tRNA-fragments in transposon control

转座子控制中的 tRNA 片段

• 批准号: 10035091
• 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/10035091
• 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; 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.

项目摘要 TRNA片段(TRF)保护宿主免受移动的遗传元件的影响，并在RNA沉默中发挥作用。我们 发现3‘-衍生tRNA片段(3’-TRF)抑制长末端重复序列(LTR)逆转录元件，这些逆转录元件使用 TRNAs的3‘端，在其高度保守的引物结合位点(PBS)启动逆转录。3‘-TRF是 在癌症和干细胞中高度表达，支持我们的中心假说，即3‘-TRFs保护基因组 在发育和疾病的表观遗传重编程过程中防止转座子损伤。我们的理由是3‘- TRF介导的对LTR逆转录元件的抑制是高度保守的，是转座子之间的古老联系， RNA干扰(RNAi)和基因组稳定性。这项提案的首要目标是确定关键因素 参与了这一新的小RNA沉默机制以及它们如何与RNAi途径相交。首先，我们 将开发对小鼠、IAP和IAP中最活跃的内源性逆转录病毒(ERV)的报告分析 ETN/MU.S.，它将决定逆转录转座的时间，并适合于筛选 调解TRF的沉默。我们假设，3‘-TRFs在缺乏 ERV的表观遗传抑制，类似于生殖系中的piRNAs，我们将在 小鼠着床前胚胎注射原核后基于荧光的ERV报道。要仔细分析 RNAi蛋白在3‘-TRF沉默中的作用，我们将确定TRF的表达，亚细胞定位， 逆转录转座率，以及候选基因敲除和基因敲除过程中的逆转录病毒中间产物。 催化缺陷的RNAi突变体将有助于解决这些酶在3‘-TRF生物发生中的作用 绑定和目标识别。最后，我们将使用RNA-蛋白质下拉列表和有针对性的CRISPR敲除 筛选逆转录转座评分以鉴定在3‘-TRF沉默中起作用的新的RNA结合蛋白 移动元素。同一细胞中抑制染色质标记和全长tRNA水平将完成 图片。3‘-TRF的生物发生和沉默是细胞决定是否保留tRNA以供翻译的根本 -和逆转录元件复制-或者产生TRF来抑制它们。LtR反转录元件的表达不仅 威胁基因组稳定性，但3‘-TRFs靶向的小鼠和人类ERV对干细胞也是必不可少的 细胞的多能性。因此，当务之急是确定逆转录调控的机制和范围 由TRF提供。以PBS为靶标的3‘-TRFs是这种高度丰富和分散的 可移动的成分，并可能实现治疗传染性LTR型逆转录病毒的创新方法，如 作为艾滋病病毒。了解tRNAs在多大程度上是RNAi途径的底物将为以下方面提供新的视角 机动部队与其东道主之间的军备竞赛。