Pluto Bioinformatics

GSE83493: hnRNP C is a key regulator of protein synthesis in mitosis

Bulk RNA sequencing

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 proteincoding transcripts (plus the five human rRNA transcripts) using bowtie2. Expression estimates were further normalized using quantile normalization. qRTPCR validation was performed using SYBR Green assays.; Results: RNAseq and Ribo/RFseq 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. SOURCE: Ran Elkon Tel Aviv University