An Integrative Approach for the Annotation of Functional smORFs

功能性 smORF 注释的综合方法

基本信息

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
项目类别：
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

项目摘要

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.

项目概要为了有效地开发疾病疗法并增强健康，准确了解需要支持生理学的生物途径。包括不同的基因、蛋白质、小分子底物和信号分子，但此后已经生长包括新的成分，例如非编码 RNA，这些成分改变了人们对这些成分如何发挥作用的理解。最近对许多生物体的转录组和蛋白质组的研究。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 (smORF)，此处定义为包含 <150 个密码子。smORF 的初始特征已显示。然而，它们在发育、新陈代谢和 DNA 修复等关键过程中发挥作用；或者可能还有更多尚未表征的应用程序的目标是注释所有人类 smORF。跨越三种细胞系（目标 1a），探索这些 smORF 在关键通路调节中的参与，包括炎症和胰岛素信号传导（目标 1b），并建立高度可信的互动伙伴选择smORF编码的蛋白质，称为微生物蛋白质，这将有助于未来的功能表征研究（目标 1a）利用 RNA-Seq 组合进行从头转录本组装，全基因组核糖体分析（Ribo-Seq），用于识别翻译的未注释 smORF，并靶向质谱法验证人 HEK293T 细胞、HeLa-S3 宫颈癌细胞、在目标 1b 中，将分析这些新鉴定的 smORF 的变化。已发表的炎症和胰岛素信号传导 RNA-Seq 研究中的 mRNA 表达，以确定 smORF 在相关疾病中发挥作用，例如糖尿病。在目标 2 中，微生物蛋白：蛋白质相互作用。将通过 FLAG 标记的微生物蛋白的免疫沉淀与质谱联用作为研究是指鉴定相关蛋白质复合物作为替代和补充方法。免疫沉淀、微生物蛋白：APEX2 融合也将用于诱导共价附着在初步实验中，2,099 个未注释的 smORF 具有细胞内的微生物蛋白结合伴侣。 Ribo-Seq 在 HEK293T 细胞中鉴定出这些 smORF，其中 50 个在小鼠中是保守的，将有助于制造。考虑到保守基因的可能性，增加了第一批用于相互作用研究的微生物蛋白鉴定出相互作用的蛋白质、直接的微生物蛋白质结合位点和生物活性。合作伙伴将通过丙氨酸扫描诱变和合成苯甲酰基苯丙氨酸来确定- 包含可光交联结合位点探针的实现将实现更大的目标。定义人类基因组的蛋白质编码能力并识别具有关键作用的其他基因在生物学和疾病中的功能。