Systematic, Genome-Scale Functional Characterization of Conserved smORFs

项目总结 短肽(10-100aa)是重要的生理、发育和代谢调节因子。 由于大小和数量丰富，检测很困难。30%的带注释的人类smORF基因包括 与疾病相关的变异被定位在外显子内，相比之下，人类基因的总体比例为15%。此外， 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。 包括人类。这些超保守的功能smORF基因我们称之为保守的smORF目录或 CSC.这些基因在500多万年的进化过程中一直是保守的，然而我们知道几乎 对它们的功能一无所知。由于一个世纪的遗传分析，模式生物的基因组 在后生动物中，果蝇拥有最完整的功能注释。功能性 来自果蝇的注释在基于假说的药物开发中发挥了重要作用 三十多年来，最近使数百个SNPs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Fly for中派生的函数批注 保守的基因可以移植到人类身上，并具有直接的临床意义。值得注意的是，只有不到10%的 果蝇中的单链ORFs已被从功能上研究，或被实验证实为生成肽。一个 基因组工程、计算、分子和功能研究的结合将用于 系统和全面地描述了CSC，这是第一个基因组规模 任何生物体中单链ORFs的特征，提供了关于SmORFs生物学功能的丰富信息 这类研究很少的蛋白质。总而言之，我们将描述和功能注释~400保守 使用CRISPR基因敲除技术进行smORFs扩增，然后进行表型鉴定和补救试验。我们将评估表型 测量活力、形态、繁殖力和生育力、寿命、新陈代谢(糖和脂) 水平)，以及一些行为表型。对于具有健壮表型的smORF，我们将尝试 通过三种方式挽救这些突变体的子集：第一，插入整个删除的RNA；第二，通过 通过增加一个停止密码子去除了smORF(S)的rna版本；最后，使用了一个微型 仅包含smORF和内源启动子的构建。我们将产生直接证据证明 使用标签表达分析和靶向MS/MS进行翻译以扫描预测的多肽 完整的胚胎和组织解剖样本。除了确认预测分子的存在外， 该数据集将为进一步开发用于计算的工具提供基本的黄金标准 功能微肽的预测。这些研究的目的是为了理解基本生活 并为促进更好的人类健康奠定基础。

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.