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Ripit sugar5/6/2023 ![]() ![]() The panoply of RBP targets within cells has recently been illuminated by approaches that generally combine enrichment of RBP-bound RNAs via immunoprecipitation (IP) with high-throughput transcript profiling methods. The post-genomic era has seen rapid advances in technologies to study nucleic acid-protein interactions on an unprecedented scale. A major goal in understanding post-transcriptional control of gene expression is therefore to identify the RNA targets directly bound to individual RBPs and their partner proteins in cells, as well as the precise binding sites of these proteins on their target RNAs. Among RNPs, messenger RNPs (mRNPs) are particularly dynamic, shedding proteins and acquiring others as they move from one cellular compartment to another and/or are acted upon by numerous macromolecular machines (e.g., the spliceosome, nuclear pore complex, and ribosome). To recognize these features, RBPs utilize a variety RNA recognition modes, with >50 different types of RNA-binding domains having been identified to date. The diverse features recognized by RBPs include common structural elements (e.g., the 7-methyl-G cap and polyA tail), short sequence motifs (e.g., exonic splicing enhancers and silencers ESEs and ESSs), particular secondary structures (e.g., double-stranded regions dsRNA) and modified nucleotides (e.g., inosine, 6-methyl-adenosine). Further, whereas some RBPs are restricted to just a few transcripts, others act on tens of thousands of distinct species. Importantly, most RBPs function within multi-protein complexes that also consist of non-RBPs. ![]() ![]() A large fraction of these engage with RNA polymerase II transcripts (precursors to messenger RNAs pre-mRNAs) to form ribonucleoprotein particles (RNPs) and exert control over post-transcriptional events such as pre-mRNA processing (splicing and polyadenylation) and intracellular localization, translation and decay of product mRNAs. To execute RNA-mediated control, the human genome encodes >1000 RNA binding proteins (RBPs) as per current estimates. Therefore, elucidating precisely when and where proteins bind to DNA and RNA is central to our understanding of the exquisite intricacies of how genetic information is decoded and regulated within cells. Nucleic acid-protein interactions govern all aspects of gene expression in every organism. It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds. Further, among current high-throughput approaches, RIPiT has the unique capacity to differentiate binding sites of RNPs with overlapping protein composition. RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. ![]()
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