{"title":"Rapid and Efficient Isolation of Total RNA-Bound Proteomes by Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP).","authors":"JohnCarlo Kristofich, Christopher V Nicchitta","doi":"10.21769/BioProtoc.5236","DOIUrl":null,"url":null,"abstract":"<p><p>The critical roles of RNA-binding proteins (RBPs) in all aspects of RNA biology fostered the development of methods utilizing ultraviolet (UV) crosslinking and method-specific RNA enrichment steps for proteome-wide identification and assessment of RBP function. Despite the substantial contributions of these UV-based RNA-centric methods to our understanding of RNA-protein interaction networks, their utility is constrained by biases in RBP recovery and significant noise contributions, which can confound meaningful interpretation. To overcome these issues, we recently developed a method termed Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP) and introduced quantitative signal-to-noise (<i>S</i>:<i>N</i>)-based metrics for the proteome-wide identification of RNA interactomes and accurate assessment of global RBP occupancy dynamics. Compared to existing methodologies, LEAP-RBP provides significant advantages in speed, cost, efficiency, and selectivity for RNA-bound proteins. In this work, we provide a step-by-step guide for the successful application of the LEAP-RBP method for both small- and large-scale investigations of RNA-bound proteomes. Key features • Unbiased and efficient isolation of total RNA-bound protein, RNA, and protein from biological samples. • Cost-effective identification of proteome-wide RNA interactomes and validation of direct RNA-binding protein functionality. • Robust and accurate assessment of context- and/or condition-dependent RBP occupancy state dynamics.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":null,"pages":null},"PeriodicalIF":1.0000,"publicationDate":"2024-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11292130/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bio-protocol","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.21769/BioProtoc.5236","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
The critical roles of RNA-binding proteins (RBPs) in all aspects of RNA biology fostered the development of methods utilizing ultraviolet (UV) crosslinking and method-specific RNA enrichment steps for proteome-wide identification and assessment of RBP function. Despite the substantial contributions of these UV-based RNA-centric methods to our understanding of RNA-protein interaction networks, their utility is constrained by biases in RBP recovery and significant noise contributions, which can confound meaningful interpretation. To overcome these issues, we recently developed a method termed Liquid Emulsion-Assisted Purification of RNA-Bound Protein (LEAP-RBP) and introduced quantitative signal-to-noise (S:N)-based metrics for the proteome-wide identification of RNA interactomes and accurate assessment of global RBP occupancy dynamics. Compared to existing methodologies, LEAP-RBP provides significant advantages in speed, cost, efficiency, and selectivity for RNA-bound proteins. In this work, we provide a step-by-step guide for the successful application of the LEAP-RBP method for both small- and large-scale investigations of RNA-bound proteomes. Key features • Unbiased and efficient isolation of total RNA-bound protein, RNA, and protein from biological samples. • Cost-effective identification of proteome-wide RNA interactomes and validation of direct RNA-binding protein functionality. • Robust and accurate assessment of context- and/or condition-dependent RBP occupancy state dynamics.