Systematic, Genome-Scale Functional Characterization of Conserved smORFs

保守 smORF 的系统、基因组规模功能表征

基本信息

批准号：
9228843
负责人：
SUSAN E CELNIKER
金额：
$ 100.27万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
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：
2017-09-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9228843
关键词：
Adipose tissue Alzheimer&apos s Disease Animal Model Animals Arthropods Autoimmune Diseases Behavior Behavioral Biological Biological Assay Biological Process CRISPR/Cas technology Catalogs Clustered Regularly Interspaced Short Palindromic Repeats Code Codon Nucleotides Collection Computer Analysis Data Data Set Detection Development Disease Dissection Drosophila genus Drosophila melanogaster Drug Targeting Evolution Exons Fertility Foundations Frameshift Mutation Gene Transfer Genes Genetic Transcription Genome Genome engineering Gold Health Human Human Genome Image In Situ Invertebrates Knock-out Life Lipids Literature Longevity Machine Learning Malignant Neoplasms Maps Mass Spectrum Analysis Measures Messenger RNA Metabolism Methods Molecular Morphology Muscle National Human Genome Research Institute Nerve Degeneration Nervous system structure Neurodegenerative Disorders Neurotransmitters Ontology Open Reading Frames Organism Peptides Phenotype Phylogeny Physiology Play Process Proteins Proteomics RNA Reproducibility Research Personnel Resources Role Sampling Scanning System Technology Terminator Codon Time Tissues Translating Translations Variant Vertebrates adipokines base clinically relevant computerized tools developmental disease drug development drug resource embryo tissue fly gene function genetic analysis genome annotation genome wide association study genome-wide human disease in situ imaging insight knock-down man mutant novel overexpression polypeptide promoter ribosome profiling sugar tool tool development translational genomics virtual

项目摘要

PROJECT SUMMARY Short peptides (10-100aa) are important regulators of physiology, development and metabolism, however their detection is difficult due to size and abundance. A stunning 30% of annotated human smORF genes include disease-associated variants mapped within exons, compared to 15% of human genes in general. Further, many smORFs are conserved across the entire metazoan phylogeny from invertebrates to vertebrates including man. These ultra-conserved functional smORF genes we call the Conserved smORF Catalog or CSC. These genes have been conserved across more than 500myr of evolution, and yet we know almost nothing at all about their functions. Due to a century of genetic analysis, the genome of the model organism Drosophila melanogaster has the most complete functional annotation among metazoans. Functional annotations derived from Drosophila have been instrumental in hypothesis-based drug development for more than thirty years, and more recently have made possible the biological interpretation of hundreds of SNPs detected in genome-wide association studies (GWAS). Hence, functional annotations derived in fly for conserved genes are transferable to human and are of direct clinical relevance. Remarkably, less than 10% of smORFs in Drosophila have been studied functionally, or experimentally verified as generating peptides. A combination of genome engineering, computational, molecular, and functional studies will be used to systematically and comprehensively characterize the CSC, representing the first genome-scale characterization of smORFs in any organism providing a wealth of information on the biological functions of this poorly studied class of proteins. In total, we will characterize and functionally annotate ~400 conserved smORFs using CRISPR knockout followed by phenotyping and rescue assays. We will assess the phenotypes of the mutants, measuring viability, morphology, fecundity and fertility, lifespan, metabolism (sugar and lipid levels), and a number of behavioral phenotypes. For smORFs with robust phenotypes, we will then attempt to rescue a subset of these mutants in three ways: first, by inserting the whole deleted RNA; second, with a version of the RNA with the smORF(s) removed by the addition a stop codon; and lastly, using a micro- construct containing only the smORF and the endogenous promoter. We will generate direct evidence for translation using tagged expression analysis and targeted MS/MS to scan for predicted polypeptides in the whole embryo and tissue dissection samples. In addition to validating the existence of the predicted molecules, this dataset will provide a foundational gold standard for further development of tools for the computational prediction of functional micropeptides. These studies are directed toward the understanding of basic life processes and lay the foundation for promoting better human health.

项目概要短肽（10-100aa）是生理、发育和代谢的重要调节剂，但是它们的由于大小和丰度，检测很困难。令人震惊的 30% 带注释的人类 smORF 基因包括与疾病相关的变异位于外显子内，而一般人类基因的变异为 15%。更远，许多 smORF 在从无脊椎动物到脊椎动物的整个后生动物系统发育中都是保守的包括男人。这些超保守的功能性 smORF 基因我们称为保守 smORF 目录或国家安全委员会。这些基因在超过 500 迈尔的进化过程中得到了保守，但我们几乎知道关于它们的功能一无所知。经过一个世纪的遗传分析，模式生物的基因组果蝇在后生动物中具有最完整的功能注释。功能性来自果蝇的注释在基于假设的药物开发中发挥了重要作用三十多年来，最近使数百个 SNP 的生物学解释成为可能在全基因组关联研究（GWAS）中检测到。因此，在飞行中派生的功能注释保守基因可转移至人类并具有直接的临床相关性。值得注意的是，不到 10% 果蝇中的 smORF 已被进行功能研究，或通过实验验证其可产生肽。一个基因组工程、计算、分子和功能研究的结合将用于系统、全面地描述了 CSC 的特征，代表了第一个基因组规模任何生物体中 smORF 的表征，提供有关其生物学功能的丰富信息这一类蛋白质的研究很少。总共，我们将表征和功能注释约 400 个保守的 smORF 使用 CRISPR 敲除，然后进行表型分析和救援分析。我们将评估表型突变体，测量活力、形态、繁殖力和生育力、寿命、代谢（糖和脂质）水平）和许多行为表型。对于具有稳健表型的 smORF，我们将尝试通过三种方式拯救这些突变体的子集：首先，插入整个删除的RNA；第二，用一个通过添加终止密码子去除 smORF 的 RNA 版本；最后，使用微型仅包含 smORF 和内源启动子的构建体。我们将生成直接证据使用标记表达分析和靶向 MS/MS 扫描预测的多肽进行翻译整个胚胎和组织解剖样本。除了验证预测分子的存在之外，该数据集将为进一步开发计算工具提供基本的黄金标准功能性微肽的预测。这些研究旨在了解基本生活过程并为促进更好的人类健康奠定基础。