@article{f11650e4c6d34386beb429bd60c21ea1,
title = "In-cell identification and measurement of RNA-protein interactions",
abstract = "Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.",
author = "Antoine Graindorge and Ines Pinheiro and Anna Nawrocka and Mallory, {Allison C.} and Peter Tsvetkov and Noa Gil and Carlo Carolis and Frank Buchholz and Igor Ulitsky and Edith Heard and Mikko Taipale and Alena Shkumatava",
note = "We thank Ines Anna Drinnenberg, Raphael Margueron, Donal O{\textquoteright}Carroll, and Dina Zielinski for comments on this paper. We also thank all members of the Shkumatava and Heard laboratories for stimulating discussions. This work was supported by grants from ERC (FLAME-337440), ATIP-Avenir, and La Fondation Bettencourt Schueller to A.S.; and by ERC (XPRESS-671027) and ANR (NoncodiX) to E.H. A.G. was supported by the ARC postdoctoral fellowship (20131200567), short-term EMBO (ESTF324-2015) and Company of Biologists travel (JCSTF-150421) fellowships, and LabEx DEEP (ANR-11-LABX-0044, ANR-10-IDEX-0001-02). I.P. was supported by the EMBO long-term fellowship (ALTF 549-2014), and Fondation pour la Recherche Medicale grant SPF 20140129387, A.N. was supported by the PSL-Biogen PhD fellowship. The imaging experiments were performed in the Cell and Tissue Imaging Facility/UMR3215 (PICT-IBiSA) of the Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR-10-INBS-04). Contributions - A.G. established the incPRINT method, performed incPRINT experiments and contributed to the generation of the HA-tagged RBM6 and ZZZ3 G6pd-Fluo cell lines and functional analyses of the Xist interaction partners. I.P. designed, executed, and analyzed most of the XCI-related experiments including FISH analyses and generation of the HA-tagged RBM6 and ZZZ3 cell lines. A.N. performed small-scale incPRINT experiments. A.C.M. contributed to the RIP and qRT-PCR experiments. P.T. generated the library of FLAG-tagged RBPs. N.G. and I.U. performed protein domain and CLIP data analyses. C.C. helped with the incPRINT equipment setup. F.B. contributed to the design and execution of the RNAi experiments. E.H. supervised the generation of the transgenic cell lines and RNA FISH experiments and contributed to the design of the Xist RNA and X inactivation assays. M.T. generated a part of the library of FLAG-tagged human proteins and supervised the initial steps of the incPRINT protocol establishment. A.G., I.P., and A.S. wrote the final version of the paper. A.S. conceived and supervised the study.",
year = "2019",
month = nov,
day = "22",
doi = "10.1038/s41467-019-13235-w",
language = "الإنجليزيّة",
volume = "10",
pages = "5317",
journal = "Nature Communications",
issn = "2041-1723",
publisher = "Nature Research",
number = "1",
}