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Fluorescence Reporter-Based Genome-Wide RNA Interference Screening to Identify Alternative Splicing Regulators

  • Ashish Misra
  • Michael R. Green
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1507)

Abstract

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 words

Alternative splicing Genome-wide RNA interference Flow cytometry RBFOX2 Pre-mRNA 

Notes

Acknowledgment

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.

References

  1. 1.
    Boutros M, Ahringer J (2008) The art and design of genetic screens: RNA interference. Nat Rev Genet 9(7):554–566. doi: 10.1038/nrg2364, nrg2364 [pii]CrossRefPubMedGoogle Scholar
  2. 2.
    Grimm S (2004) The art and design of genetic screens: mammalian culture cells. Nat Rev Genet 5(3):179–189. doi: 10.1038/nrg1291, nrg1291 [pii]CrossRefPubMedGoogle Scholar
  3. 3.
    Bernards R, Brummelkamp TR, Beijersbergen RL (2006) ShRNA libraries and their use in cancer genetics. Nat Methods 3(9):701–706. doi: 10.1038/nmeth921, nmeth921 [pii]CrossRefPubMedGoogle Scholar
  4. 4.
    Mohr S, Bakal C, Perrimon N (2010) Genomic screening with RNAi: results and challenges. Annu Rev Biochem 79:37–64. doi: 10.1146/annurev-biochem-060408-092949 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Moore MJ, Wang Q, Kennedy CJ, Silver PA (2010) An alternative splicing network links cell-cycle control to apoptosis. Cell 142(4):625–636. doi: 10.1016/j.cell.2010.07.019, S0092-8674(10)00788-9 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Misra A, Ou J, Zhu LJ, Green MR (2015) Global promotion of alternative internal exon usage by mRNA 3′ end formation factors. Mol Cell 58(5):819–831. doi: 10.1016/j.molcel.2015.03.016, S1097-2765(15)00181-1 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Whitehead KA, Langer R, Anderson DG (2009) Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 8(2):129–138. doi: 10.1038/nrd2742, nrd2742 [pii]CrossRefPubMedGoogle Scholar
  8. 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
  9. 9.
    Root DE, Hacohen N, Hahn WC, Lander ES, Sabatini DM (2006) Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat Methods 3(9):715–719. doi: 10.1038/nmeth924, nmeth924 [pii]CrossRefPubMedGoogle Scholar
  10. 10.
    Mohr SE, Perrimon N (2012) RNAi screening: new approaches, understandings, and organisms. Wiley Interdiscip Rev RNA 3(2):145–158. doi: 10.1002/wrna.110 CrossRefPubMedGoogle Scholar
  11. 11.
    Mohr SE, Smith JA, Shamu CE, Neumuller RA, Perrimon N (2014) RNAi screening comes of age: improved techniques and complementary approaches. Nat Rev Mol Cell Biol 15(9):591–600. doi: 10.1038/nrm3860, nrm3860 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wang Z, Rolish ME, Yeo G, Tung V, Mawson M, Burge CB (2004) Systematic identification and analysis of exonic splicing silencers. Cell 119(6):831–845. doi: 10.1016/j.cell.2004.11.010, S0092867404010566 [pii]CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Howard Hughes Medical Institute and Department of Molecular, Cell and Cancer BiologyUniversity of Massachusetts Medical SchoolWorcesterUSA

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