Identification of Factors Critical for SINE Retrotransposition

确定 SINE 逆转位的关键因素

• 批准号: 10095880
• 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/10095880
• 关键词: 7SL RNA; Alleles; Alu Elements; Binding; Biochemical; Biological; Biological Assay; Biology; CRISPR/Cas technology; Candidate Disease Gene; Canis familiaris; Categories; Cell Line; Cells; Cellular Assay; Chemicals; Cis-Acting Sequence; Collaborations; Complement; Cultured Cells; DNA Transposable Elements; DNA sequencing; Data; Elements; Event; Evolution; Exons; Generations; Genetic; Genome; Genomic DNA; Genomics; Goals; Hela Cells; Human; Integration Host Factors; Introns; Knock-out; Laboratories; Lead; Location; Long Interspersed Elements; Malignant Neoplasms; Maps; Mediating; Michigan; Mobile Genetic Elements; Modeling; Molecular; Mus; Mutation; Open Reading Frames; Paste substance; 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; base; causal variant; differential expression; disease phenotype; experimental study; human disease; mammalian genome; particle; 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 简短的散布元素（罪）是所有哺乳动物中存在的移动遗传元素 基因组。大多数哺乳动物的罪恶可以细分为两个一般类别：（1）从7SL派生的类别 信号识别粒子RNA（例如，人Alu元素）； （2）从转移RNA派生的那些（例如， 犬Sinec_cf元素）。 Alu和Sinec_cf元素对基因组演变产生了重大影响， 分别包括大约11％和〜15％的人类和犬基因组DNA。绝大多数 因突变过程使罪过的罪过。但是，一些人类特定的ALU元素 犬Sinec_cf元素可以通过“复制和粘贴”机制动员到新的基因组位置 称为逆转录。迄今 被认为是包括癌症在内的人类疾病的原因。 sinec_cf逆转录事件是 负责各种疾病和犬类表型差异。罪不编码蛋白质；因此， 它们被归类为“非自治”逆转录子。先前的研究，包括我们的初步数据 证明一种由自主长的散布元素1（LINE-1）逆转录子编码的蛋白质 （LINE-1 ORF2P）是ALU和SINEC_CF元素retrotransposion所需的。我们假设 Alu RNA的结构和扩展Sinec_cf RNA和未识别的宿主因子允许这些RNA 定位于核糖体，在那里他们可以与Line-1 poly（A）尾巴竞争，以使LINE-1 ORF2P结合到 促进他们的逆转录定位。在这里，我们建议将分子生物学进化的组合使用 推论，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的形状映射化学探测和已建立的培养细胞测定法，以发现顺式 人类特异性ALU和SINEC_CF逆转录所需的作用RNA结构和序列；和 （2）利用HeLa细胞分离株之间的差异，这些分离株的支持​​能力不同和Sinec_cf 逆转录置位，以识别正弦逆转录至关重要的宿主因子。该提议建立在 密歇根大学的Moran和Kidd实验室之间的成功合作 将Moran实验室在可转座元件和RNA生物学方面的专业知识与KIDD实验室的专业知识相结合 计算和统计基因组学和进化生物学方面的专业知识，以阐明正弦 逆转录机制。