Elsevier

Gene

Volume 395, Issues 1–2, 15 June 2007, Pages 8-14
Gene

Alternative splicing and nonsense-mediated mRNA decay in the regulation of a new adenomatous polyposis coli transcript

https://doi.org/10.1016/j.gene.2006.10.027Get rights and content

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.

Introduction

Familial adenomatous polyposis (FAP) is an inherited autosomal dominant precancerous condition characterized by multiple adenomatous polyps in the colon and rectum. If not treated, these polyps invariably develop to colorectal cancer, typically by the age of 40 years (Rustgi, 1994). This condition is primarily associated with germline mutations in the adenomatous polyposis coli (apc) gene (OMIM175100) (Groden et al., 1991, Kinzler et al., 1991). The apc gene, a ubiquitously expressed tumor suppressor, contains at least 21 exons (Santoro and Groden, 1997), 17 of which are coding exons and 4 are untranslated exons.

The APC protein is involved in regulating β-catenin levels, intercellular adhesion, cytoskeleton stabilization, signal transduction, apoptosis and cell-cycle control, and it probably exerts a nuclear function in chromosome segregation (Sieber et al., 2000, Fearnhead et al., 2001, Kaplan et al., 2001). Its functional domains include heptad repeats at the amino-terminal end (amino acids 6–57) that mediate homodimer formation, the armadillo repeat (amino acids 453–767) that binds to Asef, two motifs that interact with β-catenin at amino acids 1020–1169 and 1262–2033, respectively, and microtubule-, EB1- and hDLG-binding domains (Sieber et al., 2000). Alternative splicing mechanisms involving the non-coding exons 0.1, 0.2, 0.3 and BS (Horii et al., 1993, Thliveris et al., 1994) and coding exons 1, 3–4, 7, 9, 10A and 14 (Groden et al., 1991, Oshima et al., 1993, Thliveris et al., 1994, Samowitz et al., 1995, Sulekova and Ballhausen, 1995, Sulekova et al., 1995, Xia et al., 1995) lead to multiple protein variants whose molecular weight varies from 90 to 300 kDa (Kraus et al., 1996, Sieber et al., 2000).

Skipping of exon 14 results in a novel exon 13/15 connection that yields an mRNA with a novel open reading frame terminating 19 codons beyond exon 15. This transcript, co-expressed with p300apc, represents less than 5% of the p300apc (Bala et al., 1997).

It is not known whether truncated APC proteins, generated by alternative splicing, exert a biological function, or whether their expression merely reflects the altered mRNA splicing mechanism. The finding that mRNA isoforms are degraded by nonsense-mediated mRNA decay (NMD), rather than translated to make protein, shows that regulated splicing which generates mRNAs with premature termination codons (PTC+ RNAs) serves to down-regulate protein expression (Hillman et al., 2004). Since NMD does not completely ablate expression of PTC-containing mRNAs, and since the residual expression levels vary considerably between transcripts, cell types and even individuals, NMD can contribute to balancing the expression of proteins or mRNA isoforms (Holbrook et al., 2004).

In this study we characterized nine novel alternative splicing isoforms of the apc gene and evaluated the involvement of one of these transcripts, bearing an extra exon and a nonsense codon, in colorectal cancer progression and colorectal cell differentiation.

Section snippets

Patients

We studied 41 unrelated Italian FAP patients and 9 normal controls. Patients had the classical polyposis phenotype, characterized by hundreds of adenomatous polyps. They had been previously screened for mutations in the apc gene coding region (De Rosa et al., 2003, De Rosa et al., 2004). Normal colorectal mucosa and colorectal cancer tissues were sampled from a patient with a sporadic colon cancer at the “Istituto Nazionale Tumori G. Pascale” Hospital in Naples.

Cell culture

The cell lines analyzed in this

Identification and characterization of 9 novel alternative transcripts

We examined apc mRNAs from peripheral blood lymphocytes of 24 FAP patients affected by germline apc mutations, 17 FAP patients negative for germline apc mutations and 9 controls. Using RT-PCR followed by PCR with nested 5′ and 3′ primers we amplified four fragments encompassing exons 1–6, 5–9a, 8–12, and 11–15 respectively. In addition to the normal-sized transcripts, which were observed in all samples tested, 9 low-molecular-weight fragments were detected (Fig. 1a, b, c and d). Sequence

Discussion

We have identified and characterized 9 apc alternatively or aberrantly spliced transcripts, all co-expressed with p300apc at the mRNA level. Six of these transcripts (nos. 1, 2, 4, 7, 8 and 9 in Table 2), each of which results in a new exon connection with a novel prematurely terminating open reading frame, were present in normal controls and FAP patients. Three transcripts (nos. 3, 5 and 6) maintain the reading frame and occurred only in FAP patients who are carriers of an apc mutation.

Acknowledgements

We are grateful to Jean Gilder for editing the text.

Grant sponsors: MIUR, Regione Campania (L.R.N.5 del 28.03.2002) and BioGeM scarl, Italy (Project: ʽʽl Geui dell ʽUomo").

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