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, and test the functional significance of these domains in cells (Aim 1). Second, we will identify specific and non-specific RNA primary structural requirements for protein 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 local RNA structure directs the recruitment and positioning of AUF1 and HuR on ARE substrates and influences the cellular consequences of these interactions (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 steady-state 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.

Public Health Relevance

Cancer and other serious clinical syndromes result when the expression of proteins regulating cell proliferation and differentiation becomes uncontrolled. Cells use many mechanisms to limit the production of these proteins, including rapid destruction or silencing of the messenger RNAs (mRNAs) encoding them through a family of RNA sequences called AU-rich elements (AREs). The proposed studies will determine how different cellular factors recognize unique ARE sequences and target their associated mRNAs for destruction, opening the possibility that these gene-specific interactions might be exploited as targets for novel therapies to treat some cancers and chronic inflammatory diseases.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Molecular Genetics C Study Section (MGC)
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Knowlton, John R
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University of Maryland Baltimore
Schools of Medicine
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