Maturation of let-7 miRNAs

MicroRNAs(MiRNAs)作为RNA诱导沉默复合体(RISC)的一部分，通过序列互补识别信使RNAs(MRNAs)，在基因表达调控中发挥重要作用。通常，通过形成miRNA引导的RISC来靶向mRNA会导致mRNA的翻译停滞和降解。在人类中，基因组编码大约2,000个miRNAs，有可能调节超过60%的mRNAs。MiRNA水平的错误调控可以改变基因表达模式，这些变化与发育缺陷和几种人类疾病直接相关。Let-7家族的miRNAs在动物中高度保守，在许多生物过程中发挥着重要的作用。鉴于let-7 miRNAs作为发育调节因子和肿瘤抑制因子的关键作用，它们的生物发生受到严格调控。这从RNA聚合酶II转录初级miRNA(pri-miRNA)开始，经历了两个切割步骤，首先由DROSHA/DgCr8(微处理器)复合体产生前体miRNA(前miRNA)，然后由Disher产生miRNA双链。最近的一些研究导致了普遍的观点，即许多转录后机制调节miRNA的成熟，这允许miRNAs特定的时间和空间表达。此外，已确定了几种针对let-7miRNAs未成熟形式(pri-let-7和prelet-7)来控制其成熟的蛋白质，包括TRBP、Lin28(Lin28a和Lin28b)、Musashi1、TUT4、hnRNPA1和KHSRP蛋白。然而，关于这些相互作用的机制细节以及它们如何调控miRNA成熟仍有许多需要了解的地方。我们研究计划的长期目标是详细了解调节let-7miRNA成熟的具体机制。我们的工作假设是，miRNA的成熟需要蛋白质/蛋白质和蛋白质/RNA相互作用的复杂相互作用，这些相互作用针对Pri-和Pre-miRNAs，以刺激或抑制两种主要的miRNA加工酶DROSHA和DICER的活性。我们目前的重点是已知在调节let-7miRNA水平中发挥关键功能的蛋白质子集。在接下来的五年里，我们将在分子和原子水平上表征这些蛋白质调控let-7 miRNAs成熟的机制细节。我们的工作将涉及选定的蛋白质/蛋白质和蛋白质/RNA复合体的生化、生物物理和结构表征，这些复合体调节miRNA成熟的Dird和/或DROSHA裂解步骤。我们的研究将为我们理解miRNA成熟和如何控制miRNA水平提供关键和及时的见解。此外，鉴于miRNAs在医疗和农业应用方面具有巨大的潜力，我们的基础研究将提供必要的关键信息，以帮助评估和开发这些新的尖端应用。

MicroRNAs (miRNAs) play important roles in the regulation of gene expression by recognizing messenger RNAs (mRNA) through sequence complementarity as part of the RNA-induced silencing complex (RISC). Typically, targeting an mRNA by formation of the miRNA-guided RISC leads to translational arrest and degradation of the mRNA. In humans, the genome encodes around 2,000 miRNAs, which have the potential to regulate over 60% of all mRNAs. Misregulation of miRNA levels can change gene expression patterns, and these changes have been directly linked to developmental defects and several human diseases. The miRNAs of the let-7 family are highly conserved across animal species and play several important roles in many biological processes. Given the key role of let-7 miRNAs as development regulators and tumor suppressors, their biogenesis is tightly regulated. This starts with transcription by RNA polymerase II of a primary miRNA (pri-miRNA), which undergoes two cleavage steps, first by the Drosha/DGCR8 (Microprocessor) complex to yield the precursor miRNA (pre-miRNA) and then by Dicer to yield a miRNA duplex. A number of recent studies have led to the general view that numerous post-transcriptional mechanisms regulate miRNA maturation and that this allows for specific temporal and spatial expression of miRNAs. Moreover, several proteins have been identified that target immature forms of let-7 miRNAs (pri-let-7 and pre-let-7) to control their maturation, including TRBP, Lin28 (Lin28A and Lin28B), Musashi1, TUT4, hnRNPA1 and KHSRP proteins. However, much remains to be known about the mechanistic details of these interactions and how they regulate miRNA maturation. The long-term objective of our research program is to gain a detailed understanding of specific mechanisms that regulate let-7 miRNA maturation. Our working hypothesis is that miRNA maturation requires a complex interplay of protein/protein and protein/RNA interactions that target the pri- and pre-miRNAs to either stimulate or repress the activity of the two main miRNA processing enzymes, Drosha and Dicer. Our current focus is on a subset of proteins known to play critical functions in the regulation of let-7 miRNA levels. In the next five years, we will characterize the mechanistic details by which these proteins regulate the maturation of the let-7 miRNAs at both the molecular and atomic levels. Our work will involve biochemical, biophysical and structural characterization of select protein/protein and protein/RNA complexes that regulate the Dicer and/or Drosha cleavage steps of miRNA maturation. Our studies will provide critical and timely insights into our understanding of miRNA maturation and how miRNA levels are controlled. In addition, given that miRNAs have enormous potential for medical and agricultural applications, our basic research will provide crucial information needed to help evaluate and exploit these new cutting-edge applications.