Regulation of MicroRNA Populations by Oncogenic Viruses

项目摘要 MicroRNAs(MiRNAs)是一种小的非编码RNAs(NcRNAs)，它在转录后调节基因 超过一半的人类信使RNA(MRNAs)表达；异常的miRNA水平与 疾病，包括癌症。MiRNAs与ArgAerte(AGO)蛋白结合并引导它们互补 MRNAs上的序列要么导致部分碱基配对时的mRNA抑制，要么导致ago2介导的 当发生完美碱基配对时，信使核糖核酸发生切割。最近，斯泰茨博士的团队和其他人发现 MiRNA碱基配对的另一个结果是对三个疱疹病毒RNA进行配对，这些RNA表现出广泛但不完美的， 与宿主miRNAs互补，导致miRNAs降解。这个过程，是一个静止的过程 未知的机制，被称为靶向诱导的miRNA降解(TIMD)和miRNA结合 该位点被称为miRNA降解元件(miR-DE)。例如，HSUR1-a编码的ncRNA 疱疹病毒Simiri(HVS)与宿主miR-27A结合并介导其腐烂。MIR-27A是T细胞的抑制因子 人类免疫缺陷病毒(HVS)诱导的淋巴瘤的发生与其激活和水平降低有关。重要的是，虽然生物的起源 MiRNAs已被广泛研究，但对其周转情况知之甚少。含miR-DE的存在 病毒RNA本身足以引起miRNA的衰变，这表明TIMD是一种未被描述的细胞miRNA 腐烂机制。此外，其他已知的疱疹病毒miR-Des存在于mRNAs中，这意味着存在 其他病毒和人类转录本中的类似成分。在这项研究中，我打算描绘这部小说 MiRNA降解机制(目标1)，以及鉴定来自 诱发miRNA衰变的致癌病毒及其宿主(目标2)。在K99阶段，我将确定TIMD 适应时间流逝的中间体--我们的合作者马修·西蒙博士的实验室开发的一种方法 它使用单时间点高通量测序来评估RNA周转率。我将深入了解TIMD 与Ian博士合作获得Ago2-miR-27A-HSUR1络合物晶体结构的机理 马克雷。我将验证以肿瘤为靶点的致癌EB病毒编码的miR-Des预测 通过分析抑制miRNAs(miR-150和miR-125b)在B细胞中诱导miRNA衰变的能力。这就做 构建基于荧光蛋白的报告基因以响应miRNA水平的变化以执行缺失 功能遗传学筛查以确定参与TIMD的细胞酶。在R00阶段，我将描述 在基因筛查中获得了TIMD的候选基因。我将寻找其他必要的RNA基序 MIR-DE通过生物信息学和诱变发挥作用。然后我会用一条精致的生物信息学管道 搜索人类mir-des。这项研究将描绘miRNA的周转机制，这可能与 疾病，并将以生物信息识别潜在的致癌病毒和人类转录本。通向世界的道路 独立奖将使我获得必要的专业知识(如生物信息学)和专业技能 (例如在教学、写作方面)成为一流大学学术职位的有竞争力的候选人。

Project Summary MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that post-transcriptionally regulate gene expression of more than half of human messenger RNAs (mRNAs); aberrant miRNA levels are linked to disease, including cancer. miRNAs associate with Argonaute (Ago) proteins and guide them to complementary sequences on mRNAs resulting either in mRNA suppression upon partial base-paring, or in Ago2-mediated mRNA cleavage when perfect base-pairing occurs. Recently Dr. Steitz’s group and others discovered yet another outcome of miRNA base-pairing for three herpesviral RNAs that exhibit extensive, yet imperfect, complementarity to host miRNAs, and result in degradation of the miRNAs. This process, of a still uncharacterized mechanism, is known as target-induced miRNA degradation (TIMD) and the miRNA-binding site is called the miRNA-degradation element (miR-DE). For example, HSUR1 – a ncRNA encoded by Herpesvirus saimiri (HVS) binds to and mediates decay of host miR-27a. miR-27a is a repressor of T-cell activation and its decreased levels contribute to HVS-induced lymphomas. Importantly, while the biogenesis of miRNAs has been extensively studied, their turnover is poorly understood. The presence of miR-DE-containing viral RNAs alone is sufficient to induce miRNA decay, suggesting that TIMD is an undescribed cellular miRNA decay mechanism. In addition, other known herpesviral miR-DEs reside in mRNAs, implying the existence of analogous elements in other viral and human transcripts. In this study, I intend to delineate the novel miRNA degradation machinery (Aim 1), as well as to identify yet unknown transcripts derived from oncogenic viruses and their hosts that induce miRNA decay (Aim 2). In the K99 phase, I will identify TIMD intermediates by adapting TimeLapse – a method developed in the lab of our collaborator Dr. Matthew Simon that assesses RNA turnover using single time point high-throughput sequencing. I will gain insight into TIMD mechanism by obtaining a crystal structure of Ago2-miR-27a-HSUR1 complexes in collaboration with Dr. Ian MacRae. I will validate predicted miR-DEs encoded by oncogenic Epstein-Barr virus that target tumor suppressor miRNAs (miR-150 and miR-125b) by analyzing their ability to induce miRNA decay in B cells. I will construct a fluorescent protein-based reporter responding to changes in miRNA levels to perform a loss-of- function genetic screen to identify cellular enzymes participating in TIMD. In the R00 phase, I will characterize the candidates for TIMD obtained in the genetic screen. I will search for additional RNA motifs necessary for miR-DE function by using bioinformatics and mutagenesis. I will then use a refined bioinformatic pipeline to search for human miR-DEs. This study will delineate the miRNA turnover mechanism, which could be linked to disease, and will bioinformatically identify potentially oncogenic viral and human transcripts. The Pathway to Independence Award will allow me to gain necessary expertise (i.e. in bioinformatics) and professional skills (i.e. in teaching, writing) to become a competitive candidate for an academic position at a top-tier university.