Mechanisms directing oncoprotein and cytokine mRNA decay

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The stability and translation of many mRNAs encoding oncoproteins and cytokines are regulated by AU-rich elements (AREs), a diverse but evolutionarily conserved family of RNA sequences localized to their 3¿ untranslated regions. Disruption of ARE-directed regulatory mechanisms can contribute to oncogenesis and severe inflammatory syndromes. Our long-term objectives are to determine how the size and sequence diversity of AREs directs post-transcriptional regulation at the gene-specific level, and how gene-specific characteristics of AREs might ultimately be exploited as targets for novel therapies to treat some cancers and chronic inflammatory diseases. Our central hypothesis is that the metabolic fate of any ARE-containing mRNA is directed by the population of cellular trans-factors targeting each transcript; however, the biochemical basis for selecting one factor over another remains poorly defined. Recent findings indicate that some ARE-binding factors target distinct but overlapping mRNA subpopulations, and that local RNA secondary structure can influence trans-factor selectivity. Also, some factors can remodel local RNA structure or form oligomeric complexes on AREs. This project uses a series of biochemical and molecular biological strategies to define the roles of specific molecular determinants in the formation of stable, functional ribonucleoprotein (RNP) complexes on AREs. Using the ubiquitously expressed ARE-binding proteins AUF1 and HuR as model systems, we will first characterize specific protein subdomains contributing to ARE binding affinity and RNA-dependent protein oligomerization (Aim 1). Second, we will identify specific and non-specific RNA primary structural requirements for AUF1 and HuR binding, and assess the use of these sequences among the cellular mRNA subpopulation(s) interacting with these factors (Aim 2). Finally, we will determine how the energetics of local ARE structure direct the recruitment and positioning of AUF1 and HuR on RNA substrates (Aim 3). We anticipate that our approach will permit the mechanics of protein selectivity and binding to be evaluated in much greater detail than previously reported, largely through the use of steadystate and time-resolved fluorescence-based assay systems that we have adapted to study RNA-protein binding equilibria and RNA conformational events. Together, these studies will further our understanding of the relationships between ARE structure, trans-factor recognition, and the cellular functions of resulting RNP complexes.