sCLIP—an integrated platform to study RNA–protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs

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 RBP–protein interaction landscapes in health and disease.

[1]  J. Keene.  RNA regulons: coordination of post-transcriptional events , 2007, Nature Reviews Genetics.

[2]  Jernej Ule,et al.  CLIP: a method for identifying protein-RNA interaction sites in living cells. , 2005, Methods.

[3]  Richard Bonneau,et al.  The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. , 2012, Molecular cell.

[4]  Daphne H. E. W. Huberts,et al.  Moonlighting proteins: an intriguing mode of multitasking. , 2010, Biochimica et Biophysica Acta.

[5]  Bin Tian,et al.  A functional human Poly(A) site requires only a potent DSE and an A-rich upstream sequence , 2010, The EMBO journal.

[6]  J. Yong,et al.  The SMN complex: an assembly machine for RNPs. , 2006, Cold Spring Harbor symposia on quantitative biology.

[7]  Christopher R. Sibley,et al.  iCLIP: Protein–RNA interactions at nucleotide resolution , 2014, Methods.

[8]  M. Taoka,et al.  Identification of truncated forms of U1 snRNA reveals a novel RNA degradation pathway during snRNP biogenesis , 2013, Nucleic acids research.

[9]  John B. VanCuylenberg,et al.  ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3' RACE applications. , 2012, RNA.

[10]  Shimyn Slomovic,et al.  Addition of poly(A) and poly(A)-rich tails during RNA degradation in the cytoplasm of human cells , 2010, Proceedings of the National Academy of Sciences.

[11]  Sandya Liyanarachchi,et al.  Mutations in U4atac snRNA, a Component of the Minor Spliceosome, in the Developmental Disorder MOPD I , 2011, Science.

[12]  U. Fischer,et al.  The role of RNP biogenesis in spinal muscular atrophy. , 2009, Current opinion in cell biology.

[13]  Kai-Wei Chang,et al.  RNA-binding proteins in human genetic disease. , 2008, Trends in genetics : TIG.

[14]  B. Tian,et al.  Bioinformatic identification of candidate cis-regulatory elements involved in human mRNA polyadenylation. , 2005, RNA.

[15]  Dan M Roden,et al.  Defining the Cellular Phenotype of “Ankyrin-B Syndrome” Variants: Human ANK2 Variants Associated With Clinical Phenotypes Display a Spectrum of Activities in Cardiomyocytes , 2007, Circulation.

[16]  E. Wahle,et al.  Control of poly(A) tail length , 2011, Wiley interdisciplinary reviews. RNA.

[17]  A. Skoultchi,et al.  Different 3'-end processing produces two independently regulated mRNAs from a single H1 histone gene. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Yates,et al.  Overlapping and distinct functions of CstF64 and CstF64τ in mammalian mRNA 3′ processing , 2013, RNA.

[19]  E. Brown,et al.  Quantitative analysis of mRNA amplification by in vitro transcription. , 2001, Nucleic acids research.

[20]  Paul M. Jenkins,et al.  Giant ankyrin-G: A critical innovation in vertebrate evolution of fast and integrated neuronal signaling , 2014, Proceedings of the National Academy of Sciences.

[21]  Scott B. Dewell,et al.  Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP , 2010, Cell.

[22]  Z. Dominski,et al.  A Complex Containing the CPSF73 Endonuclease and Other Polyadenylation Factors Associates with U7 snRNP and Is Recruited to Histone Pre-mRNA for 3′-End Processing , 2012, Molecular and Cellular Biology.

[23]  Juliane C. Dohm,et al.  Oligoadenylation of 3′ decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells , 2016, RNA.

[24]  David Tollervey,et al.  Prp43 Bound at Different Sites on the Pre-rRNA Performs Distinct Functions in Ribosome Synthesis , 2009, Molecular cell.

[25]  Carol L. Williams,et al.  An Adenosine-Mediated Signaling Pathway Suppresses Prenylation of the GTPase Rap1B and Promotes Cell Scattering , 2013, Science Signaling.

[26]  Tyson A. Clark,et al.  HITS-CLIP yields genome-wide insights into brain alternative RNA processing , 2008, Nature.

[27]  Matthias W. Hentze,et al.  Metabolic Enzymes Enjoying New Partnerships as RNA-Binding Proteins , 2015, Trends in Endocrinology & Metabolism.

[28]  C. Bass,et al.  Cannabinoid receptor interacting protein (CRIP1a) attenuates CB1R signaling in neuronal cells. , 2015, Cellular signalling.

