Dilysine motifs in exon 2b of SMN protein mediate binding to the COPI vesicle protein α-COP and neurite outgrowth in a cell culture model of spinal muscular atrophy
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Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder that stems from low levels of survival of motor neuron (SMN) protein. The processes that cause motor neurons and muscle cells to become dysfunctional are incompletely understood. We are interested in neuromuscular homeostasis and the stresses put upon that system by loss of SMN. We recently reported that α-COP, a member of the coatomer complex of coat protein I (COPI) vesicles, is an SMN-binding partner, implicating this protein complex in normal SMN function. To investigate the functional significance of the interaction between α-COP and SMN, we constructed an inducible NSC-34 cell culture system to model the consequences of SMN depletion and find that depletion of SMN protein results in shortened neurites. Heterologous expression of human SMN, and interestingly over-expression of α-COP, restores normal neurite length and morphology. Mutagenesis of the canonical COPI dilysine motifs in exon 2b results in failure to bind to α-COP and abrogates the ability of human SMN to restore neurite outgrowth in SMN-depleted motor neuron-like NSC-34 cells. We conclude that the interaction between SMN and α-COP serves an important function in the growth and maintenance of motor neuron processes and may play a significant role in the pathogenesis of SMA.Keywords:
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Spinal muscular atrophy is a neuromuscular disease resulting from mutations in the SMN1 gene, which encodes the survival motor neuron (SMN) protein. SMN is part of a large complex that is essential for the biogenesis of spliceosomal small nuclear RNPs. SMN also colocalizes with mRNAs in granules that are actively transported in neuronal processes, supporting the hypothesis that SMN is involved in axonal trafficking of mRNPs. Here, we have performed a genome-wide analysis of RNAs present in complexes containing the SMN protein and identified more than 200 mRNAs associated with SMN in differentiated NSC-34 motor neuron-like cells. Remarkably, ∼30% are described to localize in axons of different neuron types. In situ hybridization and immuno-fluorescence experiments performed on several candidates indicate that these mRNAs colocalize with the SMN protein in neurites and axons of differentiated NSC-34 cells. Moreover, they localize in cell processes in an SMN-dependent manner. Thus, low SMN levels might result in localization deficiencies of mRNAs required for axonogenesis.
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Abstract Spinal muscular atrophy (SMA) is a heritable neurodegenerative disease affecting motor neurons that is caused by the impaired expression of the full‐length form of the survival of motor neuron protein (SMN), which may have a specialized function in neurons related to mRNA localization. We have previously shown that a population SMN complexes contain Gemin ribonucleoproteins and traffic in the form of granules to neuronal processes and growth cones of cultured neurons. A QNQKE sequence within exon 7 has been shown to be necessary for both cytoplasmic localization of SMN and axonal function. Here we show that the QNQKE sequence can influence the nucleocytoplasmic distribution of the SMN‐Gemin complex and its localization into neuronal processes. QNQKE exerted a stronger effect on SMN localization in primary neurons compared with COS‐7 cells. By using double‐label fluorescence in situ hybridization and immunofluorescence, SMN granules within neuronal processes colocalized with poly‐(A) mRNA and PABP. These findings provide further evidence in support of a neuronal function for SMN and motivation to investigate for impaired assembly and/or localization of mRNP complexes as an underlying cause of SMA. © 2007 Wiley‐Liss, Inc.
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Spinal muscular atrophy (SMA) results from reduced levels of the survival of motor neuron (SMN) protein, which has a well characterized function in spliceosomal small nuclear ribonucleoprotein assembly. Currently, it is not understood how deficiency of a housekeeping protein leads to the selective degeneration of spinal cord motor neurons. Numerous studies have shown that SMN is present in neuronal processes and has many interaction partners, including mRNA-binding proteins, suggesting a potential noncanonical role in axonal mRNA metabolism. In this study, we have established a novel technological approach using bimolecular fluorescence complementation (BiFC) and quantitative image analysis to characterize SMN-protein interactions in primary motor neurons. Consistent with biochemical studies on the SMN complex, BiFC analysis revealed that SMN dimerizes and interacts with Gemin2 in nuclear gems and axonal granules. In addition, using pull down assays, immunofluorescence, cell transfection, and BiFC, we characterized a novel interaction between SMN and the neuronal mRNA-binding protein HuD, which was dependent on the Tudor domain of SMN. A missense mutation in the SMN Tudor domain, which is known to cause SMA, impaired the interaction with HuD, but did not affect SMN axonal localization or self-association. Furthermore, time-lapse microscopy revealed SMN cotransport with HuD in live motor neurons. Importantly, SMN knockdown in primary motor neurons resulted in a specific reduction of both HuD protein and poly(A) mRNA levels in the axonal compartment. These findings reveal a noncanonical role for SMN whereby its interaction with mRNA-binding proteins may facilitate the localization of associated poly(A) mRNAs into axons.
