Genome-wide transcriptomic and translatomic profiling in halofuginone and vehicle treated mouse fibroblasts. Genome-wide transcriptomic and translatomic profiling in halofuginone and vehicle treated mouse fibroblasts

Purpose: In mammals, glutamyl-prolyl-tRNA synthetase (EPRS) catalyzes the attachment of two amino acids, glutamic acid (E) and proline (P), to their cognate tRNAs for protein synthesis. We aim to study the mechanism of EPRS in translational regulation of cardiac fibrosis and establish EPRS or its downstream target genes as potential anti-fibrosis therapeutic targets. The goals of this study are to use next generation sequencing (NGS)-derived fibroblast transcriptome profiling (RNA-seq) and translatome profiling (polysome-seq) followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods to identify the downstream effector genes that are preferentially regulated by EPRS at the transcriptional and translational levels. Results: Using a well established data analysis workflow from the Genomic Research Center from University of Rochester Medical Center, we analyzed RNA-seq and polysome-seq data. We have identified novel Pro-rich (PRR) gene pathways that are preferentially regulated by EPRS via enhanced translation elongation at Pro-rich codons and antagonized by Halofuginone. Considering the translation active state in both light and heavy polysome fractions, we redefined the 4 groups by overlapping genes from heavy and light polysome associated transcripts compared to their total RNA expression change (p value 5 ribosomes). Total RNA was extracted from the same volume of 3 samples and subjected to next generation RNA deep sequencing for translatome profiling as polysome-seq. For transcriptome profiling, total RNA extracted from the same treated cells was subjected to RNA-Seq. All the extracted RNA samples were treated with DNase I to remove potential genomic DNA followed by purification using phenol:chloroform:isoamyl alcohol for RNA-Seq. Raw reads were generated from the Illumina HiSeq2500 sequencer in the Genomic Research Center from University of Rochester Medical Center.