Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease.
Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease.
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长读蛋白质基因组学，将疾病相关 sQTL 与疾病的蛋白质亚型效应子联系起来。
DOI：
10.1101/2023.03.17.531557
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Sheynkman,GloriaM
中科院分区：
文献类型：
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作者：
Abood,Abdullah;Mesner,LarryD;Jeffery,ErinD;Murali,Mayank;Lehe,Micah;Saquing,Jamie;Farber,CharlesR;Sheynkman,GloriaM
全基因组关联研究（GWASs）鉴定的大部分位点介导选择性剪接，但短读RNA测序（RNA-seq）的技术限制阻碍了机制解释，短读RNA测序不能直接将剪接事件与全长蛋白质同工型联系起来。长读RNA-seq是表征转录异构体的有力工具，最近还用于推断蛋白质异构体的存在。在这里，我们提出了一种在疾病相关模型中整合来自GWASs、剪接定量性状位点（sQTLs）和PacBio长读RNA-seq的信息的方法，以推断sQTLs对其编码的最终蛋白质异构体产物的影响。我们使用骨矿物质密度（BMD） GWAS数据证明了我们方法的实用性。我们从基因型-组织表达（GTEx）项目中鉴定出与BMD相关的732个蛋白编码基因共定位（H4PP≥0.75）的1,863个sqtl。我们生成了人类成骨细胞的PacBio Iso-Seq数据（N= ~ 2200万个全长读取），鉴定出68,326个蛋白质编码异构体，其中17,375个（25%）未注释。通过将这些sqtl投射到蛋白质异构体上，我们将809个sqtl连接到成骨细胞中表达的441个基因的2029个蛋白质异构体上。总的来说，我们发现74个sqtl影响的亚型可能受到无义介导的衰变的影响，190个可能导致未注释的蛋白亚型的表达。最后，我们从功能上验证了共定位sQTLs inTPM2，其中sirna介导的成骨细胞敲除显示出两种otpm2亚型，它们对矿化具有相反的作用，但对整个基因的敲除没有影响。我们的方法应该是推广不同的临床特征，并提供对GWAS基因座调节的蛋白质异构体活性的见解。
A major fraction of loci identified by genome-wide association studies (GWASs) mediate alternative splicing, but mechanistic interpretation is hindered by the technical limitations of short-read RNA sequencing (RNA-seq), which cannot directly link splicing events to full-length protein isoforms. Long-read RNA-seq represents a powerful tool to characterize transcript isoforms, and recently, infer protein isoform existence. Here, we present an approach that integrates information from GWASs, splicing quantitative trait loci (sQTLs), and PacBio long-read RNA-seq in a disease-relevant model to infer the effects of sQTLs on the ultimate protein isoform products they encode. We demonstrate the utility of our approach using bone mineral density (BMD) GWAS data. We identified 1,863 sQTLs from the Genotype-Tissue Expression (GTEx) project in 732 protein-coding genes that colocalized with BMD associations (H4PP ≥ 0.75). We generated PacBio Iso-Seq data (N= ∼22 million full-length reads) on human osteoblasts, identifying 68,326 protein-coding isoforms, of which 17,375 (25%) were unannotated. By casting the sQTLs onto protein isoforms, we connected 809 sQTLs to 2,029 protein isoforms from 441 genes expressed in osteoblasts. Overall, we found that 74 sQTLs influenced isoforms likely impacted by nonsense-mediated decay and 190 that potentially resulted in the expression of unannotated protein isoforms. Finally, we functionally validated colocalizing sQTLs inTPM2, in which siRNA-mediated knockdown in osteoblasts showed twoTPM2isoforms with opposing effects on mineralization but exhibited no effect upon knockdown of the entire gene. Our approach should be to generalize across diverse clinical traits and to provide insights into protein isoform activities modulated by GWAS loci.