tRNA-fragments in transposon control

转座子控制中的 tRNA 片段

• 批准号: 10240585
• 负责人: ANDREA SCHORN
• 金额: $ 40.32万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-17 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10240585
• 关键词: Binding; Binding Proteins; Binding Sites; Biogenesis; Biological; Biological Assay; Biological Process; Candidate Disease Gene; Cell Culture Techniques; Cell Line; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complement; DNA; Data; Development; Developmental Gene; Diagnostic; Disease; Elements; Embryo; Endogenous Retroviruses; Enzymes; Epigenetic Process; Eukaryota; Family; Gene Expression; Gene Silencing; Genetic Transcription; Genome; Genome Stability; Goals; HIV; Hand; Health; Histone H3; Host Defense; Human; Human Cell Line; Injections; Knock-out; Length; Link; Long Terminal Repeats; Luciferases; Lysine; Malignant Neoplasms; Mammals; Mediating; Mobile Genetic Elements; Mus; Mutate; Oncogenes; Oncornaviruses; Plant Roots; Process; Production; Proteins; RNA; RNA Interference; RNA Interference Pathway; RNA Polymerase III; RNA-Binding Proteins; Race; Regulation; Reporter; Repression; Research; Retroelements; Retrotransposition; Retroviridae; Reverse Transcription; Role; Small RNA; Stem cell pluripotency; Structure; Supporting Cell; Testing; Time; Tissues; Transfer RNA; Translations; Untranslated RNA; Work; arm; base; blastocyst; cancer cell; cell type; endonuclease; genome-wide; implantation; in vivo; in vivo evaluation; innovation; knock-down; loss of function; luminescence; mutant; novel; piRNA; pluripotency; protein function; screening; stem cells

Project Summary tRNA fragments (tRFs) defend the host against mobile genetic elements and function in RNA silencing. We found that 3'-derived tRNA fragments (3'-tRFs) inhibit long terminal repeat (LTR)-retroelements, which use the 3'-end of tRNAs to prime reverse transcription at their highly conserved primer binding site (PBS). 3'-tRFs are highly expressed in cancer and stem cells supporting our central hypothesis that 3'-tRFs protect the genome from transposon damage during epigenetic reprogramming in development and disease. Our rationale is that 3'- tRF mediated inhibition of LTR-retroelements is highly conserved and is an ancient link between transposons, RNA interference (RNAi), and genome stability. The overarching goal of this proposal is to determine key factors involved in this novel small RNA silencing mechanism and how they intersect with the RNAi pathway. First, we will develop reporter assays for the most highly active endogenous retroviruses (ERVs) in the mouse, IAP and ETn/MusD, which will resolve the timing of retrotransposition and be suitable for screening for proteins that mediate silencing by tRFs. We hypothesize that 3'-tRFs protect the preimplantation embryo in the absence of epigenetic repression of ERVs, similar to piRNAs in the germline, and we will test the in vivo impact of tRFs in mouse preimplantation embryos after pronuclear injection of luminescence-based ERV reporters. To dissect the role of RNAi proteins in 3'-tRF silencing, we will determine tRF expression, subcellular localization, retrotransposition rates, and diagnostic retroviral intermediates during candidate knock-down and knock-out. Catalytically deficient RNAi mutants will help resolve the role of these enzymes in 3'-tRF biogenesis versus binding and target recognition. Lastly, we will use RNA-protein pull-downs and a targeted CRISPR knock-out screen that scores for retrotransposition to identify novel RNA-binding proteins that function in 3'-tRF silencing of mobile elements. Repressive chromatin marks and full-length tRNA levels in the same cells will complete the picture. 3'-tRF biogenesis and silencing are at the root of a cellular decision whether to keep tRNAs for translation - and retroelement replication - or produce tRFs to inhibit them. Expression of LTR-retroelements not only threaten genome stability, but murine and human ERVs that are targeted by 3'-tRFs are also essential for stem cell pluripotency. Therefore, it is of high priority to determine the mechanism and scope of retroelement regulation by tRFs. Targeting of the PBS by 3'-tRFs is a unique vulnerability of this highly abundant and dispersed class of mobile elements and might enable innovative approaches towards treatment of infectious LTR-retroviruses, such as HIV. Understanding to what extent tRNAs are a substrate of the RNAi pathway will add novel perspective to the arms race between mobile elements and their hosts.

项目概要 tRNA 片段 (tRF) 保护宿主免受移动遗传元件的侵害，并在 RNA 沉默中发挥作用。我们 发现 3' 衍生的 tRNA 片段 (3'-tRF) 抑制长末端重复 (LTR) 逆转录元件，该元件使用 tRNA 的 3' 末端，在其高度保守的引物结合位点 (PBS) 处引发逆转录。 3'-tRF 是 在癌症和干细胞中高表达，支持我们的中心假设：3'-tRF 保护基因组 发育和疾病过程中表观遗传重编程期间的转座子损伤。我们的理由是 3'- tRF 介导的 LTR 逆转录元件抑制是高度保守的，是转座子之间的古老联系， RNA 干扰 (RNAi) 和基因组稳定性。该提案的总体目标是确定关键因素 参与这种新颖的小RNA沉默机制以及它们如何与RNAi途径相交叉。首先，我们 将开发小鼠中活性最高的内源性逆转录病毒 (ERV)、IAP 和 ETn/MusD，它将解决逆转录转座的时间并适合筛选 通过 tRF 介导沉默。我们假设 3'-tRF 在缺乏 3'-tRF 的情况下保护植入前胚胎 ERV 的表观遗传抑制，类似于种系中的 piRNA，我们将测试 tRF 的体内影响 原核注射基于发光的 ERV 报告基因后的小鼠植入前胚胎。剖析 RNAi 蛋白在 3'-tRF 沉默中的作用，我们将确定 tRF 表达、亚细胞定位、 候选基因敲低和敲除过程中的逆转录转座率和诊断性逆转录病毒中间体。 催化缺陷的 RNAi 突变体将有助于解决这些酶在 3'-tRF 生物合成中的作用与 结合和目标识别。最后，我们将使用 RNA 蛋白下拉和靶向 CRISPR 敲除 筛选逆转录转座评分，以鉴定在 3'-tRF 沉默中发挥作用的新型 RNA 结合蛋白 移动元素。同一细胞中的抑制性染色质标记和全长 tRNA 水平将完成 图片。 3'-tRF 生物合成和沉默是细胞决定是否保留 tRNA 进行翻译的根本 - 和逆转录元件复制 - 或产生 tRF 来抑制它们。 LTR逆转录因子的表达不仅 威胁基因组稳定性，但 3'-tRF 靶向的小鼠和人类 ERV 对干细胞也至关重要 细胞多能性。因此，确定逆转录因子调控的机制和范围是当务之急。 通过 tRF。 3'-tRF 靶向 PBS 是这一高度丰富且分散的类别的独特弱点 移动元件，并可能实现治疗传染性 LTR 逆转录病毒的创新方法，例如 作为艾滋病毒。了解 tRNA 在多大程度上是 RNAi 途径的底物将为研究提供新的视角 移动元素与其主机之间的军备竞赛。