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The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase (Prmt)5 but directly requires Prmt4.

Mallappa C, Hu YJ, Shamulailatpam P, Tae S, Sif S, Imbalzano AN - Nucleic Acids Res. (2010)

Bottom Line: Both Prmts are required for myogenic microRNA induction during differentiation.By contrast, Prmt4 binds to the upstream regulatory regions of myogenic microRNAs and is required for dimethylation of the Prmt4 substrate, H3R17, at microRNA regulatory sequences.Deletion of Prmt4 does not alter MyoD binding at myogenic microRNA regulatory sequences but prevents the binding of both myogenin and the Brg1 ATPase that catalyzes SWI/SNF-dependent chromatin remodeling, resulting in an inhibition of microRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.

ABSTRACT
Myogenic microRNAs are important regulators of muscle development and differentiation. To better understand the roles of chromatin-modifying and remodeling enzymes in the activation of myogenic microRNA expression, we have functionally analyzed two different protein arginine methyltransferases, Prmt5 and Prmt4, both of which have previously been implicated in the regulation of myogenic mRNA expression. Both Prmts are required for myogenic microRNA induction during differentiation. Prmt5 is indirectly required due to the necessity of Prmt5 for expression of the transcriptional regulator, myogenin, as ectopic expression of myogenin eliminates Prmt5 dependency. By contrast, Prmt4 binds to the upstream regulatory regions of myogenic microRNAs and is required for dimethylation of the Prmt4 substrate, H3R17, at microRNA regulatory sequences. Deletion of Prmt4 does not alter MyoD binding at myogenic microRNA regulatory sequences but prevents the binding of both myogenin and the Brg1 ATPase that catalyzes SWI/SNF-dependent chromatin remodeling, resulting in an inhibition of microRNA expression.

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Expression of myogenin and Mef2D1b complements the loss of myogenic miRNA expression in a Prmt5 AS cell line. (A and B) Relative expression of myogenin and Mef2D1b upon differentiation in NIH 3T3 and in the Prmt5 AS cell line, C1, infected with retrovirus encoding myogenin and Mef2D1b. (C–F) qPCR analyses of primary transcripts of miR-1 and miR-133a in NIH 3T3 and C1 cells expressing myogenin and Mef2D1b at various times post-differentiation. The data represent the average of three independent experiments ± standard deviation. Expression at Time 0 in the empty vector (EV) control is normalized to 1. h, hours.
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Figure 3: Expression of myogenin and Mef2D1b complements the loss of myogenic miRNA expression in a Prmt5 AS cell line. (A and B) Relative expression of myogenin and Mef2D1b upon differentiation in NIH 3T3 and in the Prmt5 AS cell line, C1, infected with retrovirus encoding myogenin and Mef2D1b. (C–F) qPCR analyses of primary transcripts of miR-1 and miR-133a in NIH 3T3 and C1 cells expressing myogenin and Mef2D1b at various times post-differentiation. The data represent the average of three independent experiments ± standard deviation. Expression at Time 0 in the empty vector (EV) control is normalized to 1. h, hours.

Mentions: NIH 3T3 and C1 Prmt5 AS cells were infected with retroviruses encoding myogenin and Mef2D1b in a sequential manner and differentiated. We could detect the expression of myogenin and Mef2D1b in both the NIH 3T3 and the C1 cells from the onset of differentiation throughout the differentiation time course (Figure 3A and B), demonstrating that each of the cell lines at each of the time points assayed were equivalently expressing myogenin and Mef2D1b. We then tested the expression of primary miR-1 and miR-133a transcripts and observed that all four were robustly expressed in a manner independent of the presence of Prmt5 (Figure 3C–F). These results demonstrate that loss of miRNA expression in Prmt5 AS cell lines could be complemented by expressing myogenin and Mef2D1b, indicating that the Prmt5 requirement for myogenic miRNA expression is indirect via the induction of myogenin. In this experiment, we also noted that miRNA expression was induced earlier when cells were differentiated by myogenin and Mef2D1b than when they were differentiated by MyoD (compare Figures 1 and 3C–F). This observation argues that myogenic miRNAs may be targets for activation by myogenin because the onset of myogenic microRNA expression correlated with the introduction of myogenin. This observation is also consistent with a previous report indicating that myogenin can bind to E boxes upstream of these myogenic miRNA sequences (14).Figure 3.


