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 为了有效地开发疾病治疗方法和增强健康， 需要支持生理学的生物途径。这些通路的组成部分最初 包括不同的基因、蛋白质、小分子底物和信号分子，但此后， 包括新的成分，如非编码RNA，这已经改变了对这些基因是如何表达的理解。 路径功能。最近对许多生物体的转录组和蛋白质组的研究表明， 揭示了另一个以前被忽视的新成分--蛋白质编码小开放阅读 框架（smORFs），这里定义为含有<150个密码子。smORF的初步表征表明， 它们在发育、代谢和DNA修复等关键过程中发挥作用;然而，数百种 或者可能更多的人仍然没有特征。此应用程序的目标是注释所有人类smORF 在三种细胞系中（Aim 1a），探索这些smORF参与调控关键途径， 包括炎症和胰岛素信号传导（Aim 1b），并建立高置信度的相互作用伙伴， 选择smORF编码的蛋白质，称为微蛋白，这将有助于未来的功能 特征研究（目标2）。目的1a利用RNA-Seq的组合进行从头转录物组装， 全基因组核糖体分析，或Ribo-Seq，以鉴定翻译的未注释的smORF，并靶向 质谱法验证人HEK 293 T细胞、HeLa-S3宫颈癌细胞中的候选smORF， 和GM 12878 B-淋巴母细胞样细胞。在目标1b中，将分析这些新发现的smORF的变化 在已发表的炎症和胰岛素信号传导的RNA-Seq研究中， smORF在相关疾病如糖尿病中发挥作用。在目标2中，微蛋白：蛋白质相互作用 将通过FLAG标记的微蛋白的免疫沉淀结合质谱法进行研究， 识别相关蛋白复合物的方法。作为一种替代和补充方法， 免疫沉淀，微蛋白：APEX 2融合也将用于诱导共价连接， 微蛋白结合伴侣细胞内。在初步实验中，2，099个未注释的smORF具有 在HEK 293 T细胞中通过Ribo-Seq鉴定。在这些smORF中，有50个在小鼠中是保守的， 考虑到保守基因可能被克隆的可能性， 生物活性。在鉴定了相互作用的蛋白质之后，直接的微蛋白结合位点和 配偶体将通过丙氨酸扫描诱变和合成的苯甲酰苯丙氨酸- 含有可光交联的结合位点探针。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，并确定其他基因与关键 在生物学和疾病中的作用。