Regulation of microRNA biogenesis in stem cells

说明书(申请人提供)：microRNAs(MiRNAs)包括一组抑制靶信使RNA表达的调控RNA。MiRNAs缺失导致的早期胚胎死亡突出了它们对正常哺乳动物发育的要求，并且在癌症中观察到miRNA表达的改变。成熟的~22个核苷酸的miRNAs是由长的初级转录本通过微处理器和Dird复合体的顺序切割而产生的。MiRNA双链的一条被掺入RNA诱导沉默复合体(RISC)，并指导mRNA切割或翻译抑制。目前已鉴定出500多个miRNAs，其中许多以发育调节的方式表达。在大多数情况下，它们的表达控制发生在转录水平。然而，miRNA的加工也是可以调控的。某些miRNA前体在胚胎干细胞和原发肿瘤中高度表达，尽管相应的成熟物种无法检测到。这表明miRNA加工受阻，其机制仍然难以捉摸。我们的目标是了解在ES细胞中miRNA处理被调控的机制。我们最近发现，发育调节的RNA结合蛋白Lin28是miRNA加工的选择性抑制物。我们的 初步数据表明，Lin28是ES细胞中一个大的多亚基复合体的组成部分。为了获得 为了深入了解Lin28的功能机制，我们计划从ES细胞中分离Lin28(和Lin28b)蛋白复合体，并鉴定和功能表征复杂的成分。我们将使用各种方法来鉴定含有Lin28的核糖核蛋白复合体的RNA成分。一旦我们确认了特定多肽和RNA与Lin28的生化关联，我们将使用免疫定位和原位杂交分析来检查它们在ES细胞中的亚细胞定位。我们目前的数据表明，Lin28选择性地抑制特定miRNAs子集的处理，但这一调控途径的决定因素和机制仍不清楚。我们将进行详细的生化分析，包括体外重组实验和关键Lin28残基的突变，以从功能上确定重要的蛋白质结构域。我们试图了解加工抑制的RNA结构和序列决定因素，并将产生人工miRNA前体，并通过LIN28测量加工抑制。此外，我们计划从功能上表征Lin28复杂组件在调节ES细胞中miRNA加工过程中的作用。实现这一建议的目标将提供对干细胞如何在分子水平上进行调控的更好理解，并可能有助于新疗法的开发。 公共卫生评论：干细胞为开发抗击疾病的新方法带来了巨大的希望。然而，干细胞自我更新和分化的分子基础仍不完全清楚。这项拟议的工作将为胚胎干细胞基因调控提供新的见解，并可能导致操纵microRNAs的新疗法。这些研究与癌症、糖尿病、发育障碍和许多退行性疾病的治疗有关。

DESCRIPTION (provided by applicant): microRNAs (miRNAs) comprise a family of regulatory RNAs that repress the expression of target messenger RNAs. The early embryonic lethality caused by loss of miRNAs highlights their requirement for normal mammalian development, and altered miRNA expression is observed in cancer. Mature ~22 nucleotide miRNAs are produced from long primary transcripts through sequential cleavages by the Microprocessor and Dicer complexes. One strand of the miRNA duplex is incorporated into the RNA induced silencing complex (RISC) and directs mRNA cleavage or translational repression. Over 500 miRNAs have been identified, many of which are expressed in a developmentally regulated manner. Under most conditions, control of their expression occurs at the transcriptional level. However, it is emerging that miRNA processing can also be regulated. Certain miRNA precursors are highly expressed in embryonic stem (ES) cells, and primary tumors even though the corresponding mature species are undetectable. This suggests a block in miRNA-processing, the mechanism of which has remained elusive. Our goal is to understand the mechanism by which miRNA processing is regulated in ES cells. We recently identified the developmentally regulated RNA-binding protein Lin28 as a selective inhibitor of miRNA processing. Our preliminary data indicate that Lin28 is a component of a large multi-subunit complex in ES cells. To gain insight into the mechanism of Lin28 function we plan to isolate Lin28 (and Lin28B) protein complexes from ES cells and identify and functionally characterize complex components. We will employ a variety of approaches to identify the RNA component of Lin28-containing ribonucleoprotein complexes. Once we have confirmed the biochemical association of particular polypeptides and RNAs with Lin28 we will examine their subcellular localization in ES cells using both immunolocalization and in-situ hybridization analyses. Our current data indicate that Lin28 selectively inhibits processing of a particular subset of miRNAs, however the determinants and mechanism of this regulatory pathway remain unknown. We will perform detailed biochemical analyses including in vitro reconstitution experiments and mutagenesis of critical Lin28 residues to functionally define important protein domains. We seek to understand the RNA structural and sequence determinants of processing inhibition and will generate artificial miRNA precursors and measure processing inhibition by Lin28. Additionally, we plan to functionally characterize the role of Lin28 complex components in the regulation of miRNA processing in ES cells. Accomplishing the goals of this proposal will provide a better understanding of how stem cells are regulated at the molecular level and could aid in the development of novel therapeutics. PUBLIC HEALTH REVELANCE: Stem cells hold great promise for the development of new approaches to combat disease. However, the molecular basis for stem cell self-renewal and differentiation remains incompletely understood. The proposed work will provide novel insight into embryonic stem cell gene regulation, and may lead to new therapies to manipulate microRNAs. These studies are relevant to the treatment of cancer, diabetes, developmental disorders and numerous degenerative diseases.