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Published on: March 4, 2015
Brain- and heart-specific Patched-1 containing exon 12b is a dominant negative isoform and is expressed in
Hideki Uchikawa1, Masashi Toyoda, Kazuaki Nagao
1Department of Genetics, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
Abstract:
Mutations in the human tumor suppressor gene, Patched-1, are associated with nevoid basal cell carcinoma syndrome characterized by developmental abnormalities and tumorigenesis, such as basal cell carcinoma and medulloblastoma. During the investigation of complex alternative splicing in Patched-1, we identified an alternative exon, exon 12b, located between exon 12 and 13, both in humans and in mice. Since exon 12b has an in-frame stop codon, the mRNA isoform containing this exon (Patched12b) encodes a truncated patched-1 protein. RT-PCR and whole mount in situ hybridization revealed that mouse exon 12b was expressed in the brain and heart, particularly in the cerebellum, in both adults and embryos. We next performed a functional analysis of Patched12b using a GLI-responsive luciferase reporter. Luciferase activity was suppressed when transfected with a plasmid encoding Patched-1, but not with a plasmid for Patched12b. The suppressive activity of Patched-1 was relieved when cotransfected with a plasmid for Patched12b. This implies that the Patched12b protein has a dominant negative effect on Patched-1. Interestingly, Patched12b was found to be expressed in some of the medulloblastoma tissues and cell lines, indicating an important role in the pathogenesis of medulloblastoma as well as brain development.
Insights
A newly identified Patched-1 (Patched12b) exon creates a truncated protein that inhibits normal Patched-1 function, potentially driving medulloblastoma development.
Area of Science:
- Molecular Biology
- Developmental Biology
- Oncology
Background:
- Mutations in the Patched-1 tumor suppressor gene are linked to nevoid basal cell carcinoma syndrome.
- This syndrome involves developmental abnormalities and cancers like basal cell carcinoma and medulloblastoma.
Purpose of the Study:
- To investigate complex alternative splicing in the Patched-1 gene.
- To identify and characterize a novel alternative exon (exon 12b) and its encoded protein (Patched12b).
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) and whole mount in situ hybridization to detect exon 12b expression.
- Functional analysis using a GLI-responsive luciferase reporter assay to assess Patched12b activity.
Main Results:
- An alternative exon, 12b, was identified in Patched-1 in humans and mice, leading to a truncated Patched12b protein.
- Patched12b is expressed in the brain (cerebellum) and heart during embryonic and adult stages.
- Patched12b demonstrated a dominant-negative effect on normal Patched-1 function in reporter assays.
Conclusions:
- The Patched12b protein can inhibit the function of the normal Patched-1 protein.
- Expression of Patched12b in medulloblastoma tissues suggests its involvement in brain tumor pathogenesis and development.
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