Small Regulatory RNA Functions In The Nucleus

描述(申请人提供)：在大多数真核生物中，小的调控RNA调节基因表达。通过调节基因表达，小调节RNA在许多生物学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。小的调控RNA通过寻找和结合细胞中的同源(靶标)RNA来发挥作用。通过招募辅助蛋白来靶向RNA，小RNA能够在许多水平上控制基因的表达，包括翻译、mRNA稳定性和转录。小RNA生物学的机械基础在大多数真核生物中广泛保守。特别是，小RNA在调控大多数真核细胞的基因表达方面起着重要的作用。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线虫来了解小RNA如何调节动物细胞核中的基因表达。我们使用线虫来解决这个问题，因为有优秀的遗传工具可用，也因为在这个系统中进行RNAi实验的健壮和方便的性质。通过在线虫中使用遗传学方法，我们已经确定了一种分子途径，它使用小RNA来识别和标记新生转录产物(以及编码这些转录产物的基因)以供沉默。我们已经鉴定了辅助蛋白(称为核RNAi缺陷(NRDE)因子)，它们被小RNA招募到RNAP聚合酶II产生的新生转录本中。最后，我们证明了NRDE因子与RNA转录本的联系允许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。综上所述，我们的工作正在帮助我们理解小RNA如何调控动物细胞核中的基因表达，并可能导致对小RNA如何调控哺乳动物基因表达的深入了解。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。这些小的RNA介导的染色质修饰本质上是表观遗传的：它们改变了基因的表达，而不改变DNA序列中的潜在基因。我们已经证明，在正常的生殖过程中，内源性核小RNA和核RNAi途径在~1000个基因上调节表观遗传格局。在缺乏核RNAi机制的动物中，生殖细胞失去了不朽的特征。因此，线虫在正常的生殖过程中使用内源小RNA来调节许多基因的表观遗传状态，而这种基因沉默过程是调节重要生物过程所必需的。许多其他生物学过程，如发育、印记、X染色体失活和参变，都是由DNA和组蛋白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。鉴于真核生物中小RNA、非编码RNA和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如何调控线虫表观遗传景观的研究可能被证明在全球范围内适用于动物的不同表观遗传过程。我们尚不清楚1)NRDE因子招募到前mRNA是如何抑制RNAP II延伸以指导核RNAi的，2)RNAi引导的染色质修饰如何有助于动物的核RNAi，3)核RNAi是否/如何被调控，或4)NRDE核RNAi途径在哺乳动物中是否功能保守。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. .