Identification of Factors Critical for SINE Retrotransposition

摘要 短穿插元件(Sine)是存在于所有哺乳动物中的可移动的遗传元件 基因组。大多数哺乳动物Sine可分为两大类：(1)源自7SL的Sine 信号识别颗粒RNA(例如，人Alu元件)；以及(2)来自转移RNA的那些(例如， 犬SINEC_CF元素)。Alu和SiNEC_Cf元素对基因组进化和 分别占人类和犬类基因组DNA的11%和15%。绝大多数人 已经通过突变过程使其不能移动；然而，一些人类特有的Alu元素 犬SINEC_Cf元件可以通过复制和粘贴机制被动员到新的基因组位置 称为逆转位转位。到目前为止，超过76个独立的生殖系Alu逆转座子事件已经 被认为是包括癌症在内的人类疾病的起因。SINEC_CF转置事件包括 负责各种疾病和犬类的表型差异。正弦不编码蛋白质；因此， 它们被归类为“非自主”反转录转座子。之前的研究，包括我们的初步数据， 证明由自主长散布元件-1(LINE-1)反转录转座子编码的蛋白质 Alu和SINEC_CF元素反向转位需要(LINE-1 ORF2p)。我们假设 Alu RNA的结构，进而延伸到SINEC_CFRNA，以及未知的宿主因子(S)允许这些RNA 定位到核糖体，在那里它们可以与Line-1 Poly(A)尾部竞争Line-1 ORF2p结合到 促进他们的逆转位。在这里，我们建议使用分子生物学、进化论的组合 推断、遗传、基因组和生化方法以：(1)使用已建立的RNA二级结构 模型，基于Illumina的SHAPE-MAP化学探测，并建立了培养细胞分析，以发现顺式- 人类特异性Alu和sinec_cF逆转录转座所需的作用RNA结构和序列；以及 (2)利用支持Alu和Sinec_CF能力不同的HeLa细胞株之间的差异 逆转录转座以确定对正弦逆转录转座至关重要的宿主因子(S)。这项建议的基础是 密歇根大学莫兰和基德实验室的成功合作将 将Moran实验室在转座基因和RNA生物学方面的专业知识与Kidd实验室的 在计算和统计基因组学和进化生物学方面的专业知识，以阐明SINE 逆转位机制。

Abstract Short INterspersed Elements (SINEs) are mobile genetic elements that are present in all mammalian genomes. Most mammalian SINEs can be subdivided into two general categories: (1) those derived from 7SL signal recognition particle RNA (e.g., human Alu elements); and (2) those derived from transfer RNAs (e.g., canine SINEC_Cf elements). Alu and SINEC_Cf elements have had a major impact on genome evolution and comprise an astounding ~11% and ~15% of human and canine genomic DNA, respectively. The vast majority of SINEs have been rendered immobile by mutational processes; however, some human-specific Alu elements and canine SINEC_Cf elements can mobilize to new genomic locations by a “copy and paste” mechanism termed retrotransposition. To date, greater than 76 independent germline Alu retrotransposition events have been implicated as the cause of human diseases, including cancer. SINEC_Cf retrotransposition events are responsible for various diseases and phenotypic differences in canines. SINEs do not encode proteins; thus, they are classified as `non-autonomous' retrotransposons. Previous studies, including our preliminary data, demonstrate that a protein encoded by an autonomous Long INterspersed Element-1 (LINE-1) retrotransposon (LINE-1 ORF2p) is required for Alu and SINEC_Cf element retrotransposition. We hypothesize that the structure of Alu RNA, and by extension SINEC_Cf RNA, and unidentified host factor(s) allow these RNAs to localize to the ribosome, where they can compete with the LINE-1 poly(A) tail for LINE-1 ORF2p binding to promote their retrotransposition. Here, we propose to use a combination of molecular biological, evolutionary inference, genetic, genomic, and biochemical approaches to: (1) use established RNA secondary structure models, Illumina-based SHAPE-MaP chemical probing, and established cultured cell assays to uncover cis- acting RNA structures and sequences required for human-specific Alu and SINEC_Cf retrotransposition; and (2) exploit differences between HeLa cell isolates that differ in their ability to support Alu and SINEC_Cf retrotransposition to identify host factor(s) critical for SINE retrotransposition. This proposal builds on successful collaborations between the Moran and Kidd laboratories at the University of Michigan and will combine the Moran laboratory's expertise in transposable element and RNA biology with the Kidd laboratory's expertise in computational and statistical genomics and evolutionary biology to elucidate SINE retrotransposition mechanisms.