Different Stability and Proteasome-Mediated Degradation Rate of SMN Protein Isoforms

• Published in: PloS one Vol. 10; no. 7; p. e0134163
• Main Authors: Locatelli, Denise, Terao, Mineko, Kurosaki, Mami, Zanellati, Maria Clara, Pletto, Daniela Rita, Finardi, Adele, Colciaghi, Francesca, Garattini, Enrico, Battaglia, Giorgio Stefano
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 27.07.2015; Public Library of Science (PLoS)
• Subjects: Atrophy; Axonal transport; Biodegradation; C-Terminus; Cell Line; Cloning; Complex formation; Cytoplasm; Degeneration; Degradation; Growth factors; Humans; Immunofluorescence; Intracellular; Isoforms; Laboratories; Localization; Molecular biology; Motility; Muscular Atrophy, Spinal - genetics; Muscular Atrophy, Spinal - metabolism; Muscular Atrophy, Spinal - pathology; Neuromuscular diseases; Neurons; Pathogenesis; Proteasome Endopeptidase Complex - genetics; Proteasome Endopeptidase Complex - metabolism; Proteasomes; Protein biosynthesis; Protein interaction; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Stability; Protein Structure, Tertiary; Protein synthesis; Proteins; Proteolysis; Ribonucleoproteins (small nuclear); RNA; RNA transport; Rodents; SMN protein; Spinal muscular atrophy; Splicing; Stability; Survival of Motor Neuron 1 Protein - genetics; Survival of Motor Neuron 1 Protein - metabolism; Transcription; Ubiquitin - genetics; Ubiquitin - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0134163

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.