The RNA-binding Protein HuR Binds to Acetylcholinesterase Transcripts andRegulates Their Expression in Differentiating Skeletal MuscleCells

Abstract

During myogenic differentiation, acetylcholinesterase (AChE) transcript levels are known to increase dramatically. Although this increase can be attributed in part to increased transcriptional activity, posttranscriptional mechanisms have also been implicated in the high levels of AChE mRNA in myotubes. In this study, we observed that transfection of a luciferase reporter construct containing the full-length AChE 3-untranslated region (UTR) resulted in significantly higher (5-fold) luciferase activity in differentiated myotubes versus myoblasts. RNA-electrophoretic mobility shift assays (REMSAs) performed with a full-length AChE 3-UTR probe and the AU-rich element revealed that the intensity of RNA-binding protein complexes increased as myogenic differentiation proceeded. Using several complementary approaches including supershift REMSA, mRNA-binding protein pull-down assays, and immunoprecipitation followed by reverse transcription-PCR, we found that the mRNA-stabilizing protein HuR interacts directly with AChE transcripts. Stable overexpression of HuR in C2C12 cells increased the expression of endogenous AChE transcripts as well as that of the luciferase reporter construct containing the AChE 3-UTR. In vitro stability assays performed with protein extracts from these cells versus controls resulted in a slower rate of AChE mRNA decay. The down-regulation of HuR expression mediated through small interfering RNA further confirmed the role of HuR in the regulation of AChE mRNA levels. Taken together, these studies demonstrate that HuR interacts with the AChE 3-UTR to regulate posttranscriptionally the expression of AChE mRNA during myogenic differentiation.Neurotransmission at cholinergic synapses of the central and peripheral nervous systems is promptly terminated by the catalytic activity of the enzyme acetylcholinesterase (AChE)

ColQ Controls Postsynaptic Differentiation at the Neuromuscular Junction

“…In line with this hypothesis, members of the Hu family of RNA-binding proteins have been found to stabilize AChE mRNAs in muscle cells (Deschênes-Furry et al, 2005). Moreover, the canonical binding sequence for Hu proteins is present in the 3ЈUTR of most of AChR subunit coding transcripts.…”

Roles of the canonical myomiRs miR-1, -133 and -206 in cell development and disease

“…In contrast, muscle denervation results in strong expression of MyoD and myogenin, preceding the accumulation of nAChR, αsubunit [39] . Additionally during myogenic differentiation, acetylcholinesterase transcript levels increase dramatically (5 ×), principally due to its stabilization by binding with HuR protein [52] , consistent with a regulatory role of HuR in neuron excitability. Normally the expression of MyoD and myogenin is suppressed by activated nerve signal pathways, including by electrical conduction per se, and sets of muscle genes regulated by the MyoD family and myogenin are downregulated by increasing electrical activity and other nervederived signals.…”

Acetylcholinesterase Expression in Muscle Is Specifically Controlled by a Promoter-Selective Enhancesome in the First Intron

“…The exon 6 knock-out mouse, showing diminished brain AChE mRNA levels, as well as findings of Deschenes-Furry et al (2005) that implicate regulation by an AU-rich element in the 3Ј-UTR of the gene, provide evidence for multiple regions in the AChE gene that influence gene expression. Deletion of the 3Ј-UTR of the AChE gene produces only a modest decrease in expressed activity (Fig.…”

“…Studies in cultured muscle cells reveal evidence for mRNA stabilization (Fuentes and Taylor, 1993;Deschenes-Furry et al, 2005), but stabilization of mRNA is not sufficient to explain the large increases in activity or mRNA levels associated with myoblast to myotube differentiation. Studies using inhibitors of RNA polymerase stabilize a labile RNA in myoblasts and provide evidence for superinduction of the mRNA (Fuentes and Taylor, 1993).…”