Inducing gene expression by targeting promoter sequences using small activating RNAs


  • Ji Wang Department of Urology, University of California San Francisco
  • Robert F Place Department of Urology, University of California San Francisco
  • Victoria Portnoy Department of Urology, University of California San Francisco
  • Vera Huang Department of Urology, University of California San Francisco
  • Moo Rim Kang Department of Urology, University of California San Francisco
  • Maurice Kwok Chung Ho Biotechnology Research Institute and Division of Life Science, The Hong Kong University of Science and Technology
  • Long-Cheng LI Department of Urology, University of California San Francisco



RNAa, saRNA, transcriptional activation, gene regulation


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 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.


Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811.

Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494-498.

Song E, Lee SK, Wang J, Ince N, Ouyang N, et al. (2003) RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 9: 347-351.

Davis ME, Zuckerman JE, Choi CH, Seligson D, Tolcher A, et al. (2010) Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464: 1067-1070.

Morris KV, Chan SW, Jacobsen SE, Looney DJ (2004) Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305: 1289-1292.

Janowski BA, Huffman KE, Schwartz JC, Ram R, Hardy D, et al. (2005) Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene RNAs. Nat Chem Biol 1: 216-222.

Yue X, Schwartz JC, Chu Y, Younger ST, Gagnon KT, et al. (2010) Transcriptional regulation by small RNAs at sequences downstream from 3' gene termini. Nat Chem Biol 6: 621-629.

Li LC, Okino ST, Zhao H, Pookot D, Place RF, et al. (2006) Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci U S A 103: 17337-17342.

Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, et al. (2007) Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nat Chem Biol 3: 166-173.

Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R (2008) MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci U S A 105: 1608-1613.

Matsui M, Sakurai F, Elbashir S, Foster DJ, Manoharan M, et al. (2010) Activation of LDL receptor expression by small RNAs complementary to a noncoding transcript that overlaps the LDLR promoter. Chem Biol 17: 1344-1355.

Gagnon KT, Li L, Chu Y, Janowski BA, Corey DR (2014) RNAi factors are present and active in human cell nuclei. Cell Rep 6: 211-221.

Chu Y, Yue X, Younger ST, Janowski BA, Corey DR (2010) Involvement of argonaute proteins in gene silencing and activation by RNAs complementary to a non-coding transcript at the progesterone receptor promoter. Nucleic Acids Res 38: 7736-7748.

Turunen MP, Lehtola T, Heinonen SE, Assefa GS, Korpisalo P, et al. (2009) Efficient regulation of VEGF expression by promoter-targeted lentiviral shRNAs based on epigenetic mechanism: a novel example of epigenetherapy. Circ Res 105: 604-609.

Gagnon KT, Corey DR (2012) Argonaute and the nuclear RNAs: new pathways for RNA-mediated control of gene expression. Nucleic Acid Ther 22: 3-16.

Portnoy V, Huang V, Place RF, Li LC (2011) Small RNA and transcriptional upregulation. Wiley Interdiscip Rev RNA 2: 748-760.

Place RF, Noonan EJ, Foldes-Papp Z, Li LC (2010) Defining features and exploring chemical modifications to manipulate RNAa activity. Curr Pharm Biotechnol 11: 518-526.

Huang V, Qin Y, Wang J, Wang X, Place RF, et al. (2010) RNAa is conserved in mammalian cells. PLoS One 5: e8848.

Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD (1988) Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci U S A 85: 836-840.

Clark AJ, Archibald AL, McClenaghan M, Simons JP, Wallace R, et al. (1993) Enhancing the efficiency of transgene expression. Philos Trans R Soc Lond B Biol Sci 339: 225-232.

Stemmler MP, Hecht A, Kemler R (2005) E-cadherin intron 2 contains cis-regulatory elements essential for gene expression. Development (Cambridge, England) 132: 965-976.

Breitbart RE, Nguyen HT, Medford RM, Destree AT, Mahdavi V, et al. (1985) Intricate combinatorial patterns of exon splicing generate multiple regulated troponin T isoforms from a single gene. Cell 41: 67-82.

Leff SE, Rosenfeld MG, Evans RM (1986) Complex transcriptional units: diversity in gene expression by alternative RNA processing. Annu Rev Biochem 55: 1091-1117.

Hedman M, Hartikainen J, Yla-Herttuala S (2011) Progress and prospects: hurdles to cardiovascular gene therapy clinical trials. Gene Ther 18: 743-749.

