Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons

Abstract

MicroRNAs (miRNAs) are small regulatory RNAs that regulate gene expression posttranscriptionally by base pairing to the target mRNAs in animal cells. KRas, an oncogene known to be repressed by let-7a miRNAs, is expressed and needed for the differentiation of mammalian sympathetic neurons and PC12 cells. We documented a loss of let-7a activity during this differentiation process without any significant change in the cellular level of let-7a miRNA. However, the level of Ago2, an essential component that is associated with miRNAs to form RNP-specific miRNA (miRNP) complexes, shows an increase with neuronal differentiation. In this study, differentiation-induced phosphorylation and the subsequent loss of miRNA from Ago2 were noted, and these accounted for the loss of miRNA activity in differentiating neurons. Neuronal differentiation induces the phosphorylation of mitogen-activated protein kinase p38 and the downstream kinase mitogen-and stress-activated protein kinase 1 (MSK1). This in turn upregulates the phosphorylation of Ago2 and ensures the dissociation of miRNA from Ago2 in neuronal cells. MSK1-mediated miRNP inactivation is a prerequisite for the differentiation of neuronal cells, where let-7a miRNA gets unloaded from Ago2 to ensure the upregulation of KRas, a target of let-7a. We noted that the inactivation of let-7a is both necessary and sufficient for the differentiation of sympathetic neurons. P C12 cells derived from the pheochromocytoma of the rat adrenal medulla are widely used for studying cell signaling in response to numerous growth factors, neurotrophins, and hormones. These cells serve as a useful model to study the proliferation, differentiation, and survival of sympathetic neurons (1). PC12 cells stop dividing and differentiate to cells with neuronal extensions when cultured in the presence of nerve growth factor (NGF) and low levels of serum (2). Differentiated PC12 cells show changes in their electrical excitability, sensitivity to acetylcholine, and choline acetyltransferase activity (3). NGF-, brain-derived neurotrophic factor (BDNF)-, or cyclic AMP-treated, differentiated PC12 cells are connected to each other, although it is not clear whether newly formed neuronal extensions or neurites form functional synapses or not (4). The paradoxical finding that the Src and Ras oncogene products enhanced rather than blocked NGF-induced differentiation led to the identification of signaling pathways involving both Ras and Src as part of the total differentiation response to NGF (5). In PC12 cells, Ras accounts for the immediate effects of NGF, mediated through extracellular signal-regulated kinase (ERK) (1).MicroRNAs (miRNAs), a class of 21-nucleotide-long noncoding RNAs, can reversibly repress the translation of their target messages by binding the mRNA with imperfect complementarities in metazoan cells. miRNA-targeted messages and the components of the miRNA machinery, including the Argonaute (Ago) proteins and miRNAs, accumulate in mammalian P bodies (6-8). miRNAs can control neuronal differentiat...

P‐body‐induced inactivation of let‐7a miRNP prevents the death of growth factor‐deprived neuronal cells

“…In a previous report, we delineated how p38 activation is linked with miRNP inactivation, which causes the differentiation of mammalian cells (25). Here, we also observed how the activation of the p38 pathway is ensured by P-body components and how it may be linked with the prevention of apoptosis that is possibly caused by the reactivation of miRNPs in NGF-depleted cells.…”

“…Here, we also observed how the activation of the p38 pathway is ensured by P-body components and how it may be linked with the prevention of apoptosis that is possibly caused by the reactivation of miRNPs in NGF-depleted cells. As let-7a inactivation is known to be essential in the NGF-induced differentiation process (25), we studied the connection between let-7a activity and NGF withdrawal. Let-7a activity was observed to increase with increasing time periods of NGF withdrawal from differentiated PC12 cells (Fig.…”

MicroRNA exporter HuR clears the internalized pathogens by promoting pro‐inflammatory response in infected macrophages

“…We noted an increased association of PP2A with the endosome enriched fraction in Ld ‐infected cells (Fig N). It is important to note that substrate of PP2A, the phosphorylated Ago2, is also known to get concentrated in these fractions as we had analysed them before both in macrophage and neuronal cells [Fig L and M (Mazumder et al , ; Patranabis & Bhattacharyya, )]. Therefore, PP2A may act on its substrate phosphorylated Ago2 in the Optiprep density gradient fractions 2–4 that are positive for MVB marker protein Alix.…”

“…miRNA-mediated regulation controls varieties of genes including several cytokine encoding genes (Palanisamy et al, 2012). It has also been observed that reversible uncoupling of miRNA from Ago2 ensures switching of miRNA activity in mammalian cells (Mazumder et al, 2013;Patranabis & Bhattacharyya, 2016). It has also been noted that Ld infection controls expression of several miRNAs including those that act as repressor of pro-inflammatory cytokine mRNAs (Chakrabarty & Bhattacharyya, 2017).…”

MicroRNAs in neural development: from master regulators to fine-tuners

“…(5) Regulation of the composition and activity of the neuronal miRISC. This includes the activity-dependent control of the phosphorylation of Ago2 (Patranabis and Bhattacharyya, 2016) or its interacting partner FMRP (also known as FMR1) (Muddashetty et al, 2011), as well as the activity-regulated degradation of the miRISC protein MOV10 (Banerjee et al, 2009). such miRNA therapeutics might also prove to be applicable to neurodevelopmental disorders for which no cure is currently available.…”