RNA-binding proteins (RBPs) have essential roles in RNA-mediated gene regulation, and yet annotation of RBPs is limited mainly to those with known RNA-binding domains. To systematically identify the RBPs of embryonic stem cells (ESCs), we here employ interactome capture, which combines UV cross-linking of RBP to RNA in living cells, oligo(dT) capture and MS. From mouse ESCs (mESCs), we have defined 555 proteins constituting the mESC mRNA interactome, including 283 proteins not previously annotated as RBPs. Of these, 68 new RBP candidates are highly expressed in ESCs compared to differentiated cells, implicating a role in stem-cell physiology. Two well-known E3 ubiquitin ligases, Trim25 (also called Efp) and Trim71 (also called Lin41), are validated as RBPs, revealing a potential link between RNA biology and protein-modification pathways. Our study confirms and expands the atlas of RBPs, providing a useful resource for the study of the RNA-RBP network in stem cells.
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Smith, A.G. Embryo-derived stem cells: of mice and men. Annu. Rev. Cell Dev. Biol. 17, 435–462 (2001).
Nichols, J. & Smith, A. Naive and primed pluripotent states. Cell Stem Cell 4, 487–492 (2009).
Loh, Y.-H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38, 431–440 (2006).
Orkin, S.H. & Hochedlinger, K. Chromatin connections to pluripotency and cellular reprogramming. Cell 145, 835–850 (2011).
Kim, J., Chu, J., Shen, X., Wang, J. & Orkin, S.H. An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132, 1049–1061 (2008).
Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008).
Young, R.A. Control of the embryonic stem cell state. Cell 144, 940–954 (2011).
Hansson, J. et al. Highly coordinated proteome dynamics during reprogramming of somatic cells to pluripotency. Cell Rep. 2, 1579–1592 (2012).
Polo, J.M. et al. A molecular roadmap of reprogramming somatic cells into iPS cells. Cell 151, 1617–1632 (2012).
Sampath, P. et al. A hierarchical network controls protein translation during murine embryonic stem cell self-renewal and differentiation. Cell Stem Cell 2, 448–460 (2008).
Lu, R. et al. Systems-level dynamic analyses of fate change in murine embryonic stem cells. Nature 462, 358–362 (2009).
Gebauer, F. & Hentze, M.W. Molecular mechanisms of translational control. Nat. Rev. Mol. Cell Biol. 5, 827–835 (2004).
Abaza, I. & Gebauer, F. Trading translation with RNA-binding proteins. RNA 14, 404–409 (2008).
Glisovic, T., Bachorik, J.L., Yong, J. & Dreyfuss, G. RNA-binding proteins and post-transcriptional gene regulation. FEBS Lett. 582, 1977–1986 (2008).
Kishore, S., Luber, S. & Zavolan, M. Deciphering the role of RNA-binding proteins in the post-transcriptional control of gene expression. Brief. Funct. Genomics 9, 391–404 (2010).
Tsvetanova, N.G., Klass, D.M., Salzman, J. & Brown, P.O. Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae. PLoS ONE 5, e12671 (2010).
Scherrer, 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 5, e15499 (2010).
Castello, A. et al. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149, 1393–1406 (2012).
Baltz, A.G. et al. The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol. Cell 46, 674–690 (2012).
Choi, Y.D. & Dreyfuss, G. Isolation of the heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): a unique supramolecular assembly. Proc. Natl. Acad. Sci. USA 81, 7471–7475 (1984).
Mitchell, S.F., Jain, S., She, M. & Parker, R. Global analysis of yeast mRNPs. Nat. Struct. Mol. Biol. 20, 127–133 (2013).
König, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915 (2010).
Sugimoto, Y. et al. Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions. Genome Biol. 13, R67 (2012).
Lau, C.-K., Bachorik, J.L. & Dreyfuss, G. Gemin5-snRNA interaction reveals an RNA binding function for WD repeat domains. Nat. Struct. Mol. Biol. 16, 486–491 (2009).
Radivojac, P. et al. Intrinsic disorder and functional proteomics. Biophys. J. 92, 1439–1456 (2007).
Tompa, P. & Csermely, P. The role of structural disorder in the function of RNA and protein chaperones. FASEB J. 18, 1169–1175 (2004).
Dyson, H.J. & Wright, P.E. Intrinsically unstructured proteins and their functions. Nat. Rev. Mol. Cell Biol. 6, 197–208 (2005).
Han, T.W. et al. Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies. Cell 149, 768–779 (2012).
Kato, M. et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767 (2012).
Chi, S.W., Zang, J.B., Mele, A. & Darnell, R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460, 479–486 (2009).
Ule, J. et al. CLIP identifies Nova-regulated RNA networks in the brain. Science 302, 1212–1215 (2003).
Cho, J. et al. LIN28A is a suppressor of ER-associated translation in embryonic stem cells. Cell 151, 765–777 (2012).
