Biological functions and post-transcriptional regulation of microRNAs

miRNA是小的非编码RNA，其被加载到Argonaute蛋白中以形成miRNA诱导的沉默复合物（miRISC）的核心。miRNAs将miRISC引导至互补靶mRNA以沉默其表达。miRNA基因座中的突变破坏基因表达程序，因此可能导致包括癌症在内的各种疾病的发展。因此，了解miRNAs在正常发育中的功能和调控miRNAs的分子机制是至关重要的生物学问题。 了解miRNAs在胚胎发生中的生物学功能 虽然miRNAs在分化组织中的功能在C.在线虫和其他生物中，只有少数动物miRNA的胚胎功能被理解。此外，miRNAs如何在母合子转换（MZT）之前对基因表达的转录后调控做出贡献是完全未知的。C.由于其明确的定型细胞谱系和强大的遗传工具，线虫是研究胚胎发育的极好模式生物。我们的团队使用的是C语言。elegans阐明了miRNAs在胚胎发生中的功能，首先关注胚胎发生所需的miRNAs家族。 为了剖析这些miRNAs控制的生物学途径，我们的研究小组正在进行正向（诱变）和反向（RNAi）筛选microRNA家族突变表型的抑制因子。了解胚胎表达的microRNA家族影响的生物网络将对如何控制基因表达以协调胚胎发生产生重要的见解。 定义miRNA和Argonaute周转的分子机制 miRNA生物合成和衰变速率的平衡控制着miRNA的丰度，从而控制着基因表达程序。先前的研究已经仔细阐明了miRNA生物合成的机制。然而，我们对miRNAs和miRISC是如何组成性地或以受调控的方式转变的知之甚少。这是我们对miRNA调控的理解中的一个主要空白，因此也是基因表达调控的一个主要空白。我们正在使用深度测序来评估候选参与者在microRNA周转中的贡献，以及确定miRNA周转的新型效应子和调节子。 一个主要的未回答的问题是，miRNA及其蛋白质结合伴侣Argonaute是否一起被靶向降解，或者可以单独翻转。通过测量miRNA周转动力学的变化对Argonaute周转动力学的影响，反之亦然，我们正在研究这些事件是如何相互关联的。从长远来看，我们将因此在miRISC周转过程中建立一个事件层次。 了解miRNAs通过周转的调节可能为治疗人类疾病的新模式奠定基础。这一研究领域可能会影响癌症的治疗，因为已知miRNA在许多癌症类型中全局下调，并且miRNA生物合成酶可以作为单倍不足的肿瘤抑制因子。这些数据表明，miRNA丰度的适度增加可以逆转miRNA对癌症的贡献。因此，抑制miRNA周转是一种有吸引力的治疗策略。由于酶可能在这一过程中发挥作用，因此miRNA周转的小分子抑制剂作为靶向癌症治疗是可行的。 本财政期主要侧重于建立开展这项研究计划的基础设施。启动资金使我们能够为实验室配备C所需的所有设备。线虫遗传学、细胞生物学和分子生物学。我们还开始建立microRNA周转率和Argonaute周转率的测定方法，并建立microRNA丰度的前向筛选范式。

MiRNAs are small noncoding RNAs that are loaded into Argonaute proteins to form the core of the miRNA-Induced Silencing Complex (miRISC). MiRNAs guide miRISC to complementary target mRNAs to silence their expression. Mutations in miRNA loci disrupt gene expression programs, and thus can contribute to the development of various diseases, including cancer. Consequently, understanding both the functions of miRNAs in normal development and the molecular mechanisms that regulate miRNAs are biological questions of critical importance. Understanding the biological functions of miRNAs during embryogenesis While the functions of miRNAs in differentiated tissues are well-studied in C. elegans and other organisms, the embryonic functions of only a few animal miRNAs are understood. Moreover, how miRNAs contribute to the largely post-transcriptional control of gene expression prior to the maternal-to-zygotic transition (MZT) is completely unknown. C. elegans is an excellent model organism in which to study embryonic development due to its well-defined stereotypic cell lineage and powerful genetic tools. Our group is using use C. elegans to elucidate the functions of miRNAs in embryogenesis, first focusing on the miRNA families that are required for embryogenesis. To dissect the biological pathways controlled by these miRNAs, our group is conducting forward (mutagenesis) and reverse (RNAi) screens for suppressors of microRNA family mutant phenotypes. Understanding the biological networks impacted by the embryonically-expressed microRNA families will yield important insights into how gene expression is controlled to coordinate embryogenesis. Defining the molecular mechanisms of miRNA and Argonaute turnover The balance of the rates of miRNA biogenesis and decay control miRNA abundance, and thus gene expression programs. Previous research has carefully elucidated mechanisms of miRNA biogenesis. However, we know very little about how miRNAs and miRISC are turned over either constitutively or in a regulated manner. This is a major gap in our understanding of miRNA regulation, and thus the regulation of gene expression. We are using deep sequencing to assess the contribution of candidate players in microRNA turnover, as well as identify novel effectors and regulators of miRNA turnover. A major unanswered question is whether the miRNA and its protein binding partner Argonaute are targeted for degradation together, or can instead be turned over separately. By measuring the effect of changes in miRNA turnover kinetics on Argonaute turnover kinetics, and vice versa, we are investigating how these events are interconnected. In the long term, we will thus establish a hierarchy of events in the process of miRISC turnover. Understanding the regulation of miRNAs by turnover may lay the foundation for new modalities to treat human disease. This area of study could impact the treatment of cancer, since miRNAs are known to be globally downregulated in many cancer types, and miRNA biogenesis enzymes can act as haploinsufficient tumor suppressors. These data suggest that a modest increase in miRNA abundance could reverse the contribution of miRNAs to cancer. Thus, inhibition of miRNA turnover is an attractive therapeutic strategy. Since enzymes are likely to play a part in this process, small molecules inhibitors of miRNA turnover could be feasible as a targeted cancer therapy. This fiscal period has primarily focused on establishing the infrastructure for carrying out this research program. Start-up funds have allowed us to outfit the lab space with all equipment necessary for C. elegans genetics, cell biology, and molecular biology. We have also begun to establish assays for microRNA turnover and Argonaute turnover rates, and to establish forward screening paradigms for microRNA abundance.