sCLIP-an integrated platform to study RNA-protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs.
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 RBP-protein interaction landscapes in health and disease.
Institute for Clinical Chemistry and Laboratory Medicine, University Medical Center Mainz, Germany.|Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Germany.
Yulia
Y
Kargapolova
Institute for Clinical Chemistry and Laboratory Medicine, University Medical Center Mainz, Germany.|Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Germany.
Michal
M
Levin
Institute for Clinical Chemistry and Laboratory Medicine, University Medical Center Mainz, Germany.|German Center for Cardiovascular Research (DZHK), Germany.
Karl
K
Lackner
Institute for Clinical Chemistry and Laboratory Medicine, University Medical Center Mainz, Germany.|Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Germany.|German Center for Cardiovascular Research (DZHK), Germany.
Sven
S
Danckwardt
D017398:Alternative Splicing; D045744:Cell Line, Tumor; D039241:Cleavage Stimulation Factor; D019295:Computational Biology / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006657:Histones / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D009363:Neoplasm Proteins / Q000378:metabolism; D009447:Neuroblastoma / Q000473:pathology; D011485:Protein Binding; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D022661:RNA, Untranslated / Q000235:genetics; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014466:Ultraviolet Rays
C474466:CSTF2T protein, human; D039241:Cleavage Stimulation Factor; D018076:DNA, Complementary; D006657:Histones; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins