Allele-Selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-Coding and Noncoding RNAs, and RNA Isoforms.
Allele-Selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-Coding and Noncoding RNAs, and RNA Isoforms.
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DOI：
10.1371/journal.pone.0136798
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Sadee W
中科院分区：
文献类型：
--
作者：
Mascarenhas R;Pietrzak M;Smith RM;Webb A;Wang D;Papp AC;Pinsonneault JK;Seweryn M;Rempala G;Sadee W
mRNA翻译成蛋白质受到高度调控，但mRNA亚型、非编码RNA（ncRNA）和遗传变异体的作用仍知之甚少。已显示多聚核糖体上的mRNA水平与表达的蛋白质水平良好相关，指出多聚核糖体负载是一个关键因素。为了研究蛋白质翻译的调节和遗传因素，我们测量了淋巴母细胞系（LCL）和多聚体组分中mRNA和ncRNA（包括microRNA）的水平和等位基因比率。我们首先使用靶向测定来测量mRNA等位基因的多核糖体加载，证实了所报道的对OPRM 1和NAT 1翻译的遗传效应，并且检测到ABCB 1（MDR 1）中的rs 1045642（3435 C>T）对多核糖体加载没有影响，同时支持了先前显示3435 T等位基因的mRNA周转增加的结果。在三个LCL中使用完整转录谱的高通量测序（RNA-Seq）揭示了个体RNA类别和亚型的多聚体加载的显著差异。蛋白质编码和非编码RNA之间的相关多聚体分布表明它们之间存在相互作用。等位基因选择性多聚核糖体招聘显示出强烈的遗传影响，多种RNA，归因于RNA异构体的差异表达或差异加载到多聚核糖体，后者定义了翻译的直接遗传效应。通过胞质溶胶和多核糖体之间不同等位基因RNA比率鉴定的基因富集了影响RNA功能的已发表表达数量性状位点（eQTL），并与临床表型相关。Polysomal RNA-Seq结合等位基因比率分析提供了一种强有力的方法来研究影响蛋白质翻译的多核糖体RNA募集和调控变体。
mRNA translation into proteins is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants remains poorly understood. mRNA levels on polysomes have been shown to correlate well with expressed protein levels, pointing to polysomal loading as a critical factor. To study regulation and genetic factors of protein translation we measured levels and allelic ratios of mRNAs and ncRNAs (including microRNAs) in lymphoblast cell lines (LCL) and in polysomal fractions. We first used targeted assays to measure polysomal loading of mRNA alleles, confirming reported genetic effects on translation of OPRM1 and NAT1, and detecting no effect of rs1045642 (3435C>T) in ABCB1 (MDR1) on polysomal loading while supporting previous results showing increased mRNA turnover of the 3435T allele. Use of high-throughput sequencing of complete transcript profiles (RNA-Seq) in three LCLs revealed significant differences in polysomal loading of individual RNA classes and isoforms. Correlated polysomal distribution between protein-coding and non-coding RNAs suggests interactions between them. Allele-selective polysome recruitment revealed strong genetic influence for multiple RNAs, attributable either to differential expression of RNA isoforms or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Genes identified by different allelic RNA ratios between cytosol and polysomes were enriched with published expression quantitative trait loci (eQTLs) affecting RNA functions, and associations with clinical phenotypes. Polysomal RNA-Seq combined with allelic ratio analysis provides a powerful approach to study polysomal RNA recruitment and regulatory variants affecting protein translation.