Abstract
Regulation of calcitonin (CT)/calcitonin gene-related peptide (CGRP) RNA processing involves the use of alternative 3' terminal exons. In most tissues and cell lines, the CT terminal exon is recognized. In an attempt to define regulatory sequences involved in the utilization of the CT-specific terminal exon, we performed deletion and mutation analyses of a mini-gene construct that contains the CT terminal exon and mimics the CT processing choice in vivo. These studies identified a 127-nucleotide intron enhancer located approximately 150 nucleotides downstream of the CT exon poly(A) cleavage site that is required for recognition of the exon. The enhancer contains an essential and conserved 5' splice site sequence. Mutation of the splice site resulted in diminished utilization of the CT-specific terminal exon and increased skipping of the CT exon in both the mini-gene and in the natural CT/CGRP gene. Other components of the intron enhancer modified utilization of the CT-specific terminal exon and were necessary to prevent utilization of the 5' splice site within the intron enhancer as an actual splice site directing cryptic splicing. Conservation of the intron enhancer in three mammalian species suggests an important role for this intron element in the regulation of CT/CGRP processing and an expanded role for intronic 5' splice site sequences in the regulation of RNA processing.
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Selected References
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- Adema G. J., Bovenberg R. A., Jansz H. S., Baas P. D. Unusual branch point selection involved in splicing of the alternatively processed Calcitonin/CGRP-I pre-mRNA. Nucleic Acids Res. 1988 Oct 25;16(20):9513–9526. doi: 10.1093/nar/16.20.9513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Adema G. J., van Hulst K. L., Baas P. D. Uridine branch acceptor is a cis-acting element involved in regulation of the alternative processing of calcitonin/CGRP-l pre-mRNA. Nucleic Acids Res. 1990 Sep 25;18(18):5365–5373. doi: 10.1093/nar/18.18.5365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Amara S. G., Jonas V., Rosenfeld M. G., Ong E. S., Evans R. M. Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature. 1982 Jul 15;298(5871):240–244. doi: 10.1038/298240a0. [DOI] [PubMed] [Google Scholar]
- Boelens W. C., Jansen E. J., van Venrooij W. J., Stripecke R., Mattaj I. W., Gunderson S. I. The human U1 snRNP-specific U1A protein inhibits polyadenylation of its own pre-mRNA. Cell. 1993 Mar 26;72(6):881–892. doi: 10.1016/0092-8674(93)90577-d. [DOI] [PubMed] [Google Scholar]
- Bovenberg R. A., Moen T. C., Jansz H. S., Baas P. D. In vitro splicing analysis of mini-gene constructs of the alternatively processed human calcitonin/CGRP-I pre-mRNA. Biochim Biophys Acta. 1989 Jul 7;1008(2):223–233. doi: 10.1016/0167-4781(80)90013-5. [DOI] [PubMed] [Google Scholar]
- Cote G. J., Abruzzese R. V., Lips C. J., Gagel R. F. Transfection of calcitonin gene regulatory elements into a cell culture model of the C cell. J Bone Miner Res. 1990 Feb;5(2):165–171. doi: 10.1002/jbmr.5650050210. [DOI] [PubMed] [Google Scholar]
- Cote G. J., Nguyen I. N., Lips C. J., Berget S. M., Gagel R. F. Validation of an in vitro RNA processing system for CT/CGRP precursor mRNA. Nucleic Acids Res. 1991 Jul 11;19(13):3601–3606. doi: 10.1093/nar/19.13.3601. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cote G. J., Stolow D. T., Peleg S., Berget S. M., Gagel R. F. Identification of exon sequences and an exon binding protein involved in alternative RNA splicing of calcitonin/CGRP. Nucleic Acids Res. 1992 May 11;20(9):2361–2366. doi: 10.1093/nar/20.9.2361. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Crenshaw E. B., 3rd, Russo A. F., Swanson L. W., Rosenfeld M. G. Neuron-specific alternative RNA processing in transgenic mice expressing a metallothionein-calcitonin fusion gene. Cell. 1987 May 8;49(3):389–398. doi: 10.1016/0092-8674(87)90291-1. [DOI] [PubMed] [Google Scholar]
