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.

摘要 西内斯是一种移动的遗传元件，存在于所有哺乳动物的染色体中 基因组大多数哺乳动物的西内斯可分为两大类：（1）7SL衍生的SINE 信号识别颗粒RNA（例如，人Alu元件）;和（2）来源于转移RNA的那些（例如， 犬SINEC_Cf元件）。Alu和SINEC_Cf元件对基因组进化具有重大影响， 分别占人类和犬类基因组DNA的约11%和约15%。绝大多数 的西内斯已经被突变过程变得不动;然而，一些人类特异性的Alu元件 犬SINEC_Cf元件可以通过“复制粘贴”机制移动到新的基因组位置 称为反转录转位。到目前为止，超过76个独立的生殖系Alu逆转录转座事件， 被认为是人类疾病包括癌症的原因。SINEC_Cf反转录转座事件是 导致犬的各种疾病和表型差异。西内斯不编码蛋白质;因此， 它们被归类为“非自主”反转录转座子。以前的研究，包括我们的初步数据， 证明了由自主的长间隔元件-1（LINE-1）反转录转座子编码的蛋白质 （LINE-1 ORF 2 p）是Alu和SINEC_Cf元件反转录转座所必需的。我们假设 Alu RNA和延伸SINEC_Cf RNA的结构以及未鉴定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 ORF 2 p与 促进它们的反转录转座在这里，我们建议使用分子生物学，进化 推理、遗传学、基因组学和生物化学方法：（1）使用已建立RNA二级结构 模型，基于Illumina的SHAPE-MaP化学探测，并建立了培养细胞测定，以揭示顺式- 人特异性Alu和SINEC_Cf反转录转座所需的作用RNA结构和序列;和 (2)利用HeLa细胞分离株之间的差异，其支持Alu和SINEC_Cf的能力不同 逆转录转座，以鉴定对SINE逆转录转座至关重要的宿主因子。该提案基于 密歇根大学莫兰和基德实验室之间的成功合作， 联合收割机结合了莫兰实验室在转座因子和RNA生物学方面的专业知识和基德实验室的 在计算和统计基因组学和进化生物学的专业知识，以阐明SINE 反转录转座机制