A ribosome scanning model has been proposed to explain translation initiation of eukaryotic mRNAs. In this model, binding of the 43S ternary ribosomal subunit near or at the 5' end of the mRNA is facilitated by a concerted interaction between the m/7GpppN cap-structure at the end of the mRNA and the cap binding protein complex eIF-4F. However, certain viral and cellular mRNAs have been identified that can contain internal ribosome entry site elements that recruit directly 43S subunits onto mRNAs. The goal of this proposal is to study the molecular events leading to 5' cap- dependent and 5' cap-independent translational initiation in mRNA molecules. First, it will be tested whether a local critical concentration of translation initiation factors can mediate the recruitment of 43S subunits onto mRNAs. Specifically, it will be tested whether hybridization of """"""""initiation competent"""""""" RNAs to """"""""initiation non- competent"""""""" RNAs will result in the translational activation of the latter RNA species. Secondly, the mechanism by which an appropriate AUG codon is selected as start codon for translation will be investigated. Specifically, a putative role of the La autoantigen in AUG start site selection will be tested by monitoring the effects of La and La-associated factors on the translation of mRNAs containing the AUG embedded in different sequence motifs. Third, the mechanism by which a novel """"""""cap- binding"""""""" RNA (SELEX RNA) inhibits translation of capped but not of uncapped mRNAs will be examined. Fourth, the SELEX RNA will be used as a tool in the identification and characterization of naturally occurring mRNAs that can be translated cap-independently. Specifically, SELEX RNAs will be added to an in vitro translation system, resulting in the dissociation from polysomes of mRNAs that are translated cap-dependently. RNAs that are still associated with polysomes in the presence of SELEX RNAs will be isolated using differential mRNA method and characterized for their ability to be translated cap-independently. Lastly, the mechanism will be determined by which HAP4 mRNA of Saccharomyces cerevisiae is translationally initiated when the organism encounters nonfermentable carbon sources. Overexpression of the cap binding protein 4E can lead to cell transformation. Thus, regulating the rates of translational initiation of certain mRNAs, such as those encoding growth factors, is essential for normal cell growth.
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