Allele-selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-coding and Noncoding RNAs, and RNA Isoforms

Purpose: mRNA translation into protein is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants has yet to be systematically studied. Using high-throughput sequencing (RNA-seq), we have measured cellular levels of mRNAs and ncRNAs, and their isoforms, in lymphoblast cell lines (LCL) and in polysomal fractions, the latter shown to yield strong correlations of mRNAs with expressed protein levels. Analysis of allelic RNA ratios at heterozygous SNPs served to reveal genetic factors in ribosomal loading. Methods: RNA-seq was performed on cytosolic extracts and polysomal fractions (3 ribosomes or more) from three lymphoblastoid cell lines. As each RNA fraction was amplified (NuGen kit), and relative contributions from various RNA classes differed between cytosol and polysomes, the fraction of any given RNA species loaded onto polysomes was difficult to quantitate. Therefore, we focused on relative recovery of the various RNA classes and rank order of single RNAs compared to total RNA. Results: RNA-seq of coding and non-coding RNAs (including microRNAs) in three LCLs revealed significant differences in polysomal loading of individual RNAs and isoforms, and between RNA classes. Moreover, correlated distribution between protein-coding and non-coding RNAs suggests possible interactions between them. Allele-selective RNA recruitment revealed strong genetic influence on polysomal loading for multiple RNAs. Allelic effects can be attributed to generation of different RNA isoforms before polysomal loading or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Several variants and genes identified by this approach are also associated with RNA expression and clinical phenotypes in various databases. Conclusions: These results provide a novel approach using complete transcriptome RNA-seq to study polysomal RNA recruitment and regulatory variants affecting protein translation. Overall design: cells from 3 samples were grown to 5x105 cells/mL density in T75 tissue culture flask and harvested, total RNA and polysome bound RNA was sequenced by Ion Proton

研究目的：mRNA翻译为蛋白质的过程受到严密调控，但mRNA同工型（isoforms）、非编码RNA（noncoding RNAs, ncRNAs）以及遗传变异的系统性研究仍有待开展。本研究借助转录组高通量测序（RNA-seq），对淋巴母细胞样细胞系（lymphoblastoid cell lines, LCL）及其多聚体组分（polysomal fractions）中的mRNA、ncRNA及其同工型的细胞水平进行了定量检测；其中多聚体组分已被证实可反映mRNA与表达蛋白水平间的强相关性。通过分析杂合单核苷酸多态性（single nucleotide polymorphisms, SNPs）位点的等位基因RNA比例，我们得以揭示核糖体加载过程中的遗传调控因子。

研究方法：我们对来自3株淋巴母细胞样细胞系的胞质提取物及多聚体组分（含3个及以上核糖体）进行了RNA-seq测序。由于各RNA组分均经扩增（采用NuGen试剂盒），且胞质与多聚体中各类RNA的相对占比存在差异，因此难以定量测定任一特定RNA物种在多聚体上的加载比例。故此，本研究聚焦于各类RNA的相对回收率，以及单条RNA相较于总RNA的丰度排序。

研究结果：对3株LCL中编码RNA与非编码RNA（包括微小RNA（microRNAs））的RNA-seq分析显示，单个RNA及其同工型的多聚体加载情况存在显著差异，且不同RNA类别间也存在显著区别。此外，编码RNA与非编码RNA的分布模式具有相关性，提示二者间可能存在相互作用。等位基因选择性RNA招募实验表明，多种RNA的多聚体加载过程受到强烈的遗传影响。等位基因效应可归因于多聚体加载前产生的不同RNA同工型，或是直接发生在多聚体加载环节的差异——后者定义了对翻译过程的直接遗传调控效应。本研究通过该方法鉴定出的若干变异与基因，在多个数据库中均与RNA表达及临床表型存在关联。

研究结论：本研究结果提供了一种全新的研究策略，即通过完整转录组RNA-seq来探究多聚体RNA招募过程以及影响蛋白质翻译的调控变异。

总体实验设计：将3份样本的细胞在T75组织培养瓶中培养至密度为5×10^5 cells/mL后收获，分别对总RNA与多聚体结合RNA进行Ion Proton测序。