An Integrative Approach for the Annotation of Functional smORFs

功能性 smORF 注释的综合方法

• 批准号: 9329058
• 负责人: Thomas Farid Martinez
• 金额: $ 5.67万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9329058
• 关键词: Alanine; Binding; Binding Sites; Biochemistry; Biological; Biology; Cell Line; Cells; Cervix carcinoma; Code; Codon Nucleotides; Coupled; Critical Pathways; DNA Repair; Data; Data Set; Development; Diabetes Mellitus; Disease; Future; Gene Proteins; Genes; Genetic Transcription; Genome; Goals; HeLa S3; Health; Human; Human Biology; Human Cell Line; Human Genome; Immunoprecipitation; Inflammation; Insulin Signaling Pathway; Investigation; Knowledge; Malignant Epithelial Cell; Mass Spectrum Analysis; Mediating; Metabolism; Methods; Mus; Mutagenesis; Open Reading Frames; Organism; Pathway interactions; Phenylalanine; Physiological; Physiology; Play; Prevention; Process; Proteins; Proteome; Proteomics; Publishing; Regulation; Role; Scanning; Signaling Molecule; Testing; Transcript; Translating; Untranslated RNA; Validation; base; disease diagnosis; experimental study; genome annotation; genome-wide; human disease; improved; insight; insulin signaling; mRNA Expression; protein complex; ribosome profiling; small molecule; therapy development; transcriptome; transcriptome sequencing

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.

项目摘要 为了有效发展疾病疗法并改善健康，对 需要基础生理的生物学途径。这些途径的组成部分最初是 包括不同的基因，蛋白质，小分子底物和信号分子，但此后已生长 包括包括新组件（例如非编码RNA），这些组件改变了对这些方式的理解 途径功能。许多生物体的转录组和蛋白质组的最新投资已有 揭示了以前被忽略的另一个新组件 - 蛋白编码小的开放阅读 帧（SMORF），在此定义为包含<150个密码子。 Smorfs的初始表征已显示 它们在诸如发展，代谢和DNA修复等关键过程中发挥作用；但是，数百个 或更多可能仍然没有特征。该应用的目标是注释所有人类smorfs 在三个细胞系（AIM 1A）中，探索这些SMORF的参与临界途径的调节， 包括炎症和胰岛素信号传导（AIM 1B），并建立高信任的互动伙伴 选定的SMORF编码蛋白，称为微蛋白，这将有助于将来的功能 表征研究（目标2）。 AIM 1A利用RNA-Seq组合从头转录组件， 全基因组核糖体分析或核糖表，以识别翻译的未经通知的smorfs并靶向 质谱法以验证人HEK293T细胞中的候选smorfs，HeLa-S3宫颈癌细胞， 和GM12878 B蛋白母细胞细胞。在AIM 1B中，将分析这些新鉴定的Smorfs以进行更改 在发表的炎症和胰岛素信号的RNA-seq研究中，mRNA表达以确定 Smorfs在相关疾病（例如糖尿病）中发挥作用。在AIM 2中，微蛋白：蛋白质相互作用 将通过对质谱耦合的标志标记的微蛋白免疫沉淀作为一个 鉴定相关蛋白质复合物的手段。作为一种替代和补充方法 免疫沉淀，微蛋白：APEX2融合也将用于诱导共价附着 微蛋白结合伴侣细胞内。在初步实验中，有2,099个未经通知的smorfs具有 在HEK293T细胞中通过Ribo-Seq鉴定。在这些smorf中，有50个是在小鼠中保守的，将有助于使 鉴于保守基因的可能性是 生物活性。鉴定相互作用蛋白质，直接微蛋白结合位点和 伴侣将通过丙氨酸扫描诱变和合成苯甲酸苯丙氨酸 - 包含可光叠链接结合位点探针。实现这些目标将实现更大的目标 定义人类基因组的蛋白质编码能力并鉴定具有关键的其他基因 生物学和疾病的功能。