Abstract
The key pathogenic steps leading to spinal muscular atrophy (SMA), a genetic disease characterized by selective motor neuron degeneration, are not fully clarified. The full-length SMN protein (FL-SMN), the main protein product of the disease gene SMN1, plays an established role in the cytoplasm in snRNP biogenesis ultimately leading to mRNA splicing within the nucleus. It is also involved in the mRNA axonal transport. However, to what extent the impairment of these two SMN functions contributes to SMA pathogenesis remains unknown. A shorter SMN isoform, axonal-SMN or a-SMN, with more specific axonal localization, has been discovered, but whether it might act in concert with FL-SMN in SMA pathogenesis is not known. As a first step in defining common or divergent intracellular roles of FL-SMN vs a-SMN proteins, we here characterized the turn-over of both proteins and investigated which pathway contributed to a-SMN degradation. We performed real time western blot and confocal immunofluorescence analysis in easily controllable in vitro settings. We analyzed co-transfected NSC34 and HeLa cells and cell clones stably expressing both a-SMN and FL-SMN proteins after specific blocking of transcript or protein synthesis and inhibition of known intracellular degradation pathways. Our data indicated that whereas the stability of both FL-SMN and a-SMN transcripts was comparable, the a-SMN protein was characterized by a much shorter half-life than FL-SMN. In addition, as already demonstrated for FL-SMN, the Ub/proteasome pathway played a major role in the a-SMN protein degradation. We hypothesize that the faster degradation rate of a-SMN vs FL-SMN is related to the protection provided by the protein complex in which FL-SMN is assembled. The diverse a-SMN vs FL-SMN C-terminus may dictate different protein interactions and complex formation explaining the different localization and role in the neuronal compartment, and the lower expression and stability of a-SMN.
Highlights
Spinal Muscular Atrophy or spinal muscular atrophy (SMA) is a severe autosomal recessive disease characterized by selective motor neuron degeneration
NSC34 cells were harvested at different time points (0, 1, 3, 5, 7 hrs) after CHX and the protein levels examined through quantitative western blot analysis
We analyze the intracellular fate of full-length SMN protein (FL-SMN) and a-SMN mRNAs and proteins to investigate the stability of both proteins and the pathways contributing to their degradation
Summary
Spinal Muscular Atrophy or SMA is a severe autosomal recessive disease characterized by selective motor neuron degeneration. SMA is genetically determined by disruptions of the Survival of Motor Neuron 1 (SMN1) gene, first reported in 1995 [4]. The different disease severity of affected patients is related to the presence, peculiar to the human species, of the almost identical SMN2 copy gene. In contrast to SMN1, which produces the functional “full-length” FL-SMN protein, the SMN2 gene mainly encodes an exon 7 truncated SMN form (Δ7-SMN), unstable and rapidly degraded, and only low amounts of FL-SMN [5,6,7,8,9,10]. The a-SMN protein is mainly produced by the SMN1 gene through an intron-retention event [13]. In comparison to FL-SMN, the a-SMN protein is more selectively expressed in axons and stimulates axon growth when over-expressed in vitro [13,14]
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