An Integrative Approach for the Annotation of Functional smORFs

项目摘要 为了有效地开发疾病的治疗方法，增进健康，准确地理解 作为生理学基础的生物途径是必需的。这些路径的组件最初是 包括不同的基因、蛋白质、小分子底物和信号分子，但自那以来一直在增长 包括新的组件，如非编码RNA，这些组件改变了对这些的理解 通路起作用了。最近对许多生物体的转录本和蛋白质组的研究已经 揭示了另一个以前被忽视的新成分--蛋白质编码的小开放阅读 框架(SmORF)，这里定义为包含&lt；150个密码子。SmORF的初步表征显示 它们在发育、新陈代谢和DNA修复等关键过程中发挥作用；然而，数百 或者，可能还有更多的人没有特征。这个应用程序的目标是注释所有人类smORF 跨越三个细胞系(目标1a)，探索这些smORF参与关键途径的调控， 包括炎症和胰岛素信号转导(目标1b)，并建立高度信任的相互作用伙伴 选定的smORF编码的蛋白质，称为微蛋白，将有助于未来的功能 特征研究(目标2)。AIM 1a利用RNA-Seq的组合进行从头转录组装， 全基因组核糖体图谱，或Ribo-Seq，以识别翻译的、未注释的smORF和靶向 人HEK293T细胞、HeLa-S3宫颈癌细胞中候选smORF的质谱学验证 和GM12878 B淋巴母细胞。在目标1b中，将分析这些新发现的smORF的变化 在已发表的关于炎症和胰岛素信号转导的RNA-Seq研究中确定 这些单链ORF在糖尿病等相关疾病中发挥作用。在目标2中，微蛋白：蛋白质相互作用 将通过免疫沉淀FLAG标记的微蛋白连接到质谱学进行研究 识别相关蛋白质复合体的方法。作为替代和补充方法 免疫沉淀，微蛋白：APEX2融合也将被用来诱导共价结合 微蛋白在细胞内结合伙伴。在初步实验中，2099个未注释的smORF具有 经Ribo-Seq在HEK293T细胞中鉴定。在这些smORF中，有50个在小鼠体内保守，将有助于 增加第一批用于相互作用研究的微蛋白，考虑到保守基因可能是 生物活性的。在鉴定了相互作用的蛋白质之后，直接的微蛋白结合部位和 合作伙伴将通过丙氨酸扫描突变和合成的苯甲酰苯丙氨酸- 含有可光交联性结合位点探针。实现这些目标将实现更大的目标 确定人类基因组的蛋白质编码能力，并确定其他关键基因 在生物学和疾病中的作用。

Project Summary In order to effectively develop therapies for disease and enhance health, an accurate understanding of the biological pathways that underpin physiology is required. The components of these pathways originally included different genes, proteins, small molecule substrates, and signaling molecules, but have since grown to include new components, such as non-coding RNAs, that have changed the understanding of how these pathways function. Recent investigations of transcriptomes and proteomes across many organisms have revealed yet another new component that was previously overlooked—protein-coding small open reading frames (smORFs), defined here as containing <150 codons. Initial characterization of smORFs has shown them to function in critical processes such as development, metabolism, and DNA repair; however, hundreds or possibly more remain uncharacterized. The goals of this application are to annotate all human smORFs across three cell lines (Aim 1a), explore these smORFs' involvement in the regulation of critical pathways, including inflammation and insulin signaling (Aim 1b), and to establish high confidence interacting partners of selected smORF-encoded proteins, referred to as microproteins, which will aid in future functional characterization studies (Aim 2). Aim 1a utilizes a combination of RNA-Seq for de novo transcript assembly, genome-wide ribosome profiling, or Ribo-Seq, to identify translated non-annotated smORFs, and targeted mass spectrometry to validate candidate smORFs in human HEK293T cells, HeLa-S3 cervical carcinoma cells, and GM12878 B-lymphoblastoid cells. In Aim 1b, these newly identified smORFs will be analyzed for changes in mRNA expression across published RNA-Seq studies of inflammation and insulin signaling to determine which smORFs play a role in associated diseases, such as diabetes. In Aim 2, microprotein:protein interactions will be investigated by immunoprecipitation of FLAG-tagged microproteins coupled to mass spectrometry as a means to identify associated protein complexes. As an alternative and complementary method to immunoprecipitation, microprotein:APEX2 fusions will also be used to induce covalent attachment to microprotein binding partners intracellularly. In preliminary experiments, 2,099 non-annotated smORFs have been identified by Ribo-Seq in HEK293T cells. Of these smORFs, 50 are conserved in mice and will help make up the initial batch of microproteins for interaction studies, given the likelihood of conserved genes to be biologically active. Following identification of interacting proteins, the direct microprotein binding sites and partners will be determined by alanine scanning mutagenesis and a synthetic benzoyl phenylalanine- containing photocrosslinkable binding site probe. Achieving these objectives will accomplish the larger goal of defining the protein-coding capacity of the human genome and identifying additional genes with critical functions in biology and disease.