KH-type splicing regulatory protein interacts with survival motor neuron protein and is misregulated in spinal muscular atrophy.

KH-type splicing regulatory protein (KSRP) is closely related to chick zipcode-binding protein 2 and rat MARTA1, which are involved in neuronal transport and localization of beta-actin and microtubule-associated protein 2 mRNAs, respectively. KSRP is a multifunctional RNA-binding protein that has been implicated in transcriptional regulation, neuro-specific alternative splicing and mRNA decay. More specifically, KSRP is an essential factor for targeting AU-rich element-containing mRNAs to the exosome. We report here that KSRP is arginine methylated and interacts with the Tudor domain of SMN, the causative gene for spinal muscular atrophy (SMA), in a CARM1 methylation-dependent fashion. These two proteins colocalize in granule-like foci in the neurites of differentiating neuronal cells and the CARM1 methyltransferase is required for normal localization of KSRP in neuronal cells. Strikingly, this interaction is abrogated by naturally-occurring Tudor domain mutations found in human patients affected with severe Type I SMA, a strong indication of its functional significance to the etiology of the disease. We also report for the first time that Q136E and I116F Tudor mutations behave similarly to the previously characterized E134K mutation, and cause loss of Tudor interactions with several cellular methylated proteins. Finally, we show that KSRP is misregulated in the absence of SMN, and this correlated with increased mRNA stability of its mRNA target, p21(cip1/waf1), in spinal cord of mild SMA model mice. Our results suggest SMN can act as a molecular chaperone for methylated proteins involved in RNA metabolism and provide new insights into the pathophysiology of SMA.

The RNA-binding protein HuD binds acetylcholinesterase mRNA in neurons and regulates its expression after axotomy.

After axotomy, expression of acetylcholinesterase (AChE) is greatly reduced in the superior cervical ganglion (SCG); however, the molecular events involved in this response remain unknown. Here, we first examined AChE mRNA levels in the brain of transgenic mice that overexpress human HuD. Both in situ hybridization and reverse transcription-PCR demonstrated that AChE transcript levels were increased by more than twofold in the hippocampus of HuD transgenic mice. Additionally, direct interaction between the HuD transgene product and AChE mRNA was observed. Next, we examined the role of HuD in regulating AChE expression in intact and axotomized rat SCG neurons. After axotomy of the adult rat SCG neurons, AChE transcript levels decreased by 50 and 85% by the first and fourth day, respectively. In vitro mRNA decay assays indicated that the decrease in AChE mRNA levels resulted from changes in the stability of presynthesized transcripts. A combination of approaches performed using the region that directly encompasses an adenylate and uridylate (AU)-rich element within the AChE 3'-untranslated region demonstrated a decrease in RNA-protein complexes in response to axotomy of the SCG and, specifically, a decrease in HuD binding. After axotomy, HuD transcript and protein levels also decreased. Using a herpes simplex virus construct containing the human HuD sequence to infect SCG neurons in vivo, we found that AChE and GAP-43 mRNA levels were maintained in the SCG after axotomy. Together, the results of this study demonstrate that AChE expression in neurons of the rat SCG is regulated via post-transcriptional mechanisms that involve the AU-rich element and HuD.

The RNA-binding protein HuD: a regulator of neuronal differentiation, maintenance and plasticity.

mRNA stability is increasingly recognized as being essential for controlling the expression of a wide variety of transcripts during neuronal development and synaptic plasticity. In this context, the role of AU-rich elements (ARE) contained within the 3' untranslated region (UTR) of transcripts has now emerged as key because of their high incidence in a large number of cellular mRNAs. This important regulatory element is known to significantly modulate the longevity of mRNAs by interacting with available stabilizing or destabilizing RNA-binding proteins (RBP). Thus, in parallel with the emergence of ARE, RBP are also gaining recognition for their pivotal role in regulating expression of a variety of mRNAs. In the nervous system, the member of the Hu family of ARE-binding proteins known as HuD, has recently been implicated in multiple aspects of neuronal function including the commitment and differentiation of neuronal precursors as well as synaptic remodeling in mature neurons. Through its ability to interact with ARE and stabilize multiple transcripts, HuD has now emerged as an important regulator of mRNA expression in neurons. The present review is designed to provide a comprehensive and updated view of HuD as an RBP in the nervous system. Additionally, we highlight the role of HuD in multiple aspects of a neuron's life from early differentiation to changes in mature neurons during learning paradigms and in response to injury and regeneration. Finally, we describe the current state of knowledge concerning the molecular and cellular events regulating the expression and activity of HuD in neurons.

In vivo post-transcriptional regulation of GAP-43 mRNA by overexpression of the RNA-binding protein HuD.

