2004 Fiscal Year Final Research Report Summary
Development of a new treatment with splicing modulators for spinal muscular atrophy
| Project/Area Number |
15591106
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pediatrics
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| Research Institution | Kobe University |
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Principal Investigator |
NSIHIO Hisahide Kobe University, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (80189258)
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| Co-Investigator(Kenkyū-buntansha) |
MATUO Masahumi Kobe University, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (10157266)
TAKESIMA Ysuhiro Kobe University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (40281141)
LEE Miyojin Kobe University, Graduate School of Medicine, Assistant Professor, 大学院・医学系研究科, 講師 (20273766)
AYAKI Hitosi Kobe University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (80222701)
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| Project Period (FY) |
2003 – 2004
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| Keywords | spinal muscular atrophy / the SMN1 gene / the SMN2 gene / hnRNP C1 / C2 / in vitro splicing assay / peptide nucleic acid |
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| Research Abstract |
1 (Purpose) The SMN1 gene is a responsible gene for the most common form of spinal muscular atrophy (SMA). The SMN2 gene is an almost identical gene to SMN1 and codes the same protein as SMN1 does. However, main product of SMN1 is a transcript including exon 7, while that of SMN2 is a transcript lacking exon7. That is the reason why SMN2 cannot compensate the loss of SMN1 in most SMA patients. A single nucleotide change in exon 7, 6C in SMN1 and 6T in SMN2, makes the splicing difference between them. The purpose of the study is to clarify the inclusion mechanism of exon 7 into the transcript of SMN1.
2 (Identification of SMN1 pre-mRNA binding proteins) To obtain the SMN1 pre-mRNA proteins, HeLa cell nuclear extract proteins bound to fluorescent die-labeled RNA probe were isolated by gel-shift method. Subsequent mass-spectrometry analysis proved that the isolated proteins were mixture of hnRNPC1/C2.
3 (In vitro splicing assay) In vitro splicing assay with mini-genes with SMN1 or SMN2 exon 7 sequences was performed. The presence of anti- hnRNPC1/C2 antibody reduced dose-dependently the splicing efficiency with SMN1 mini-gene, but did not influence on the efficiency of SMN2 mini-gene. The findings suggested that hnRNPC1/C2 has a specific role in the exon 7 inclusion into SMN1 transcript.
4 (Splicing modulation by PNA) A peptide nucleic acid (PNA) with SMN2 exon 7 binding domain and serine-arginine repeat domain was synthesized. The presence of the synthesized PNA facilitated in vitro splicing reaction of the SMN2 mini-gene. The synthesized PNA may also facilitate in the cells a transcription of full-length SMN2 mRNA to produce functional SMN protein, which suggests a splicing modulation therapy for SMA patients.
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Research Products
(6 results)
