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| Status |
Public on Sep 09, 2021 |
| Title |
Genome-wide transcriptomic and translatomic profiling in halofuginone and vehicle treated mouse fibroblasts |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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 <0.05 for differential expressed genes). 1371 genes were downregulated at translation (indicated by polysome-seq) and steady-state mRNA (indicated by RNA-seq) levels, and 775 genes were upregulated at both levels. There are a small number of genes whose gene regulation was changed in the opposite direction at the mRNA and translation levels. These observations indicate a coordinated regulatory effect between steady-state mRNA level and translation efficiency under the EPRS inhibitory condition. KEGG analysis of the genes that are reduced at RNA and translation levels revealed multiple pro-fibrotic pathways, including ECM-receptor interaction and proteoglycans. In contrast, the genes that are induced at RNA and translation levels are enriched in general translation factors, including ribosome and aminoacyl-tRNA biosynthesis, which suggests a compensatory amino acid starvation response upon EPRS inhibition. Altered expression of typical Pro-rich collagen and other genes was confirmed with qRT-PCR.
Conclusions: Our study represents the first detailed analysis of fibroblast translatome in comparison with transcriptome under EPRS inhibitory condition (halofuginone treatment), with biological triplicates, generated by RNA-seq and polysome-seq technology. Our results show that the next generation sequencing offers a comprehensive qualitative and quantitative evaluation of mRNA content and its translation state within cells treated with translation inhibitory drug halofuginone (EPRS inhibitor). Based on our results, we conclude that Pro-rich motif bearing genes are preferential targets of EPRS, and mild inhibition of EPRS leads to inefficient translation of these PRR genes.
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| Overall design |
We treated NIH/3T3 mouse fibroblasts with halofuginone (EPRS-specific inhibitor; 100 nM) or vehicle (PBS) in biological triplicates. For polysome-Seq, all the fractions were pooled into 3 samples, polysome free (free mRNP, 40S, 60S subunit), light polysomes (monosome, disome, trisome, etc.), and heavy polysomes (>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.
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| Contributor(s) |
Wu J, Venkata Subbaiah KC, Xie LH, Jiang F, Mickelsen D, Myers JR, Wilson Tang WH, Yao P |
| Citation(s) |
32611237 |
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| Submission date |
Sep 04, 2019 |
| Last update date |
Sep 09, 2021 |
| Contact name |
Peng Yao |
| E-mail(s) |
[email protected]
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| Phone |
2166507876
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| Organization name |
Aab Cardiovascular Research Institute
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| Department |
Medicine
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| Lab |
Peng Yao lab
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| Street address |
601 Elmwood Avenue
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| City |
Rochester |
| State/province |
NY |
| ZIP/Postal code |
14534 |
| Country |
USA |
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| Platforms (1) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
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| Samples (24)
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| Relations |
| BioProject |
PRJNA563844 |
| SRA |
SRP220286 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE136838_deSeq2_counts.txt.gz |
952.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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