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）捍卫宿主免受RNA沉默中的移动遗传元素和功能。我们 发现3'衍生的tRNA片段（3'-trf）抑制了使用长期重复（LTR）的retroements，使用该元素 在高度保守的底漆结合位点（PBS）处，TRNA的3' - 末端逆转录。 3'-trf是 在癌症和干细胞中高度表达了我们的中心假设，即3'-TRF保护基因组 来自发育和疾病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重新元素抑制是高度保守的，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 参与了这种新型的小RNA沉默机制，以及它们如何与RNAi途径相交。首先，我们 将对小鼠，IAP和 ETN/MUSD，它将解决逆转录的时机，适合筛选蛋白质 通过TRF介导沉默。我们假设3'-TRF在没有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 小鼠基于发光的ERV报告者的核注射后植入前胚胎。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， 候选和淘汰赛期间的逆转录率和诊断逆转录病毒中间体。 催化不足的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-蛋白下拉和有针对性的CRIS淘汰赛 屏幕得分为逆转录定位以识别在3'-trf沉默中起作用的新型RNA结合蛋白 移动元素。抑制性染色质标记和同一细胞中的全长tRNA水平将完成 图片。 3'-trf生物发生和沉默是蜂窝决定是否保留TRNA的根源 - 和恢复复制 - 或产生TRF以抑制它们。 LTR重新元素的表达不仅 威胁基因组稳定性，但是由3'-trf靶向的鼠和人类ERV也对STEM至关重要 细胞多能。因此，确定重新元素调节的机制和范围是优先的 由TRF。 3'-trf对PBS的靶向是这种高度丰富且分散的类别的独特漏洞 移动元素，并可能使创新的方法用于治疗感染性LTR逆转录病毒，例如 作为艾滋病毒。理解RNAi途径的底物在多大程度上将增加新的视角 移动元素与主机之间的武器竞赛。