Toru Takarada1, Mawaddah Ar Rochmah2, Nur Imma Fatimah Harahap2, Masakazu Shinohara2, Toshio Saito3, Kayoko Saito4, Poh San Lai5, Yoshihiro Bouike6, Yasuhiro Takeshima7, Hiroyuki Awano8, Ichiro Morioka8, Kazumoto Iijima8, Hisahide Nishio9, Atsuko Takeuchi1. 1. Analytical Laboratory, Kobe Pharmaceutical University, Kobe, Japan. 2. Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan. 3. Department of Neurology, Toneyama National Hospital, Osaka, Japan. 4. Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan. 5. Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 6. Faculty of Nutrition, Kobe Gakuin University, Kobe, Japan. 7. Department of Pediatrics, Hyogo College of Medicine, Nishinomiya, Japan. 8. Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan. 9. 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.
Abstract
BACKGROUND AND PURPOSE: 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. PATIENTS AND 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.
BACKGROUND AND PURPOSE: 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. PATIENTS AND 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: Amanda C Raimer; Suhana S Singh; Maina R Edula; Tamara Paris-Davila; Vasudha Vandadi; Ashlyn M Spring; A Gregory Matera Journal: Dis Model Mech Date: 2020-05-22 Impact factor: 5.758
Authors: Marianna A Maretina; Galina Y Zheleznyakova; Kristina M Lanko; Anna A Egorova; Vladislav S Baranov; Anton V Kiselev Journal: Curr Genomics Date: 2018-08 Impact factor: 2.236