CARM1 Regulates Proliferation of PC12 Cells by Methylating HuD

Abstract

HuD is an RNA-binding protein that has been shown to induce neuronal differentiation by stabilizing labile mRNAs carrying AU-rich instability elements. Here, we show a novel mechanism of arginine methylation of HuD by coactivator-associated arginine methyltransferase 1 (CARM1) that affected mRNA turnover of p21 cip1/waf1 mRNA in PC12 cells. CARM1 specifically methylated HuD in vitro and in vivo and colocalized with HuD in the cytoplasm. Inhibition of HuD methylation by CARM1 knockdown elongated the p21 cip1/waf1 mRNA half-life and resulted in a slow growth rate and robust neuritogenesis in response to nerve growth factor (NGF). Methylation-resistant HuD bound more p21 cip1/waf1 mRNA than did the wild type, and its overexpression upregulated p21 cip1/waf1 protein expression. These results suggested that CARM1-methylated HuD maintains PC12 cells in the proliferative state by committing p21 cip1/waf1 mRNA to its decay system. Since the methylated population of HuD was reduced in NGF-treated PC12 cells, downregulation of HuD methylation is a possible pathway through which NGF induces differentiation of PC12 cells.

Emerging complexity of the HuD/ELAVl4 gene; implications for neuronal development, function, and dysfunction

“…On the other hand, inhibition of HuD methylation or knockdown of CARM1 resulted in increased p21 cip1/waf1 mRNA levels, cell-cycle withdrawal, and neuronal differentiation. Surprisingly, although reduced CARM1 levels also increased GAP-43 mRNA expression, they had no effect on other known HuD-targeted mRNAs, including Tau and p27, suggesting that stabilization of these other target mRNAs requires additional posttranslational modifications of HuD and/or other trans-acting factors (Fujiwara et al 2006;Hubers et al 2010). These findings provide evidence that methylation of HuD by CARM1 may be a universal switch in neuronal precursors that regulates the transition from proliferation to neuronal differentiation.…”

“…Using a variety of complementary techniques, including an in vitro protein methylation assay, CARM1 was demonstrated to interact with and methylate an amino acid located in the hinge region of rat (Arg 236 ) (Fujiwara et al 2006) and mouse (Arg 248 ) (Hubers et al 2010) HuD (Fig. 1C).…”

“…Currently, there are only three known pathways that regulate the abundance or function of HuD: protein kinase C (PKC) Pascale et al 2005;Lim and Alkon 2012); coactivatorassociated arginine methyltransferase 1 (CARM1) (Fujiwara et al 2006;Hubers et al 2010); and protein kinase B (PKB/ AKT) (Fujiwara et al 2011a). …”

The RNA-Binding Protein HuD Binds Acetylcholinesterase mRNA in Neurons and Regulates its Expression after Axotomy

“…In addition to decreased HuD expression, the observed decrease in HuD binding may also result from post-translational modification of HuD, or from interactions with other RNA-binding proteins or structural proteins (Kasashima et al, 1999(Kasashima et al, , 2002Pascale et al, 2004). For instance, during neuronal differentiation, HuD binding activity can be controlled by the protein kinase C signaling pathway (Mobarak et al, 2000;Pascale et al, 2005) or through methylation by the methyltransferase CARM1 (coactivator-associated methyltransferase 1) (Fujiwara et al, 2006). The Hu family of RNA-binding proteins are known to interact with several other proteins, and they are found in ribonucleoprotein complexes often associated with the cytoskeleton Pascale et al, 2004).…”

Mouse Prickle1 and Prickle2 are expressed in postmitotic neurons and promote neurite outgrowth

“…Preparation of total RNA from cultured mammalian cells was as described [20]. The total RNA extract was reverse transcribed by using oligo(dT) [12][13][14][15][16][17][18] 0 (reverse complement of bases 1833-1852).…”