Role of the Hu family of RNA-binding proteins in regulating acetylcholinesterase expression in excitatory cells

Abstract

Post-transcriptional mechanisms regulating acetylcholinesterase (AChE) expression in muscle and neurons are relatively ill-defined, even though several studies have implicated mRNA stability as a means of controlling expression. We have, therefore, undertaken studies examining the role of post-transcriptional mechanisms in regulating AChE mRNA levels during neuronal differentiation, myogenic differentiation and in vivo following axotomy of the rat superior cervical ganglion (SCG). Specifically, using a combination of molecular techniques we have investigated the role of AChE's 3'-untranslated region (UTR) and have identified both cis- and trans-acting factors that modulate AChE mRNA levels. During neuronal differentiation of PC12 cells we determined that post-transcriptional mechanisms were predominantly responsible for the sustained increase in AChE mRNA since a modest increase in gene transcription occurred early and transiently. We examined the role of the AU-rich element (ARE) found in the 3'-UTR and of the RNA-binding protein (RBP) HuD in modulating AChE mRNA levels. Using a combination of complementary binding assays and transfection assays we demonstrated direct binding of HuD to the ARE and the stabilizing effect of this interaction. Given these results, we examined whether the ubiquitous Hu family protein, HuR, is implicated in post-transcriptional regulation of AChE during C2C12 myogenesis. Using similar approaches, we observed that the ARE and interactions with RBP complexes increased with differentiation and are important elements to AChE expression. We showed that HuR associates with the ARE and is involved in increased AChE transcript stability measured during myogenesis. Finally, we examined the relationship between HuD and AChE mRNA in neurons in vivo following SCG axotomy. Initially, we demonstrated that human HuD expressed in the hippocampus of HuD transgenic mice could bind AChE transcripts and increase AChE mRNA levels. Following SCG axotomy, we observed dramatic decreases in AChE mRNA levels and interactions with RBP complexes, including HuD. HuD protein and mRNA levels were also decreased as a result of axotomy. Exogenous expression of HuD in the SCG prior to axotomy prevented the decrease in ACNE mRNA. Accordingly, during the course of these studies we have identified an ARE in the AChE 3'-UTR and its binding partners HuD and HuR, as essential mediators of post-transcriptional mechanisms regulating AChE expression in neurons and muscle, respectively.