Alternative splicing events in the coding region of the cytochrome P450 aromatase gene in male rat germ cells.
Expression of cytochrome P450 mRNA in rat germ cells was characterized by reverse transcription PCR with various primers located at the 3'-end of the coding region. At least two unusual isoforms (Ex10-S and INT) of P450 aromatase (P450arom) mRNA were expressed. Analysis of their sequences demonstrated that an alternative splicing event occurred first at the exon-intron boundary of the GT consensus sequence of the last coding exon, and second in the internal 5' donor inside exon 9 used as a minor cryptic splicing site. These isoforms lacked the last coding exon which contained the heme-binding domain; in addition, for the Ex10-S transcript, the catalytic domain was also absent because of a frameshift in the open reading frame. The deduced amino acid sequences led to truncated P450arom polypeptides without the heme-binding domain, which were probably unable to convert androgens into estrogens. Adult rat germ cells are able to express P450arom mRNA, which is then translated into a biologically active enzyme which is involved in estrogen production. Moreover, for the first time, we report the existence of alternative splicing events of P45Oarom mRNA in pachytene spermatocytes and round spermatids, which probably cannot encode functional aromatase molecules.
Laboratoire de Biochimie-IRBA, CHRU Clémenceau, CNRS EP 9, Université, Esplanade de la Paix, Caen, France.
J
J
Levallet
H
H
Mittre
B
B
Delarue
S
S
Carreau
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001141:Aromatase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers; D005091:Exons; D007438:Introns; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013094:Spermatozoa / Q000201:enzymology*
D017931:DNA Primers; D007527:Isoenzymes; D001141:Aromatase