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SCLIP – An integrated platform to study RNA-protein interactome(s) in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs [CLIP-Seq]

ABSTRACT: RNA-binding proteins (RBPs) are central for gene expression by controlling the RNA fate from birth to decay. Various disorders arising from perturbations of RNA-protein interactions document their critical function. However, deciphering their function is complex, limiting the general functional elucidation of this growing class of proteins and their contribution to (patho)physiology. Here we present sCLIP, a simplified and robust platform for genome-wide interrogation of RNA-protein interactomes based on crosslinking-immunoprecipitation and high-throughput sequencing. sCLIP exploits linear amplification of the immunoprecipitated RNA improving the complexity of the sequencing-library despite significantly reducing the amount of input material and omitting several purification steps. Additionally it permits a radiolabel-free visualization of immunoprecipitated RNA. In a proof of concept, we identify that CSTF2tau binds many previously not recognized RNAs including histone, snoRNA and snRNAs. CSTF2tau-binding is associated with internal oligoadenylation resulting in shortened snRNA isoforms subjected to rapid degradation. We provide evidence for a new mechanism whereby CSTF2tau controls the abundance of snRNAs resulting in alternative splicing of several RNAs including ANK2 with critical roles in tumorigenesis and cardiac function. Combined with a bioinformatic pipeline sCLIP thus uncovers new functions for established RBPs and fosters the illumination of RNA-protein interaction landscapes in health and disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE92995 | GEO | 2017/03/01

SECONDARY ACCESSION(S): PRJNA359390

REPOSITORIES: GEO

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Project description:The general role of RNA in the cell beyond protein biosynthesis is only beginning to emerge with little knowledge about the structural or organizational functions of RNA. In turn, functional non-coding RNAs (ncRNAs) are often identified by their regulation or impact on cellular phenotypes. However, the huge number and diversity of ncRNAs highly complicate the task of their molecular characterization and rationalization into cellular processes. For the vast majority of ncRNAs as well as for non-coding functions of known mRNAs, the mechanisms underlying their modes of action as well as their impact on protein complexes are unknown. Thus, the greatest challenge in the field of RNA biology today is the elucidation of molecular mechanisms and functional interaction partners of RNA molecules. Since RNAs do not act alone, but often interact with specific protein partners, proteomic approaches have been developed to study RNA-binding proteins (RBPs) and aim at revealing the molecular mechanisms underlying RNA function. Large-scale studies aiming at globally identifying RNA-binding proteins principally focused on poly(A)-RNA transcripts. A notable limitation of such approaches is that many ncRNAs are not polyadenylated, so that their interaction partners were not detected in these screens. Most importantly however, the overlap between the numerous studies aiming to identify RBPs is limited and their specificity remains unclear. Moreover, the previous studies were based on the identification of RBPs omitting their participation in protein-protein complexes and more interestingly in RNA-dependent protein-protein complexes. Here, we introduce the concept of "RNA dependence" to overcome these challenges. We define a protein as RNA-dependent if its interactome (hence likely its function) depends on RNA without necessarily directly binding to RNA. Based on this new concept, we developed a proteome-wide screening approach to gain mechanistic insight into the function of RNAs - both coding and non-coding - in RNA-protein complexes and their impact on the function of such complexes.

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SCLIP – An integrated platform to study RNA-protein interactome(s) in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs [CLIP-Seq]

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