U543140.pdf (34.91 MB)
Alternative splicing of tropomyosin pre-mRNA: Control in non-muscle cells.
thesisposted on 2015-11-19, 09:08 authored by Ian R. Graham
Alternative splicing of tropomyosin pre-mRNA: control in non-muscle cells. Ian R. Graham The human tropomyosin gene hTMnm contains a pair of mutually exclusive exons, NM and SK, which are used in non-muscle and skeletal muscle cells, respectively. I have undertaken an analysis of the factors affecting the splicing of these exons in the non-muscle cell line COS-1. I used a strategy involving mutation of the gene, followed by recloning of the appropriate region into a mammalian expression vector containing a tropomyosin cDNA clone. The wild-type and mutant mini-genes were transfected into the cell line, and the RNA produced after 48 hours' expression was isolated, then analysed by S1 nuclease protection mapping, and by a reverse transcriptase-polymerase chain reaction (RT-PCR) process. The results showed that exons NM and SK are not in competition in this non-muscle cell line; rather, I have shown that exon SK is not recognised as a splicing substrate when any other exons are present that can be used instead. Improvement, by mutation, of the branchpoint associated with exon SK restored use of that exon, as did replacement of the extreme 5' and 3' ends of the exon with the corresponding sequences from exon NM. The observation that exon SK is still overlooked by the cell's splicing apparatus, when it is placed in the exact context normally occupied by exon NM, strongly suggests that the exon itself is contributing to its deficiency. I have proposed a model in which the poor branchpoint sequence and elements within exon SK are responsible for preventing its recognition in the non-muscle cell, which is overcome, in skeletal muscle, by stimulation of the exon 4 to exon SK splice. Additionally, by studying the alternative splicing of the chick a-actinin gene, I have attempted to compare the regulation of splicing in smooth and skeletal muscle.
Date of award1992-01-01
Awarding institutionUniversity of Leicester