BIOCHEMICAL AND TRANSLATION CONTROL OF CYTOKINE MRNA DEC

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This proposal is directed to an emerging but poorly understand area of genetic control, the selective cytoplasmic degradation of short-lived mRNAs in mammalian cells, which is regulated by an AU-rich element (ARE) found in the 3' non-coding region (3'NCR) of the mRNA (ARE- mRNA). The mechanisms by which the ARE promotes rapid mRNA decay, the interplay between translation and rapid ARE-mRNA decay, and the cellular proteins that promote selective rapid degradation of ARE- mRNAs are the subject of this application. Aim 1 will characterize the role translation plays in promoting rapid degradation of mRNAs containing the GM-CSF ARE. Translation of ARE-mRNAs has been shown to accelerate or activate their rapid decay. Studies are proposed to investigate the mechanism by which translation activates or promotes rapid decay of ARE-mRNAs. One group of studies will investigate how translation of the ARE-mRNA promotes its own rapid decay. Other studies will address whether short-lived trans-acting cellular proteins must be continuously synthesized to promote ARE- mRNA turnover. Aim 2 will investigate the molecular mechanism by which ARE-mRNAs are targeted for rapid degradation. Essentially nothing is known about the fundamental molecular mechanisms that control rapid degradation of ARE mRNAs in mammalian cells. The specific ARE binding protein family known as AUF1 promotes the rapid decay of ARE-mRNAs. AUF1 bound to mRNA forms a complex in vivo with a number of proteins, including translation initiation factor eIF4G, poly(A) binding protein (PABP) and heat shock proteins hsp70 and hsc70. Experiments are proposed to understand how binding of AUF1 to the ARE facilities the rapid decay of ARE-mRNAs, and the roles of eIF4G, PAPB and hsp- hsc70 in ARE-mRNA decay. Aim 3 will identify unknown AUF1 binding proteins and examine their roles in regulating rapid decay of ARE mRNAs. Although studies have identified several AUF1 binding proteins, still other binding prote3ins have been detected in vivo but not identified Experiments are proposed to genetically identify remaining AUF1 binding proteins using a yeast 2- hybrid analysis, and to biochemically investigate possible roles of these proteins in promoting or inhibiting decay of ARE-mRNAs.