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.

项目摘要短肽（10-100AA）是生理，发育和代谢的重要调节剂，但是它们由于大小和丰度，很难检测。令人惊叹的30％注释的人Smorf基因包括疾病相关的变体映射在外显子内，而通常的人类基因的15％。更远，从无脊椎动物到脊椎动物的整个后代系统发育中，许多smorf是保守的包括男人。这些超保存的功能性SMORF基因，我们称为保守的SMORF目录或 CSC。这些基因在500mr的进化中得到了保存，但我们几乎知道对他们的功能一无所有。由于一个世纪的遗传分析，模型生物的基因组果蝇中，果蝇在后生动物中具有最完整的功能注释。功能从果蝇中得出的注释已经在基于假设的药物开发中发挥了作用超过三十年，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，fly的功能注释保守基因可转移到人类，并且具有直接的临床相关性。值得注意的是，不到10％果蝇中的SMORF已在功能上进行了研究，或者在实验上证实了为产生肽。一个基因组工程，计算，分子和功能研究的组合将用于系统，全面地表征CSC，代表第一个基因组规模在任何有机体中的smorf的表征，提供有关生物学功能的大量信息这种研究不佳的蛋白质类别。总共，我们将表征并在功能上注释〜400保守 Smorfs使用CRISPR淘汰赛，然后进行表型和救援测定法。我们将评估表型突变体的生存能力，形态，生育和生育，寿命，代谢（糖和脂质）水平）和许多行为表型。对于具有健壮表型的Smorfs，我们将尝试以三种方式营救这些突变体的子集：首先，插入整个已删除的RNA；第二，与加法终止密码子删除了带有SMORF的RNA的版本；最后，使用微型仅包含smorf和内源启动子的构造。我们将为直接证据使用标记的表达分析和针对MS/MS进行翻译以扫描预测的多肽整个胚胎和组织解剖样品。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的工具提供基本的黄金标准功能菌根的预测。这些研究是针对理解基本生活的过程并为促进更好的人类健康奠定基础。