Sequence and spatial requirements for regulated muscle-specific processing of the sarco/endoplasmic reticulum Ca(2+)-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 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.
Mertens
L
L
Laboratory of Physiology, University of Leuven (KULeuven), Belgium.
Van den Bosch
L
L
Verboomen
H
H
Wuytack
F
F
De Smedt
H
H
Eggermont
J
J
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D002454:Cell Differentiation; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009474:Neurons / Q000201:enzymology; D011061:Poly A / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D017384:Sequence Deletion
D017931:DNA Primers; D012333:RNA, Messenger; D011061:Poly A; D000252:Calcium-Transporting ATPases