Characterizing Protein Arginine Methyltransferase Expression, Localization, and Function During Myogenesis
Notice bibliographique
Résumé
Protein arginine methyltransferases (PRMTs) play a critical role in the development of skeletal muscle. Despite the importance of PRMTs, the expression, localization, and function of these enzymes, specifically PRMT1, PRMT4, and PRMT5, during muscle development remains poorly understood. Therefore, the purpose of our study was to investigate quantitative, temporal, spatial, and functional metrics of PRMT biology in skeletal muscle cells during myogenesis. C 2 C 12 skeletal muscle differentiation was employed as an in vitro model of skeletal muscle development. Cells were assessed during the myoblast stage, and during days 1, 3, 5, and 7 of differentiation. RT‐qPCR was used to examine transcript levels, while Western blotting was used to assess protein expression, localization, and function throughout the experimental timecourse. In situ immunofluorescence techniques were employed to investigate cell morphology and maturity. The progression of myogenesis throughout the experimental timecourse was confirmed via significant increases in embryonic myosin heavy chain and myogenin expression, two molecular markers of muscle development. PRMT1 transcript levels progressively increased throughout differentiation and were 50–120% higher (p < 0.05) at day 5 and 7 compared to all preceding timepoints. Similarly, PRMT1 protein expression gradually increased during myogenesis and was 1.5‐fold higher (p < 0.05) at day 5 of differentiation, as compared to the myoblast stage. Interestingly, PRMT1 protein expression followed a similar pattern of expression as myogenin, suggesting a co‐regulation between these molecules during myogenic differentiation. PRMT4 transcript and protein levels were significantly lower (~20–50%) at day 5 and 7 of differentiation compared to the myoblast stage. In contrast, PRMT5 transcript and protein expression progressively increased during the timecourse and was significantly higher by day 5 of differentiation compared to the myoblast stage. Collectively, these data demonstrate PRMT‐specific patterns of expression during myogenesis. Monomethylarginine content, a marker of global PRMT activity, was similar across the timecourse. This suggests that, despite enzyme‐specific differentiation‐induced alterations in PRMT expression, PRMTs are active methyl donors throughout myogenesis. Cell fractionation analyses revealed a 3‐fold greater (p < 0.05) PRMT protein content within the cytosol, as compared to the myonuclei at all timepoints of differentiation. Furthermore, PRMT1 and PRMT5 protein levels gradually increased in the cytosol during myogenesis and were significantly higher compared to the myoblast stage by day 7 of differentiation. In contrast, PRMT4 protein expression progressively increased in the myonuclei throughout the timecourse, consistent with its reported transcriptional coactivator functions. To specifically examine the role of PRMT1 during myogenic differentiation, cells were treated with TC‐E 5003 (TCE), a selective inhibitor of PRMT1. In comparison to vehicle‐treated cells, TCE‐treated cells exhibited a deficiency in muscle differentiation, as indicated by significant reductions in myoblast fusion, and decreased myotube surface area. Altogether, our data reveal that PRMT expression, localization, and function are dynamic during myogenesis. This study expands our understanding of PRMT biology during skeletal muscle remodelling. Support or Funding Information Natural Sciences and Engineering Research Council of Canada and Canada Research Chairs
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,001 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».