Recognition of Chromosomal DNA by Double-Stranded RNA

描述(由申请人提供)：此竞争修订正在提交通知编号(NOT-OD-09-058)和通知标题：NIH宣布恢复法案资金可用于竞争修订申请。背景资料。短干扰RNA(SiRNAs)识别mRNA的能力得到了广泛的重视。SiRNA正在进入第一阶段试验，但它们最终影响人类健康的潜力尚不清楚。家长提案GM 77253的总体目标是研究与基因启动子互补的双链RNA的作用。通用汽车77253有四个目标： I.评估ArgAerte蛋白在RNA介导的基因激活中的作用 二、表征agRNAs转录沉默的机制 三、定义针对基因启动子的规则 四、识别抑制转录的内源性miRNAs 显著扩大研究范围的理由。最近的转录组研究发现，许多非编码转录本与基因的3‘端重叠。这些转录本的功能尚不清楚，3‘-UTR下游的调控潜力也几乎没有引起人们的注意。在追求AIM II的同时，我们观察到启动子靶向的RNA与我们的靶基因孕酮受体5‘端重叠的非编码转录本结合。我们假设，靶序列超过基因3‘末端的RNA也可能能够结合非编码转录本并调节基因表达。我们鉴定了孕激素受体(PR)基因的转录，并鉴定出与该基因3‘端重叠的非编码转录本。与非编码转录物互补的小RNA抑制T47D细胞(PR高表达细胞系)的PR转录，并激活MCF7细胞(PR低表达细胞系)的PR转录。RNAs将ArgAerte招募到3‘-非编码转录本上，改变RNA聚合酶的水平，调节PR Pre-mRNA和成熟mRNA的转录。与BRCA1 3‘mRNA末端以外的序列互补的RNA也抑制基因的表达。我们的结果将RNA介导的基因调控的可能性扩展到了基因3‘端的下游区域。我们建议对3‘-靶向RNA的作用机制进行表征，以了解它们如何在与远离基因启动子的mRNA和靶序列互补的情况下调节转录。 公共卫生相关性：基因表达调控是生物过程的基础。我们的研究调查了位于基因末端之外的染色体DNA区域。这些区域几乎没有受到关注，我们将调查基因调控是否可以延伸到这些区域。

DESCRIPTION (provided by applicant): This competitive revision is being submitted for Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availablity of Recovery Act Funds for Competitive Revision Applications. Background. The ability of short interfering RNAs (siRNAs) to recognize mRNA is widely appreciated. siRNAs are entering Phase 1 trials, but their ultimate potential to impact human health is unclear. The overall goal of the parent proposal GM 77253 was to investigate the action of duplex RNAs that are complementary to gene promoters. GM 77253 had four Aims: i. Evaluate involvement of argonaute proteins in RNA-mediated gene activation ii. Characterize the mechanism of transcriptional silencing by agRNAs iii. Define rules for targeting gene promoters iv. Identify endogenous miRNAs that inhibit transcription Rationale for a significant expansion of research scope. Recent transcriptome studies have revealed many noncoding transcripts overlapping the 3' terminus of genes. The function of these transcripts is unknown and the potential for regulation downstream from the 3'-UTR has attracted little attention. While pursuing Aim ii, we observed that promoter-targeted RNAs were binding to a noncoding transcript that overlaps the 5' terminus of our target gene progesterone receptor. We hypothesized that RNAs that target sequences past the 3'-terminus of genes might also be able to bind noncoding transcripts and modulate gene expression. We characterized transcription at the progesterone receptor (PR) locus and identified noncoding transcripts that overlap the 3' end of the gene. Small RNAs complementary to a noncoding transcript inhibit PR transcription in T47D cells (a cell line with high PR expression) and activate PR transcription in MCF7 cells (a cell line with low PR expression). The RNAs recruit argonaute to the 3'- noncoding transcript, alter levels of RNA polymerase, and modulate transcription of PR pre-mRNA and mature mRNA. RNAs complementary to sequences beyond the 3' mRNA terminus of BRCA1 also inhibit gene expression. Our results extend the potential for RNA-mediated gene regulation to regions downstream from the 3' end of genes. We propose to characterize the mechanism of action of 3'-targeted RNAs to understand how they can modulate transcription even though they are not complementary to mRNA and target sequences that are far distant from gene promoters. PUBLIC HEALTH RELEVANCE: Regulation of gene expression is fundamental to biological processes. Our research investigates regions of chromosomal DNA that lie beyond the termini of genes. These regions have received little attention and we will investigate whether gene regulation can extend to these regions.