A splicing silencer that regulates smooth muscle specific alternative splicing is active in multiple cell types.
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.
Gromak
Natalia
N
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Smith
Christopher W J
CW
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D051743:CCAAT-Enhancer-Binding Protein-delta; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D005091:Exons / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009928:Organ Specificity; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors; D014335:Tropomyosin / Q000235:genetics*
D022762:CCAAT-Enhancer-Binding Proteins; C497252:CEBPD protein, human; C497253:Cebpd protein, mouse; C497254:Cebpd protein, rat; D012322:RNA Precursors; D012333:RNA, Messenger; D014157:Transcription Factors; D014335:Tropomyosin; D051743:CCAAT-Enhancer-Binding Protein-delta