Fluorescence Reporter-Based Genome-Wide RNA Interference Screening to Identify Alternative Splicing Regulators.
Alternative splicing is a regulated process that leads to inclusion or exclusion of particular exons in a pre-mRNA transcript, resulting in multiple protein isoforms being encoded by a single gene. With more than 90 % of human genes known to undergo alternative splicing, it represents a major source for biological diversity inside cells. Although in vitro splicing assays have revealed insights into the mechanisms regulating individual alternative splicing events, our global understanding of alternative splicing regulation is still evolving. In recent years, genome-wide RNA interference (RNAi) screening has transformed biological research by enabling genome-scale loss-of-function screens in cultured cells and model organisms. In addition to resulting in the identification of new cellular pathways and potential drug targets, these screens have also uncovered many previously unknown mechanisms regulating alternative splicing. Here, we describe a method for the identification of alternative splicing regulators using genome-wide RNAi screening, as well as assays for further validation of the identified candidates. With modifications, this method can also be adapted to study the splicing regulation of pre-mRNAs that contain two or more splice isoforms.
Howard Hughes Medical Institute and Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA. ashish.misra@umassmed.edu.
Ashish
A
Misra
Howard Hughes Medical Institute and Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA. michael.green@umassmed.edu.
Michael R
MR
Green
D017398:Alternative Splicing; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D015894:Genome, Human; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins
Alternative splicing; Flow cytometry; Genome-wide; Pre-mRNA; RBFOX2; RNA interference