The role of cis-acting sequences in the regulation of α-tropomyosin alternative splicing

• Main Author: Grellscheid, S. N.
• Published: University of Cambridge 2003
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599698

The main investigation was a study of the possible role of a zero length exon (ZLE) observed 234 nucleotides downstream of exon 3, in α-TM splicing. The ZLE overlaps with the DRE, and consists of potential branchpoints, a polypyrimidine tract and a 3’ splice site (3’ss) GAG, followed immediately by a 5’ splice site (5’ss). In addition, a pseudo 5’ss is located 107 nucleotides downstream of the 3’ss. All of these sequences are conserved between rat mouse and human α-TM genes. While the initial aim was to study the role of the ZLE, results from this study suggest that the ZLE is part of a 107 nucleotide pseudoexon, formed by the ZLE 3’ss and the downstream pseudo 5’ss. RT-PCR analysis of RNA from various rat tissues allowed identification of splicing intermediates involving the 3’ss of the pseudoexon and exons 2 or 3. Transfection studies showed that activation of the ZLE 3’ss or repression of the ZLE 5’ss resulted in inclusion of the pseudoexon, together with exon 3. Blocking the ZLE in the endogenous gene using an anti-sense approach resulted in an increase in levels of exon 2 products. Exon 3 transcripts including the pseudoexon contain a premature termination codon, and therefore, are expected to be degraded by nonsense mediated decay. This may represent a mechanism of negative regulation of α-TM exon 3. In a study of the optimum distance required between the branch point and the 3’ splice site in the α-TM system, inhibition of splicing of an alternative exon may require sub-optimal recognition of some key sequences, creating a capacity for regulation. The typical distance between the branchpoint and 3’ss AG is 18-40 nucleotides. In α-TM exons 2 and 3, this distance is unusually long. The resulting sub-optimal recognition of the polypyrimidine tract by U2AF, may be essential to allow competing regulatory factors to bind.