CHARACTERIZATION AND REGULATION OF 2 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.