The application's long-term objectives are to provide a molecular basis for the complex biological regulation of cAMP- and phorbol ester-inducible genes at the postranscriptional level. The somatic lactate dehydrogenase A and B subunits (LDH) were chosen as a model system. The LDH-regulating mechanism involves effector agents which activate the protein kinases A and C-mediated signal transduction pathways leading to a posttranscriptional regulation of LDH A but not of LDH B subunit mRNA levels. This proposal aims to delineate (a) the posttranscriptional mechanisms controlling LDH A and B mRNA stability/instability, and (b) the mechanisms whereby protein kinases A and C, regulate the turnover of LDH A which cAMP and phorbol ester both cause a cooperative stabilization of LDH A mRNA.
The Specific Aims i nclude (a) identification and functional analysis of cis-regulatory modules in LDH A and B mRNA regions that control mRNA stability/instability; (b) identification and functional analysis of trans- regulatory RNA-binding proteins and/or RNA/protein complex-associated proteins which are instrumental in controlling mRNA half-life; (c) identification of the functional role of protein kinases A and C in controlling the functional activity of mRNA-binding proteins and/or associated proteins. We propose to identify specific domains in the mRNAs controlling mRNA stability and that are the target for regulatory phosphoproteins by measuring the rate of decay of wild-type and mutated 32P-labeled LDH A and B mRNAs in cytoplasmic extracts from untreated and stimulated cells. Proteins which specifically bind to LDH mRNA regulatory domains will be identified by UV-crosslinking of 32P-labeled RNA/protein complexes, RNAase digestion, and identification of proteins by SDS/PAGE. Furthermore, mRNA regions that show regulatory activity will be functionally evaluated by transfection assay. The assay will use chimeric constructs containing various domains of the LDH mRNAs linked to reporter genes of known half-lives. The studies will add to our understanding of the molecular basis of second messenger gene regulation, specifically at the posttranscriptional level which, so far, has received little experimental attention. Our laboratory was the first to identify a postranscriptional regulation of gene activity in the cAMP-mediated signal transduction pathway.
We aim to continue and expand this work. Over-all, the studies will contribute to our understanding of the detailed mechanism of gene regulation by cAMP and phorbol ester at the level of mRNA turnover, of the regulation of intermediary metabolism, and for recognition of abnormal states involving the LDH isozyme system.
