Identification of Factors Critical for SINE Retrotransposition

确定 SINE 逆转位的关键因素

基本信息

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
项目类别：
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

项目摘要

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）；和（2）源自转移 RNA 的那些（例如，犬 SINEC_Cf 元件）。Alu 和 SINEC_Cf 元件对基因组进化产生了重大影响。分别包含令人震惊的约 11% 和约 15% 的人类和犬类基因组 DNA。的 SINE 已因突变过程而变得不可移动；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迄今为止，已有超过 76 个独立种系 Alu 逆转录事件。 SINEC_Cf 逆转录转座事件被认为是人类疾病的病因。导致犬科动物的各种疾病和表型差异，因此，SINE 不编码蛋白质；它们被归类为“非自主”反转录转座子，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 元件逆转录所必需的。 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 结合在这里，我们建议结合分子生物学、进化论来促进它们的逆转录。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； (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。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。