Kenneth R Lyon, Tatsuya Morisaki, Timothy J Stasevich
{"title":"Imaging and Quantifying Ribosomal Frameshifting Dynamics with Single-RNA Precision in Live Cells.","authors":"Kenneth R Lyon, Tatsuya Morisaki, Timothy J Stasevich","doi":"10.1007/978-1-0716-4248-1_9","DOIUrl":null,"url":null,"abstract":"<p><p>Recent advances in fluorescence microscopy have now made it possible to measure the translation dynamics of individual RNA in living cells and in multiple colors. Here we describe a protocol that exploits these recent advances to simultaneously image the translation of two open reading frames encoded on a single reporter RNA yet frameshifted with respect to each other. This enables precise measurements of frameshifting dynamics and efficiency from specific frameshift stimulatory sequences, all with single-RNA precision.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2875 ","pages":"99-110"},"PeriodicalIF":0.0000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11633442/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in molecular biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1007/978-1-0716-4248-1_9","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
Abstract
Recent advances in fluorescence microscopy have now made it possible to measure the translation dynamics of individual RNA in living cells and in multiple colors. Here we describe a protocol that exploits these recent advances to simultaneously image the translation of two open reading frames encoded on a single reporter RNA yet frameshifted with respect to each other. This enables precise measurements of frameshifting dynamics and efficiency from specific frameshift stimulatory sequences, all with single-RNA precision.
期刊介绍:
For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.