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Nucleic Acids Research, 2002, Vol. 30, No. 16 3548-3557
© 2002 Oxford University Press

A splicing silencer that regulates smooth muscle specific alternative splicing is active in multiple cell types

Natalia Gromak and Christopher W. J. Smith*

Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK

*To whom correspondence should be addressed. Tel: +44 1223 333655; Fax: +44 1223 766002; Email: cwjs1{at}mole.bio.cam.ac.uk

Alternative splicing of {alpha}-tropomyosin ({alpha}-TM) involves mutually exclusive selection of exons 2 and 3. Selection of exon 2 in smooth muscle (SM) cells is due to inhibition of exon 3, which requires both binding sites for polypyrimidine tract-binding protein as well as UGC (or CUG) repeat elements on both sides of exon 3. Point mutations or substitutions of the UGC-containing upstream regulatory element (URE) with other UGC elements disrupted the {alpha}-TM splicing pattern in transfected cells. Multimerisation of the URE caused enhanced exon skipping in SM and various non-SM cells. In the presence of multiple UREs the degree of splicing regulation was decreased due to the high levels of exon skipping in non-SM cell lines. These results suggest that the URE is not an intrinsically SM- specific element, but that its functional strength is fine tuned to exploit differences in the activities of regulatory factors between SM and other cell types. Co-transfection of tropomyosin reporters with members of the CUG-binding protein family, which are candidate URE-binding proteins, indicated that these factors do not mediate repression of tropomyosin exon 3.


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