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Regulation of PP2Cm expression by miRNA-204/211 and miRNA-22 in mouse and human cells.

Pan BF, Gao C, Ren SX, Wang YB, Sun HP, Zhou MY - Acta Pharmacol. Sin. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

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miR-204 and miR-211 directly target recognition motifs in PPM1K 3′UTR. (A) Schematic diagram showing the seed sequence of miR-204 and miR-211 and putative conserved recognition motif in human PPM1K 3′UTR. (B) Luciferase assay results from HeLa cells co-transfected with miRNA mimics and luciferase reporter vectors. Luc-human 3′UTR-D is the mutated reporter vector where the conserved recognition motif was deleted in human_PPM1K 3′UTR. (C) Schematic diagram showing the seed sequences of miR-204 and miR-211, with putative matched conserved and poorly conserved sequences in mouse Ppm1k 3′UTR. (D) Luciferase assay results from HeLa cells. Luc-mouse 3′UTR-D1 is the mouse Ppm1k 3′UTR reporter vector with conserved site deleted. Luc-mouse 3′UTR-D2 is the mouse Ppm1k 3′UTR reporter vector with less-conserved site deleted. Luc-mouse 3′UTR-DD is the mouse Ppm1k 3′UTR reporter vector with both sites deleted. Data are mean±SEM for three individual samples (bP<0.05).
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fig3: miR-204 and miR-211 directly target recognition motifs in PPM1K 3′UTR. (A) Schematic diagram showing the seed sequence of miR-204 and miR-211 and putative conserved recognition motif in human PPM1K 3′UTR. (B) Luciferase assay results from HeLa cells co-transfected with miRNA mimics and luciferase reporter vectors. Luc-human 3′UTR-D is the mutated reporter vector where the conserved recognition motif was deleted in human_PPM1K 3′UTR. (C) Schematic diagram showing the seed sequences of miR-204 and miR-211, with putative matched conserved and poorly conserved sequences in mouse Ppm1k 3′UTR. (D) Luciferase assay results from HeLa cells. Luc-mouse 3′UTR-D1 is the mouse Ppm1k 3′UTR reporter vector with conserved site deleted. Luc-mouse 3′UTR-D2 is the mouse Ppm1k 3′UTR reporter vector with less-conserved site deleted. Luc-mouse 3′UTR-DD is the mouse Ppm1k 3′UTR reporter vector with both sites deleted. Data are mean±SEM for three individual samples (bP<0.05).

Mentions: TargetScan predicted numerous putative recognition motifs for miR-204 and miR-211 in the human and mouse PPM1K 3′UTRs. One conserved 8-mer seed match motif was identified in genes from both species, and one less-conserved 7-mer-m8 or 7-mer-1A seed match motif was identified in the mouse gene (Figure 1A). To validate the function of these putative miRNA recognition motifs, we generated mutant reporter constructs with targeted deletion of these motifs. For human PPM1K 3′UTR (Figure 3A), the deletion of the conserved recognition motifs (1545-1551 base pairs) completely abolished the inhibitory effect of miR-204 and miR-211 (Figure 3B). For mouse Ppm1k 3′UTR (Figure 3C), deletion of either the conserved motif (1363-1370 base pairs) or the less-conserved motif (661-667 base pairs) partially abolished the inhibitory effect of miR-204 and miR-211. However, the simultaneous deletion of both sites completely eliminated the effect of miR-204 and miR-211 on the Ppm1k 3′UTR (Figure 3D). These data suggest that the putative recognition motifs within the PPM1K 3′UTR are functional binding sites for miR-204/211 and are required for their inhibitory effect on PP2Cm expression.


Regulation of PP2Cm expression by miRNA-204/211 and miRNA-22 in mouse and human cells.

Pan BF, Gao C, Ren SX, Wang YB, Sun HP, Zhou MY - Acta Pharmacol. Sin. (2015)

miR-204 and miR-211 directly target recognition motifs in PPM1K 3′UTR. (A) Schematic diagram showing the seed sequence of miR-204 and miR-211 and putative conserved recognition motif in human PPM1K 3′UTR. (B) Luciferase assay results from HeLa cells co-transfected with miRNA mimics and luciferase reporter vectors. Luc-human 3′UTR-D is the mutated reporter vector where the conserved recognition motif was deleted in human_PPM1K 3′UTR. (C) Schematic diagram showing the seed sequences of miR-204 and miR-211, with putative matched conserved and poorly conserved sequences in mouse Ppm1k 3′UTR. (D) Luciferase assay results from HeLa cells. Luc-mouse 3′UTR-D1 is the mouse Ppm1k 3′UTR reporter vector with conserved site deleted. Luc-mouse 3′UTR-D2 is the mouse Ppm1k 3′UTR reporter vector with less-conserved site deleted. Luc-mouse 3′UTR-DD is the mouse Ppm1k 3′UTR reporter vector with both sites deleted. Data are mean±SEM for three individual samples (bP<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4816230&req=5

fig3: miR-204 and miR-211 directly target recognition motifs in PPM1K 3′UTR. (A) Schematic diagram showing the seed sequence of miR-204 and miR-211 and putative conserved recognition motif in human PPM1K 3′UTR. (B) Luciferase assay results from HeLa cells co-transfected with miRNA mimics and luciferase reporter vectors. Luc-human 3′UTR-D is the mutated reporter vector where the conserved recognition motif was deleted in human_PPM1K 3′UTR. (C) Schematic diagram showing the seed sequences of miR-204 and miR-211, with putative matched conserved and poorly conserved sequences in mouse Ppm1k 3′UTR. (D) Luciferase assay results from HeLa cells. Luc-mouse 3′UTR-D1 is the mouse Ppm1k 3′UTR reporter vector with conserved site deleted. Luc-mouse 3′UTR-D2 is the mouse Ppm1k 3′UTR reporter vector with less-conserved site deleted. Luc-mouse 3′UTR-DD is the mouse Ppm1k 3′UTR reporter vector with both sites deleted. Data are mean±SEM for three individual samples (bP<0.05).
Mentions: TargetScan predicted numerous putative recognition motifs for miR-204 and miR-211 in the human and mouse PPM1K 3′UTRs. One conserved 8-mer seed match motif was identified in genes from both species, and one less-conserved 7-mer-m8 or 7-mer-1A seed match motif was identified in the mouse gene (Figure 1A). To validate the function of these putative miRNA recognition motifs, we generated mutant reporter constructs with targeted deletion of these motifs. For human PPM1K 3′UTR (Figure 3A), the deletion of the conserved recognition motifs (1545-1551 base pairs) completely abolished the inhibitory effect of miR-204 and miR-211 (Figure 3B). For mouse Ppm1k 3′UTR (Figure 3C), deletion of either the conserved motif (1363-1370 base pairs) or the less-conserved motif (661-667 base pairs) partially abolished the inhibitory effect of miR-204 and miR-211. However, the simultaneous deletion of both sites completely eliminated the effect of miR-204 and miR-211 on the Ppm1k 3′UTR (Figure 3D). These data suggest that the putative recognition motifs within the PPM1K 3′UTR are functional binding sites for miR-204/211 and are required for their inhibitory effect on PP2Cm expression.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

Show MeSH