Sequence and Spatial Requirements for Regulated Muscle-specific Processing of the Sarco/Endoplasmic Reticulum Ca-ATPase 2 Gene Transcript

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca ATPase (SERCA2) requires activation of an otherwise inefficient splicing process at the 3′-end of the primary gene transcript. The sequence and topology requirements for this regulated splicing event were studied in the BC 3 H1 myogenic cell line using a minigene containing the 3′-end of the SERCA2 gene. In undifferentiated BC 3 H1 cells, the splice process is made inefficient by the presence of a weak muscle-type 5′-donor site (5′D1) and a long terminal intron. Both optimizing the 5′D1 and decreasing the length of the muscle-specific intron, induced muscle-type splicing in undifferentiated myogenic cells. Moreover, the induction of muscle-type transcripts was only observed when two competing processing sites, the polyadenylation site (pA u ) used in non-muscle cells and the second neuronal 5′-donor site (5′D2), were weak. Indeed, making 5′D2 consensus induced neuronal-type splicing in undifferentiated myocytes and prevented the appearance of muscle-type transcripts. Similarly, replacing the polyadenylation site (pA u ) with a strong site almost completely inhibited muscle-type splicing after myogenic differentiation. We conclude that weak processing sites and a long terminal intron are required for tissue-dependent mRNA processing of the SERCA2 transcript.