Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M.; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H.; Lin, Yongshun; Guček, Marjan; Jun, Zhu; Murphy, Elizabeth

doi:10.1093/cvr/cvw081

Cited by 31 publications

(27 citation statements)

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“…One of the alternatively spliced genes was cytosolic purine 5′-nucleotidase (NT5C2), which is shown in the Sashimi plot analysis and was verified with quantitative PCR analysis ( Figure 5, A and B). Interestingly, we previously found that DMOG increased exon skipping of NT5C2, which is involved in the NAD salvage pathway (17). Here, we found that the effects of DMOG on exon skipping were abolished for NT5C2 in OGFOD1-KO, which would be consistent with a role for OGFOD1 in mediating these effects on exon skipping.…”

Section: Resultssupporting

confidence: 87%

“…Because OGFOD1 is known to influence translation (10), we hypothesized that there might be differences in protein synthesis in OGFOD1-KO versus WT cardiomyocytes. To address this hypothesis, we used a previously developed labeling method in iPSC-CMs (17). We cultured OGFOD1-KO and WT cardiomyocytes in media containing light amino acids and then switched them to media containing heavy amino acids and followed the incorporation of the heavy label as a measure of protein synthesis and the loss of the light label as a measure of protein degradation.…”

Section: Resultsmentioning

confidence: 99%

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

Stoehr¹,

Kennedy²,

Yang³

et al. 2019

JCI Insight

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

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

Stoehr¹,

Kennedy²,

Yang³

et al. 2019

JCI Insight

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“…Messenger RNA processing and RNA splicing processes are some of the most enriched cellular pathways with proline hydroxylation substrates in Hela cells, which agrees with previous studies that have linked alternative RNA splicing and microRNA processing with hypoxia and oxygen sensing [20, 21, 66, 67]. Our data suggests a potentially critical role of proline hydroxylation pathways on the RNA processing in cancer cells.…”

Section: Discussionsupporting

confidence: 92%

Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

et al. 2016

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Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells.

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“…Transcription of MYC is upregulated in our infarcted hearts, so it is possible that multiple activation pathways are contributing to the upregulation of Pkm2 expression. Interestingly, mass spectroscopy analysis of prolyl hydroxylation sites regulated by PHD2 in human induced-pluripotent cardiomyocytes found both Pkm2 and hnRNPA1 to be modified (44), suggesting the expression of Pkm2 may also be modulated by posttranslational modifications in response to oxygen levels.…”

Section: Discussionmentioning

confidence: 99%

HIF1 mediates a switch in pyruvate kinase isoforms after myocardial infarction

Williams

Khadka

Tang

et al. 2018

Physiological Genomics

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Alternative splicing of RNA is an underexplored area of transcriptional response. We expect that early changes in alternatively spliced genes may be important for responses to cardiac injury. Hypoxia inducible factor 1 (HIF1) is a key transcription factor that rapidly responds to loss of oxygen through alteration of metabolism and angiogenesis. The goal of this study was to investigate the transcriptional response after myocardial infarction (MI) and to identify novel, hypoxia-driven changes, including alternative splicing. After ligation of the left anterior descending artery in mice, we observed an abrupt loss of cardiac contractility and upregulation of hypoxic signaling. We then performed RNA sequencing on ischemic heart tissue 1 and 3 days after infarct to assess early transcriptional changes and identified 89 transcripts with altered splicing. Of particular interest was the switch in Pkm isoform expression (pyruvate kinase, muscle). The usually predominant Pkm1 isoform was less abundant in ischemic hearts, while Pkm2 and associated splicing factors (hnRNPA1, hnRNPA2B1, Ptbp1) rapidly increased. Despite increased Pkm2 expression, total pyruvate kinase activity remained reduced in ischemic myocardial tissue. We also demonstrated HIF1 binding to PKM by chromatin immunoprecipitation, indicating a direct role for HIF1 in mediating this isoform switch. Our study provides a new, detailed characterization of the early transcriptome after MI. From this analysis, we identified an HIF1-mediated alternative splicing event in the PKM gene. Pkm1 and Pkm2 play distinct roles in glycolytic metabolism and the upregulation of Pkm2 is likely to have important consequences for ATP synthesis in infarcted cardiac muscle.

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Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes

Abstract: This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing.

Cited by 31 publications

References 50 publications

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

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

Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

HIF1 mediates a switch in pyruvate kinase isoforms after myocardial infarction

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