SEQUENCE AND SPATIAL REQUIREMENTS FOR REGULATED MUSCLE-SPECIFIC PROCESSING OF THE SARCOPLASMIC ENDOPLASMIC RETICULUM CA2+-ATPASE 2 GENE TRANSCRIPT

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca2+ 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.