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: 10308710
• 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/10308710
• 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; Lead; Link; Literature; Macaca mulatta; 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; Supervision; Testing; Transcript; Translating; Translations; Untranslated RNA; Untranslated Regions; Vertebrates; base; 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在理解病因方面非常有用 遗传疾病和基本生物学研究。尽管是最准确，最全面的集合 注释的基因组特征，新兴证据表明，翻译区域数量越来越多 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）代表当前文献中缺少重要的生物学。例如，肌酸蛋白，一个 在“非编码”区域发现了保守的46个氨基酸微肽，后来被证明 在控制小鼠的骨骼肌方面起作用。这些潜在功能的新型Torf通常很小，并且 因此，大多数编码基因注释程序都忽略了。为了克服这一挑战，努力利用 功能性基因组学数据集识别人类基因组的新编码区域已开始揭示 这种先前不足的基因组特征类别。特别是，申请人以前开发了 计算方法，ribohmm，它利用功能中翻译区域特有的模式 基因组学数据，例如Ribo-Seq数据，以识别全基因组。使用ribohmm系统地 在人淋巴细胞细胞系中的注释TORF，还发现了7,273个新型Torfs，此外还 已知的编码基因。这些新颖的托尔夫在先前注释的转录组区域中发现了 非编码（例如未翻译区域和lincrnas）。尽管新开发的方法，例如ribohmm， 现在可以系统地识别成千上万的先前被忽视的Torfs，这是这些的生物学相关性 翻译事件尚不清楚。当前建议的目的是评估功能相关性 这些新发现的Torfs。将评估生物学重要性的三个主要方面。首先，损失 功能影响。 Torf缺失对细胞生存能力和合成适应性影响的影响与良好 将使用合并的CRISPR辍学屏幕评估表征的编码基因（AIM 1）。第二，能力 编码蛋白质/肽。将在质量中评估TORF产生稳定蛋白/肽的能力 专为检测小型ORF的翻译产物而设计的光谱研究（AIM 2）。第三，进化 保护。将使用新的对齐方式仔细评估这些本地净化选择的强度 基于黑猩猩和恒河猕猴的独立注释的小说Torfs创建。完成 拟议的目标将提供对新型TORF的生物学相关性的首次系统评估。影响 这些新的功能注释可能从提供GWAS命中的新解释到重新评估 “非编码RNA”功能。拟议研究的结果将指导该小组的未来研究指示 以前被忽视的基因组特征。考虑到大量意外的Torf和先前的示例 在重要的生物学途径中扮演关键角色的被忽视的托尔夫（Torfs） 对基本和翻译的生物医学研究具有很大的影响。