Staufen1 interacts with mRNAs with expanded CUG repeats and promotes their nuclear export and translation, while also promoting alternative splicing of other mRNAs.

A therapeutic strategy to treat Duchenne muscular dystrophy (DMD) involves identifying compounds that can elevate utrophin A expression in muscle fibers of affected patients. The dystrophin homologue utrophin A can functionally substitute for dystrophin when its levels are enhanced in the mdx mouse model of DMD. Utrophin A expression in skeletal muscle is regulated by mechanisms that promote the slow myofiber program. Since activation of peroxisome proliferator-activated receptor (PPAR) beta/delta promotes the slow oxidative phenotype in skeletal muscle, we initiated studies to determine whether pharmacological activation of PPARbeta/delta provides functional benefits to the mdx mouse. GW501516, a PPARbeta/delta agonist, was found to stimulate utrophin A mRNA levels in C2C12 muscle cells through an element in the utrophin A promoter. Expression of PPARbeta/delta was greater in skeletal muscles of mdx versus wild-type mice. We treated 5-7-week-old mdx mice with GW501516 for 4 weeks. This treatment increased the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated utrophin A mRNA levels leading to its increased expression at the sarcolemma. Expression of alpha1-syntrophin and beta-dystroglycan was restored to the sarcolemma. Improvement of mdx sarcolemmal integrity was evidenced by decreased intracellular IgM staining and decreased in vivo Evans blue dye (EBD) uptake. GW501516 treatment also conferred protection against eccentric contraction (ECC)-induced damage of mdx skeletal muscles, as shown by a decreased contraction-induced force drop and reduction of dye uptake during ECC. These results demonstrate that pharmacological activation of PPARbeta/delta might provide functional benefits to DMD patients through enhancement of utrophin A expression.

In addition to showing differences in the levels of contractile proteins and metabolic enzymes, fast and slow muscles also differ in their expression profile of structural and synaptic proteins. Because utrophin is a structural protein expressed at the neuromuscular junction, we hypothesize that its expression may be different between fast and slow muscles. Western blots showed that, compared with fast extensor digitorum longus (EDL) muscles, slow soleus muscles contain significantly more utrophin. Quantitative RT-PCR revealed that this difference is accompanied by a parallel increase in the expression of utrophin transcripts. Interestingly, the higher levels of utrophin and its mRNA appear to occur in extrasynaptic regions of muscle fibers as shown by immunofluorescence and in situ hybridization experiments. Furthermore, nuclear run-on assays showed that the rate of transcription of the utrophin gene was nearly identical between EDL and soleus muscles, indicating that increased mRNA stability accounts for the higher levels of utrophin in slow muscles. Direct plasmid injections of reporter gene constructs showed that cis-acting elements contained within the utrophin 3'-untranslated region (3'-UTR) confer greater stability to chimeric LacZ transcripts in soleus muscles. Finally, we observed a clear difference between EDL and soleus muscles in the abundance of RNA-binding proteins interacting with the utrophin 3'-UTR. Together, these findings highlight the contribution of posttranscriptional events in regulating the expression of utrophin in muscle.

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-We examined whether calcineurin-NFAT (nuclear factors of activated T cells) signaling plays a role in specifically directing the expression of utrophin in the synaptic compartment of muscle fibers. Immunofluorescence experiments revealed the accumulation of components of the calcineurin-NFAT signaling cascade within the postsynaptic membrane domain of the neuromuscular junction. RT-PCR analysis using synaptic vs. extrasynaptic regions of muscle fibers confirmed these findings by showing an accumulation of calcineurin transcripts within the synaptic compartment. We also examined the effect of calcineurin on utrophin gene expression. Pharmacological inhibition of calcineurin in mice with either cyclosporin A or FK506 resulted in a marked decrease in utrophin A expression at synaptic sites, whereas constitutive activation of calcineurin had the opposite effect. Mutation of the previously identified NFAT binding site in the utrophin A promoter region, followed by direct gene transfer studies in mouse muscle, led to an inhibition in the synaptic expression of a lacZ reporter gene construct. Transfection assays performed with cultured myogenic cells indicated that calcineurin acted additively with GA binding protein (GABP) to transactivate utrophin A gene expression. Because both GABP-and calcineurin-mediated pathways are targeted by peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣), we examined whether this coactivator contributes to utrophin gene expression. In vitro and in vivo transfection experiments showed that PGC-1␣ alone induces transcription from the utrophin A promoter. Interestingly, this induction is largely potentiated by coexpression of PGC-1␣ with GABP. Together, these studies indicate that the synaptic expression of utrophin is also driven by calcineurin-NFAT signaling and occurs in conjunction with signaling events that involve GABP and PGC-1␣. synaptic gene expression; Duchenne muscular dystrophy; nuclear respiratory factor 2 THE NEUROMUSCULAR JUNCTION (NMJ) serves as an excellent model system for examining the events regulating the formation, maintenance, and plasticity of synapses, primarily because of its simplicity and accessibility in the peripheral nervous system. In particular, considerable emphasis has been placed on the elucidation of the mechanisms responsible for the accumulation of synaptic proteins at the postsynaptic membrane. Transcripts encoding numerous synaptic proteins, including the acetylcholine receptor (AChR) subunits, acetylcholinesterase (AChE), and utrophin, have been shown to accumulate at the NMJ as a result of the local transcriptional induction of their genes within subsynaptic nuclei (for review, see Refs. 6,39,49,55). A current model used to explain this local transcriptional control posits that nerve-derived factors such as neuregulin bind to receptors located on the postsynaptic membrane and initiate a series of signaling events that ultimately result in the local activation of synaptic genes. In this context, several studies have highlighted the...

