title: Different Stability and Proteasome-Mediated Degradation Rate of SMN Protein Isoforms.
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.
authors.affiliation: Molecular Neuroanatomy and Pathogenesis Unit, IRCCS Neurological Institute "C. Besta", Milano, Italy.
authors.forename: Denise
authors.initials: D
authors.lastname: Locatelli
authors.affiliation: Laboratory of Molecular Biology, IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy.
authors.forename: Mineko
authors.initials: M
authors.lastname: Terao
authors.affiliation: Laboratory of Molecular Biology, IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy.
authors.forename: Mami
authors.initials: M
authors.lastname: Kurosaki
authors.affiliation: Molecular Neuroanatomy and Pathogenesis Unit, IRCCS Neurological Institute "C. Besta", Milano, Italy.
authors.forename: Maria Clara
authors.initials: MC
authors.lastname: Zanellati
authors.affiliation: Molecular Neuroanatomy and Pathogenesis Unit, IRCCS Neurological Institute "C. Besta", Milano, Italy.
authors.forename: Daniela Rita
authors.initials: DR
authors.lastname: Pletto
authors.affiliation: Molecular Neuroanatomy and Pathogenesis Unit, IRCCS Neurological Institute "C. Besta", Milano, Italy.
authors.forename: Adele
authors.initials: A
authors.lastname: Finardi
authors.affiliation: Molecular Neuroanatomy and Pathogenesis Unit, IRCCS Neurological Institute "C. Besta", Milano, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Colciaghi
authors.affiliation: Laboratory of Molecular Biology, IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Garattini
authors.affiliation: Molecular Neuroanatomy and Pathogenesis Unit, IRCCS Neurological Institute "C. Besta", Milano, Italy.
authors.forename: Giorgio Stefano
authors.initials: GS
authors.lastname: Battaglia
mesh_terms: D002460:Cell Line; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; C526471:SMN1 protein, human; D055533:Survival of Motor Neuron 1 Protein; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex