![]() Given that pyrimidine-rich U2AF2 binding motifs also exist throughout introns, the question arises how U2AF2 is specifically recruited to 3′ splice sites. In vitro iCLIP provides the intrinsic binding landscape of U2AF2Ĭonsistent with U2AF2 playing a key role in 3′ splice site definition, iCLIP data from HeLa cells show that U2AF2 binding is highly enriched at 3′ splice sites and depleted from the intron body ( Supplemental Fig. Our study thus offers a blueprint for high-throughput in vitro experiments to study interactive RBP binding in mRNP assembly and the regulatory code of splicing. Based on these data, we employ mathematical modeling and machine learning to systematically compare in vitro and in vivo iCLIP landscapes and to identify regulatory RBPs controlling U2AF2 binding in vivo. Here, we develop in vitro iCLIP to close the gap between in vitro and in vivo approaches. However, they often employ short, artificially designed oligonucleotides as substrates, making it difficult to directly compare in vitro to in vivo binding. Depending on the setup, these approaches allow determination of the consensus motif of an RBP, measuring its affinity to a large number of short RNA substrates, or studying the impact of additional factors on RBP binding. Given that RBP binding to RNA is highly modulated by the presence of other RBPs in vivo, different high-throughput assays have been developed to study the intrinsic binding of isolated RBPs in vitro, including RNAcompete, RNA Bind-n-Seq, RNA-MaP, and RNA-MITOMI ( Ray et al. Despite these individual examples, we still lack a comprehensive understanding of the factors that shape U2AF2 binding and enable targeted splice site recognition.Ī state-of-the-art approach to study protein–RNA interactions in vivo is individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), which allows quantitative mapping of RBP binding at high resolution ( König et al. U2AF2 binding comprises a major regulatory step during spliceosome assembly and is modulated by several RBPs, e.g., by means of direct competition, cooperative recruitment, or modulation of RNA secondary structure ( Zuo and Maniatis 1996 Saulière et al. Together with SF1 and U2AF1, which recognize the branch point and the AG dinucleotide of the 3′ splice site, respectively, U2AF2 is essential to recruit the small nuclear ribonucleoprotein (snRNP) U2, a subunit of the spliceosome ( Berglund et al. U2AF2 binds to a uridine/cytidine-rich sequence element upstream of the 3′ splice site, referred to as the polypyrimidine tract (Py-tract) ( Singh et al. How these RBPs act together on the cis-regulatory elements to assemble pre-ribonucleoprotein complexes (pre-mRNPs) and determine the splicing outcome is commonly referred to as the “splicing code.” Understanding the splicing code remains one major goal in RNA biology.Ī central player in 3′ splice site definition is the U2 Auxiliary Factor 2 (U2AF2 also referred to as U2AF65). Spliceosome activity is regulated by a large set of trans-acting RNA-binding proteins (RBPs) that bind to cis-regulatory elements in the pre-mRNA and guide splice site recognition ( Fu and Ares 2014 Vuong et al. Splicing is catalyzed by a large multi-subunit complex called the spliceosome that recognizes the 5′ and 3′ splice sites as well as the branch point of each intron ( Matera and Wang 2014). The splicing reaction, i.e., the removal of introns and joining of exons in different combinations, allows for the production of distinct mature mRNA isoforms and is the main source of proteome diversity in eukaryotes ( Nilsen and Graveley 2010). Most eukaryotic genes are transcribed into long pre-mRNAs that consist of multiple exons and introns. Our study offers a blueprint for the high-throughput characterization of in vitro mRNP assembly and in vivo splicing regulation. Using machine learning, we identify and experimentally validate novel trans-acting RBPs (including FUBP1, CELF6, and PCBP1) that modulate U2AF2 binding and affect splicing outcomes. ![]() We find that trans-acting RBPs extensively regulate U2AF2 binding in vivo, including enhanced recruitment to 3′ splice sites and clearance of introns. We measure U2AF2 affinities at hundreds of binding sites and compare in vitro and in vivo binding landscapes by mathematical modeling. We establish “in vitro iCLIP” experiments, in which recombinant RBPs are incubated with long transcripts, to study how U2AF2 recognizes RNA sequences and how this is modulated by trans-acting RBPs. The RNA-binding protein (RBP) U2AF2 is central to splicing decisions, as it recognizes 3′ splice sites and recruits the spliceosome. Alternative splicing generates distinct mRNA isoforms and is crucial for proteome diversity in eukaryotes.
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