The ribosomal prolyl-hydroxylase OGFOD1 decreases during cardiac differentiation and modulates translation and splicing
Andrea Stoehr, Leslie Kennedy, Yanqin Yang, Sajni Patel, Yongshun Lin, Kaari L. Linask, Maria Fergusson, Jun Zhu, Marjan Gucek, Jizhong Zou, Elizabeth Murphy
Andrea Stoehr, Leslie Kennedy, Yanqin Yang, Sajni Patel, Yongshun Lin, Kaari L. Linask, Maria Fergusson, Jun Zhu, Marjan Gucek, Jizhong Zou, Elizabeth Murphy
View: Text | PDF
Research Article Cardiology

The ribosomal prolyl-hydroxylase OGFOD1 decreases during cardiac differentiation and modulates translation and splicing

Abstract

The mechanisms regulating translation and splicing are not well understood. We provide insight into a new regulator of translation, 2-oxoglutarate and iron dependent oxygenase domain–containing protein 1 (OGFOD1), which is a prolyl-hydroxylase that catalyzes the posttranslational hydroxylation of Pro62 in the small ribosomal protein S23. We show that deletion of OGFOD1 in an in vitro model of human cardiomyocytes decreases translation of specific proteins (e.g., RNA-binding proteins) and alters splicing. RNA-Seq showed poor correlation between changes in mRNA and protein synthesis, suggesting that posttranscriptional regulation was the primary cause for the observed differences. We found that loss of OGFOD1 and the resultant alterations in protein translation modulated the cardiac proteome, shifting it toward higher protein amounts of sarcomeric proteins, such as cardiac troponins, titin, and cardiac myosin-binding protein C. Furthermore, we found a decrease of OGFOD1 during cardiomyocyte differentiation. These results suggest that loss of OGFOD1 modulates protein translation and splicing, thereby leading to alterations in the cardiac proteome, and highlight the role of altered translation and splicing in regulating the proteome.

Authors

Andrea Stoehr, Leslie Kennedy, Yanqin Yang, Sajni Patel, Yongshun Lin, Kaari L. Linask, Maria Fergusson, Jun Zhu, Marjan Gucek, Jizhong Zou, Elizabeth Murphy

×

Figure 2

Analysis of incorporated heavy amino acid label into WT and OGFOD1-KO iPSC-CMs as a measure of protein synthesis.

Options: View larger image (or click on image) Download as PowerPoint
Analysis of incorporated heavy amino acid label into WT and OGFOD1-KO iP...
Human iPSC-CMs were analyzed 16 days after initiation of the differentiation. Cardiomyocytes were adapted to dialyzed FBS containing light medium (12C6–Lys, 12C614N4–Arg) for 3 days and then switched to heavy label (13C6 l-lysine-2HCl, 13C615N4 l-arginine-HCl). For the analysis of protein stability, samples were harvested 24 hours after media switch. Samples underwent tryptic digestion and mass spectrometry analysis. Quantitative analysis was performed by QUOIL (QUantification withOut Isotope Labeling). (A) Volcano plot analysis for heavy proteins showing basal differences in protein synthesis in KO vs. WT. n = 3 biological samples per group. (B) Protein class analysis was performed for proteins with significant differences in protein synthesis with PANTHER. Protein classes are shown as percentage of total number of identified protein classes. (C) Volcano plot analysis for heavy proteins showing basal differences in protein synthesis in WT Ctr versus dimethyloxalylglycine (DMOG, 1 mM). n = 3 biological samples per group. (D) Venn diagram overlap of proteins with significant differences in protein synthesis in OGFOD1-KO vs. WT and WT ± DMOG. (E) KEGG pathway enrichment analysis was performed with DAVID showing pathways for common proteins with decrease in synthesis. Volcano plots: The horizontal line represents the threshold of P = 0.05; the vertical lines represent the threshold for the log2FC. The x axis depicts the log2 difference in protein synthesis.