Small Regulatory RNA Functions In The Nucleus

描述（由申请人提供）：在大多数真核生物中，小调控rna调节基因表达。通过调节基因表达，小调控rna在许多生物过程中发挥关键作用，包括发育、基因组防御、肿瘤发生和抗病毒免疫。小调控rna通过在细胞中寻找并结合同源（靶）rna来起作用。通过向靶rna招募辅助蛋白，小rna能够在许多水平上控制基因表达，包括；翻译，mRNA稳定性和转录。小RNA生物学的机制基础在大多数真核生物中广泛保守。特别是，小rna在调节大多数真核生物细胞核内的基因表达方面发挥着重要作用。我的实验室已经建立了系统，使我们能够研究小rna如何以及为什么调节动物细胞核中的基因。我们正在使用模式生物秀丽隐杆线虫来了解小rna如何调节动物细胞核中的基因表达。我们之所以使用秀丽隐杆线虫来解决这个问题，是因为现有的优秀遗传工具，以及在这个系统中进行RNAi实验的强大和方便的性质。在秀丽隐杆线虫中使用遗传方法，我们已经确定了一种分子途径，该途径使用小rna识别和标记新生转录本（以及编码这些转录本的基因）以使其沉默。我们已经确定了辅助蛋白（称为核RNAi缺陷（NRDE）因子），它们被小rna招募到RNAP聚合酶II产生的新生转录物中。最后，我们已经证明NRDE因子与RNA转录物的关联允许NRDE因子在转录的延伸阶段抑制RNA聚合酶II。我们已经确定的一些NRDE因子在哺乳动物中是保守的。总之，我们的工作正在帮助我们了解小rna如何调节动物细胞核中的基因表达，并可能导致对小rna如何调节哺乳动物基因表达的见解。我们也有兴趣了解为什么小rna调节动物细胞核中的基因表达。在大多数真核细胞中，小的调控rna指导DNA和组蛋白的共价修饰。这些小rna介导的染色质修饰本质上是表观遗传的：它们改变基因表达而不改变潜在的DNA序列。我们已经证明，内源性核小rna和核RNAi途径在正常繁殖过程中调控了约1000个基因的表观遗传景观。在缺乏核RNAi机制的动物体内，生殖细胞失去了不朽的特性。因此，秀丽隐杆线虫在正常繁殖过程中使用内源性小rna调节许多基因的表观遗传“状态”，这种基因沉默过程是介导重要生物学过程所必需的。许多其他的生物过程，如发育、印记、x染色体失活和参数化，都是由DNA和组蛋白上的表观遗传修饰指导的。有趣的是，非编码rna也参与了这些过程中的许多，如果不是全部的话。鉴于小rna、非编码rna和真核生物表观遗传过程之间存在广泛的联系，我们相信，我们探索小非编码rna如何调节秀丽隐杆线虫表观遗传景观的研究可能被证明是全球适用于动物的各种表观遗传过程。我们尚不清楚1)NRDE因子在pre-mRNA上的募集如何抑制RNAP II的延伸以指导核RNAi， 2) RNAi引导的染色质修饰如何促进动物核RNAi， 3)核RNAi是否/如何被调节，或4)NRDE核RNAi途径在哺乳动物中是否在功能上保守。我们提出的实验就是为了回答这些问题。

DESCRIPTION (provided by applicant): Small regulatory RNAs regulate gene expression in most eukaryotes. By regulating gene expression, small regulatory RNAs, play key roles in many biological processes that include development, genome defense, oncogenesis, and antiviral immunity. Small regulatory RNAs act by seeking out and binding homologous (target) RNAs in cells. By recruiting accessory proteins to target RNAs, small RNAs are able to control gene expression at many levels that include; translation, mRNA stability, and transcription. The mechanistic underpinnings of small RNA biology are widely conserved in most eukaryotes. In particular, small RNAs play an important role in regulating gene expression within most eukaryote nuclei. My lab has established systems that are allowing us to study how and why small RNAs regulate genes in animal nuclei. We are using the model organism C. elegans to understand how small RNAs regulate gene expression in animal nuclei. We are using C. elegans to address this question because of the excellent genetic tools that are available, and because of the robust and facile nature of conducting RNAi experiments in this system. Using genetic approaches in C. elegans, we have identified a molecular pathway that uses small RNAs to recognize and mark nascent transcripts (and the genes that encode these transcripts) for silencing. We have identified accessory proteins (termed the nuclear RNAi defective (NRDE) factors), which are recruited by small RNAs to nascent transcripts emanating from RNAP Polymerase II. Finally, we have shown that the association of the NRDE factors with RNA transcripts allows that NRDE factors to inhibit RNA Polymerase II during the elongation phase of transcription. Some of the NRDE factors that we have identified are conserved in mammals. In summary, our work is helping us understand how small RNAs regulate gene expression in animal nuclei, and may lead to insights into how small RNAs regulate gene expression in mammals. We are also interested in understanding why small RNAs regulate gene expression in animal nuclei. Small regulatory RNAs direct the covalent modification of DNA and histones proteins in most eukaryotic cells. These small RNA-mediated chromatin modifications are epigenetic in nature: they alter gene expression without changing the underlying in DNA sequence. We have shown that endogenous nuclear small RNAs, and the nuclear RNAi pathway, regulate the epigenetic landscape at ~1000 genes during the normal course of reproduction. In animals that lack the nuclear RNAi machinery, germ cells loose their immortal character. Thus, C. elegans uses endogenous small RNAs to regulate epigenetic "states" at many genes during the normal course of reproduction and this gene-silencing process is required to mediate important biological processes. Many other biological processes such as development, imprinting, X-chromosome inactivation, and paramutation are directed by epigenetic modifications on DNA and histones. Interestingly, non-coding RNAs also contribute to many, if not all, of these processes. Given the widespread connections that exist between small RNAs, non-coding RNAs, and epigenetic processes in eukaryotes, we believe that our research exploring how small non-coding RNAs regulate epigenetic landscapes in C. elegans may prove to be globally applicable to diverse epigenetic processes in animals. We do not yet understand 1) how the recruitment of NRDE factors to pre-mRNA inhibits RNAP II elongation to direct nuclear RNAi, 2) how RNAi-guided chromatin modifications contribute to nuclear RNAi in animals, 3) if/how nuclear RNAi is regulated, or 4) if the NRDE nuclear RNAi pathway is functionally conserved in mammals. Our proposed experiments are designed to answer these questions. .