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Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay
Published on: August 26, 2018
Differential expression and splicing isoform analysis of human Tcf-4 transcription factor in brain tumors
Shen-Long Howng1, Feng-Hsiang Huang, Shiuh-Lin Hwang
1Department of Neurosurgery, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC.
Abstract:
Tcf family transcription factors are the downstream effectors of the Wnt signal transduction pathway that regulates developmental and oncogenetic processes among species. The alternative splicing of consecutive exons in the 3' part of the Tcf-4 gene and an error-prone A9 repeat in exon 17 have recently been examined in several tumors. To further understand the roles played by differential expression and splicing isoforms of human Tcf-4 in brain tumorigenesis, the expression of the 3' part of the Tcf-4 gene (exons 10-17) was analyzed by RT-PCR, nested RT-PCR and DNA sequencing. The results showed that at least 13 of the Tcf-4 alternative splicing isoforms were found and most of them were overexpressed in various brain tumors. L3 isoform was particularly dominant in metastasis. Several novel splicing isoforms were identified. One that contains different combinations of exon 16 (10-11-12-16-17, S5) was found in normal brain and pituitary adenoma but not in astrocytoma, meningioma or metastasis, whereas the other that contains part of intron 16, designated exon-16', was found in metastasis. Overall 23% of sequencing analysis in brain tumors exhibited frameshift mutations in an A9 repeat region of exon 17. These mutants exhibited 2-base or 1-base deletion (A7, A8) and 1-base insertion (A10). Nonetheless, in vitro functional assay showed that these mutants did not affect the transactivity of Tcf-4 comparing to wild-type Tcf-4. Collectively, our data suggest that a large number of alternative splicing isoforms may together with variable mutations of A9 repeat region maintain balanced pools of Tcf-4 isoforms during brain tumorigenesis.
Insights
The study identified numerous Tcf-4 alternative splicing isoforms and mutations in brain tumors, suggesting their role in tumorigenesis. Some isoforms, like L3, were dominant in metastasis, while mutations in the A9 repeat region did not affect Tcf-4 transactivity.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- Tcf family transcription factors are key Wnt pathway effectors regulating development and oncogenesis.
- Alternative splicing and mutations in the Tcf-4 gene, particularly in its 3' region and A9 repeat, are implicated in various tumors.
Purpose of the Study:
- To investigate the role of differential expression and splicing isoforms of human Tcf-4 in brain tumorigenesis.
- To analyze the expression of the 3' part of the Tcf-4 gene (exons 10-17) in brain tumors.
Main Methods:
- RT-PCR, nested RT-PCR, and DNA sequencing were employed to analyze Tcf-4 gene expression and identify splicing isoforms and mutations.
- In vitro functional assays were conducted to assess the impact of identified mutations on Tcf-4 transactivity.
Main Results:
- At least 13 Tcf-4 alternative splicing isoforms were identified, with most overexpressed in brain tumors.
- The L3 isoform was dominant in metastasis, and novel isoforms (S5, exon-16') were identified with differential expression patterns.
- 23% of brain tumor analyses revealed frameshift mutations in the Tcf-4 exon 17 A9 repeat region, including deletions and insertions.
Conclusions:
- A significant number of alternative splicing isoforms, along with A9 repeat mutations, may contribute to maintaining Tcf-4 isoform balance during brain tumorigenesis.
- The identified Tcf-4 isoforms and mutations provide insights into the molecular mechanisms underlying brain tumor development.