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Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic cause of infant mortality. SMN protein is present at high levels in both axons and growth cones, and loss of its function disrupts axonal extension and pathfinding. SMN is known to associate with the RNA-binding protein hnRNP-R, and together they are responsible for the transport and/or local translation of β-actin mRNA in the growth cones of motor neurons. However, the full complement of SMN-interacting proteins in neurons remains unknown. Here we used mass spectrometry to identify HuD as a novel neuronal SMN-interacting partner. HuD is a neuron-specific RNA-binding protein that interacts with mRNAs, including candidate plasticity-related gene 15 (cpg15). We show that SMN and HuD form a complex in spinal motor axons, and that both interact with cpg15 mRNA in neurons. CPG15 is highly expressed in the developing ventral spinal cord and can promote motor axon branching and neuromuscular synapse formation, suggesting a crucial role in the development of motor axons and neuromuscular junctions. Cpg15 mRNA previously has been shown to localize into axonal processes. Here we show that SMN deficiency reduces cpg15 mRNA levels in neurons, and, more importantly, cpg15 overexpression partially rescues the SMN-deficiency phenotype in zebrafish. Our results provide insight into the function of SMN protein in axons and also identify potential targets for the study of mechanisms that lead to the SMA pathology and related neuromuscular diseases.
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Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by a loss of α motoneurons in the spinal cord. SMA is caused by low levels of the ubiquitously expressed survival motor neuron (Smn) protein. As it is unclear how low levels of Smn specifically affect motoneurons, we have modeled SMA in zebrafish, a vertebrate model organism with well-characterized motoneuron development. Using antisense morpholinos to reduce Smn levels throughout the entire embryo, we found motor axon–specific pathfinding defects. Reduction of Smn in individual motoneurons revealed that smn is acting cell autonomously. These results show for the first time, in vivo, that Smn functions in motor axon development and suggest that these early developmental defects may lead to subsequent motoneuron loss.
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Spinal muscular atrophy (SMA) is an autosomal recessive disease of childhood due to loss of the telomeric survival motor neuron gene, SMN1 . The general functions of the main SMN1 protein product, full-length SMN (FL-SMN), do not explain the selective motoneuronal loss of SMA. We identified axonal-SMN (a-SMN), an alternatively spliced SMN form, preferentially encoded by the SMN1 gene in humans. The a-SMN transcript and protein are down-regulated during early development in different tissues. In the spinal cord, the a-SMN protein is selectively expressed in motor neurons and mainly localized in axons. Forced expression of a-SMN stimulates motor neuron axonogenesis in a time-dependent fashion and induces axonal-like growth in non-neuronal cells. Exons 2b and 3 are essential for the axonogenic effects. This discovery indicates an unexpected complexity of the SMN gene system and may help in understanding the pathogenesis of SMA.
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J. Neurochem. (2012) 121 , 465–474. Abstract The axonal survival of motor neuron (a‐SMN) protein is a truncated isoform of SMN1 , the spinal muscular atrophy (SMA) disease gene. a‐SMN is selectively localized in axons and endowed with remarkable axonogenic properties. At present, the role of a‐SMN in SMA is unknown. As a first step to verify a link between a‐SMN and SMA, we investigated by means of over‐expression experiments in neuroblastoma‐spinal cord hybrid cell line (NSC34) whether SMA pathogenic mutations located in the N‐terminal part of the protein affected a‐SMN function. We demonstrated here that either SMN1 missense mutations or small intragenic re‐arrangements located in the Tudor domain consistently altered the a‐SMN capability of inducing axonal elongation in vitro . Mutated human a‐SMN proteins determined in almost all NSC34 motor neurons the growth of short axons with prominent morphologic abnormalities. Our data indicate that the Tudor domain is critical in dictating a‐SMN function possibly because it is an association domain for proteins involved in axon growth. They also indicate that Tudor domain mutations are functionally relevant not only for FL‐SMN but also for a‐SMN, raising the possibility that also a‐SMN loss of function may contribute to the pathogenic steps leading to SMA.
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Spinal muscular atrophy (SMA), an inherited disease of motor neuron dysfunction, results from insufficient levels of the survival motor neuron (SMN) protein. Movement of the SMN protein as granules within cultured axons suggests that the pathogenesis of SMA may involve defects in neuronal transport, yet the nature of axon transport vesicles remains enigmatic. Here we show that SMN directly binds to the α-subunit of the coat protein I (COPI) vesicle coat protein. The α-COP protein co-immunoprecipitates with SMN, small nuclear ribonucleoprotein-associated assembly factors and β-actin mRNA. Although typically Golgi associated, in neuronal cells α-COP localizes to lamellipodia and growth cones and moves within the axon, with a subset of these granules traveling together with SMN. Depletion of α-COP resulted in mislocalization of SMN and actin at the leading edge at the lamellipodia. We propose that neurons utilize the Golgi-associated COPI vesicle to deliver cargoes necessary for motor neuron integrity and function.
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