title: Inducing gene expression by targeting promoter sequences using small activating RNAs.
abstract: Vector-based systems comprised of exogenous nucleic acid sequences remain the standard for ectopic expression of a particular gene. Such systems offer robust overexpression, but have inherent drawbacks such as the tedious process of construction, excluding sequences (e.g. introns and untranslated regions) important for gene function and potential insertional mutagenesis of host genome associated with the use of viral vectors. We and others have recently reported that short double-stranded RNAs (dsRNAs) can induce endogenous gene expression by targeting promoter sequences in a phenomenon referred to as RNA activation (RNAa) and such dsRNAs are termed small activating RNAs (saRNAs). To date, RNAa has been successfully utilized to induce the expression of different genes such as tumor suppressor genes. Here, we describe a detailed protocol for target selection and dsRNA design with associated experiments to facilitate RNAa in cultured cells. This technique may be applied to selectively activate endogenous gene expression for studying gene function, interrogating molecular pathways and reprogramming cell fate.
authors.affiliation: Department of Urology and Helen-Diller Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA.
authors.forename: Ji
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Urology and Helen-Diller Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Place
authors.affiliation: Department of Urology and Helen-Diller Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA.
authors.forename: Victoria
authors.initials: V
authors.lastname: Portnoy
authors.affiliation: Department of Urology and Helen-Diller Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA.
authors.forename: Vera
authors.initials: V
authors.lastname: Huang
authors.affiliation: Department of Urology and Helen-Diller Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA.
authors.forename: Moo Rim
authors.initials: MR
authors.lastname: Kang
authors.affiliation: Department of Urology and Helen-Diller Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA.
authors.forename: Mika
authors.initials: M
authors.lastname: Kosaka
authors.affiliation: Biotechnology Research Institute and Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
authors.forename: Maurice Kwok Chung
authors.initials: MKC
authors.lastname: Ho
authors.affiliation: Department of Urology and Helen-Diller Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA.|Laboratory of Molecular Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Long-Cheng
authors.initials: LC
authors.lastname: Li
keywords: RNAa; gene regulation; saRNA; transcriptional activation
references.citation: Circ Res. 2009 Sep 11;105(6):604-9
references.pmid: 19696410
references.citation: Bioinformatics. 2002 Nov;18(11):1427-31
references.pmid: 12424112
references.citation: Nature. 2010 Apr 15;464(7291):1067-70
references.pmid: 20305636
references.citation: Silence. 2010 Jul 08;1(1):14
references.pmid: 20615220
references.citation: Philos Trans R Soc Lond B Biol Sci. 1993 Feb 27;339(1288):225-32
references.pmid: 8097052
references.citation: Nature. 1998 Feb 19;391(6669):806-11
references.pmid: 9486653
references.citation: Nucleic Acids Res. 2008 Jan;36(Database issue):D97-101
references.pmid: 17942421
references.citation: Mol Cell Biol. 2001 Jun;21(11):3830-9
references.pmid: 11340174
references.citation: Development. 2005 Mar;132(5):965-76
references.pmid: 15673570
references.citation: Nat Genet. 1993 Aug;4(4):332-3
references.pmid: 8401577
references.citation: Science. 2004 Aug 27;305(5688):1289-92
references.pmid: 15297624
references.citation: Proc Natl Acad Sci U S A. 1988 Feb;85(3):836-40
references.pmid: 3422466
references.citation: Cell. 1985 May;41(1):67-82
references.pmid: 2986851
references.citation: Nat Med. 2003 Mar;9(3):347-51
references.pmid: 12579197
references.citation: Mol Cancer Ther. 2008 Mar;7(3):698-703
references.pmid: 18347154
references.citation: Cell Rep. 2014 Jan 16;6(1):211-21
references.pmid: 24388755
references.citation: Curr Pharm Biotechnol. 2010 Aug;11(5):518-26
references.pmid: 20662764
references.citation: Chem Biol. 2010 Dec 22;17(12):1344-55
references.pmid: 21168770
references.citation: Wiley Interdiscip Rev RNA. 2011 Sep-Oct;2(5):748-60
references.pmid: 21823233
references.citation: Nature. 2001 May 24;411(6836):494-8
references.pmid: 11373684
references.citation: Cloning Stem Cells. 2007 Winter;9(4):523-34
references.pmid: 18154513
references.citation: Nat Biotechnol. 2004 Mar;22(3):326-30
references.pmid: 14758366
references.citation: Proc Natl Acad Sci U S A. 2006 Jan 31;103(5):1412-7
references.pmid: 16432200
references.citation: Methods Mol Biol. 2011;764:301-15
references.pmid: 21748649
references.citation: Proc Natl Acad Sci U S A. 2008 Feb 5;105(5):1608-13
references.pmid: 18227514
references.citation: PLoS One. 2010 Jan 22;5(1):e8848
references.pmid: 20107511
references.citation: J Sex Med. 2011 Oct;8(10):2773-80
references.pmid: 21819543
references.citation: Genomics. 1992 Aug;13(4):1095-107
references.pmid: 1505946
references.citation: Biochemistry. 1998 Oct 20;37(42):14719-35
references.pmid: 9778347
references.citation: Nucleic Acids Res. 2010 Nov;38(21):7736-48
references.pmid: 20675357
references.citation: Cancer Res. 2010 Dec 15;70(24):10182-91
references.pmid: 21159640
references.citation: Stem Cells Dev. 2015 Feb 1;24(3):345-53
references.pmid: 25232932
references.citation: PLoS Comput Biol. 2008 Nov;4(11):e1000216
references.pmid: 18989395
references.citation: Genes Dev. 2003 Dec 15;17(24):3011-6
references.pmid: 14681208
references.citation: Nat Chem Biol. 2005 Sep;1(4):216-22
references.pmid: 16408038
references.citation: Nat Chem Biol. 2010 Aug;6(8):621-9
references.pmid: 20581822
references.citation: Biochem J. 2012 May 1;443(3):821-8
references.pmid: 22339500
references.citation: Annu Rev Biochem. 1986;55:1091-117
references.pmid: 3017190
references.citation: Nat Genet. 2005 Aug;37(8):906-10
references.pmid: 16025112
references.citation: Mol Cell Probes. 2001 Oct;15(5):307-11
references.pmid: 11735303
references.citation: Nucleic Acid Ther. 2012 Feb;22(1):3-16
references.pmid: 22283730
references.citation: Genome Res. 2011 May;21(5):775-89
references.pmid: 21372179
references.citation: Cancer Res. 2012 Oct 1;72(19):5069-79
references.pmid: 22869584
references.citation: Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17337-42
references.pmid: 17085592
references.citation: Gene Ther. 2011 Aug;18(8):743-9
references.pmid: 21490683
references.citation: Nat Chem Biol. 2007 Mar;3(3):166-73
references.pmid: 17259978