Wong, D.J. et al. Module map of stem cell genes guides creation of epithelial cancer stem cells. Cell Stem Cell 2, 333–344 (2008).
Ben-Porath, I. et al. An embryonic stem cell–like gene expression signature in poorly differentiated aggressive human tumors. Nat. Genet. 40, 499–507 (2008).
Bhattacharya, B. et al. Gene expression in human embryonic stem cell lines: unique molecular signature. Blood 103, 2956–2964 (2004).
Ivanova, N. et al. Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538 (2006).
Liu, Z., Scannell, D.R., Eisen, M.B. & Tjian, R. Control of embryonic stem cell lineage commitment by core promoter factor, TAF3. Cell 146, 720–731 (2011).
Wong, R.C.-B. et al. L1TD1 is a marker for undifferentiated human embryonic stem cells. PLoS ONE 6, e19355 (2011).
Närvä, E. et al. RNA-binding protein L1TD1 interacts with LIN28 via RNA and is required for human embryonic stem cell self-renewal and cancer cell proliferation. Stem Cells 30, 452–460 (2012).
Iwabuchi, K.A. et al. ECAT11/L1td1 is enriched in ESCs and rapidly activated during iPSC generation, but it is dispensable for the maintenance and induction of pluripotency. PLoS ONE 6, e20461 (2011).
Kim, J. et al. A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs. Cell 143, 313–324 (2010).
Chang, H.-M. et al. Trim71 cooperates with microRNAs to repress Cdkn1a expression and promote embryonic stem cell proliferation. Nat. Commun. 3, 923 (2012).
Loedige, I. & Filipowicz, W. TRIM-NHL proteins take on miRNA regulation. Cell 136, 818–820 (2009).
Loedige, I., Gaidatzis, D., Sack, R., Meister, G. & Filipowicz, W. The mammalian TRIM-NHL protein TRIM71/LIN-41 is a repressor of mRNA function. Nucleic Acids Res. 41, 518–532 (2013).
Hatakeyama, S. TRIM proteins and cancer. Nat. Rev. Cancer 11, 792–804 (2011).
Tian, L. et al. Characterization and potential function of a novel pre-implantation embryo-specific RING finger protein: TRIML1. Mol. Reprod. Dev. 76, 656–664 (2009).
Ding, L. et al. A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity. Cell Stem Cell 4, 403–415 (2009).
Hu, G. et al. A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal. Genes Dev. 23, 837–848 (2009).
Chia, N.-Y. et al. A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Nature 468, 316–320 (2010).
Benz, C., Mulindwa, J., Ouna, B. & Clayton, C. The Trypanosoma brucei zinc-finger protein ZC3H18 is involved in differentiation. Mol. Biochem. Parasitol. 177, 148–151 (2011).
Beekman, R. et al. Sequential gain of mutations in severe congenital neutropenia progressing to acute myeloid leukemia. Blood 119, 5071–5077 (2012).
Gewurz, B.E. et al. Genome-wide siRNA screen for mediators of NF-κB activation. Proc. Natl. Acad. Sci. USA 109, 2467–2472 (2012).
Denis, C.L. & Chen, J. The CCR4–NOT complex plays diverse roles in mRNA metabolism. Prog. Nucleic Acid Res. Mol. Biol. 73, 221–250 (2003).
Zheng, X. et al. Cnot1, Cnot2, and Cnot3 maintain mouse and human ESC identity and inhibit extraembryonic differentiation. Stem Cells 30, 910–922 (2012).
Kabe, Y. et al. The role of human MBF1 as a transcriptional coactivator. J. Biol. Chem. 274, 34196–34202 (1999).
Takemaru, K.-i., Li, F.Q., Ueda, H. & Hirose, S. Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein. Proc. Natl. Acad. Sci. USA 94, 7251–7256 (1997).
Takemaru, K.-i., Harashima, S., Ueda, H. & Hirose, S. Yeast coactivator MBF1 mediates GCN4-dependent transcriptional activation. Mol. Cell Biol. 18, 4971–4976 (1998).
Brendel, C., Gelman, L. & Auwerx, J. Multiprotein bridging factor-1 (MBF-1) is a cofactor for nuclear receptors that regulate lipid metabolism. Mol. Endocrinol. 16, 1367–1377 (2002).
Dragoni, I., Mariotti, M., Consalez, G.G., Soria, M.R. & Maier, J.a. EDF-1, a novel gene product down-regulated in human endothelial cell differentiation. J. Biol. Chem. 273, 31119–31124 (1998).
Yasuhara, N. et al. Triggering neural differentiation of ES cells by subtype switching of importin-α. Nat. Cell Biol. 9, 72–79 (2007).
Nisole, S., Stoye, J.P. & Saïb, A. TRIM family proteins: retroviral restriction and antiviral defence. Nat. Rev. Microbiol. 3, 799–808 (2005).
Urano, T. et al. Efp targets 14–3-3σ for proteolysis and promotes breast tumour growth. Nature 417, 871–875 (2002).
