Translation of mRNA is tightly regulated, requiring the ordered assembly of initiation factors and ribosomal subunits. Certain viral and cellular mRNAs circumvent traditional requirements for translation initiation by utilizing structured RNA elements termed internal ribosome entry sites (IRESs) to direct ribosomes to the translational start site. IRES structure is intimately linked to function, but the three-dimensional organization of cellular IRESs is poorly understood. This proposal aims to obtain a crystallographic structure of the IRES element in Cyr61 mRNA in complex with proteins required for its function, providing the first high-resolution structure of a cellular IRES element. Cyr61 is an angiogenic inducer suggested to play a critical role in tumor progression. Dicistronic reporter assays will define a minimal functional IRES and trans-acting factors required for activity will be identified. Chemical and enzymatic probing will determine the secondary structure of the Cyr61 IRES and identify structural elements amenable to crystallization. Results from this work will yield a detailed mechanistic understanding of the Cyr61 IRES and may elucidate mechanisms of cellular IRES activity, possibly identifying strategies to therapeutically manipulate Cyr61 production. ? ? ?
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