Regulation of MicroRNA Populations by Oncogenic Viruses

项目摘要 microRNAs（miRNAs）是一类转录后调节基因表达的小分子非编码RNA（ncRNAs）， 超过一半的人类信使RNA（mRNA）表达;异常的miRNA水平与 疾病，包括癌症。miRNAs与Argonaute（Ago）蛋白相关，并引导它们与其他蛋白互补。 mRNA上的序列导致部分碱基配对时mRNA的抑制，或Ago2介导的 当完美碱基配对发生时，mRNA裂解。最近Steitz博士的团队和其他人发现 三种疱疹病毒RNA的miRNA碱基配对的另一个结果是， 在一些实施方案中，miRNA与宿主miRNA互补，并导致miRNA的降解。这一过程， 一种未表征的机制，被称为靶诱导的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 ncRNA由 松鼠猴疱疹病毒（HVS）与宿主miR-27 a结合并介导宿主miR-27 a的衰变。miR-27 a是T细胞增殖抑制因子 激活及其降低的水平有助于HVS诱导的淋巴瘤。重要的是，虽然生物起源 miRNAs已被广泛研究，但其周转率知之甚少。含有miR-DE的存在 单独的病毒RNA足以诱导miRNA降解，这表明TIMD是一种未描述的细胞miRNA 衰变机制此外，其他已知的疱疹病毒miR-DE存在于mRNA中，这意味着 在其他病毒和人类转录物中的类似元件。在这项研究中，我打算描绘小说 miRNA降解机制（Aim 1），以及鉴定来自于 致癌病毒及其宿主诱导miRNA衰变（Aim 2）。在K99阶段，我将确定TMD 中间体通过适应TimeLapse-在我们的合作者Matthew Simon博士的实验室开发的方法 它使用单时间点高通量测序来评估RNA周转。我将深入了解TMD 通过与Ian博士合作获得Ago2-miR-27a-HSUR1复合物的晶体结构， 麦克雷我将验证预测的由致癌EB病毒编码的靶向肿瘤的miR-DE 抑制性miRNA（miR-150和miR-125 B b），通过分析它们在B细胞中诱导miRNA衰变的能力。我会 构建一个基于荧光蛋白的报告基因，响应miRNA水平的变化， 功能基因筛选以鉴定参与TIMD的细胞酶。在R00阶段，我将描述 在遗传筛选中获得的TIMD候选者。我将寻找其他必要的RNA基序 miR-DE功能的研究。然后我将使用一个精炼的生物信息学管道， 寻找人类的miR-DE这项研究将描述miRNA的周转机制，这可能与 疾病，并将生物信息学识别潜在的致癌病毒和人类转录本。的途径 独立奖将使我获得必要的专业知识（即生物信息学）和专业技能 (i.e.在教学，写作），成为一个有竞争力的候选人，在一流大学的学术职位。

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.