REGULATION OF SPLICING IS RESPONSIBLE FOR THE EXPRESSION OF THE MUSCLE-SPECIFIC 2A ISOFORM OF THE SARCO ENDOPLASMIC-RETICULUM CA2+-ATPASE/

Tissue-specific alternative processing of sarco/endoplasmic reticulum Ca2(+)-ATPase 2 (SERCA2) transcripts generates functionally different Ca2+ pump isoforms in muscle compared with non-muscle tissues. In non- muscle cells, the SERCA2 pre-mRNA can be polyadenylated at a site loca ted between the donor and acceptor splice site of an intron which is o nly removed in muscle tissues. To define the cis-active elements invol ved in differential processing, we constructed a minigene (pCM beta SE RCA2) containing the 3' end of the SERCA2 gene. When stably transfecte d into a myogenic cell line, minigene transcripts were differentially processed depending on the differentiation state of the cells. This pr oves that the essential elements required for regulated processing are present in the construct. Furthermore, co transfection of the pCM bet a SERCA2 minigene and a myogenin expressionvector in a fibroblast cell line induced muscle-specific splicing of transcripts from pCM beta SE RCA2. This shows that trans-acting factor(s) responsible for muscle-sp ecific processing can be induced by one of the important regulatory ge nes of muscle differentiation. Inactivation of the non-muscle poly(A) site did not induce splicing in non-muscle cells. This excludes a simp le competition model between splicing and polyadenylation, but it is c onsistent with splicing being very inefficient in non-muscle cells. Mo reover, splicing could be induced in non-muscle eels by optimizing the muscle-specific donor splice site and/or by shortening the intron len gth. We therefore propose that expression of the muscle-specific SERCA 2a isoform is the result of activation of an otherwise inefficient spl icing process.