Characterization and regulation of two testicular inhibin/activin beta B-subunit messenger ribonucleic acids that are transcribed from alternate initiation sites.
We and others have shown that the inhibin/activin beta B-subunit gene is expressed differently in the gonads. Two species of 4.8- and 3.7-kilobase (kb) beta B-subunit messenger RNA (mRNA) with equal concentrations were identified in the testis, whereas 1 predominant 4.8-kb and a minor 3.7-kb mRNA were observed in the ovary. In this study, we analyzed the structures of these 2 mRNAs in rat testis and showed that both 4.8- and 3.7-kb beta B-subunit mRNAs were terminated at the region proximal to 2.2 kb down-stream from the translation stop codon. However, only 4.8-kb mRNA could be detected when RNA probes prepared from the 5'-region 1 kb up-stream from the translation start site were used for Northern blot analysis. Our observations suggested that the 2 heterogeneously sized beta B-subunit mRNAs are transcribed from different initiation sites. Transcription of the 4.8-kb mRNA was initiated at 3 adjacent nucleotides, GGA, 1.1 kb up-stream from the translation start codon ATG, whereas multiple transcription initiation sites spreading over 150 nucleotides upstream from the ATG codon were previously identified for 3.7-kb mRNA. Neither of the 2 transcripts contained TATA and CAAT boxes in their promoters. The 5'-flanking DNAs required for transcription of the 4.8- and 3.7-kb mRNA were examined by their ability to induce transient expression of the chloramphenicol acetyltransferase (CAT) gene in MA-10 Leydig tumor cells. A marked increase in CAT activity was detected when the 5'-flanking DNA for the 4.8- or 3.7-kb transcript was progressively shortened from its 5'-end. Maximal CAT activity was observed when -409 and -139 basepair beta B-subunit DNA up-stream from the 4.8- and 3.7-kb transcription initiation site, respectively, were fused to the CAT gene, suggesting the presence of a negative regulatory element(s) at the up-stream regions of these promoters. Although putative AP-2 sites were identified, treatment of the transfected cells with cAMP and/or phorbol 12-myristate 13-acetate did not apparently change CAT activity driven by either the 4.8- or 3.7-kb promoter. Our results concluded that 1) the two inhibin/activin beta B-subunit mRNAs were transcribed from different initiation sites; 2) both promoters may be controlled by up-stream negative regulatory elements; and 3) neither of these promoters is responsive to cAMP and/or phorbol esters under the conditions employed.
Feng
Z M
ZM
Population Council, New York, New York 10021.
Wu
A Z
AZ
Chen
C L
CL
D028341:Activins; D000818:Animals; D001483:Base Sequence; D018387:Codon, Initiator; D000242:Cyclic AMP / Q000494:pharmacology; D005260:Female; D007265:Inhibins / Q000235:genetics*; D008297:Male; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013737:Testis / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic
D018387:Codon, Initiator; D015335:Molecular Probes; D009842:Oligopeptides; D010455:Peptides; D012333:RNA, Messenger; C044147:activin B; D028341:Activins; C048617:myelopeptides; D007265:Inhibins; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate