Biogenesis and function of the small temporal RNA let-7

小颞RNA let-7 的生物发生和功能

• 批准号: 6691716
• 负责人: PHILLIP D ZAMORE
• 金额: $ 31.01万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6691716
• 关键词: Biogenesis; function; small; temporal; RNA

DESCRIPTION (provided by applicant): Two related classes of 21-23 nt RNAs -- small interfering RNAs (siRNAs) and small temporal RNAs (stRNAs) -- provide nucleic acid specificity determinants for the post-transcriptional control of mRNA expression, siRNAs are double-stranded and act in the RNA interference (RNAi) pathway to target mRNAs for endonucleolytic cleavage, silencing their expression by triggering mRNA destruction. In contrast, stRNAs are single-stranded and are thought to regulate mRNA translation without altering mRNA stability. Current evidence suggests that despite their different modes of target gene regulation, the RNAi and stRNA pathways are remarkably similar. In fact, the multi-domain RNase III enzyme, Dicer, is required to generate both siRNAs and stRNAs. Dicer cleaves long, double-stranded RNA to generate siRNAs that mediate RNAi, whereas it acts on small (ca. 70 nt) stem-loop precursor RNAs to produce stRNAs. How Dicer generates stRNAs and why these stRNAs mediate translational control rather than mRNA degradation via the RNAi pathway remains undiscovered. Furthermore, the absence of an in vitro system that recapitulates translational control by stRNAs has hamstrung efforts to understand the biochemical mechanism by which they regulate gene expression. The experiments proposed here seek to define the biochemical mechanism by which stRNAs are generated, to determine what differentiates the siRNA and stRNA fates, and to test specific hypotheses that seek to explain how an stRNA bound to a 3' UTR sequence represses mRNA translation. Specifically, experiments are proposed to (1) identify the proteins required for the production of the stRNA let-7 from its 72 nt precursor stem-loop RNA; (2) to determine the sequence or structural features of pre-let-7 RNA required for the asymmetric production of mature let-7; (3) to determine why stRNAs do not trigger cleavage of their target mRNAs; (4) to re-engineer pre-stRNAs to generate functional siRNAs instead of stRNAs; and (5) to investigate the mechanism by which stRNAs regulate expression of their mRNA targets.

DESCRIPTION (provided by applicant): Two related classes of 21-23 nt RNAs -- small interfering RNAs (siRNAs) and small temporal RNAs (stRNAs) -- provide nucleic acid specificity determinants for the post-transcriptional control of mRNA expression, siRNAs are double-stranded and act in the RNA interference (RNAi) pathway to target mRNAs for endonucleolytic cleavage, silencing their expression by触发mRNA破坏。相反，strnas是单链，被认为可以调节mRNA翻译而不改变mRNA稳定性。当前的证据表明，尽管其靶基因调节模式不同，但RNAi和StRNA途径非常相似。实际上，需要多域RNase III酶（dicer）同时产生siRNA和strNA。 DICER切开长，双链RNA以产生介导RNAi的siRNA，而其作用于小（大约70 nt）茎环前体RNA产生strnas。 DICER如何产生strnas，以及为什么这些StrNA通过RNAi途径介导转化控制而不是mRNA降解。此外，缺乏通过Strnas概括翻译控制的体外系统陷入了努力，以理解它们调节基因表达的生化机制。此处提出的实验试图定义生成StrNA的生化机制，以确定siRNA和StRNA命运的区别是什么，并测试特定的假设，这些假设试图解释与3'UTR序列结合的strNA如何抑制mRNA的抑制。具体而言，提出实验以（1）从其72 nt前体的茎环RNA中识别出StRNA Let-7所需的蛋白质； （2）确定成熟let-7的不对称产生所需的前7个RNA的序列或结构特征； （3）确定为什么strnas不会触发其靶标mRNA的切割； （4）重新设计器预先刺激以产生功能性siRNA而不是strnas； （5）研究StrNA调节其mRNA靶标的表达的机制。