Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Apr 1;363(Pt 1):1–5. doi: 10.1042/0264-6021:3630001

Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA.

Malgorzata Kisielow 1, Sandra Kleiner 1, Michiaki Nagasawa 1, Amir Faisal 1, Yoshikuni Nagamine 1
PMCID: PMC1222444  PMID: 11903040

Abstract

Many eukaryotic genes are expressed as multiple isoforms through the differential utilization of transcription/translation initiation sites or alternative splicing. The conventional approach for studying individual isoforms in a clean background (i.e. without the influence of other isoforms) has been to express them in cells or whole organisms in which the target gene has been deleted; this is time-consuming. Recently an efficient post-transcriptional gene-silencing method has been reported that employs a small interfering double-stranded RNA (siRNA). On the basis of this method we report a rapid alternative approach for isoform-specific gene expression. We show how the adaptor protein ShcA can be suppressed and expressed in an isoform-specific manner in a human cell line. ShcA exists in three isoforms, namely p66, p52 and p46, which differ only in their N-terminal regions and are derived from two different transcripts, namely p66 and p52/p46 mRNAs. An siRNA with a sequence shared by the two transcripts suppressed all of them. However, another siRNA whose sequence was present only in p66 mRNA suppressed only the p66 isoform, suggesting that the siRNA signal did not propagate to other regions of the target mRNA. The expression of individual isoforms was achieved by first down-regulating all isoforms by the common siRNA and then transfecting with an expression vector for each isoform that harboured silent mutations at the site corresponding to the siRNA. This allowed functional analysis of individual ShcA isoforms and may be more generally applicable for studying genes encoding multiple proteins.

Full Text

The Full Text of this article is available as a PDF (209.3 KB).

Selected References

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

  1. Andreadis A., Gallego M. E., Nadal-Ginard B. Generation of protein isoform diversity by alternative splicing: mechanistic and biological implications. Annu Rev Cell Biol. 1987;3:207–242. doi: 10.1146/annurev.cb.03.110187.001231. [DOI] [PubMed] [Google Scholar]
  2. Bernstein E., Caudy A. A., Hammond S. M., Hannon G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001 Jan 18;409(6818):363–366. doi: 10.1038/35053110. [DOI] [PubMed] [Google Scholar]
  3. Bonfini L., Migliaccio E., Pelicci G., Lanfrancone L., Pelicci P. G. Not all Shc's roads lead to Ras. Trends Biochem Sci. 1996 Jul;21(7):257–261. [PubMed] [Google Scholar]
  4. Bosher J. M., Labouesse M. RNA interference: genetic wand and genetic watchdog. Nat Cell Biol. 2000 Feb;2(2):E31–E36. doi: 10.1038/35000102. [DOI] [PubMed] [Google Scholar]
  5. Carthew R. W. Gene silencing by double-stranded RNA. Curr Opin Cell Biol. 2001 Apr;13(2):244–248. doi: 10.1016/s0955-0674(00)00204-0. [DOI] [PubMed] [Google Scholar]
  6. Cogoni C., Macino G. Post-transcriptional gene silencing across kingdoms. Curr Opin Genet Dev. 2000 Dec;10(6):638–643. doi: 10.1016/s0959-437x(00)00134-9. [DOI] [PubMed] [Google Scholar]
  7. Cogoni C., Romano N., Macino G. Suppression of gene expression by homologous transgenes. Antonie Van Leeuwenhoek. 1994;65(3):205–209. doi: 10.1007/BF00871948. [DOI] [PubMed] [Google Scholar]
  8. Elbashir S. M., Harborth J., Lendeckel W., Yalcin A., Weber K., Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24;411(6836):494–498. doi: 10.1038/35078107. [DOI] [PubMed] [Google Scholar]
  9. Fagard M., Vaucheret H. Systemic silencing signal(s). Plant Mol Biol. 2000 Jun;43(2-3):285–293. doi: 10.1023/a:1006404016494. [DOI] [PubMed] [Google Scholar]
  10. Fire A., Xu S., Montgomery M. K., Kostas S. A., Driver S. E., Mello C. C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998 Feb 19;391(6669):806–811. doi: 10.1038/35888. [DOI] [PubMed] [Google Scholar]
  11. Gorman C., Bullock C. Site-specific gene targeting for gene expression in eukaryotes. Curr Opin Biotechnol. 2000 Oct;11(5):455–460. doi: 10.1016/s0958-1669(00)00127-0. [DOI] [PubMed] [Google Scholar]
  12. Johnson J. E., Wold B. J., Hauschka S. D. Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice. Mol Cell Biol. 1989 Aug;9(8):3393–3399. doi: 10.1128/mcb.9.8.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Luzi L., Confalonieri S., Di Fiore P. P., Pelicci P. G. Evolution of Shc functions from nematode to human. Curr Opin Genet Dev. 2000 Dec;10(6):668–674. doi: 10.1016/s0959-437x(00)00146-5. [DOI] [PubMed] [Google Scholar]
  15. Migliaccio E., Giorgio M., Mele S., Pelicci G., Reboldi P., Pandolfi P. P., Lanfrancone L., Pelicci P. G. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature. 1999 Nov 18;402(6759):309–313. doi: 10.1038/46311. [DOI] [PubMed] [Google Scholar]
  16. Migliaccio E., Mele S., Salcini A. E., Pelicci G., Lai K. M., Superti-Furga G., Pawson T., Di Fiore P. P., Lanfrancone L., Pelicci P. G. Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway. EMBO J. 1997 Feb 17;16(4):706–716. doi: 10.1093/emboj/16.4.706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Müller U. Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech Dev. 1999 Apr;82(1-2):3–21. doi: 10.1016/s0925-4773(99)00021-0. [DOI] [PubMed] [Google Scholar]
  18. Nakamura T., Muraoka S., Sanokawa R., Mori N. N-Shc and Sck, two neuronally expressed Shc adapter homologs. Their differential regional expression in the brain and roles in neurotrophin and Src signaling. J Biol Chem. 1998 Mar 20;273(12):6960–6967. doi: 10.1074/jbc.273.12.6960. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Tan S. S. Liver-specific and position-effect expression of a retinol-binding protein-lacZ fusion gene (RBP-lacZ) in transgenic mice. Dev Biol. 1991 Jul;146(1):24–37. doi: 10.1016/0012-1606(91)90443-7. [DOI] [PubMed] [Google Scholar]
  21. Tuschl T., Zamore P. D., Lehmann R., Bartel D. P., Sharp P. A. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 1999 Dec 15;13(24):3191–3197. doi: 10.1101/gad.13.24.3191. [DOI] [PMC free article] [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]
  23. van der Krol A. R., Mur L. A., Beld M., Mol J. N., Stuitje A. R. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell. 1990 Apr;2(4):291–299. doi: 10.1105/tpc.2.4.291. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES