Multiple APC messenger RNA isoforms encoding exon 15 short open reading frames are expressed in the context of a novel exon 10A-derived sequence.

Intragenic splice mechanisms affecting the coding exons 8 to 15 of the human adenomatous polyposis coli (APC) gene have been analyzed. Three mechanisms within this gene area were found to contribute to mRNA heterogeneity: (i) facultative expression of exon 9-encoded sequences; (ii) in-frame insertion of a 54-nucleotide sequence encoded by a novel exon located 1.6 kb down-stream from exon 10, provisionally designated APC exon 10A; (iii) skipping of exon 14, resulting in a novel exon 13/15 connection. Interestingly the latter event provided the mRNA with a novel open reading frame, which was terminated after 19 codons of exon 15-derived sequences. Combinatorial joining of these segments yielded 7 different transcripts in addition to an mRNA species resulting from an exon 10/15 connection, as determined by cloning and sequence analysis. RT-PCR expression analyses were carried out to demonstrate that this complexity of splice variants is indeed synthesized in cell lines derived from various tissues. Furthermore, in accordance with our findings at the transcript level, we provide Western blot analyses demonstrating that moderate steady-state levels of genuine APC-specific low m.w. polypeptide chains exist. These APC "light chains", however, are not identical with polypeptide chains, which have been reported to accompany apoptosis and necrosis, since the molecules described here are definitively co-expressed with p300apc at the transcript and protein levels.

Immunochemical identification of novel high-molecular-weight protein isoforms of the adenomatous polyposis coli (APC) gene.

Mapping analyses of monoclonal antibodies (MAbs) directed against the amino-terminus of the adenomatous polyposis coli (APC) gene product revealed that epitopes recognized by the MAbs FE9, CF11 and AC4 constitute different peptide sequences encoded by the APC exons 1, 2 and 3, respectively. The protein pattern detected with these specificity-defined immunoreagents, however, differed depending on the particular antibody used on Western blots of cellular urea extracts. APC exon 15-positive "classic" p300apc polypeptide chains were identified by the MAb FE9, MAb CF11 and the C-terminus-specific MAb IE1, but only weak signals were obtained with the AC4 MAb, which defines an exon 3-encoded epitope. In contrast with this immunoreactivity, 2 novel high m.w. products of approx. 150/160 and 200 kDa were exclusively recognized by the AC4 MAb, which was shown to bind to the APC exon 3-encoded peptide sequence SRESTGYL. A molecular form of some 400 kDa was identified to represent a disulfide-bound oligomer of the p150/160apc molecules. The novel APC-related molecules did not contain exon 1- and exon 15-encoded epitopes, as confirmed with the help of the FE9 and IE1 MAbs, respectively. This observation was corroborated by the fact that these novel proteins were not truncated in a collection of familial adenomatous polyposis patients with stop mutations in exon 15. We conclude, that APC MAb AC4-reactive p150/160 and p200 polypeptide chains represent novel genuine products of the APC gene devoid of exon 1- and exon 15-encoded protein interaction domains.