hnRNP C is a key regulator of protein synthesis in mitosis

Purpose: The goal of this study is to characterize the role of hnRNP C in translation regulation of a specific set of mRNAs during mitosis. Methods: mitotic HeLa S3 cells expressing a Dox-inducible shRNA against hnRNP C were used and profiles of total mRNAs and ribosome footprints (RFs) of either untreated (wild type) cells or hnRNP C-knockdown cells were generated by deep sequencing, using Illumina HiSeq 2500. The sequence reads that passed quality filters were aligned to a reference set of human curated protein-coding transcripts (plus the five human rRNA transcripts) using bowtie2. Expression estimates were further normalized using quantile normalization. qRT–PCR validation was performed using SYBR Green assays. Results: RNA-seq and Ribo/RF-seq datasets were combined according to gene ID. Only genes with expression level of at least 1.0 RPKM in both datasets were included in subsequent analyses. The combined dataset includes 10,996 genes. Transcripts encoding components of the translation machinery (Gene Ontology Biological Process [GOBP] “Translation” annotation) show reduced ribosome occupancy during mitosis under hnRNP C knockdown (p-value = 1.07 x 10-13) although their total mRNA levels are increased (p-value = 1.78 x 10-5). Translation efficiency (TE) calculations of ribosome occupancy per mRNA level showed a statistically-significant reduction for mRNAs encoding ribosomal proteins (Gene Ontology Cellular Compartment [GOCC] “Ribosome” annotation; p-value = 9.92 x 10-12). In particular, the TE of 5' TOP-containing mRNAs was found to be significantly reduced under hnRNP C knockdown (p-value = 5.16 x 10-4). Conclusions: Our study represents the first detailed analysis of the translation effeciency of mRNAs in wild type and hnRNP C knockdown HeLa S3 cells during mitosis using RNA-Seq and Ribo-Seq. Our results show that the translation of a specific subset of mRNAs (5' TOP mRNAs) is regulated by hnRNP C in mitosis. Overall design: Profiles of total mRNAs and RFs of either wild type or hnRNP C knockdown cells in mitosis were generated by deep sequencing Illumina HighSeq 2500.

研究目的：本研究旨在阐明异质核核糖核蛋白C（heterogeneous nuclear ribonucleoprotein C, hnRNP C）在有丝分裂过程中对特定信使RNA（mRNA）群体的翻译调控作用。
实验方法：本研究采用表达多西环素诱导型抗hnRNP C短发夹RNA（short hairpin RNA, shRNA）的有丝分裂HeLa S3细胞；使用Illumina HiSeq 2500测序平台，通过深度测序获取未处理（野生型）细胞及hnRNP C敲低细胞的总mRNA与核糖体足迹（ribosome footprints, RFs）谱。将通过质量过滤的序列读段利用bowtie2比对至人类注释编码蛋白转录本参考集（包含5个人类核糖体RNA转录本），随后采用分位数归一化对表达量进行标准化校正。采用SYBR Green染料法完成定量实时聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）验证。
实验结果：将RNA测序（RNA-seq）与核糖体足迹测序（Ribo/RF-seq）数据集按基因ID进行整合。仅保留两个数据集中共表达量至少为1.0 RPKM的基因用于后续分析，最终整合数据集包含10996个基因。翻译机器组分编码转录本（注释为基因本体生物过程（Gene Ontology Biological Process, GOBP）“Translation”）在hnRNP C敲低的有丝分裂细胞中核糖体占用率降低（p值=1.07×10^-13），但其总mRNA水平却升高（p值=1.78×10^-5）。基于每mRNA分子核糖体占用率计算的翻译效率（translation efficiency, TE）显示，编码核糖体蛋白的mRNA（注释为基因本体细胞组分（Gene Ontology Cellular Compartment, GOCC）“Ribosome”）的翻译效率存在统计学显著降低（p值=9.92×10^-12）。尤为关键的是，携带5'端寡聚嘧啶序列（5'-terminal oligopyrimidine, 5' TOP）的mRNA的翻译效率在hnRNP C敲低条件下显著降低（p值=5.16×10^-4）。
研究结论：本研究首次通过RNA-seq与Ribo-seq技术，对有丝分裂期野生型及hnRNP C敲低的HeLa S3细胞中的mRNA翻译效率开展了系统性详细分析。研究结果证实，特定mRNA亚群（5' TOP mRNA）的翻译过程在有丝分裂期间受hnRNP C调控。
整体实验设计：采用Illumina HiSeq 2500深度测序，获取有丝分裂期野生型及hnRNP C敲低细胞的总mRNA与RFs谱。