Expanding the known coding genome: identifying biological function for novel tORFs

扩展已知的编码基因组：识别新型 tORF 的生物学功能

• 批准号: 10531851
• 负责人: Sidney Wang
• 金额: $ 30.33万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10531851
• 关键词: 5' Untranslated Regions; Active Sites; Amino Acids; Area; Biological; Biological Process; Biology; Biomedical Research; CRISPR library; CRISPR screen; Catalogs; Categories; Cell Culture Techniques; Cell Survival; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Codon Nucleotides; Computing Methodologies; Culture Media; DNA Sequence; Data; Dropout; Drosophila genus; Evaluation; Event; Evolution; Foundations; Frequencies; Future; Genes; Genetic; Genetic Diseases; Genetic Predisposition to Disease; Genetic Screening; Genetic study; Genome; Genomic Segment; Genomics; Goals; Guide RNA; Human; Human Genome; Initiator Codon; Learning; Link; Literature; Macaca mulatta; Maps; Mass Spectrum Analysis; Medicine; Mendelian disorder; Messenger RNA; Methods; Modernization; Molecular; Molecular Weight; Mus; Natural Selections; Open Reading Frames; Pan Genus; Pathway interactions; Pattern; Peptides; Periodicity; Play; Preparation; Primates; Production; Proteins; Proteomics; Protocols documentation; Public Health; Research; Research Design; Ribosomes; Role; Sampling; Sequence Alignment; Shotguns; Signal Transduction; Skeletal Muscle; Testing; Transcript; Translating; Translations; Untranslated RNA; Untranslated Regions; Vertebrates; design; experimental study; fitness; follow-up; functional genomics; genetic variant; genome annotation; genome wide association study; genome-wide; genomic data; improved; loss of function; lymphoblastoid cell line; muscle physiology; novel; programs; reverse genetics; ribosome profiling; tandem mass spectrometry; transcriptome

Abstract Annotations of coding genes in the human genome have been tremendously useful in understanding etiology of genetic disorders and in basic biology research. Despite being the most accurate and comprehensive set of genomic features annotated, emerging evidence has indicated that an increasing number of translated regions are missing from the current annotation. These overlooked genomic regions, or formally translated open reading frames (tORFs), represents important biology missing from the current literature. For example, myoregulin, a conserved 46 amino acid micro-peptide was discovered in a “non-coding” region, and was later demonstrated to function in regulating skeletal muscles in mice. These potentially functional novel tORFs are often small, and therefore overlooked by most coding gene annotation programs. To overcome this challenge, efforts leveraging functional genomics datasets to identify novel coding regions across the human genome have begun to reveal this previously underappreciated class of genomic features. In particular, the applicants previously developed a computational method, riboHMM, which leverages patterns specific to the translated regions in functional genomics data, such as ribo-seq data, in order to identify tORFs genome-wide. Using riboHMM to systematically annotate tORFs in human lymphoblastoid cell lines, 7,273 novel tORFs were found, in addition to the tORFs of known coding genes. These novel tORFs were found in regions of the transcriptome previously annotated as non-coding (e.g. Untranslated Regions and lincRNAs). Although newly developed methods, such as riboHMM, can now systematically identify thousands of previously overlooked tORFs, the biological relevance of these translation events remains unclear. The objective of the current proposal is to evaluate functional relevance for these newly discovered tORFs. Three major aspects of biological importance will be evaluated. First, loss of function impact. Effects of tORF deletion on cell viability and synthetic fitness impact in combination with well- characterized coding genes will be evaluated using pooled CRISPR dropout screens (Aim 1). Second, ability to encode protein/peptide. The ability of tORFs to produce stable protein/peptide will be evaluated in mass spectrometry studies designed for detecting translation products of small ORFs (Aim 2). Third, evolution conservation. The strength of purifying selection on these loci will be carefully evaluated using new alignments created based on independently annotated novel tORFs in chimpanzee and rhesus macaque. The completion of the proposed aims will provide the first systematic evaluation of biological relevance for novel tORFs. Impacts of these new functional annotations could range from providing new interpretations for GWAS hits to reevaluating “non-coding RNA” function. Results from the proposed study will guide future research directions on this group of previously overlooked genomic features. Given the sheer number of unexplored tORFs and the prior examples of overlooked tORFs that turned out to play critical roles in important biological pathways, the findings here will have far reaching implications for both basic and translational biomedical research.

抽象的 人类基因组中编码基因的注释对于理解疾病的病因学非常有用 尽管是最准确和最全面的一组，但在遗传性疾病和基础生物学研究中。 注释的基因组特征，新出现的证据表明，越来越多的翻译区域 当前注释中缺少这些被忽视的基因组区域，或正式翻译的开放阅读。 框架（tORF）代表了当前文献中缺失的重要生物学，例如肌调节蛋白。 保守的46个氨基酸微肽在“非编码”区域被证明，后来被证实 这些具有潜在功能的新型 tORF 通常很小，并且具有调节小鼠骨骼肌的功能。 因此被大多数编码基因注释程序所忽视，为了克服这一挑战，要利用努力。 用于识别人类基因组新编码的功能基因组学数据集已经开始揭示 特别是，申请人之前开发了这一先前未被充分重视的基因组特征。 计算方法 riboHMM，它利用功能中翻译区域的特定模式 基因组学数据，例如 ribo-seq 数据，以便使用 riboHMM 系统地识别全基因组的 tORF。 注释人淋巴母细胞系中的 tORF，除了 tORF 之外，还发现了 7,273 个新的 tORF。 这些新的 tORF 是在先前注释为的转录组区域中发现的。 非编码（例如非翻译区和 lincRNA）。尽管新开发的方法，例如 riboHMM， 现在可以系统地识别数千个以前被忽视的 tORF，这些 tORF 的生物学相关性 当前提案的目标是评估翻译事件的功能相关性。 这些新发现的 tORF 的生物学重要性将在三个方面进行评估。 tORF 缺失对细胞活力的影响以及与良好结合的综合适应度影响。 将使用汇集的 CRISPR 缺失筛选来评估特征编码基因（目标 1）。 编码蛋白质/肽的tORF产生稳定蛋白质/肽的能力将被大规模评估。 旨在检测小 ORF 翻译产物的光谱研究（目标 2）。 将使用新的比对仔细评估这些基因座的纯化选择的强度。 基于黑猩猩和恒河猴中独立注释的新颖 tORF 创建。 拟议目标将首次对新型 tORF 影响的生物学相关性进行系统评估。 这些新的功能注释的范围可能从为 GWAS 命中提供新的解释到重新评估 “非编码RNA”功能。拟议研究的结果将指导该小组未来的研究方向。 考虑到大量未探索的 tORF 和之前的例子。 被忽视的 tORF 在重要的生物途径中发挥着关键作用，这里的发现将 对基础和转化生物医学研究都具有深远的影响。