Identification of Factors Critical for SINE Retrotransposition

确定 SINE 逆转位的关键因素

• 批准号: 10527360
• 负责人: Jeffrey M Kidd
• 金额: $ 47.58万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-02 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10527360
• 关键词: 7SL RNA; Alleles; Alu Elements; Binding; Biochemical; Biological; Biological Assay; Biology; Candidate Disease Gene; Canis familiaris; Categories; Cell Line; Cell Separation; Cells; Cellular Assay; Chemicals; Cis-Acting Sequence; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complement; Cultured Cells; DNA Transposable Elements; DNA sequencing; Data; Disease; Elements; Event; Evolution; Exons; Generations; Genetic; Genome; Genomic DNA; Genomics; Goals; Hela Cells; Human; Immobilization; Integration Host Factors; Introns; Knock-out; Laboratories; Long Interspersed Elements; Malignant Neoplasms; Maps; Mediating; Michigan; Mobile Genetic Elements; Modeling; Molecular; Mus; Mutation; Open Reading Frames; Paste substance; Phenotype; Poly(A) Tail; Process; Proteins; RNA; RNA Sequences; Retrotransposition; Retrotransposon; Ribonucleoproteins; Ribosomes; Short Interspersed Nucleotide Elements; Signal Recognition Particle; Structural Models; Structure; Technology; Testing; Transfer RNA; Universities; Untranslated RNA; candidate identification; causal variant; differential expression; experimental study; genetic element; genomic locus; human disease; mammalian genome; particle; permissiveness; transcriptome sequencing; whole genome

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.

抽象的 短散布元件 (SINE) 是存在于所有哺乳动物中的可移动遗传元件 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，人类Alu元件）； (2) 源自转移 RNA 的那些（例如， 犬 SINEC_Cf 元素）。 Alu 和 SINEC_Cf 元素对基因组进化和 分别包含令人震惊的人类和犬类基因组 DNA 的约 11% 和约 15%。绝大多数 的 SINE 已因突变过程而变得无法移动；然而，一些人类特有的 Alu 元素 犬类 SINEC_Cf 元件可以通过“复制和粘贴”机制移动到新的基因组位置 称为逆转录转座。迄今为止，已有超过 76 个独立种系 Alu 逆转录转座事件 被认为是导致人类疾病（包括癌症）的原因。 SINEC_Cf 逆转录转座事件是 造成犬科动物的各种疾病和表型差异。 SINE 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， 证明由自主长插入元件 1 (LINE-1) 逆转录转座子编码的蛋白质 (LINE-1 ORF2p) 是 Alu 和 SINEC_Cf 元件逆转录转座所必需的。我们假设 Alu RNA 的结构，以及扩展的 SINEC_Cf RNA 和未识别的宿主因子允许这些 RNA 定位于核糖体，在那里它们可以与 LINE-1 Poly(A) 尾竞争 LINE-1 ORF2p 结合 促进它们的逆转录转座。在这里，我们建议结合使用分子生物学、进化论 推理、遗传、基因组和生化方法：(1) 使用已建立的 RNA 二级结构 模型、基于 Illumina 的 SHAPE-MaP 化学探测，并建立培养细胞测定来揭示顺式 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；和 (2) 利用 HeLa 细胞分离株之间支持 Alu 和 SINEC_Cf 的能力不同的差异 逆转录转座以确定对 SINE 逆转录转座至关重要的宿主因子。该提案建立在 密歇根大学莫兰和基德实验室之间的成功合作，并将 将 Moran 实验室在转座元件和 RNA 生物学方面的专业知识与 Kidd 实验室的技术相结合 计算和统计基因组学以及进化生物学方面的专业知识来阐明 SINE 逆转录转座机制。