The expression pattern of Staufen1 during mouse skeletal muscle development is described. Sustained expression of Staufen1 in myoblasts prevents normal differentiation by causing decreases in the expression of key myogenic markers by an SMD-independent mechanism and by promoting the translational regulation of c-myc.

Expression of acetylcholinesterase (AChE) is greatly enhanced during neuronal differentiation, but the nature of the molecular mechanisms remains to be fully defined. In this study, we observed that nerve growth factor treatment of PC12 cells leads to a progressive increase in the expression of AChE transcripts, reaching approximately 3.5-fold by 72 h. Given that the AChE 3'-untranslated region (UTR) contains an AU-rich element, we focused on the potential role of the RNA-binding protein HuD in mediating the increase in AChE mRNA seen in differentiating neurons. Using PC12 cells engineered to stably express HuD or an antisense to HuD, our studies indicate that HuD can regulate the abundance of AChE transcripts in neuronal cells. Furthermore, transfection of a reporter construct containing the AChE 3'-UTR showed that this 3'-UTR can increase expression of the reporter gene product in cells expressing HuD but not in cells expressing the antisense. RNA gel shifts and Northwestern blots revealed an increase in the binding of several protein complexes in differentiated neurons. Immunoprecipitation experiments demonstrated that HuD can bind directly AChE transcripts. These results show the importance of post-transcriptional mechanisms in regulating AChE expression in differentiating neurons and implicate HuD as a key trans-acting factor in these events.

J. Neurochem. (2012) 120, 230–238. Abstract Brain‐derived neurotrophic factor (BDNF) is required for efficient skeletal‐muscle regeneration and perturbing its expression causes abnormalities in the proliferation and differentiation of skeletal muscle cells. In this study, we investigated the mechanism of BDNF suppression that occurs during myogenic differentiation. BDNF is expressed at the mRNA level as two isoforms that differ in the length of their 3′UTRs as a result of alternative cleavage and polyadenylation. Sequence analysis revealed the presence of three miR‐206 target sites in the long BDNF 3′UTR (BDNF‐L), whereas only one site was found in the short mRNA BDNF 3′UTR (BDNF‐S). miR‐206 is known to regulate the differentiation of C2C12 myoblasts and its expression is induced during the transition from myoblasts to myotubes. We thus examined whether miR‐206‐mediated suppression is responsible for the expression pattern of BDNF during myogenic differentiation. BDNF‐L was suppressed to a greater extent than BDNF‐S during differentiation of C2C12 myoblasts. Transfection of a miR‐206 precursor decreased activity of reporters representative of the BDNF‐L 3′UTR, but not BDNF‐S 3′UTR, and repressed endogenous BDNF mRNA levels. This suppression was found to be dependent on the presence of multiple miR‐206 target sites in the BDNF‐L 3′UTR. Conversely, suppression of miR‐206 levels resulted in de‐repression of BDNF 3′UTR reporter activity and increased endogenous BDNF‐L mRNA levels. A receptor for BDNF, p75NTR, was also suppressed during differentiation and in response to miR‐206, but this appeared to not be entirely mediated via a miR‐206 target site its 3′UTR. Based on these observations, BDNF represents a novel target through which miR‐206 controls the initiation and maintenance of the differentiated state of muscle cells. These results further suggest that miR‐206 might play a role in regulating retrograde signaling of BDNF at the neuromuscular junction.