Biological functions and post-transcriptional regulation of microRNAs

microRNA的生物学功能和转录后调控

• 批准号: 9554532
• 负责人: Katherine McJunkin
• 金额: $ 134.98万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9554532
• 关键词: Address; Animal Model; Animals; Area; Behavior; Binding Proteins; Biochemical; Biogenesis; Biological; Biological Assay; Biological Process; Biology; CRISPR/Cas technology; Caenorhabditis elegans; Cell Lineage; Cellular biology; Complex; Data; Development; Disease; Embryo; Embryonic Development; Enzymes; Equilibrium; Equipment; Event; Exhibits; Family; Foundations; Funding; Gene Expression; Gene Expression Regulation; Genetic; Kinetics; Malignant Neoplasms; Measures; Mediating; Messenger RNA; MicroRNAs; Modality; Molecular; Molecular Biology; Mutagenesis; Mutation; Organism; Pathway interactions; Phenotype; Physiological Processes; Plants; Play; Post-Transcriptional Regulation; Process; Proteins; RNA interference screen; Regulation; Research; Research Infrastructure; Role; Techniques; Therapeutic; Tissue Differentiation; Tumor Suppressor Proteins; Untranslated RNA; cancer therapy; cancer type; deep sequencing; forward genetics; genome editing; human disease; insight; mutant; next generation sequencing; novel; programs; screening; small molecule inhibitor; targeted cancer therapy; tool

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.

MiRNAs是一种小的非编码RNA，它被装载到ArgAerte蛋白中，形成miRNA诱导的沉默复合体(MiRISC)的核心。MiRNAs引导miRISC与互补的靶mRNAs结合，使其表达沉默。MiRNA基因座的突变扰乱了基因表达程序，从而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节miRNAs的分子机制都是至关重要的生物学问题。 了解miRNAs在胚胎发育过程中的生物学功能 虽然在线虫和其他生物中对miRNAs在分化组织中的功能进行了很好的研究，但只有少数动物的miRNAs的胚胎功能被了解。此外，在母体到受精卵的转变(MZT)之前，miRNAs如何在很大程度上对基因表达进行转录后控制是完全未知的。线虫是研究胚胎发育的良好模式生物，因为它有明确的定型细胞谱系和强大的遗传工具。我们的团队正在使用线虫来阐明miRNAs在胚胎发生中的功能，首先专注于胚胎发生所需的miRNA家族。 为了剖析由这些miRNAs控制的生物通路，我们小组正在对microRNA家族突变表型的抑制子进行正向(突变)和反向(RNAi)筛选。了解受胚胎表达的microRNA家族影响的生物网络将为如何控制基因表达以协调胚胎发生提供重要的见解。 确定miRNA和ArgAerte周转的分子机制 MiRNA生物发生和衰退速率的平衡控制miRNA的丰度，从而控制基因表达程序。以前的研究已经仔细地阐明了miRNA生物发生的机制。然而，我们对miRNAs和miRISC是如何被结构性地或以受监管的方式移交的知之甚少。这是我们对miRNA调控，从而对基因表达调控的理解的一个主要差距。我们正在使用深度测序来评估候选角色在microRNA周转中的贡献，以及识别miRNA周转的新效应器和调节器。 一个主要的悬而未决的问题是，miRNA和它的蛋白质结合伙伴ArgAerte是一起降解的目标，还是可以单独降解。通过测量miRNA周转动力学的变化对ArgAerte周转动力学的影响，以及反之亦然，我们正在研究这些事件是如何相互联系的。因此，从长远来看，我们将在miRISC的更替过程中建立一个事件等级。 了解miRNAs的周转调节可能为治疗人类疾病的新方法奠定基础。这一研究领域可能会影响癌症的治疗，因为众所周知，在许多癌症类型中，miRNAs在全球范围内下调，而miRNA生物发生酶可以作为单倍体不足的肿瘤抑制因子。这些数据表明，miRNA丰度的适度增加可能会逆转miRNAs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一种靶向癌症治疗可能是可行的。 本财政期间主要侧重于为开展这一研究计划建立基础设施。启动资金使我们能够为实验室空间配备线虫遗传学、细胞生物学和分子生物学所需的所有设备。我们还开始建立microRNA周转率和ArgAerte周转率的分析方法，并建立microRNA丰度的正向筛选范例。