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. 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

研究背景：mRNA翻译为蛋白质的过程受到严格调控，但mRNA异构体、非编码RNA（noncoding RNAs, ncRNAs）以及遗传变异的作用尚未得到系统研究。本研究借助高通量测序（high-throughput sequencing, RNA-seq）技术，对淋巴母细胞系（lymphoblast cell lines, LCL）及多聚核糖体组分（polysomal fractions）中的mRNA、ncRNA及其异构体的细胞水平进行了检测；其中多聚核糖体组分已被证实可使mRNA与表达的蛋白质水平呈现强相关性。通过分析杂合单核苷酸多态性（single nucleotide polymorphism, SNPs）位点的等位基因RNA比值，可揭示核糖体装载过程中的遗传调控因子。

实验方法：对3株淋巴母细胞样细胞系的胞浆提取物及多聚核糖体组分（结合3个及以上核糖体的组分）开展RNA测序。由于各RNA组分均通过NuGen试剂盒（NuGen kit）进行扩增，且胞浆与多聚核糖体中各类RNA的相对占比存在差异，因此难以定量某一特定RNA物种在多聚核糖体上的装载比例。据此，本研究聚焦于各类RNA的相对回收量，以及与总RNA相比的单RNA丰度排序。

实验结果：对3株淋巴母细胞系中编码RNA与非编码RNA（含微小RNA，microRNAs）进行RNA测序后发现，单个RNA及其异构体的多聚核糖体装载情况、以及不同RNA类别之间，均存在显著差异。此外，编码蛋白RNA与非编码RNA的分布呈现相关性，提示二者之间可能存在相互作用。等位基因选择性RNA招募实验显示，遗传因素对多种RNA的多聚核糖体装载具有显著影响。等位基因效应可归因于多聚核糖体装载前产生的不同RNA异构体，或是多聚核糖体上的差异化装载，后者定义了对翻译过程的直接遗传调控效应。通过本研究方法鉴定出的若干变异与基因，在多个公共数据库中均与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基于完整转录组RNA测序的全新方法，用于研究多聚核糖体RNA招募及影响蛋白质翻译的调控变异。本研究将3份样本的细胞在T75组织培养瓶中培养至5×10^5个细胞/mL的密度后收获，通过Ion Proton测序平台对总RNA及结合多聚核糖体的RNA进行了测序。