[29]  Mihaela Zavolan,et al.  Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length. , 2012, Cell reports.

[30]  M. Hentze,et al.  p38 MAPK controls prothrombin expression by regulated RNA 3' end processing. , 2011, Molecular cell.

[31]  S. Yehudai-Resheff,et al.  Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence. , 2000, Nucleic acids research.

[32]  Jeroen Krijgsveld,et al.  The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs , 2015, Nature Communications.

[33]  Blaz Zupan,et al.  iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution , 2011, Journal of visualized experiments : JoVE.

[34]  S. Ackerman,et al.  Mutation of a U2 snRNA Gene Causes Global Disruption of Alternative Splicing and Neurodegeneration , 2012, Cell.

[35]  W. Huber,et al.  Detecting differential usage of exons from RNA-seq data , 2012, Genome research.

[36]  Jeroen Krijgsveld,et al.  The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease , 2016, Cell reports.

[37]  H. Le Hir,et al.  CLIP-seq to discover transcriptome-wide imprinting of RNA binding proteins in living cells. , 2015, Methods in molecular biology.

[38]  M. Griswold,et al.  Loss of polyadenylation protein τCstF-64 causes spermatogenic defects and male infertility , 2007, Proceedings of the National Academy of Sciences.

[39]  R. Lothe,et al.  Four DNA methylation biomarkers in biliary brush samples accurately identify the presence of cholangiocarcinoma , 2015, Hepatology.

[40]  T. Hashimshony,et al.  CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification. , 2012, Cell reports.

[41]  M. Ruepp,et al.  Interactions of CstF-64, CstF-77, and symplekin: Implications on localisation and function , 2011, Molecular biology of the cell.

[42]  Carol L. Williams,et al.  Splice Variants of SmgGDS Control Small GTPase Prenylation and Membrane Localization , 2010, The Journal of Biological Chemistry.

[43]  Kees-Jan Françoijs,et al.  Linear amplification for deep sequencing , 2011, Nature Protocols.

[44]  Howard Y. Chang,et al.  Dissecting noncoding and pathogen RNA–protein interactomes , 2015, RNA.

[45]  Ji Wan,et al.  Transcriptome-wide analyses of CstF64–RNA interactions in global regulation of mRNA alternative polyadenylation , 2012, Proceedings of the National Academy of Sciences.

[46]  Li Yang,et al.  Regulation of alternative splicing in Drosophila by 56 RNA binding proteins , 2015, Genome research.

[47]  Ryan T Fuchs,et al.  Structural bias in T4 RNA ligase-mediated 3′-adapter ligation , 2012, Nucleic acids research.

[48]  Yan Yan,et al.  FLASH, a proapoptotic protein involved in activation of caspase-8, is essential for 3' end processing of histone pre-mRNAs. , 2009, Molecular cell.

[49]  S. Miyano,et al.  Expression and clinical significance of genes frequently mutated in small cell lung cancers defined by whole exome/RNA sequencing. , 2015, Carcinogenesis.

[50]  Gene W. Yeo,et al.  Robust transcriptome-wide discovery of RNA binding protein binding sites with enhanced CLIP (eCLIP) , 2016, Nature Methods.

[51]  Howard Y. Chang,et al.  irCLIP platform for efficient characterization of protein–RNA interactions , 2016, Nature Methods.

[52]  Jeroen Krijgsveld,et al.  The RNA-binding protein repertoire of embryonic stem cells , 2013, Nature Structural &Molecular Biology.

[53]  Norman E. Davey,et al.  Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins , 2012, Cell.

[54]  J. Schott,et al.  Exon organization and novel alternative splicing of the human ANK2 gene: implications for cardiac function and human cardiac disease. , 2008, Journal of molecular and cellular cardiology.

[55]  Bernd Fischer,et al.  RNA-binding proteins in Mendelian disease. , 2013, Trends in genetics : TIG.

[56]  S. Danckwardt,et al.  Processing and transcriptome expansion at the mRNA 3′ end in health and disease: finding the right end , 2016, Pflügers Archiv - European Journal of Physiology.

[57]  Gioele La Manno,et al.  Quantitative single-cell RNA-seq with unique molecular identifiers , 2013, Nature Methods.

[58]  Laurent Lestrade,et al.  snoRNA-LBME-db, a comprehensive database of human H/ACA and C/D box snoRNAs , 2005, Nucleic Acids Res..