Boguski MS, Lowe TM, Tolstoshev CM (1993) dbEST--database for "expressed sequence tags". Nat Genet 4: 332-333.

Wakaguri H, Yamashita R, Suzuki Y, Sugano S, Nakai K (2008) DBTSS: database of transcription start sites, progress report 2008. Nucleic Acids Res 36: D97-101.

Larsen F, Gundersen G, Lopez R, Prydz H (1992) CpG islands as gene markers in the human genome. Genomics 13: 1095-1107.

Li LC, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18: 1427-1431.

Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, et al. (2004) Rational siRNA design for RNA interference. Nat Biotechnol 22: 326-330.

Ting AH, Schuebel KE, Herman JG, Baylin SB (2005) Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat Genet 37: 906-910.

Xia T, SantaLucia J, Jr., Burkard ME, Kierzek R, Schroeder SJ, et al. (1998) Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry 37: 14719-14735.

Wang J, Place RF, Huang V, Wang X, Noonan EJ, et al. (2010) Prognostic Value and Function of KLF4 in Prostate Cancer: RNAa and Vector-Mediated Overexpression Identify KLF4 as an Inhibitor of Tumor Cell Growth and Migration. Cancer Res 70: 10182-10191.

Chen Z, Place RF, Jia ZJ, Pookot D, Dahiya R, et al. (2008) Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells. Mol Cancer Ther 7: 698-703.

Wang J, Huang V, Ye L, Barcena A, Lin G, et al. (2015) Identification of Small Activating RNAs that Enhance Endogenous OCT4 Expression in Human Mesenchymal Stem Cells. Stem Cells Dev 24: 345-353.

Wang J, Huang V, Ye L, Barcena A, Lin G, et al. (2014) Identification of Small Activating RNAs that Enhance Endogenous OCT4 Expression in Human Mesenchymal Stem Cells. Stem Cells Dev.

Yamashita R, Sathira NP, Kanai A, Tanimoto K, Arauchi T, et al. (2011) Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis. Genome Res 21: 775-789.

Saxonov S, Berg P, Brutlag DL (2006) A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc Natl Acad Sci U S A 103: 1412-1417.

Anderson JD, Widom J (2001) Poly(dA-dT) promoter elements increase the equilibrium accessibility of nucleosomal DNA target sites. Mol Cell Biol 21: 3830-3839.

Field Y, Kaplan N, Fondufe-Mittendorf Y, Moore IK, Sharon E, et al. (2008) Distinct modes of regulation by chromatin encoded through nucleosome positioning signals. PLoS computational biology 4: e1000216.

Chen R, Wang T, Rao K, Yang J, Zhang S, et al. (2011) Up-regulation of VEGF by Small Activator RNA in Human Corpus Cavernosum Smooth Muscle Cells. J Sex Med.

Nakayama A, Sato M, Shinohara M, Matsubara S, Yokomine T, et al. (2007) Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system. Cloning Stem Cells 9: 523-534.

Kang MR, Yang G, Place RF, Charisse K, Epstein-Barash H, et al. (2012) Intravesical delivery of small activating RNA formulated into lipid nanoparticles inhibits orthotopic bladder tumor growth. Cancer Res 72: 5069-5079.

Vaishnaw AK, Gollob J, Gamba-Vitalo C, Hutabarat R, Sah D, et al. (2010) A status report on RNAi therapeutics. Silence 1: 14.

Yi R, Qin Y, Macara IG, Cullen BR (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17: 3011-3016.

Schwartz JC, Corey DR (2011) Practical Considerations for Analyzing Antigene RNAs (agRNAs): RNA Immunoprecipitation of Argonaute Protein. Methods Mol Biol 764: 301-315.

Wang X, Wang J, Huang V, Place RF, Li LC (2012) Induction of NANOG expression by targeting promoter sequence with small activating RNA antagonizes retinoic acid-induced differentiation. Biochem J 443: 821-828.

Selvey S, Thompson EW, Matthaei K, Lea RA, Irving MG, et al. (2001) Beta-actin--an unsuitable internal control for RT-PCR. Mol Cell Probes 15: 307-311.


Additional Files



How to Cite

Wang J, Place RF, Portnoy V, Huang V, Kang MR, Ho MKC, LI L-C. Inducing gene expression by targeting promoter sequences using small activating RNAs. J Biol Methods [Internet]. 2015Mar.11 [cited 2022Jan.27];2(1):e14. Available from: