Bang-fen Pan1, Chen Gao2, Shu-xun Ren2, Yi-bin Wang1,2, Hai-peng Sun1,2, Mei-yi Zhou1. 1. Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. 2. Division of Molecular Medicine, Department of Anesthesiology, Medicine and Physiology, Molecular Biology Institute, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California, Los Angeles, CA 90095, USA.
Abstract
AIM: The mitochondrial targeted 2C-type serine/threonine protein phosphatase (PP2Cm) is encoded by the gene PPM1K and is highly conserved among vertebrates. PP2Cm plays a critical role in branched-chain amino acid catabolism and regulates cell survival. Its expression is dynamically regulated by the nutrient environment and pathological stresses. However, little is known about the molecular mechanism underlying the regulation of PPM1K gene expression. In this study, we aimed to reveal how PPM1K expression is affected by miRNA-mediated post-transcriptional regulation. METHODS: Computational analysis based on conserved miRNA binding motifs was applied to predict the candidate miRNAs that potentially affect PPM1K expression. Dual-luciferase reporter assay was performed to verify the miRNAs' binding sites in the PPM1K gene and their influence on PPM1K 3'UTR activity. We further over-expressed the mimics of these miRNAs in human and mouse cells to examine whether miRNAs affected the mRNA level of PPM1K. RESULTS: Computational analysis identified numerous miRNAs potentially targeting PPM1K. Luciferase reporter assays demonstrated that the 3'UTR of PPM1K gene contained the recognition sites of miR-204 and miR-211. Overexpression of these miRNAs in human and mouse cells diminished the 3'UTR activity and the endogenous mRNA level of PPM1K. However, the miR-22 binding site was found only in human and not mouse PPM1K 3'UTR. Accordingly, PPM1K 3'UTR activity was suppressed by miR-22 overexpression in human but not mouse cells. CONCLUSION: These data suggest that different miRNAs contribute to the regulation of PP2Cm expression in a species-specific manner. miR-204 and miR-211 are efficient in both mouse and human cells, while miR-22 regulates PP2Cm expression only in human cells.
AIM: The mitochondrial targeted 2C-type serine/threonine protein phosphatase (PP2Cm) is encoded by the gene PPM1K and is highly conserved among vertebrates. PP2Cm plays a critical role in branched-chain amino acid catabolism and regulates cell survival. Its expression is dynamically regulated by the nutrient environment and pathological stresses. However, little is known about the molecular mechanism underlying the regulation of PPM1K gene expression. In this study, we aimed to reveal how PPM1K expression is affected by miRNA-mediated post-transcriptional regulation. METHODS: Computational analysis based on conserved miRNA binding motifs was applied to predict the candidate miRNAs that potentially affect PPM1K expression. Dual-luciferase reporter assay was performed to verify the miRNAs' binding sites in the PPM1K gene and their influence on PPM1K 3'UTR activity. We further over-expressed the mimics of these miRNAs in human and mouse cells to examine whether miRNAs affected the mRNA level of PPM1K. RESULTS: Computational analysis identified numerous miRNAs potentially targeting PPM1K. Luciferase reporter assays demonstrated that the 3'UTR of PPM1K gene contained the recognition sites of miR-204 and miR-211. Overexpression of these miRNAs in human and mouse cells diminished the 3'UTR activity and the endogenous mRNA level of PPM1K. However, the miR-22 binding site was found only in human and not mousePPM1K 3'UTR. Accordingly, PPM1K 3'UTR activity was suppressed by miR-22 overexpression in human but not mouse cells. CONCLUSION: These data suggest that different miRNAs contribute to the regulation of PP2Cm expression in a species-specific manner. miR-204 and miR-211 are efficient in both mouse and human cells, while miR-22 regulates PP2Cm expression only in human cells.
Authors: Christopher B Newgard; Jie An; James R Bain; Michael J Muehlbauer; Robert D Stevens; Lillian F Lien; Andrea M Haqq; Svati H Shah; Michelle Arlotto; Cris A Slentz; James Rochon; Dianne Gallup; Olga Ilkayeva; Brett R Wenner; William S Yancy; Howard Eisenson; Gerald Musante; Richard S Surwit; David S Millington; Mark D Butler; Laura P Svetkey Journal: Cell Metab Date: 2009-04 Impact factor: 27.287
Authors: Gaia Novarino; Paul El-Fishawy; Hulya Kayserili; Nagwa A Meguid; Eric M Scott; Jana Schroth; Jennifer L Silhavy; Majdi Kara; Rehab O Khalil; Tawfeg Ben-Omran; A Gulhan Ercan-Sencicek; Adel F Hashish; Stephan J Sanders; Abha R Gupta; Hebatalla S Hashem; Dietrich Matern; Stacey Gabriel; Larry Sweetman; Yasmeen Rahimi; Robert A Harris; Matthew W State; Joseph G Gleeson Journal: Science Date: 2012-09-06 Impact factor: 47.728