Regulation of alternative processing of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2) gene transcripts during muscle differentiation.

The Ca2+ transport ATPases of the sarcoplasmic or endoplasmic reticulum (SERCA) mediate the uptake of Ca2+ into intracellular stores. These Ca2+ pumps are encoded by three different genes. Alternative processing of the SERCA2 messenger generates two different protein isoforms. These two isoforms differ in their C-terminal part and this divergence is responsible for the functional difference between SERCA2a and SERCA2b. The aim of this study was to characterise the cis-active elements and the trans-acting factors required for the generation of the muscle-specific messenger during myogenic differentiation. A competition model between muscle-type splicing and non-muscle polyadenylation was excluded as inactivation of the non-muscle polyadenylation site did not induce muscle-type splicing in non-muscle or in undifferentiated muscle cells. We therefore propose that the expression of the SERCA2a isoform is due to the regulation of the muscle-specific splice process during myogenic differentiation. We characterised the spatial and sequence requirements essential for tissue-specific transcript processing. It was demonstrated that the processing signals in the transcript, i.e. both donor splice sites and the polyadenylation site located in the muscle-specific intron, have to be weak. An exception to this rule is the 3' acceptor splice site that has to be strong in order to get muscle-specific splicing. We also found an inverse relationship between intron length and splice efficiency as shortening the terminal intron resulted in muscle-specific splicing in non-muscle and in undifferentiated muscle cells. Finally, it was demonstrated that sequences around the muscle-specific acceptor region as well as in the muscle-specific exon are required to prevent muscle-specific splicing in undifferentiated muscle cells. Especially a region upstream of the 3' acceptor contains important sequence information required for the inhibition of muscle-specific splicing. It was demonstrated that the trans-acting factors regulating the alternative SERCA2 splicing are muscle specific. First, myogenin, a transcription factor that plays a key role in muscle differentiation, induced muscle-specific SERCA2 splicing in a fibroblast cell line. Second, changes in general splice and polyadenylation efficiency, as observed during B-cell maturation, did not affect SERCA2 splicing. Finally, expression and overexpression studies did not support the hypothesis that changes in the level of the alternative splice factor ASF or other arginine and serine rich proteins are involved in the regulation of muscle-specific splicing. We conclude that muscle-specific SERCA2a expression is a tightly regulated process dependent on the inhibition of the muscle-specific splice process at the 3' end of the primary transcript. During differentiation this inhibition is overcome by the downregulation of an inhibitory factor and/or the expression of a positive trans-acting factor.