tRNA-fragments in transposon control

项目摘要 tRNA片段（tRFs）保护宿主免受移动的遗传元件的侵害，并在RNA沉默中起作用。我们 发现3 '-衍生的tRNA片段（3'-tRFs）抑制长末端重复序列（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）引发逆转录。3 '-tRF是 在癌症和干细胞中高度表达，支持我们的核心假设，即3 '-tRFs保护基因组 在发育和疾病中的表观遗传重编程过程中免受转座子损伤。我们的理由是，3 '- tRF介导的LTR-逆转录元件的抑制是高度保守的并且是转座子之间的古老联系， RNA干扰（RNAi）和基因组稳定性。本提案的总体目标是确定关键因素 参与这种新的小RNA沉默机制以及它们如何与RNAi途径交叉。一是 将开发小鼠、IAP和 ETn/MusD，这将解决逆转录转座的时间，并适用于筛选蛋白质， 通过tRFs介导沉默。我们推测，在缺乏3 '-tRFs的情况下， ERV的表观遗传抑制，类似于生殖系中的piRNA，我们将测试tRF在ERV中的体内影响。 小鼠植入前胚胎在原核注射基于发光的ERV报告基因后。解剖 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， 逆转录转座率和候选物敲低和敲除期间的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， 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 对逆转录转座进行评分以鉴定在3 '-tRF沉默中起作用的新型RNA结合蛋白的筛选 移动的元素。抑制性染色质标记和相同细胞中的全长tRNA水平将完成 picture. 3 '-tRF生物合成和沉默是细胞决定是否保留tRNA用于翻译的根本 - 和逆转录因子复制-或产生tRF来抑制它们。LTR-逆转录元件的表达不仅 威胁到基因组的稳定性，但3 '-tRF靶向的鼠和人ERV也是干细胞的关键。 细胞多能性。因此，确定追溯性规制的机制和范围是当务之急 tRFs的。通过3 '-tRF靶向PBS是这类高度丰富和分散的细胞的独特脆弱性。 移动的元素，并可能使创新的方法来治疗传染性LTR-逆转录病毒，如 艾滋病病毒。了解tRNA在多大程度上是RNAi途径的底物，将为RNAi的研究提供新的视角。 移动的分子和他们的宿主之间的军备竞赛。

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.