title: SMA mutations in SMN Tudor and C-terminal domains destabilize the protein.
abstract: OBJECTIVE
  Most spinal muscular atrophy (SMA) patients are homozygous for survival of motor neuron 1 gene (SMN1) deletion. However, some SMA patients carry an intragenic SMN1 mutation. Such patients provide a clue to understanding the function of the SMN protein and the role of each domain of the protein. We previously identified mutations in the Tudor domain and C-terminal region of the SMN protein in three Japanese SMA patients. To clarify the effect of these mutations on protein stability, we conducted expression assays of SMN with mutated domains.
  
  
  METHODS
  Patients A and B carried a mutation in SMN1 exon 3, which encodes a Tudor domain, c.275G>C (p.Trp92Ser). Patient C carried a mutation in SMN1 exon 6, which encodes a YG-box; c.819_820insT (p.Thr274Tyrfs). We constructed plasmid expression vectors containing wild-type and mutant SMN1 cDNAs. After transfection of HeLa cells with the expression plasmids, RNA and protein were isolated and analyzed by reverse-transcription PCR and western blot analysis.
  
  
  RESULTS
  The abundance of wild-type and mutant SMN1 transcripts in HeLa cells was almost the same. However, western blot analysis showed lower levels of mutant SMN proteins compared with wild-type SMN. In mutant SMN proteins, it is noteworthy that the level of the p.Thr274Tyrfs mutant was much reduced compared with that of the p.Trp92Ser mutant.
  
  
  CONCLUSIONS
  SMN mutations may affect the stability and levels of the protein.
authors.affiliation: Analytical Laboratory, Kobe Pharmaceutical University, Kobe, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Takarada
authors.affiliation: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Mawaddah
authors.initials: M
authors.lastname: Ar Rochmah
authors.affiliation: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Nur Imma Fatimah
authors.initials: NIF
authors.lastname: Harahap
authors.affiliation: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Shinohara
authors.affiliation: Department of Neurology, Toneyama National Hospital, Osaka, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Saito
authors.affiliation: Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Saito
authors.affiliation: Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Poh San
authors.initials: PS
authors.lastname: Lai
authors.affiliation: Faculty of Nutrition, Kobe Gakuin University, Kobe, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Bouike
authors.affiliation: Department of Pediatrics, Hyogo College of Medicine, Nishinomiya, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takeshima
authors.affiliation: Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Awano
authors.affiliation: Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Morioka
authors.affiliation: Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Kazumoto
authors.initials: K
authors.lastname: Iijima
authors.affiliation: Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan; Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan. Electronic address: nishio@med.kobe-u.ac.jp.
authors.forename: Hisahide
authors.initials: H
authors.lastname: Nishio
authors.affiliation: Analytical Laboratory, Kobe Pharmaceutical University, Kobe, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Takeuchi
mesh_terms: D002648:Child; D002675:Child, Preschool; D005260:Female; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007223:Infant; D008297:Male; D009134:Muscular Atrophy, Spinal / Q000235:genetics; D009154:Mutation; D000072417:Protein Domains; D055550:Protein Stability; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics* / Q000378:metabolism*
chemical_list: C526471:SMN1 protein, human; D055533:Survival of Motor Neuron 1 Protein
keywords: C-terminal region; SMN mutation; SMN stability; Tudor domain; YG box