Levels Of Survival Motor Neuron Protein Research Articles

Degradation of survival motor neuron ( SMN) protein is mediated via the ubiquitin/proteasome pathway

Homozygous deletion or mutation in the survival motor neuron ( SMN) 1 gene causes proximal spinal muscular atrophy (SMA), whereas SMN2 acts as a modifying gene that can influence the severity of SMA. It has been suggested that restoration of the SMN protein level in neuronal cells may prevent cell loss and may be helpful for treatment of SMA. Recent studies indicate that the ubiquitin/proteasome pathway is a major system for proteolysis of intracellular proteins. In this study, we investigate whether SMN protein is degraded via the ubiquitin/proteasome pathway. Primary fibroblasts were established from the skin biopsies of SMA patients and the effect of a proteasome inhibitor MG132 and lysosome inhibitor NH 4Cl on SMN protein level was examined. We found that MG132, but not NH 4Cl, significantly increased the amount and nuclear accumulation of SMN protein in SMA patient’s fibroblasts. Immunoprecipitation/western blot analysis indicated that SMN protein was ubiquitinated in cells. In vitro protein ubiquitination assay also demonstrated that SMN protein could be conjugated with ubiquitin. Taken together, we have provided clear evidences that degradation of SMN protein is mediated via the ubiquitin/proteasome pathway and suggest that proteasome inhibitors may up-regulate SMN protein level and may be useful for the treatment of SMA.

Monitoring of recombinant survival motor neuron protein using fiber-optic surface plasmon resonance.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of the survival motor neuron 1 (SMN1) gene. A nearly identical copy gene exists known as SMN2, however, due to an aberrant splicing event, the SMN2 gene fails to produce sufficient full-length protein to protect against disease development in the absence of SMN1. While a number of compounds have recently been identified that can stimulate full-length survival motor neuron (SMN) expression from the nearly identical copy SMN2, one of the difficulties has been the lack of a highly reproducible and quantitative means to measure the levels of SMN protein. To develop a technique that allows the rapid and highly sensitive measurement of SMN protein, a Surface Plasmon Resonance (SPR) application has been developed. The ability to quantify unassociated SMN protein and monitor the binding of SMN with other proteins in solution using a SPR sensor in less than 15 min and at low ng mL(-1) levels in HEPES Buffer Saline (HBS) has been achieved. The detection limit for the specific binding of SMN in HBS pH 7.4 solution is 0.99 ng mL(-1) with non-specific binding accounting for approximately 30% of the signal. Quantification of SMN is based on an immunoassay performed on the gold surface of the SPR sensor. 16-mercaptohexadecanoic acid (MHA) was reacted with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) to form a pre-activated thiol (MHA-NHS). Antibodies for SMN were then coupled to the sensor with the pre-activated thiol. Sensor specificity was examined with mixtures of myoglobin (MG) and SMN. SMN sensor response decreases by more than 60% when MG was added to SMN. The decrease in sensor response can be attributed to non-specific binding of SMN to MG, verified with a sensor for MG.

HnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-beta1.

Proximal spinal muscular atrophy (SMA) is a common motor neuron disease caused by homozygous loss of the survival motor neuron gene (SMN1). SMN2, a nearly identical copy of the gene and present in all SMA patients, fails to provide protection from SMA, due to the disruption of an exonic splicing enhancer (ESE) by a single translationally silent nucleotide exchange, which causes alternative splicing of SMN2 exon 7. Identification of splicing factors that stimulate exon 7 inclusion and thereby produce sufficient amounts of full-length transcripts from the SMN2 gene is of great importance for therapy approaches. Here, by use of in vivo splicing assays, we identified the protein hnRNP-G and its paralogue RBM as two novel splicing factors that promote the inclusion of SMN2 exon 7. Moreover, hnRNP-G and RBM non-specifically bind RNA, but directly and specifically bind Htra2-beta1, an SR-like splicing factor which we have previously shown to stimulate inclusion of exon 7 through a direct interaction with the AG-rich ESE in SMN2 exon 7 pre-mRNA. By using deletion mutants of hnRNP-G, we show that the specific protein-protein interaction of hnRNP-G with Htra2-beta1 mediates the inclusion of SMN2 exon 7 rather than the non-specific interaction of hnRNP-G with SMN pre-mRNA. Additionally, we show for the first time that recombinant trans-acting splicing factors such as hnRNP-G and Htra2-beta1 are also effective on endogenous SMN2 transcripts and increase the endogenous SMN protein level. Finally, we suggest a model of how the exon 7 mRNA processing is regulated by the splicing factors identified so far.

Identification of survival motor neuron as a transcriptional activator-binding protein.

Spinal muscular atrophy (SMA) is an inherited neuro-muscular disease characterized by specific degeneration of spinal cord anterior horn cells and subsequent muscle atrophy. Survival motor neuron ( SMN ), located on chromosome 5q13, is the SMA-determining gene. In the nucleus, SMN is present in large foci called gems, the function of which is not yet known, while cytoplasmic SMN has been implicated in snRNP biogenesis. In SMA patients, SMN protein levels and the number of gems generally correlate with disease severity, suggesting a critical nuclear function for SMN. In a screen for proteins associated with the nuclear transcription activator 'E2' of papillomavirus, two independent SMN cDNAs were isolated. The E2 and SMN proteins were found to associate specifically in vitro and in vivo. Expression of SMN enhanced E2-dependent transcriptional activation, and patient-derived SMN missense mutations reduced E2 gene expression. Our results demonstrate that SMN interacts with a nuclear transcription factor and imply that SMN may serve a role in regulating gene expression. These observations suggest that SMA may in part result from abnormal gene expression and that E2 may influence viral gene expression through SMN interaction.