HuR mediates changes in the stability of AChR β-subunit mRNAs after skeletal muscle denervation

Abstract

Acetylcholine receptors (AChRs) are heteromeric membrane proteins essential for neurotransmission at the neuromuscular junction. Previous work showed that muscle denervation increases expression of AChR mRNAs due to transcriptional activation of AChR subunit genes. However, it remains possible that post-transcriptional mechanisms are also involved in controlling the levels of AChR mRNAs following denervation. We examined whether post-transcriptional events indeed regulate ACh β-subunitmRNAsin response to denervation. First, in vitro stability assays revealed that the half-life of AChR β-subunit mRNAs was increased in the presence of denervated muscle protein extracts. A bioinformatics analysis revealed the existence of a conserved AU-rich element (ARE) in the 3’-untranslated region (UTR) of AChR β-subunit mRNA. Furthermore, denervation of mouse muscle injected with a luciferase reporter construct containing the AChR β-subunit 3′UTR, caused an increase in luciferase activity. By contrast, mutation of this ARE prevented this increase. We also observed that denervation increased expression of the RNA-binding protein human antigen R (HuR) and induced its translocation to the cytoplasm. Importantly, HuR binds to endogenous AChR β-subunit transcripts in cultured myotubes and in vivo, and this binding is increased in denervated versus innervated muscles. Finally, p38MAPK,a pathwayknownto activate HuR, was induced following denervation as a result of MKK3/6 activation and a decrease in MKP-1 expression, thereby leading to an increase in the stability of AChR β-subunit transcripts. Together, these results demonstrate the important contribution of post-transcriptional events in regulating AChR β-subunit mRNAs and point toward a central role for HuR in mediating synaptic gene expression.