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

摘要 人类基因组中编码基因的注释在理解人类遗传性疾病的病因学方面非常有用。 遗传疾病和基础生物学研究。尽管是最准确和最全面的一套 基因组特征注释，新出现的证据表明，越来越多的翻译区， 当前注释中缺少。这些被忽视的基因组区域，或正式翻译开放阅读 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， 在“非编码”区发现了一个保守的46个氨基酸的微肽，后来证明， 调节小鼠骨骼肌的功能。这些潜在的功能性新型tORF通常很小， 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， 用于识别人类基因组中新编码区的功能基因组学数据集已经开始揭示 这类以前被低估的基因组特征。特别地，申请人先前开发了一种 计算方法，riboHMM，它利用特定的模式，翻译区的功能， 基因组学数据，如核糖核酸测序数据，以确定全基因组的tORF。使用riboHMM系统地 在人类淋巴母细胞样细胞系中注释tORF，发现了7，273个新的tORF，除了 已知的编码基因这些新的tORF发现于先前注释为 非编码区（例如非翻译区和lincRNA）。虽然新开发的方法，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， 翻译事件仍不清楚。本提案的目的是评价以下方面的功能相关性： 这些新发现的tORF将评估生物学重要性的三个主要方面。第一，损失 功能影响。tORF缺失对细胞活力和合成适合度影响的影响与良好的 将使用合并的CRISPR脱落筛选（Aim 1）来评估表征的编码基因。第二，能力 编码蛋白质/肽。tORF产生稳定蛋白质/肽的能力将以质量评估 设计用于检测小ORF的翻译产物的光谱学研究（Aim 2）。第三，进化 节约在这些位点上的纯化选择的强度将使用新的比对进行仔细评估 基于黑猩猩和恒河猴中独立注释的新tORF创建。完成 提出的目标将提供新的tORF的生物学相关性的第一个系统评价。影响 这些新的功能注释的范围可以从为GWAS命中提供新的解释到重新评估 “非编码RNA”功能。从拟议的研究结果将指导未来的研究方向对这一群体 以前被忽视的基因组特征考虑到大量未探测的tORF和之前的例子 被忽视的tORF在重要的生物学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有深远的影响。

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.