An alternative splicing site modifies the carboxyl-terminal trans-membrane domains of the Na+/Ca2+ exchanger.
The 6-kilobase (kb) cDNA of pTB11 clone and its 5' fragment of 3.7 kb encoding the canine heart Na+/Ca2+ exchanger (Nicoll, D.A., Longoni, S., and Philipson, K.D. (1990) Science 250, 562-565) were transiently expressed in 293 cells to investigate the role of the 3'-"untranslated" region. Both fragments yielded high levels of expressed protein that were well incorporated in the membranes. Cells expressing the 6-kb cDNA produced rearranged transcripts of smaller than expected size. A 120-kDa polypeptide was produced in cells expressing the modified exchanger, and Ca2+ uptake was higher in this type of transfected cells. A constant stretch of nucleotides located at the 3' end of the 6 kb cDNA was found to be connected, by alternative RNA splicing, to four different upstream sequence positions. The deduced hydrophobic sequence of the spliced-in exon could replace the IX or the XI trans-membrane domain of the exchanger protein in two spliced isoforms. The new exon sequence was not completely included in the pTB11 insert, i.e. these two products were artificially truncated. The RNA processing of these two alternative 5'-splicing sites also occurred in tissues, as shown by RNase protection analysis. In a third type of isoform the splicing took place downstream of the originally proposed stop codon, whereas in a fourth type a stop codon was introduced after the V hydrophobic segment in the large intracellular loop.
Gabellini
N
N
Department of Biological Chemistry, University of Padova, Italy.
Iwata
T
T
Carafoli
E
E
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics*; D002478:Cells, Cultured; D003062:Codon; D018076:DNA, Complementary / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012333:RNA, Messenger / Q000032:analysis; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D003062:Codon; D018076:DNA, Complementary; D012333:RNA, Messenger; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium