Post-transcriptional gene regulation

 描述(申请人提供)：大部分真核基因调控发生在转录后，通过不同的mRNA稳定性和/或翻译效率。这项建议的研究试图回答转录后基因控制的三个相互关联的领域中的基本问题：microRNAs、RNA干扰和信使核糖核酸聚(A)尾。MicroRNAs(MiRNAs)是~22-NT的RNA，与mRNAs配对，指导其失稳和翻译抑制。已在人类中发现了600多个miRNA基因，由于大多数人类基因是miRNAs的保守靶标，因此miRNAs在哺乳动物发育和人类疾病(包括病毒感染和癌症)中发挥重要作用也就不足为奇了。将使用分子、计算和结构方法来确定1)microRNA生物发生机制如何识别要加工成microRNA的细胞转录本，2)miRNA靶识别的生化基础和预测最受抑制靶的改进方法，3)报告基因的mRNAs与内源基因的mRNAs被不同地抑制的原因，以及4)长的非编码RNA调节miRNA的机制和生物学功能。这些研究的结果有望加强对这类重要的基因调控分子的基本了解，并为许多生物学家提供有用的资源，包括那些研究miRNAs在人类疾病中的作用的生物学家。RNA干扰(RNAi)是许多真核生物用来沉默转座子和病毒的一种基因调控途径。在这个途径中，类似于miRNAs的短干扰RNA(SiRNAs)被装载到ArgAerte中，ArgAerte是一种效应蛋白，可以切割与siRNA具有广泛互补性的转录产物。将使用遗传、结构、生化和分子方法1)鉴定和研究酵母中有效的RNAi所需的额外蛋白质；2)确定siRNA-ArgAerte复合体的形成方式以及它如何识别信使核糖核酸靶标；3)研究斑马鱼ArgAerte的异常活动。这些结果有望为保护许多真核生物免受转座子和病毒攻击的基础的基因沉默途径提供机械性的见解，并对生物学家利用这一途径研究基因功能以及利用它治疗病人具有实际意义。MRNA聚(A)尾对于mRNA的稳定性和翻译效率是重要的，而后生动物miRNAs通常通过招募酶来缩短聚(A)尾。Poly(A)-尾长和翻译效率的关系随着胚胎的发育而变化。分子、计算、生化和遗传方法将被用来确定尾长和翻译效率之间的耦合是如何在原肠形成之前建立的，以及为什么它在原肠形成后消失。结果是 预计将提供对翻译控制和胚胎发育的基本洞察，对人类生育、发育缺陷或其他疾病具有潜在影响。OMB编号0925-0001/0002(08/12版批准至2015年8月31日)页面续格式页面

 DESCRIPTION (provided by applicant): Much of eukaryotic gene regulation occurs post-transcriptionally, through differential mRNA stability and/or translational efficiency. The researc of this proposal seeks to answer fundamental questions within three interrelated areas of post-transcriptional gene control: microRNAs, RNA interference, and mRNA poly (A) tails. MicroRNAs (miRNAs) are ~22-nt RNAs that pair to mRNAs to direct their destabilization and translational repression. More than 600 miRNA genes have been identified in humans, and because most human genes are conserved targets of miRNAs, it is no surprise that miRNAs play important roles in mammalian development and human diseases, including viral infections and cancers. Molecular, computational, and structural approaches will be used to determine 1) how the microRNA-biogenesis machinery recognizes the cellular transcripts that are to be processed into microRNAs, 2) the biochemical basis of miRNA-target recognition and improved methods for predicting the most repressed targets, 3) the reason that mRNAs from reporter genes are repressed differently than those from endogenous genes, and 4) the mechanism and the biological function of the regulation of a miRNA by a long noncoding RNA. Results of these studies are expected to enhance the fundamental understanding of this important class of gene-regulatory molecules and provide resources helpful for many biologists, including those studying the roles of miRNAs in human diseases. RNA interference (RNAi) is a gene-regulatory pathway that many eukaryotic species use to silence transposons and viruses. In this pathway, short interfering RNAs (siRNAs) resembling miRNAs are loaded into Argonaute, which is an effector protein that cleaves transcripts with extensive complementarity to the siRNA. Genetic, structural, biochemical, and molecular approaches will be used to 1) identify and study additional proteins required for efficient RNAi in yeast, 2) determine how the siRNA-Argonaute complex forms and how it recognizes mRNA targets, and 3) investigate the unusual activities of zebrafish Argonaute. Results are expected to provide mechanistic insight into this gene-silencing pathway fundamental for defending many eukaryotic species against transposons and viruses, with practical implications for biologists using this pathway to study gene function, as well as those harnessing it to treat patients. mRNA poly (A) tails are important for mRNA stability and translational efficiency, and metazoan miRNAs usually act by recruiting enzymes that shortening poly (A) tails. The relationship between poly (A)-tail length and translational efficiency changes as the embryo develops. Molecular, computational, biochemical, and genetic approaches will be used to determine how coupling between tail length and translational efficiency is established before gastrulation and why it disappears after gastrulation. Results are expected to provide fundamental insight into translational control and embryonic development, with potential implications for human fertility, developmental defects, or other diseases. OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015) Page Continuation Format Page