Biogenesis and function of the small temporal RNA let-7

描述(由申请人提供)：两类相关的21-23个核苷酸RNAs--小干扰RNAs(SiRNAs)和小临时RNAs(StRNAs)--为mRNA表达的转录后控制提供核酸特异性决定因素，siRNAs是双链的，并在RNA干扰(RNAi)途径中作用于靶向mRNAs进行内核切割，通过触发mRNAs破坏来沉默其表达。相反，stRNAs是单链的，被认为在不改变mRNA稳定性的情况下调节mRNA的翻译。目前的证据表明，尽管RNAi和stRNA的靶基因调控模式不同，但它们的途径非常相似。事实上，多结构域RNaseIII酶DICER是产生siRNAs和stRNAs所必需的。Dier裂解长的双链RNA以产生介导RNAi的siRNAs，而它作用于小的(约70nT)茎环前体RNA来产生stRNAs。Dier是如何产生stRNAs的，以及为什么这些stRNAs通过RNAi途径介导翻译控制而不是mRNA降解，这一点尚不清楚。此外，缺乏一个概括stRNAs翻译控制的体外系统，阻碍了理解stRNAs调控基因表达的生化机制的努力。这里提出的实验试图定义stRNA产生的生化机制，确定是什么区别了siRNA和stRNA的命运，并检验了试图解释与3‘UTR序列结合的stRNA如何抑制mRNA翻译的特定假设。具体地说，建议进行以下实验：(1)从其72个核苷酸的前体茎环RNA中鉴定生产stRNA let-7所需的蛋白质；(2)确定不对称生产成熟let-7所需的prelet-7 RNA的序列或结构特征；(3)确定为什么stRNAs不会触发其目标mRNAs的切割；(4)重新设计prestRNAs以产生功能强大的siRNAs而不是stRNAs；以及(5)研究stRNAs调节其信使核糖核酸靶标表达的机制。

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.