Weinberg Marc S, Barichievy Samantha, Schaffer Lana, Han Jiang, Morris Kevin V
Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, 7 York Rd Parktown 2193, South Africa.
Nucleic Acids Res. 2007;35(21):7303-12. doi: 10.1093/nar/gkm847. Epub 2007 Oct 24.
Transcriptional gene silencing (TGS) can be achieved by small RNAs targeted to upstream promoter regions. Previously we characterized siRNAs targeted to the HIV-1 long terminal repeat (LTR) promoter at site 247, and found that a 21-base antisense strand of siRNA-247 (LTR-247as) suppressed LTR-mediated expression. To characterize the specificity of LTR-247as, vectors expressing antisense RNAs targeted to a region spanning 50 bases up- and downstream of the 247 target site were generated. LTR-247as+7, a approximately 22 base antisense RNA that is shifted by only seven bases upstream of LTR-247as, showed a significant increase in LTR-driven reporter gene expression that was independent of cell type and active chromatin methyl-marks. Promoter-targeting siRNAs have been recently shown to induce gene activation. However, here we demonstrate gene activation via a sequence-specific off-target effect. Microarray analysis of LTR-247as+7-treated cultures resulted in the deregulation of approximately 185 genes. A gene of unknown function, C10orf76, was responsive to inhibition by LTR-247as+7 and the loss of C10orf76 resulted in the upregulation of several genes that were activated by LTR-247as+7. These data suggest caution when using short antisense RNAs or siRNAs designed to target promoter sequences, since promoter-targeted RNAs may have unintended inhibitory effects against factors with suppressive gene activity.
转录基因沉默（TGS）可通过靶向上游启动子区域的小RNA来实现。此前我们对靶向HIV-1长末端重复序列（LTR）启动子247位点的小干扰RNA（siRNA）进行了表征，发现siRNA-247（LTR-247as）的21个碱基反义链可抑制LTR介导的表达。为了表征LTR-247as的特异性，构建了表达靶向247靶点上下游各50个碱基区域的反义RNA的载体。LTR-247as+7是一种约22个碱基的反义RNA，仅比LTR-247as的上游偏移7个碱基，它显示出LTR驱动的报告基因表达显著增加，且与细胞类型和活性染色质甲基化标记无关。最近研究表明，靶向启动子的siRNA可诱导基因激活。然而，在此我们证明基因激活是通过序列特异性脱靶效应实现的。对用LTR-247as+7处理的培养物进行微阵列分析，结果显示约185个基因失调。一个功能未知的基因C10orf76对LTR-247as+7的抑制有反应，C10orf76的缺失导致几个被LTR-247as+7激活的基因上调。这些数据表明，在使用设计用于靶向启动子序列的短反义RNA或siRNA时需谨慎，因为靶向启动子的RNA可能对具有抑制基因活性的因子产生意外的抑制作用。
