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Characterization of RET proto-oncogene 3' splicing variants and polyadenylation sites: a novel C-terminus for RET
1Department of Pathology, Queen's University, Kingston, ON, Canada.
Abstract:
The RET proto-oncogene, which encodes a receptor tyrosine kinase, displays multiple alternative splicing variants. Splicing of sequences 3' of exon 19 to generate several coding and untranslated region (UTR) sequences has been previously reported. We have sequenced the full length RET coding region and characterized the transcripts and 3' UTRs generated by alternative splicing of the RET 3' terminus. These analyses were performed using both RET cDNA cloned from a pheochromocytoma library and reverse transcriptase PCR products generated using RNA from a neuroblastoma cell line (LA-N-2). Three different carboxyl termini were identified. In addition to the nine and 51 terminal amino acid forms already known, we identified a third with 43 terminal amino acids predicted to encode a novel RET protein isoform. A total of 3621 base pairs of DNA 3' of exon 19, which spans the alternatively spliced exons and RET UTRs, was sequenced. Four polyadenylation sites were identified. The observed combinations of polyadenylation sites and 3' coding sequence suggest that RET transcripts with up to 10 different 3' sequences and up to 40 different full length RET transcripts may exist.
Insights
Researchers identified a novel RET protein isoform by analyzing alternative splicing variants of the RET proto-oncogene. This discovery expands our understanding of RET transcript diversity and potential protein functions.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- The RET proto-oncogene encodes a receptor tyrosine kinase involved in various cellular processes.
- Alternative splicing of RET transcripts generates diverse coding and untranslated region (UTR) sequences.
- Previous studies reported splicing variants 3' of exon 19, but a comprehensive characterization was lacking.
Purpose of the Study:
- To sequence the full-length RET coding region.
- To characterize RET transcripts and 3' UTRs generated by alternative splicing.
- To identify novel RET protein isoforms and alternative splicing events.
Main Methods:
- Sequencing of RET cDNA from a pheochromocytoma library.
- Reverse transcriptase PCR (RT-PCR) using RNA from a neuroblastoma cell line (LA-N-2).
- Analysis of alternatively spliced exons and 3' UTRs downstream of exon 19.
Main Results:
- Identification of three distinct carboxyl termini for RET, including a novel 43-amino acid form.
- Sequencing of 3621 base pairs downstream of exon 19, revealing alternatively spliced exons and UTRs.
- Discovery of four polyadenylation sites, suggesting the potential for up to 10 different 3' RET sequences and 40 full-length transcripts.
Conclusions:
- A novel RET protein isoform, resulting from alternative splicing, has been identified.
- The RET gene exhibits extensive transcript diversity due to alternative splicing of its 3' terminus and multiple polyadenylation sites.
- This complexity in RET gene expression may contribute to its role in various cellular functions and diseases.