Cyclic AMP regulates expression of the RI alpha subunit of cAMP-dependent protein kinase through an alternatively spliced 5 ' UTR

The present study examines novel mechanisms that regulate levels of the RI alpha -subunit of cAMP-dependent protein kinase. We found that RI alpha pro tein is induced threefold by 8-(4-chlorophenyl)thio-cAMP in hormone respons ive rat Sertoli cells, while total RI alpha mRNA is not correspondingly ind uced. Two RI alpha mRNA isoforms with different 5' untranslated sequences ( RI alpha 1a and RI alpha 1b) are produced from the RI alpha gene in Sertoli cells. Deletion/mutation analysis of the cAMP-response-element-containing promoter upstream of the RI alpha exon 1b revealed that while mutation of t he cAMP response element had no effects on cAMP-mediated induction, a 73-bp region of the RI alpha exon 1b itself conferred a fivefold to eightfold in duction of reporter activity to homologous and heterologous promoters. The responsiveness of this region was dependent on a sense orientation downstre am of the promoter start sites and had no effect on reporter mRNA, indicati ng that the cAMP-mediated induction occurs at the post-transcriptional leve l. Modeling of the RI alpha 1b 5' UTR secondary structure revealed a 5' CAP -proximal, strong stem-loop presenting an element similar to multiple start -site element downstream-1 (GCTCGG) in the loop region. RNA-EMSAs performed with the labeled RI alpha lb 5' UTR showed stabilization of a protein/RNA complex in extracts from 8-(4-chlorophenyl)thio-cAMP stimulated Sertoli cel ls. This complex was abolished by mutation of the multiple start-site eleme nt downstream-1-like element. Our findings indicate that there is a cAMP-me diated induction of RI alpha expression at the post-transcriptional level, dependent on the 5' UTR of RI alpha 1b mRNA.