- Emeson R. B., Hedjran F., Yeakley J. M., Guise J. W., Rosenfeld M. G. Alternative production of calcitonin and CGRP mRNA is regulated at the calcitonin-specific splice acceptor. Nature. 1989 Sep 7;341(6237):76–80. doi: 10.1038/341076a0. [DOI] [PubMed] [Google Scholar]
- Furth P. A., Choe W. T., Rex J. H., Byrne J. C., Baker C. C. Sequences homologous to 5' splice sites are required for the inhibitory activity of papillomavirus late 3' untranslated regions. Mol Cell Biol. 1994 Aug;14(8):5278–5289. doi: 10.1128/mcb.14.8.5278. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gunderson S. I., Beyer K., Martin G., Keller W., Boelens W. C., Mattaj L. W. The human U1A snRNP protein regulates polyadenylation via a direct interaction with poly(A) polymerase. Cell. 1994 Feb 11;76(3):531–541. doi: 10.1016/0092-8674(94)90116-3. [DOI] [PubMed] [Google Scholar]
- Karin M., Richards R. I. Human metallothionein genes--primary structure of the metallothionein-II gene and a related processed gene. Nature. 1982 Oct 28;299(5886):797–802. doi: 10.1038/299797a0. [DOI] [PubMed] [Google Scholar]
- Lou H., Cote G. J., Gagel R. F. The calcitonin exon and its flanking intronic sequences are sufficient for the regulation of human calcitonin/calcitonin gene-related peptide alternative RNA splicing. Mol Endocrinol. 1994 Dec;8(12):1618–1626. doi: 10.1210/mend.8.12.7535892. [DOI] [PubMed] [Google Scholar]
- Lutz C. S., Alwine J. C. Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal. Genes Dev. 1994 Mar 1;8(5):576–586. doi: 10.1101/gad.8.5.576. [DOI] [PubMed] [Google Scholar]
- Mayeda A., Helfman D. M., Krainer A. R. Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF. Mol Cell Biol. 1993 May;13(5):2993–3001. doi: 10.1128/mcb.13.5.2993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Niwa M., MacDonald C. C., Berget S. M. Are vertebrate exons scanned during splice-site selection? Nature. 1992 Nov 19;360(6401):277–280. doi: 10.1038/360277a0. [DOI] [PubMed] [Google Scholar]
- Roesser J. R., Liittschwager K., Leff S. E. Regulation of tissue-specific splicing of the calcitonin/calcitonin gene-related peptide gene by RNA-binding proteins. J Biol Chem. 1993 Apr 15;268(11):8366–8375. [PubMed] [Google Scholar]
- Rosenfeld M. G., Amara S. G., Evans R. M. Alternative RNA processing: determining neuronal phenotype. Science. 1984 Sep 21;225(4668):1315–1320. doi: 10.1126/science.6089345. [DOI] [PubMed] [Google Scholar]
- Wassarman K. M., Steitz J. A. Association with terminal exons in pre-mRNAs: a new role for the U1 snRNP? Genes Dev. 1993 Apr;7(4):647–659. doi: 10.1101/gad.7.4.647. [DOI] [PubMed] [Google Scholar]
- Yeakley J. M., Hedjran F., Morfin J. P., Merillat N., Rosenfeld M. G., Emeson R. B. Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements. Mol Cell Biol. 1993 Oct;13(10):5999–6011. doi: 10.1128/mcb.13.10.5999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhuang Y., Weiner A. M. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. doi: 10.1016/0092-8674(86)90064-4. [DOI] [PubMed] [Google Scholar]
- Zuo P., Manley J. L. The human splicing factor ASF/SF2 can specifically recognize pre-mRNA 5' splice sites. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3363–3367. doi: 10.1073/pnas.91.8.3363. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Boer J. G., Drobetsky E. A., Grosovsky A. J., Mazur M., Glickman B. W. The Chinese hamster aprt gene as a mutational target. Its sequence and an analysis of direct and inverted repeats. Mutat Res. 1989 Aug;226(4):239–244. doi: 10.1016/0165-7992(89)90076-6. [DOI] [PubMed] [Google Scholar]
- van Oers C. C., Adema G. J., Zandberg H., Moen T. C., Baas P. D. Two different sequence elements within exon 4 are necessary for calcitonin-specific splicing of the human calcitonin/calcitonin gene-related peptide I pre-mRNA. Mol Cell Biol. 1994 Feb;14(2):951–960. doi: 10.1128/mcb.14.2.951. [DOI] [PMC free article] [PubMed] [Google Scholar]