title: Dicer-independent processing of short hairpin RNAs.
abstract: Short hairpin RNAs (shRNAs) are widely used to induce RNA interference (RNAi). We tested a variety of shRNAs that differed in stem length and terminal loop size and revealed strikingly different RNAi activities and shRNA-processing patterns. Interestingly, we identified a specific shRNA design that uses an alternative Dicer-independent processing pathway. Detailed analyses indicated that a short shRNA stem length is critical for avoiding Dicer processing and activation of the alternative processing route, in which the shRNA is incorporated into RISC and processed by the AGO2-mediated slicer activity. Such alternatively processed shRNAs (AgoshRNAs) yield only a single RNA strand that effectively induces RNAi, whereas conventional shRNA processing results in an siRNA duplex of which both strands can trigger RNAi. Both the processing and subsequent RNAi activity of these AgoshRNAs are thus mediated by the RISC-component AGO2. These results have important implications for the future design of more specific RNAi therapeutics.
authors: Department of Medical Microbiology, Laboratory of Experimental Virology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
authors: Ying Poi
authors: YP
authors: Liu
authors: Nick C T
authors: NC
authors: Schopman
authors: Ben
authors: B
authors: Berkhout
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism; D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism
chemical_list: D060565:Argonaute Proteins; D034741:RNA, Small Interfering; D043244:Ribonuclease III
references: J Gene Med. 2008 Oct;10(10):1059-70
references: 18642399
references: Cancer Sci. 2005 Feb;96(2):111-5
references: 15723655
references: J Gene Med. 2004 Jul;6(7):715-23
references: 15241778
references: Circulation. 2008 Sep 30;118(14 Suppl):S226-33
references: 18824759
references: Cell. 2005 Nov 18;123(4):607-20
references: 16271386
references: Nature. 2004 Nov 11;432(7014):235-40
references: 15531877
references: PLoS One. 2009;4(4):e5382
references: 19401777
references: Nat Struct Mol Biol. 2012 Dec;19(12):1324-31
references: 23142987
references: Nucleic Acids Res. 2005 Feb 01;33(2):796-804
references: 15687388
references: RNA. 2012 May;18(5):945-57
references: 22461413
references: Mol Ther. 2009 Oct;17(10):1712-23
references: 19672247
references: Nature. 2000 Mar 16;404(6775):293-6
references: 10749213
references: Nature. 2001 Jan 18;409(6818):363-6
references: 11201747
references: Mol Cell. 2004 Jul 23;15(2):185-97
references: 15260970
references: EMBO J. 2002 Sep 2;21(17):4663-70
references: 12198168
references: Cell. 2007 Jul 27;130(2):287-97
references: 17662943
references: RNA. 2002 Jun;8(6):842-50
references: 12088155
references: Nat Struct Mol Biol. 2012 Jan 10;19(2):145-51
references: 22233755
references: Nucleic Acids Res. 2003 Jan 15;31(2):700-7
references: 12527779
references: Nature. 2004 Nov 11;432(7014):231-5
references: 15531879
references: Antiviral Res. 2010 May;86(2):204-11
references: 20188764
references: Cell. 2012 Nov 9;151(4):900-911
references: 23141545
references: J Cell Sci. 2010 Jun 1;123(Pt 11):1819-23
references: 20484662
references: Mol Cell. 2009 Nov 13;36(3):431-44
references: 19917251
references: Cell. 2005 Nov 18;123(4):621-9
references: 16271385
references: RNA. 2010 Jan;16(1):43-56
references: 19917635
references: Science. 2009 Aug 21;325(5943):965
references: 19556464
references: Mol Ther. 2006 Dec;14(6):883-92
references: 16959541
references: Nat Biotechnol. 2003 Jun;21(6):635-7
references: 12754523
references: Nat Methods. 2006 Sep;3(9):701-6
references: 16929315
references: Nucleic Acids Res. 2004 Sep 08;32(16):4776-85
references: 15356295
references: Nature. 2010 Jun 3;465(7298):584-9
references: 20424607
references: Nature. 2001 May 24;411(6836):494-8
references: 11373684
references: RNA. 2012 Nov;18(11):2083-92
references: 22984192
references: J Biol Chem. 2010 Oct 15;285(42):31930-43
references: 20584909
references: Oligonucleotides. 2007 Summer;17(2):223-36
references: 17638526
references: RNA Biol. 2011 Jan-Feb;8(1):158-77
references: 21282978
references: RNA. 2007 Oct;13(10):1765-74
references: 17698642
references: Methods Enzymol. 2007;427:123-38
references: 17720482
references: Nat Genet. 2003 Jul;34(3):263-4
references: 12796781
references: Nucleic Acids Res. 2008 May;36(9):2811-24
references: 18346971
references: Nucleic Acids Res. 2012 Oct;40(18):9255-71
references: 22810205
references: Science. 2010 Jun 25;328(5986):1694-8
references: 20448148
references: Cell. 2002 Sep 6;110(5):563-74
references: 12230974
references: Nature. 2009 Jun 18;459(7249):1010-4
references: 19458619
references: Nat Biotechnol. 2005 Feb;23(2):227-31
references: 15619616
references: Cell. 2001 Nov 2;107(3):309-21
references: 11701122
references: Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
references: 12824337
references: BMC Genomics. 2009 Sep 04;10:413
references: 19732433
references: J RNAi Gene Silencing. 2008 May 27;4(1):295-301
references: 19771239
references: Nature. 2005 Mar 31;434(7033):666-70
references: 15800629
references: Nucleic Acids Res. 2012 Oct;40(19):9850-62
references: 22844086
references: Science. 2001 Oct 26;294(5543):858-62
references: 11679671
references: BMC Mol Biol. 2011 Aug 08;12:34
references: 21819628
references: Mol Ther. 2009 Jan;17(1):169-75
references: 19002161
references: Retrovirology. 2006 Jan 06;3:2
references: 16398936
references: Science. 2004 Jan 2;303(5654):95-8
references: 14631048
references: Nature. 2008 Dec 18;456(7224):921-6
references: 19092929
references: Nature. 1998 Feb 19;391(6669):806-11
references: 9486653
references: Nat Struct Mol Biol. 2011 Jun;18(6):650-7
references: 21552258
references: Clin Biochem. 2010 Feb;43(3):324-7
references: 19782670
references: Mol Ther. 2008 Mar;16(3):557-564
references: 28178502
references: Cell. 2009 Jan 23;136(2):215-33
references: 19167326
references: Cell. 2003 Oct 17;115(2):209-16
references: 14567918
references: Nat Biotechnol. 2008 Dec;26(12):1379-82
references: 19029911
references: Nat Struct Mol Biol. 2010 Jan;17(1):17-23
references: 19966796
references: Plant Cell. 1990 Apr;2(4):279-289
references: 12354959
references: Proc Natl Acad Sci U S A. 2010 Aug 24;107(34):15163-8
references: 20699384
references: Science. 2004 Sep 3;305(5689):1437-41
references: 15284456
references: Genes Dev. 2003 Dec 15;17(24):3011-6
references: 14681208
references: Cell. 2003 Oct 17;115(2):199-208
references: 14567917
references: RNA. 2010 Jan;16(1):106-17
references: 19952116
references: Science. 2002 Apr 19;296(5567):550-3
references: 11910072
references: Nature. 2010 Jun 10;465(7299):818-22
references: 20505670
references: RNA. 2005 May;11(5):674-82
references: 15811921