Exon-specific RNAi: a tool for dissecting the functional relevance of alternative splicing (original) (raw)

Abstract

The goal of functional genomics is to determine the function of each protein encoded by an organism. Typically, this is done by inactivating individual genes and, subsequently, analyzing the phenotype of the modified organisms. In higher eukaryotes, where a tremendous amount of alternative splicing occurs, such approaches are not feasible because they have the potential to simultaneously affect multiple proteins that could have quite distinct and important functions. Thus, it is necessary to develop techniques that inactivate only a subset of proteins synthesized from genes encoding alternatively spliced mRNAs. Here we demonstrate that RNA interference (RNAi) can be used to selectively degrade specific alternatively spliced mRNA isoforms in cultured Drosophila cells. This is achieved by treating the cells with double-stranded RNA corresponding to an alternatively spliced exon. This technique may prove to be a powerful tool to assess the function of proteins synthesized from alternatively spliced mRNAs. In addition, these results have implications regarding the mechanism of RNAi in Drosophila.

Full Text

The Full Text of this article is available as a PDF (502.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams Melissa D., Sekelsky Jeff J. From sequence to phenotype: reverse genetics in Drosophila melanogaster. Nat Rev Genet. 2002 Mar;3(3):189–198. doi: 10.1038/nrg752. [DOI] [PubMed] [Google Scholar]
  2. Bernstein E., Denli A. M., Hannon G. J. The rest is silence. RNA. 2001 Nov;7(11):1509–1521. [PMC free article] [PubMed] [Google Scholar]
  3. Black D. L. Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell. 2000 Oct 27;103(3):367–370. doi: 10.1016/s0092-8674(00)00128-8. [DOI] [PubMed] [Google Scholar]
  4. Celotto A. M., Graveley B. R. Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated. Genetics. 2001 Oct;159(2):599–608. doi: 10.1093/genetics/159.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cho J. H., Bandyopadhyay J., Lee J., Park C. S., Ahnn J. Two isoforms of sarco/endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans. Gene. 2000 Dec 31;261(2):211–219. doi: 10.1016/s0378-1119(00)00536-9. [DOI] [PubMed] [Google Scholar]
  6. Fraser A. G., Kamath R. S., Zipperlen P., Martinez-Campos M., Sohrmann M., Ahringer J. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature. 2000 Nov 16;408(6810):325–330. doi: 10.1038/35042517. [DOI] [PubMed] [Google Scholar]
  7. Graveley B. R. Alternative splicing: increasing diversity in the proteomic world. Trends Genet. 2001 Feb;17(2):100–107. doi: 10.1016/s0168-9525(00)02176-4. [DOI] [PubMed] [Google Scholar]
  8. Gönczy P., Echeverri C., Oegema K., Coulson A., Jones S. J., Copley R. R., Duperon J., Oegema J., Brehm M., Cassin E. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature. 2000 Nov 16;408(6810):331–336. doi: 10.1038/35042526. [DOI] [PubMed] [Google Scholar]
  9. Hammond S. M., Boettcher S., Caudy A. A., Kobayashi R., Hannon G. J. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science. 2001 Aug 10;293(5532):1146–1150. doi: 10.1126/science.1064023. [DOI] [PubMed] [Google Scholar]
  10. Hutvágner György, Zamore Phillip D. RNAi: nature abhors a double-strand. Curr Opin Genet Dev. 2002 Apr;12(2):225–232. doi: 10.1016/s0959-437x(02)00290-3. [DOI] [PubMed] [Google Scholar]
  11. Kumar A., Snyder M. Emerging technologies in yeast genomics. Nat Rev Genet. 2001 Apr;2(4):302–312. doi: 10.1038/35066084. [DOI] [PubMed] [Google Scholar]
  12. Lipardi C., Wei Q., Paterson B. M. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell. 2001 Nov 2;107(3):297–307. doi: 10.1016/s0092-8674(01)00537-2. [DOI] [PubMed] [Google Scholar]
  13. Maeda I., Kohara Y., Yamamoto M., Sugimoto A. Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Curr Biol. 2001 Feb 6;11(3):171–176. doi: 10.1016/s0960-9822(01)00052-5. [DOI] [PubMed] [Google Scholar]
  14. Modrek Barmak, Lee Christopher. A genomic view of alternative splicing. Nat Genet. 2002 Jan;30(1):13–19. doi: 10.1038/ng0102-13. [DOI] [PubMed] [Google Scholar]
  15. Mourrain P., Béclin C., Elmayan T., Feuerbach F., Godon C., Morel J. B., Jouette D., Lacombe A. M., Nikic S., Picault N. Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell. 2000 May 26;101(5):533–542. doi: 10.1016/s0092-8674(00)80863-6. [DOI] [PubMed] [Google Scholar]
  16. Nishikura K. A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell. 2001 Nov 16;107(4):415–418. doi: 10.1016/s0092-8674(01)00581-5. [DOI] [PubMed] [Google Scholar]
  17. Nykänen A., Haley B., Zamore P. D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell. 2001 Nov 2;107(3):309–321. doi: 10.1016/s0092-8674(01)00547-5. [DOI] [PubMed] [Google Scholar]
  18. Schmucker D., Clemens J. C., Shu H., Worby C. A., Xiao J., Muda M., Dixon J. E., Zipursky S. L. Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell. 2000 Jun 9;101(6):671–684. doi: 10.1016/s0092-8674(00)80878-8. [DOI] [PubMed] [Google Scholar]
  19. Schneider I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 1972 Apr;27(2):353–365. [PubMed] [Google Scholar]
  20. Sijen T., Fleenor J., Simmer F., Thijssen K. L., Parrish S., Timmons L., Plasterk R. H., Fire A. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell. 2001 Nov 16;107(4):465–476. doi: 10.1016/s0092-8674(01)00576-1. [DOI] [PubMed] [Google Scholar]
  21. Zamore P. D. RNA interference: listening to the sound of silence. Nat Struct Biol. 2001 Sep;8(9):746–750. doi: 10.1038/nsb0901-746. [DOI] [PubMed] [Google Scholar]
  22. Zamore P. D., Tuschl T., Sharp P. A., Bartel D. P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000 Mar 31;101(1):25–33. doi: 10.1016/S0092-8674(00)80620-0. [DOI] [PubMed] [Google Scholar]