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对于理解霍奇金淋巴瘤的病因学非常有用。 遗传性疾病和基础生物学研究。尽管它是最准确和最全面的一套 有注释的基因组特征，新出现的证据表明，越来越多的翻译区域 在当前批注中缺失。这些被忽视的基因组区域，或正式翻译的公开阅读 框架(Torf)，代表了当前文献中缺失的重要生物学。例如，肌调节素，一种 在一个“非编码区”发现了一个保守的46个氨基酸的微肽，后来证实它是 对小鼠骨骼肌的调节作用。这些具有潜在功能的小说Torf通常很小，而且 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，利用 识别人类基因组中新编码区的功能基因组数据集已经开始揭示 这一类以前被低估的基因组特征。特别是，申请者之前开发了一个 计算方法riboHMM，它利用泛函中特定于翻译区域的模式 基因组学数据，例如RIBO-SEQ数据，以便在全基因组范围内识别TORF。利用RiboHMM系统地 在人类淋巴母细胞系中发现了7,273个新的TURF，此外还发现了 已知的编码基因。这些新的Torf是在转录组中发现的，以前注释为 非编码(例如，未翻译区域和连接RNA)。尽管新开发的方法，如rboHMM， 现在可以系统地识别数以千计以前被忽视的TORF，这些TURF的生物学相关性 翻译事件仍不清楚。当前提案的目标是评价以下项目的职能相关性 这些新发现的Torf。将对生物学重要性的三个主要方面进行评估。第一，损失 功能影响。TORF缺失对细胞活力和合成适合度的影响 将使用汇集的CRISPR辍学筛选(目标1)来评估特定的编码基因。第二，有能力 编码蛋白质/多肽。TORF产生稳定蛋白质/多肽的能力将进行大规模评估 为检测小ORF翻译产物而设计的光谱研究(目标2)。第三，进化 保护环境。将使用新的比对来仔细评估在这些基因座上的净化选择的强度 根据黑猩猩和恒河猴独立注释的小说Torf创作。完成度 将首次系统地评估新的TORF的生物学相关性。影响 这些新的功能注释的范围可能从为GWASHITS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未来对这一群体的研究方向 以前被忽视的基因组特征。考虑到大量未探索的Torf和前面的示例 在被证明在重要的生物途径中发挥关键作用的被忽视的TORF中，这里的发现将 对基础生物医学研究和翻译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.