The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase (Prmt)5 but directly requires Prmt4.

Mallappa C, Hu YJ, Shamulailatpam P, Tae S, Sif S, Imbalzano AN - Nucleic Acids Res. (2010)

Expression of myogenin and Mef2D1b complements the loss of myogenic miRNA expression in a Prmt5 AS cell line. (A and B) Relative expression of myogenin and Mef2D1b upon differentiation in NIH 3T3 and in the Prmt5 AS cell line, C1, infected with retrovirus encoding myogenin and Mef2D1b. (C–F) qPCR analyses of primary transcripts of miR-1 and miR-133a in NIH 3T3 and C1 cells expressing myogenin and Mef2D1b at various times post-differentiation. The data represent the average of three independent experiments ± standard deviation. Expression at Time 0 in the empty vector (EV) control is normalized to 1. h, hours.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3045594&req=5

Figure 3: Expression of myogenin and Mef2D1b complements the loss of myogenic miRNA expression in a Prmt5 AS cell line. (A and B) Relative expression of myogenin and Mef2D1b upon differentiation in NIH 3T3 and in the Prmt5 AS cell line, C1, infected with retrovirus encoding myogenin and Mef2D1b. (C–F) qPCR analyses of primary transcripts of miR-1 and miR-133a in NIH 3T3 and C1 cells expressing myogenin and Mef2D1b at various times post-differentiation. The data represent the average of three independent experiments ± standard deviation. Expression at Time 0 in the empty vector (EV) control is normalized to 1. h, hours.
Mentions: NIH 3T3 and C1 Prmt5 AS cells were infected with retroviruses encoding myogenin and Mef2D1b in a sequential manner and differentiated. We could detect the expression of myogenin and Mef2D1b in both the NIH 3T3 and the C1 cells from the onset of differentiation throughout the differentiation time course (Figure 3A and B), demonstrating that each of the cell lines at each of the time points assayed were equivalently expressing myogenin and Mef2D1b. We then tested the expression of primary miR-1 and miR-133a transcripts and observed that all four were robustly expressed in a manner independent of the presence of Prmt5 (Figure 3C–F). These results demonstrate that loss of miRNA expression in Prmt5 AS cell lines could be complemented by expressing myogenin and Mef2D1b, indicating that the Prmt5 requirement for myogenic miRNA expression is indirect via the induction of myogenin. In this experiment, we also noted that miRNA expression was induced earlier when cells were differentiated by myogenin and Mef2D1b than when they were differentiated by MyoD (compare Figures 1 and 3C–F). This observation argues that myogenic miRNAs may be targets for activation by myogenin because the onset of myogenic microRNA expression correlated with the introduction of myogenin. This observation is also consistent with a previous report indicating that myogenin can bind to E boxes upstream of these myogenic miRNA sequences (14).Figure 3.

Bottom Line: Both Prmts are required for myogenic microRNA induction during differentiation.By contrast, Prmt4 binds to the upstream regulatory regions of myogenic microRNAs and is required for dimethylation of the Prmt4 substrate, H3R17, at microRNA regulatory sequences.Deletion of Prmt4 does not alter MyoD binding at myogenic microRNA regulatory sequences but prevents the binding of both myogenin and the Brg1 ATPase that catalyzes SWI/SNF-dependent chromatin remodeling, resulting in an inhibition of microRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.

ABSTRACT
Myogenic microRNAs are important regulators of muscle development and differentiation. To better understand the roles of chromatin-modifying and remodeling enzymes in the activation of myogenic microRNA expression, we have functionally analyzed two different protein arginine methyltransferases, Prmt5 and Prmt4, both of which have previously been implicated in the regulation of myogenic mRNA expression. Both Prmts are required for myogenic microRNA induction during differentiation. Prmt5 is indirectly required due to the necessity of Prmt5 for expression of the transcriptional regulator, myogenin, as ectopic expression of myogenin eliminates Prmt5 dependency. By contrast, Prmt4 binds to the upstream regulatory regions of myogenic microRNAs and is required for dimethylation of the Prmt4 substrate, H3R17, at microRNA regulatory sequences. Deletion of Prmt4 does not alter MyoD binding at myogenic microRNA regulatory sequences but prevents the binding of both myogenin and the Brg1 ATPase that catalyzes SWI/SNF-dependent chromatin remodeling, resulting in an inhibition of microRNA expression.

Show MeSH
Related in: MedlinePlus