Abstract

Familial adenomatous polyposis (FAP) is a rare precancerous condition caused by mutations in the adenomatous polyposis coli (apc) gene. Alternative splicing mechanisms involving non-coding and coding exons result in multiple protein variants whose molecular weight ranges between 90 and 300 kDa. We examined the apc 5′ coding region and identified nine new transcripts generated from alternative and/or aberrant splicing. Three of these preserve the reading frame and the corresponding proteins include the catalytic domains and the sequences required for β-catenin regulation. The other six transcripts create a frameshift that produces a premature stop codon; one of these has an additional 77-nucleotide-long exon (1A) between exons 1 and 2 that leads to a frameshift and a premature stop codon in exon 2. Quantitative PCR analysis suggests that the expression of this transcript is regulated during colorectal cancer tumorigenesis and differentiation. Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA surveillance mechanism that detects and degrades mRNAs that have premature termination codons (PTCs). Expression of splicing variants containing PTCs and their subsequent degradation via NMD seems to be a general mechanism of gene regulation. Incubation of Caco2 cell lines with cycloheximide, a chemical inhibitor of translation that is known to inhibit also NMD, indicates that the apc mRNA isoform that includes exon 1A is degraded by NMD, thereby suggesting that regulated unproductive splicing and NMD degradation could modulate APC protein expression.

Keywords: Familial adenomatous polyposis; Splicing machinery; Colorectal cancer progression; Colon differentiation

Abbreviations: FAP, familial adenomatous polyposis; APC, adenomatous polyposis coli; NMD, nonsense-mediated mRNA decay; PTC, premature termination codon; GSH-synthetase, glutathione-synthetase.

Article Outline

1. Introduction

2. Material and methods

2.1. Patients

2.2. Cell culture

2.3. RT-PCR

2.4. Real-time PCR quantification

3. Results

3.1. Identification and characterization of 9 novel alternative transcripts

3.2. Characterization of a messenger containing a novel extra exon (exon 1A)

3.3. The alternative splicing containing exon 1A is overexpressed during colon carcinogenesis

3.4. Alternatively spliced mRNA containing exon 1A is a target of the NMD

4. Discussion

Acknowledgements

References