7/9/2023 0 Comments Accounting canada iclipFor discussion of additional protein-centric methods, we refer the readers to recent reviews 12, 13, 14. Finally, we discuss the applications of these techniques to obtain a systems-level view of RNP assembly and dynamics in multiple model organisms and review strategies for method optimization and quality assessment of the data. We also cover the identification of RBP binding sites by tagging RBPs with enzymes that naturally act on RNA, where the resulting RNA modifications can be identified by high-throughput sequencing 10, as well as the use of subcellular compartment-specific proximity labelling to study localized transcriptomes 11. This Primer focuses on experimental and computational aspects of CLIP methods that have been broadly adopted and have generated widely used data sets. The development of dedicated bioinformatics workflows has allowed the determination of binding sites and consensus motifs to better understand post-transcriptional regulation 9. CLIP techniques have been further developed to identify cross-link sites with nucleotide resolution, either through analysis of mutations in reads (photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP)) 7 or by capturing cDNAs that terminate at the cross-linked peptide during reverse transcription (individual-nucleotide resolution CLIP (iCLIP)) 8. The development of high-throughput sequencing of RNA isolated by CLIP (HITS-CLIP) has enabled a transcriptome-wide view of RNA binding sites 6. CLIP uses a limited RNase treatment of cross-linked RNPs to isolate RNA fragments occupied by the RBP and sequencing of these fragments can identify RBP binding sites, which allows inference of RBP function through determining the location of binding sites relative to, for example, other RBP binding sites or cis-acting elements (Box 1). The covalent cross-links allow stringent purification of the RNA–protein complexes, which is followed by a series of steps to determine the interactions of a specific protein across the transcriptome. CLIP techniques are more widely used and rely on the irradiation of cells by UV light, which causes proteins in the immediate vicinity of the irradiated bases to irreversibly cross-link to the RNA by a covalent bond 5 (Fig. RIP approaches purify the RNA–protein complexes under native conditions 2, 3 or using formaldehyde cross-linking 4. The most common protein-centric strategies are based on the immunopurification of an RBP and its associated RNAs, and can be broadly categorized as RNA immunoprecipitation (RIP) or cross-linking and immunoprecipitation (CLIP) approaches. These approaches can be protein-centric, describing the compendium of RNA sites bound by a specific RBP, or RNA-centric, identifying the RNA-bound proteome. Numerous methods can characterize the RNA interactions that coordinate RNP assembly. Ribonucleoprotein (RNP) complexes are key to every step of RNA processing and function, and understanding the roles that RNA-binding proteins (RBPs) play requires methods that identify the set of RNAs that they bind in cells during specific developmental stages, activities or disease states. The protein complement decorating an RNA molecule changes dynamically in space and time, orchestrating RNA processing and function in the nucleus and cytoplasm 1. Proteins begin to interact with nascent RNAs as soon as transcription is initiated. Finally, we present open questions in the field and give directions for further development and applications. We discuss the prospect of integrating data obtained by CLIP with complementary methods to gain a comprehensive view of RNP assembly and remodelling, unravel the spatial and temporal dynamics of RNPs in specific cell types and subcellular compartments and understand how defects in RNPs can lead to disease. We outline the various applications of CLIP and available databases for data sharing. We summarize the main challenges of computational CLIP data analysis, how to handle various sources of background and how to identify functionally relevant binding regions. In this Primer, we discuss the main variants of these protein-centric methods and the strategies for their optimization and quality assessment, as well as RNA-centric methods that identify the protein partners of a specific RNA. To identify the sites bound by a specific RNA-binding protein on endogenous RNAs, cross-linking and immunoprecipitation (CLIP) and complementary, proximity-based methods have been developed. Dynamic RNP assembly, largely directed by cis-acting elements on the RNA, coordinates all processes in which the RNA is involved. RNA molecules start assembling into ribonucleoprotein (RNP) complexes during transcription.
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