-bound/release ratios; Figure 2E), while a random distribution of peptides within and outside of classical RBDs would yield a horizontal line (i.e., within/outside ratios do not vary in accordance with the RNA-bound/released ratios; Figure 2E). As shown in Figure 2F, the relative fraction of peptides mapping within classical RBDs increases in parallel with the RNA-bound/released ratios. Thus, RBDmap correctly assigns RNA-binding activity to well-established RBDs. Unexpected initially, helicase domains are underrepresented in the RNA-bound fraction (Figure 2C). However, the high number of released helicase peptides likely reflects (1) the transitory and dynamic interactions that helicases establish with RNA, (2) the large protein segments of the domain situated far from the RNA, and (3) the predominance of interactions with the phospho-sugar backbone over nucleotide bases (Figures S2C–S2E) (Bono et al., 2006Bono F. Ebert J. Lorentzen E. Conti E. The crystal structure of the exon junction complex reveals how it maintains a stable grip on mRNA.Cell. 2006; 126: 713-725Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Nevertheless, high-confidence RBDpeps are found at the exit of the helicase tunnel, as discussed below (Figures S2C–S2E). For direct validation of the RBDmap data, we selected all those RBPs for which protein-RNA co-structures are available within the Protein Data Bank (PDB) repository. These were “digested” in silico with either LysC or ArgC, and the predicted proteolytic fragments were considered as “proximal” to RNA when the distance to the closest RNA molecule is 4.3 Å or less; otherwise, they were categorized as non-proximal (Figure 3A). About half of all LysC and ArgC fragments are proximal to RNA by this criterion, reflecting that many RBP structures are incomplete and focused on the RBDs (average protein coverage ∼50%). By contrast, 70.3% (LysC) and 81% (ArgC), respectively, of RBDpeps qualify as proximal, showing that RBPmap highly significantly enriches for peptides in close proximity to the RNA (Figure 3A). Several factors suggest that the pool of peptides classified as proximal in the analyzed structures even underestimates the performance of RBDmap: (1) in several structures of RBPs that harbor two or more RBDs, only one of the RBDs displays the interaction with RNA (e.g., PDB 3NNC) (Teplova et al., 2010Teplova M. Song J. Gaw H.Y. Teplov A. Patel D.J. Structural insights into RNA recognition by the alternate-splicing regulator CUG-binding protein 1.Structure. 2010; 18: 1364-1377Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). At least in some of these cases, structures lack RNA contacts of RBDs that likely occur in vivo. (2) Proteins are normally co-crystallized with short nucleic acids (5 to 8 nucleotides), and their physiological RNA partners likely establish additional interactions with the RBP. (3) RNA-protein co-structures usually reflect one interaction state, while protein-RNA interactions are typically more dynamic in vivo (Ozgur et al., 2015Ozgur S. Buchwald G. Falk S. Chakrabarti S. Prabu J.R. Conti E. The conformational plasticity of eukaryotic RNA-dependent ATPases.FEBS J. 2015; 282: 850-863Crossref PubMed Scopus (65) Google Scholar, Safaee et al., 2012Safaee N. Kozlov G. Noronha A.M. Xie J. Wilds C.J. Gehring K. Interdomain allostery promotes assembly of the poly(A) mRNA complex with PABP and eIF4G.Mol. Cell. 2012; 48: 375-386Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). RBDmap also correctly assigns RNA-binding regions within large protein complexes such as the nuclear cap-binding complex. The small nuclear cap-binding protein (NCBP) 2 (or CBP20) directly contacts mRNA via the cap structure (m7GpppG), while the larger NCBP1 (CBP80) interacts with NCBP2 (Mazza et al., 2002Mazza C. Segref A. Mattaj I.W. Cusack S. Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex.EMBO J. 2002; 21: 5548-5557Crossref PubMed Scopus (161) Google Scholar). In agreement, RBDmap defines the RNA-binding region of NCBP2 within the m7GpppG-binding pocket and no RBDpep is assigned to the large NCBP1 (Figure S3A). Moreover, RBDmap defines the corresponding RNA-binding sites within NCBP2 (Mazza et al., 2002Mazza C. Segref A. Mattaj I.W. Cusack S. Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex.EMBO J. 2002; 21: 5548-5557Crossref PubMed Scopus (161) Google Scholar) and its cytoplasmic counterpart eIF4E (Brown et al., 2007Brown C.J. McNae I. Fischer P.M. Walkinshaw M.D. Crystallographic and mass spectrometric characterisation of eIF4E with N7-alkylated cap derivatives.J. Mol. Biol. 2007; 372: 7-15Crossref PubMed Scopus (58) Google Scholar) (Figure S3B), in spite of their low sequence identity. The glutamyl-prolyl-tRNA synthetase (EPRS) represents a large non-canonical RBP that harbors two tRNA synthase domains separated by three WHEP motifs (Figures S3C and S3D). The first and second WHEP motif bind the GAIT RNA element present in the 3′ UTRs of a number of pro-inflammatory mRNAs (Jia et al., 2008Jia J. Arif A. Ray P.S. Fox P.L. WHEP domains direct noncanonical function of glutamyl-Prolyl tRNA synthetase in translational control of gene expression.Mol. Cell. 2008; 29: 679-690Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), in com" @default.
• W2480300184 created "2016-08-23" @default.
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• W2480300184 date "2016-08-01" @default.
• W2480300184 modified "2023-10-15" @default.
• W2480300184 title "Comprehensive Identification of RNA-Binding Domains in Human Cells" @default.
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