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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：
9548692
负责人：
SUSAN E CELNIKER
金额：
$ 100.25万
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/9548692
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- 100 aa）是重要的生理、发育和代谢调节因子，但其由于大小和丰度，检测是困难的。令人震惊的30%的注释人类smORF基因包括疾病相关的变异定位在外显子内，而一般人类基因的15%。此外，本发明还许多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这些超保守的功能性smORF基因我们称之为保守smORF目录或 CSC。这些基因在超过500万年的进化过程中一直是保守的，然而我们几乎知道，完全不了解它们的功能。经过世纪的基因分析，果蝇是后生动物中功能注释最完整的。功能来自果蝇的注释有助于基于假设的药物开发，三十多年来，以及最近，已经使数百个SNP的生物学解释成为可能全基因组关联研究（GWAS）。因此，在fly for中派生的函数注释保守基因可转移到人类，并具有直接的临床相关性。值得注意的是，果蝇中的smORF已被功能性地研究，或被实验验证为产生肽。一基因组工程，计算，分子和功能研究的组合将用于系统、全面地描述了CSC，代表了第一个基因组尺度任何生物体中smORFs的表征提供了关于这类研究很少的蛋白质。总之，我们将描述和功能注释~400保守使用CRISPR敲除的smORF，随后进行表型分析和拯救测定。我们将评估的突变体，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：第一，通过插入整个缺失的RNA;第二，通过添加终止密码子去除smORF的RNA版本;最后，使用微- 构建体仅含有smORF和内源启动子。我们会提供直接证据使用标记的表达分析和靶向MS/MS进行翻译，以扫描所述蛋白质中的预测多肽。整个胚胎和组织解剖样本。除了验证预测分子的存在外，该数据集将为进一步开发计算工具提供基础黄金标准。功能性多肽的预测。这些研究的目的是为了了解基本的生命这一进程为促进更好的人类健康奠定基础。