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.
Key wordsAlternative splicing Genome-wide RNA interference Flow cytometry RBFOX2 Pre-mRNA
We thank Sara Deibler for editorial assistance. This work was supported by NIH grant R01 GM035490 to M.R.G. M.R.G. is also an investigator of the Howard Hughes Medical Institute.
- 8.Birmingham A, Selfors LM, Forster T, Wrobel D, Kennedy CJ, Shanks E, Santoyo-Lopez J, Dunican DJ, Long A, Kelleher D, Smith Q, Beijersbergen RL, Ghazal P, Shamu CE (2009) Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 6(8):569–575. doi: 10.1038/nmeth.1351, nmeth.1351 [pii]CrossRefPubMedPubMedCentralGoogle Scholar