Sequence and Spatial Requirements for Regulated Muscle-specific Processing of the Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 Gene Transcript

Abstract

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 BC3H1 myogenic cell line using a minigene containing the 3'-end of the SERCA2 gene. In undifferentiated BC3H1 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 (pAu) 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 (pAu) 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.

Sequence elements surrounding the acceptor site suppress alternative splicing of the sarco/endoplasmic reticulum Ca2+-ATPase 2 gene transcript

“…In previous experiments [7], we demonstrated that weak 5h splice donor sites and a long terminal intron are prerequisites for regulated muscle-specific splicing (Figure 1), but that there are no specific negative cis-acting sequences present in the optional terminal intron. In those experiments we did not analyse the sequence of the branchpoint and 3h acceptor upstream of the SERCA2 optional exon 25, as it seemed to fit reasonably well with the mammalian consensus sequence.…”

“…BC $ H1 mouse myoblasts (ATCC ; CRL1443) were cultured and stably transfected using the calcium phosphate co-precipitation method as described previously [7]. Briefly, 24 h before transfection exponentially growing cells were seeded at a density of 50 000\cm#.…”

“…Total RNA was isolated from undifferentiated and differentiated cells by the Chirgwin procedure [25]. RT-PCR (25 cycles ; 1 min at 95 mC, 1 min at 55 mC and 1 min at 72 mC) to detect class-1, class-2\3 and class-4 messengers was performed as described by Mertens et al [7], and the amplification products were separated by electrophoresis on a 6 % polyacrylamide gel. The relative positions of the primers used to amplify fragments of the different SERCA2 transcripts are shown in Figure 1.…”

“…The P5 primer (5h-CAACGACGGCACGAACGGGCAGAT3h) is located in the λ-phage DNA sequence published by Sanger et al [27] (position 12971-12994). The neuronal-specific class-4 mRNA was amplified using the U4 sense and P2 antisense primer in the first PCR (25 cycles), and the S2 sense primer and the same antisense primer in the second PCR (10 cycles) [7].…”

“…(2) Different structural mRNA elements are necessary for regulated splicing to occur ( Figure 1). These include weak 5h splice sites, a weak internal polyadenylation site and the presence of a long terminal intron [7]. (3) This structural organization is not sufficient for splice regulation.…”