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PMID: 11959843 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.

Genes & development ·Vol. 16 ·No. 8 ·2002-04-15 ·Pages 948-58

Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS

Abstract

RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (approximately 70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.

MeSH Terms
3T3 Cells Animals COS Cells Cell Line Conserved Sequence Drosophila Endoribonucleases/metabolism Gene Expression/drug effects Gene Silencing/drug effects,physiology Humans Mice RNA/metabolism,pharmacology RNA Polymerase III/metabolism RNA Processing, Post-Transcriptional/physiology RNA, Messenger/metabolism Ribonuclease III Substrate Specificity Transfection
Chemicals
RNA, Messenger RNA RNA Polymerase III Endoribonucleases Ribonuclease III
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Paddison Patrick J
Watson School of Biological Sciences, Cold Spring Harbor, New York 11724, USA.
Caudy Amy A
Bernstein Emily
Hannon Gregory J
Conklin Douglas S
References (40)
40 references, click to expand
  1. Post-transcriptional gene silencing by double-stranded RNA.
    Nat Rev Genet. 2001 Feb;2(2):110-9 PMID: 11253050
  2. Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action.
    Apoptosis. 2000 Apr;5(2):107-14 PMID: 11232238
  3. Reconstitution of transcription from the human U6 small nuclear RNA promoter with eight recombinant polypeptides and a partially purified RNA polymerase III complex.
    J Biol Chem. 2001 Jun 8;276(23):20727-34 PMID: 11279001
  4. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing.
    Cell. 2001 Jul 13;106(1):23-34 PMID: 11461699
  5. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.
    Science. 2001 Aug 3;293(5531):834-8 PMID: 11452083
  6. Argonaute2, a link between genetic and biochemical analyses of RNAi.
    Science. 2001 Aug 10;293(5532):1146-50 PMID: 11498593
  7. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.
    Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9742-7 PMID: 11481446
  8. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans.
    Science. 2001 Sep 21;293(5538):2269-71 PMID: 11486053
  9. Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells.
    Mol Cell Biol. 2001 Nov;21(22):7807-16 PMID: 11604515
  10. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.
    Genes Dev. 2001 Oct 15;15(20):2654-9 PMID: 11641272
  11. Identification of novel genes coding for small expressed RNAs.
    Science. 2001 Oct 26;294(5543):853-8 PMID: 11679670
  12. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.
    Science. 2001 Oct 26;294(5543):858-62 PMID: 11679671
  13. An extensive class of small RNAs in Caenorhabditis elegans.
    Science. 2001 Oct 26;294(5543):862-4 PMID: 11679672
  14. ATP requirements and small interfering RNA structure in the RNA interference pathway.
    Cell. 2001 Nov 2;107(3):309-21 PMID: 11701122
  15. The rest is silence.
    RNA. 2001 Nov;7(11):1509-21 PMID: 11720281
  16. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate.
    EMBO J. 2001 Dec 3;20(23):6877-88 PMID: 11726523
  17. Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines.
    Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14428-33 PMID: 11724966
  18. Stable suppression of gene expression by RNAi in mammalian cells.
    Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1443-8 PMID: 11818553
  19. cis-acting elements required for RNA polymerase II and III transcription in the human U2 and U6 snRNA promoters.
    Nucleic Acids Res. 1990 May 25;18(10):2891-9 PMID: 2349089
  20. Multiple cis-acting elements are required for RNA polymerase III transcription of the gene encoding H1 RNA, the RNA component of human RNase P.
    J Biol Chem. 1991 Dec 5;266(34):22796-9 PMID: 1720774
  21. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans.
    Cell. 1993 Dec 3;75(5):855-62 PMID: 8252622
  22. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed.
    Cell. 1995 May 19;81(4):611-20 PMID: 7758115
  23. Interactions between the double-stranded RNA binding motif and RNA: definition of the binding site for the interferon-induced protein kinase DAI (PKR) on adenovirus VA RNA.
    RNA. 1995 Mar;1(1):7-20 PMID: 7489491
  24. A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation.
    Genes Dev. 1996 Dec 1;10(23):3041-50 PMID: 8957004
  25. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.
    Cell. 1997 Mar 7;88(5):593-602 PMID: 9054499
  26. Role of the double-stranded RNA-activated protein kinase (PKR) in cell regulation.
    Biochem Soc Trans. 1997 May;25(2):509-13 PMID: 9191145
  27. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
    Nature. 1998 Feb 19;391(6669):806-11 PMID: 9486653
  28. Targeted mRNA degradation by double-stranded RNA in vitro.
    Genes Dev. 1999 Dec 15;13(24):3191-7 PMID: 10617568
  29. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes.
    Nat Genet. 2000 Feb;24(2):180-3 PMID: 10655066
  30. Specific interference with gene function by double-stranded RNA in early mouse development.
    Nat Cell Biol. 2000 Feb;2(2):70-5 PMID: 10655585
  31. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.
    Nature. 2000 Feb 24;403(6772):901-6 PMID: 10706289
  32. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.
    Nature. 2000 Mar 16;404(6775):293-6 PMID: 10749213
  33. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor.
    Mol Cell. 2000 Apr;5(4):659-69 PMID: 10882102
  34. Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA.
    RNA. 2000 Jul;6(7):1069-76 PMID: 10917601
  35. Heritable gene silencing in Drosophila using double-stranded RNA.
    Nat Biotechnol. 2000 Aug;18(8):896-8 PMID: 10932163
  36. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference.
    Development. 2000 Oct;127(19):4147-56 PMID: 10976047
  37. Total silencing by intron-spliced hairpin RNAs.
    Nature. 2000 Sep 21;407(6802):319-20 PMID: 11014180
  38. Role for a bidentate ribonuclease in the initiation step of RNA interference.
    Nature. 2001 Jan 18;409(6818):363-6 PMID: 11201747
  39. RNA interference is mediated by 21- and 22-nucleotide RNAs.
    Genes Dev. 2001 Jan 15;15(2):188-200 PMID: 11157775
  40. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
    Nature. 2001 May 24;411(6836):494-8 PMID: 11373684
Article Info
Journal
Genes & development
Abbr.
Genes Dev
ISSN
0890-9369
DOI
10.1101/gad.981002
Published
2002-04-15
Pages
948-58
Language
English
Region
United States
NLM ID
8711660
PMCID
PMC152352
Subset
IM
Grants
NIGMS NIH HHS · R01 GM062534 · United States
NIGMS NIH HHS · R01-GM62534 · United States
External Links
PubMed source DOI: 10.1101/gad.981002 PMC Full Text View journal details
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