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A single nucleotide polymorphism in an exon dictates allele dependent differential splicing of episialin mRNA

Abstract

The episialin gene (MUC1) encodes an epithelial mucin containing a variable number of repeats with a length of twenty amino acids, resulting in many different alleles that can be subdivided into two size classes. The episialin pre-mRNA uses either one of two neighbouring splice acceptor sites for exon 2, which mainly encodes the repeats. Using the genetic polymorphism of the episialin gene to identify different alleles, we show here that the splice site recognition is allele dependent and is based on a single A/G nucleotide difference in exon 2 eight nucleotides downstream of the second splice acceptor site. Transfection experiments confirm that this polymorphic nucleotide regulates the splice site selection. The identity of this nucleotide is in most cases correlated with one of the size classes of the alleles, indicating that mutations altering the number of repeats seldom arise by unequal cross-over between the repeat regions.

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Selected References

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  1. Eperon L. P., Graham I. R., Griffiths A. D., Eperon I. C. Effects of RNA secondary structure on alternative splicing of pre-mRNA: is folding limited to a region behind the transcribing RNA polymerase? Cell. 1988 Jul 29;54(3):393–401. doi: 10.1016/0092-8674(88)90202-4. [DOI] [PubMed] [Google Scholar]
  2. Ge H., Noble J., Colgan J., Manley J. L. Polyoma virus small tumor antigen pre-mRNA splicing requires cooperation between two 3' splice sites. Proc Natl Acad Sci U S A. 1990 May;87(9):3338–3342. doi: 10.1073/pnas.87.9.3338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gendler S. J., Lancaster C. A., Taylor-Papadimitriou J., Duhig T., Peat N., Burchell J., Pemberton L., Lalani E. N., Wilson D. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem. 1990 Sep 5;265(25):15286–15293. [PubMed] [Google Scholar]
  4. Gendler S., Taylor-Papadimitriou J., Duhig T., Rothbard J., Burchell J. A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J Biol Chem. 1988 Sep 15;263(26):12820–12823. [PubMed] [Google Scholar]
  5. Hilkens J., Buijs F. Biosynthesis of MAM-6, an epithelial sialomucin. Evidence for involvement of a rare proteolytic cleavage step in the endoplasmic reticulum. J Biol Chem. 1988 Mar 25;263(9):4215–4222. [PubMed] [Google Scholar]
  6. Hilkens J., Buijs F., Hilgers J., Hageman P., Calafat J., Sonnenberg A., van der Valk M. Monoclonal antibodies against human milk-fat globule membranes detecting differentiation antigens of the mammary gland and its tumors. Int J Cancer. 1984 Aug 15;34(2):197–206. doi: 10.1002/ijc.2910340210. [DOI] [PubMed] [Google Scholar]
  7. Jeffreys A. J., Neumann R., Wilson V. Repeat unit sequence variation in minisatellites: a novel source of DNA polymorphism for studying variation and mutation by single molecule analysis. Cell. 1990 Feb 9;60(3):473–485. doi: 10.1016/0092-8674(90)90598-9. [DOI] [PubMed] [Google Scholar]
  8. Lan M. S., Batra S. K., Qi W. N., Metzgar R. S., Hollingsworth M. A. Cloning and sequencing of a human pancreatic tumor mucin cDNA. J Biol Chem. 1990 Sep 5;265(25):15294–15299. [PubMed] [Google Scholar]
  9. Ligtenberg M. J., Vos H. L., Gennissen A. M., Hilkens J. Episialin, a carcinoma-associated mucin, is generated by a polymorphic gene encoding splice variants with alternative amino termini. J Biol Chem. 1990 Apr 5;265(10):5573–5578. [PubMed] [Google Scholar]
  10. Linsley P. S., Kallestad J. C., Horn D. Biosynthesis of high molecular weight breast carcinoma associated mucin glycoproteins. J Biol Chem. 1988 Jun 15;263(17):8390–8397. [PubMed] [Google Scholar]
  11. Morgenstern J. P., Land H. A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells. Nucleic Acids Res. 1990 Feb 25;18(4):1068–1068. doi: 10.1093/nar/18.4.1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Siddiqui J., Abe M., Hayes D., Shani E., Yunis E., Kufe D. Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2320–2323. doi: 10.1073/pnas.85.7.2320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Smith C. W., Patton J. G., Nadal-Ginard B. Alternative splicing in the control of gene expression. Annu Rev Genet. 1989;23:527–577. doi: 10.1146/annurev.ge.23.120189.002523. [DOI] [PubMed] [Google Scholar]
  14. Swallow D. M., Gendler S., Griffiths B., Corney G., Taylor-Papadimitriou J., Bramwell M. E. The human tumour-associated epithelial mucins are coded by an expressed hypervariable gene locus PUM. Nature. 1987 Jul 2;328(6125):82–84. doi: 10.1038/328082a0. [DOI] [PubMed] [Google Scholar]
  15. Watakabe A., Inoue K., Sakamoto H., Shimura Y. A secondary structure at the 3' splice site affects the in vitro splicing reaction of mouse immunoglobulin mu chain pre-mRNAs. Nucleic Acids Res. 1989 Oct 25;17(20):8159–8169. doi: 10.1093/nar/17.20.8159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wreschner D. H., Hareuveni M., Tsarfaty I., Smorodinsky N., Horev J., Zaretsky J., Kotkes P., Weiss M., Lathe R., Dion A. Human epithelial tumor antigen cDNA sequences. Differential splicing may generate multiple protein forms. Eur J Biochem. 1990 May 20;189(3):463–473. doi: 10.1111/j.1432-1033.1990.tb15511.x. [DOI] [PubMed] [Google Scholar]
  17. Zhuang Y., Weiner A. M. The conserved dinucleotide AG of the 3' splice site may be recognized twice during in vitro splicing of mammalian mRNA precursors. Gene. 1990 Jun 15;90(2):263–269. doi: 10.1016/0378-1119(90)90189-x. [DOI] [PubMed] [Google Scholar]
  18. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]