Müge KOVANCILAR, Mehmet Nejat DALAY, Uğur DELİGEZER*

Testing the putative effect of Dicer-substrate siRNAs in regulating gene expression at transcriptional level

RNA interference (RNAi) pathway is a gene silencing process during which small double-stranded RNA (dsRNA) molecules trigger the degradation of homologous RNA targets. Small interfering RNAs (siRNAs) are the mediators of the RNAi that can be induced in vitro and in vivo by direct application of chemically synthesized siRNAs. Recently, promoter-targeted small non-coding RNAs have been described to be capable of regulating gene expression at the transcriptional level. In the present study we tested the hypothesis that Dicer-substrate siRNAs (D-siRNAs), which trigger gene silencing through intrinsic RNAi pathway and are therefore more potent in gene knockdown than traditionally used 21-23mer siRNAs, may also display regulatory effects at the transcriptional level. Synthetic 27mer D-siRNAs targeting the promoter/early coding region of the CDKN2A gene were designed and transfected into HeLa cells, and 48 h post-transfection the CDKN2A expression was analyzed in quantitative real-time PCR using the house-keeping G6PDH gene as reference. In comparison to the mutant version, the CDKN2A gene was effectively knocked-down by the D-siRNA, but no evidence of transcriptional regulation was found. We conclude that the D-siRNA-induced suppression is likely to occur after transcription.

Anahtar Kelimeler:

Key words: RNA interference, Dicer-substrate siRNAs, transcriptional silencing, CDKN2A

Testing the putative effect of Dicer-substrate siRNAs in regulating gene expression at transcriptional level

Keywords:

Key words: RNA interference, Dicer-substrate siRNAs, transcriptional silencing, CDKN2A,

PDF

___

Bernstein E, Caudy AA, Hammond SM et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 4: 363-366, 2001. 2. Morris KV, Chan SW, Jacobsen SE et al. Small interfering RNA- induced transcriptional gene silencing in human cells. Science 305: 1289-1292, 2004. 3. Morris KV. siRNA-mediated transcriptional gene silencing: the potential mechanism and a possible role in the histone code. Cell Mol Life Sci 62: 3057-3066, 2005.
Li LC, Okino ST, Zhao H et al. Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci USA 103: 17337-17342, 2006.
Janowski BA, Younger ST, Hardy DB et al. Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nat Chem Biol 3: 166-173, 2007.
Weinberg MS, Villeneuve LM, Ehsani A et al. Th e antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. RNA 12: 256-262, 2006.
Han J, Kim D, Morris KV. Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells. Proc Natl Acad Sci USA 104: 12422-12427, 2007.
Napoli S, Pastori C, Magistri M et al. Promoter-specifi c transcriptional interference and c-myc gene silencing by siRNAs in human cells. EMBO J 28: 1708-1719, 2009.
Morris KV, Santoso S, Turner AM et al. Bidirectional transcription directs both transcriptional gene activation and suppression in human cells. PLoS Genet. E4: e1000258, 2008.
Kim DH, Behlke MA, Rose SD et al. Synthetic dsRNA Dicer- substrates enhance RNAi potency and effi cacy. Nat Biotechnol 23: 226-226, 2005.
Rose SD, Kim DH, Amarzguioui M et al. Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res 33: 4140-4156, 2005.
Hefner E, Clark K, Whitman C et al. Increased potency and longevity of gene silencing using validated Dicer substrates. J Biomol Tech 19: 231-237, 2008.
Gonzalgo ML, Hayashida T, Bender CM et al. Th e role of DNA methylation in expression of the p19/p16 locus in human bladder cancer cell lines. Cancer Res 58: 1245-1252, 1998.
Deligezer U, Esin Akisik E, Dalay N. A novel application of melting curves: utility of peak area calculation for relative methylation quantifi cation. Clin Chem Lab Med 45: 867-873, 2007.
Deligezer U, Esin Akisik E, Erten N et al. Sequence-specifi c histone methylation is detectable on circulating nucleosomes. Clin Chem 54: 1125-1131, 2008.
Guillerm G, Gyan E, Wolowiec D et al. p16(INK4a) and p15(INK4b) gene methylations in plasma cells from monoclonal gammopathy of undetermined signifi cance. Blood 98: 244-246, 2001.
Wei Y, Xia W, Zhang Z et al. Loss of trimethylation at lysine 27 of histone H3 is a predictor of poor outcome in breast, ovarian, and pancreatic cancers. Mol Carcinogenesis 47: 701-706, 2008.