HuD is a neuronal-specific RNA-binding protein that binds to and stabilizes the mRNAs of growth-associated protein-43 (GAP-43) and other neuronal proteins. HuD expression increases during brain development, nerve regeneration, and learning and memory, suggesting that this protein is important for controlling gene expression during developmental and adult plasticity. To examine the function of HuD in vivo, we generated transgenic mice overexpressing human HuD under the control of the calcium-calmodulin-dependent protein kinase IIalpha promoter. The transgene was expressed at high levels throughout the forebrain, including the hippocampal formation, amygdala and cerebral cortex. Using quantitative in situ hybridization, we found that HuD overexpression led to selective increases in GAP-43 mRNA in hippocampal dentate granule cells and neurons in the lateral amygdala and layer V of the neorcortex. In contrast, GAP-43 pre-mRNA levels were unchanged or decreased in the same neuronal populations. Comparison of the levels of mature GAP-43 mRNA and pre-mRNA in the same neurons of transgenic mice suggested that HuD increased the stability of the transcript. Confirming this, mRNA decay assays revealed that the GAP-43 mRNA was more stable in brain extracts from HuD transgenic mice than non-transgenic littermates. In conclusion, our results demonstrate that HuD overexpression is sufficient to increase GAP-43 mRNA stability in vivo.

Role of ELAV-like RNA-binding proteins HuD and HuR in the post-transcriptional regulation of acetylcholinesterase in neurons and skeletal muscle cells.

Over the last few years, several laboratories have focused their attention on elucidating the molecular events that control the expression and localization of acetylcholinesterase (AChE) in neurons and skeletal muscle cells. In this context, results from a number of studies have clearly shown the important contribution of transcriptional events in regulating AChE expression. Specifically, these studies have highlighted the roles of several cis- and trans-acting factors that control transcription of the AChE gene in these excitable cells. However, it has also become apparent that changes in the transcriptional activity of the AChE gene cannot fully account for the alterations seen in the overall abundance of AChE transcripts in neurons and muscle cells placed under a variety of experimental conditions. This indicates, therefore, that post-transcriptional mechanisms also play a significant role in controlling AChE mRNA expression. With this in mind, we have recently begun to address this issue in greater detail. Here, we provide a summary of our most recent findings dealing with the post-transcriptional regulation of AChE. Together, our studies have shown so far the important contribution of an AU-rich element located in the 3'UTR of AChE transcripts and of the stabilizing RNA-binding proteins of the ELAV-like family in regulating AChE expression in differentiating neuronal and muscle cells.

The RNA-binding protein HuR binds to acetylcholinesterase transcripts and regulates their expression in differentiating skeletal muscle cells.

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.

Impaired fast axonal transport in neurons of the sciatic nerves from dystonia musculorum mice.

Dystonia musculorum (dt) mice suffer from a severe sensory neuropathy caused by mutations in the gene encoding the cytoskeletal cross-linker protein dystonin/bullous pemphigoid antigen 1 (Bpag1). Loss of function of dystonin/Bpag1 within neurons leads to a loss in the maintenance of cytoskeletal organization and to the development of focal axonal swellings prior to death of the neuron. In the present study, we demonstrate that neurons within the sciatic nerves of dt27J mice undergo axonal degeneration as has been previously reported for the dorsal roots. Furthermore, ultrastructural studies reveal a perturbed organization of the neurofilament and microtubule networks within the axons of sciatic nerves in dt27J mice. The disrupted cytoskeletal organization suggested that axonal transport is affected in dt mice. To address this, we assessed fast axonal transport by measuring the rate of accumulation of acetylcholinesterase (AChE) proximal and distal to a surgically introduced ligature on the sciatic nerves of normal and dt27J mice. Our findings demonstrate that axonal transport of AChE in both orthograde and retrograde directions is markedly affected, and allow us to conclude that axonal transport defects do exist in the sciatic nerves of dt27J mice.

Expression of utrophin A mRNA correlates with the oxidative capacity of skeletal muscle fiber types and is regulated by calcineurin/NFAT signaling.

Utrophin levels have recently been shown to be more abundant in slow vs. fast muscles, but the nature of the molecular events underlying this difference remains to be fully elucidated. Here, we determined whether this difference is due to the expression of utrophin A or B, and examined whether transcriptional regulatory mechanisms are also involved. Immunofluorescence experiments revealed that slower fibers contain significantly more utrophin A in extrasynaptic regions as compared with fast fibers. Single-fiber RT-PCR analysis demonstrated that expression of utrophin A transcripts correlates with the oxidative capacity of muscle fibers, with cells expressing myosin heavy chain I and IIa demonstrating the highest levels. Functional muscle overload, which stimulates expression of a slower, more oxidative phenotype, induced a significant increase in utrophin A mRNA levels. Because calcineurin has been implicated in controlling this slower, high oxidative myofiber program, we examined expression of utrophin A transcripts in muscles having altered calcineurin activity. Calcineurin inhibition resulted in an 80% decrease in utrophin A mRNA levels. Conversely, muscles from transgenic mice expressing an active form of calcineurin displayed higher levels of utrophin A transcripts. Electrophoretic mobility shift and supershift assays revealed the presence of a nuclear factor of activated T cells (NFAT) binding site in the utrophin A promoter. Transfection and direct gene transfer studies showed that active forms of calcineurin or nuclear NFATc1 transactivate the utrophin A promoter. Together, these results indicate that expression of utrophin A is related to the oxidative capacity of muscle fibers, and implicate calcineurin and its effector NFAT in this mechanism.

Post-transcriptional regulation of acetylcholinesterase mRNAs in nerve growth factor-treated PC12 cells by the RNA-binding protein HuD.

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.