Gack, M.U. et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446, 916–920 (2007).
Suzuki, T. et al. Estrogen-responsive finger protein as a new potential biomarker for breast cancer. Clin. Cancer Res. 11, 6148–6154 (2005).
Sakuma, M. et al. Expression of estrogen-responsive finger protein (Efp) is associated with advanced disease in human epithelial ovarian cancer. Gynecol. Oncol. 99, 664–670 (2005).
Nakayama, H., Sano, T., Motegi, A., Oyama, T. & Nakajima, T. Increasing 14-3-3 sigma expression with declining estrogen receptor alpha and estrogen-responsive finger protein expression defines malignant progression of endometrial carcinoma. Pathol. Int. 55, 707–715 (2005).
Rybak, A. et al. The let-7 target gene mouse lin-41 is a stem cell specific E3 ubiquitin ligase for the miRNA pathway protein Ago2. Nat. Cell Biol. 11, 1411–1420 (2009).
Chen, J., Lai, F. & Niswander, L. The ubiquitin ligase mLin41 temporally promotes neural progenitor cell maintenance through FGF signaling. Genes Dev. 26, 803–815 (2012).
Maller Schulman, B.R., Liang, X. & Stahlhut, C. The let-7 microRNA target gene, Mlin41/Trim71 is required for mouse embryonic survival and neural tube closure. Cell Cycle 7, 3935–3942 (2008).
Slack, F.J. et al. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol. Cell 5, 659–669 (2000).
Kanamoto, T., Terada, K., Yoshikawa, H. & Furukawa, T. Cloning and regulation of the vertebrate homologue of lin-41 that functions as a heterochronic gene in Caenorhabditis elegans. Dev. Dyn. 235, 1142–1149 (2006).
Lin, Y.-C. et al. Human TRIM71 and its nematode homologue are targets of let-7 microRNA and its zebrafish orthologue is essential for development. Mol. Biol. Evol. 24, 2525–2534 (2007).
Cano, F., Miranda-Saavedra, D. & Lehner, P.J. RNA-binding E3 ubiquitin ligases: novel players in nucleic acid regulation. Biochem. Soc. Trans. 38, 1621–1626 (2010).
Vinuesa, C.G. et al. A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature 435, 452–458 (2005).
Yu, D. et al. Roquin represses autoimmunity by limiting inducible T-cell co-stimulator messenger RNA. Nature 450, 299–303 (2007).
Glasmacher, E. et al. Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression. Nat. Immunol. 11, 725–733 (2010).
Castello, A. et al. System-wide identification of RNA-binding proteins by interactome capture. Nat. Protoc. 8, 491–500 (2013).
Boersema, P.J., Raijmakers, R., Lemeer, S., Mohammed, S. & Heck, A.J.R. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494 (2009).
Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
Jain, E. et al. Infrastructure for the life sciences: design and implementation of the UniProt website. BMC Bioinformatics 10, 136 (2009).
Gentleman, R.C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).
Smyth, G.K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, 3 (2004).
Lunde, B.M., Moore, C. & Varani, G. RNA-binding proteins: modular design for efficient function. Nat. Rev. Mol. Cell Biol. 8, 479–490 (2007).
Dosztányi, Z., Csizmok, V., Tompa, P. & Simon, I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics 21, 3433–3434 (2005).
Guttman, M. et al. Ab initio reconstruction of cell type–specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat. Biotechnol. 28, 503–510 (2010).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
We thank H. Chang for teaching and sharing Python codes, A. Cho, Y.C. Chang and H. Kim for technical help, and all members of our laboratories for helpful discussion. We gratefully acknowledge the EMBL Proteomics Core Facility for technical support. We are grateful to G. Dreyfuss (University of Pennsylvania), F.G. Wulczyn (Charité–Universitätsmedizin Berlin), S.H. Baek (Seoul National University), H.-Y. Kao (Case Western Reserve University), D.-E. Zhang (The Scripps Research Institute), D. Rimm (Yale School of Medicine), K. Helin (Københavns Universitet) and Y. Kawakami (University of Minnesota) for valuable plasmids and antibodies. This work was supported by the Research Center Program (EM1202) of the Institute for Basic Science (S.C.K., H.Y., K.T.Y. and V.N.K.) and the BK21 Research Fellowships (S.C.K. and H.Y.) from the Ministry of Education, Science and Technology of Korea. Work in the group of M.W.H. was funded by an ERC Advanced grant (ERC-2011-ADG_20110310) to M.W.H.
The authors declare no competing financial interests.
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Kwon, S., Yi, H., Eichelbaum, K. et al. The RNA-binding protein repertoire of embryonic stem cells. Nat Struct Mol Biol 20, 1122–1130 (2013). https://doi.org/10.1038/nsmb.2638
Identification, quantification and bioinformatic analysis of RNA-dependent proteins by RNase treatment and density gradient ultracentrifugation using R-DeeP
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