Alternative pre-messenger RNA splicing of murine N-CAM and human tropomyosin in non-muscle and muscle cells.
thesisposted on 19.11.2015, 09:08 by Marion. Hamshere
A quantitative RT-PCR method for analysis of alternative isoforms of RNA, and a method for the transient expression of mini-genes in differentiated muscle cells have been developed. This has enabled the analysis of endogenous RNA transcribed from rodent N-CAM and from human tropomyosin mini-genes which were expressed in COS cells and mouse C-2 myoblasts and myotubes. The sequences of the previously unreported mouse homologues of human exons MSD1b and MSD1c of N-CAM have been determined, and deposited in the EMBL data base. The tissue- and stage-specific alternative splicing patterns of exons within the muscle-specific domain (MSD) of N-CAM have also been established; the exons were normally incorporated as a unit in muscle cells, but were not included in transcripts derived from non-muscle myoblasts and neural cells. The triplet AAG exon was also included in a stage- and tissue-specific manner, but independently of inclusion of other exons of the MSD. Transfection of C-2 myoblasts with mutant mini-gene constructs of human tropomyosin determined the cis-acting elements which regulate the mutually exclusive alternative splicing of the central exons (NM and SK) in both non-muscle and muscle cells. In non-muscle, these were found to be due either to cis-acting repressor sequences within the SK exon or cis-acting activator sequences within the NM exon. In differentiated cells, exclusion of the NM exon is not via cis-acting repressor sequences within the NM exon, but because the upstream (NM) exon site is dormant and is therefore skipped by the splicing machinery. The evolution of alternative pre-mRNA splicing has also been discussed, and on this basis and from analysis of the data presented here, I conclude that regulation of alternative pre-mRNA splicing of transcripts from different genes may be founded upon a common mechanism which is largely dependent upon the presence of sub- optimal splice-signals and the potential for variation in the relative concentrations of certain splicing factors.