Genetic & Developmental Analysis of Internal Initiation of Translation in Drosophila

9304983 Cavener The cap-dependent scanning model for translation initiation in eukaryotes was previously argued to be used by all eukaryotic mRNAs. Recently the poliovirus, EMCV, immunoglobulin binding protein, and Drosophila Antennapedia mRNAs were shown to use a novel cap-independent mechanism for translation initiation in cultured cells. These mRNAs contain internal ribosome entry the sites (IRES) in their 5' untranslated region (UTR) leader sequences. This proposal will examine the developmental regulation of internal initiation using transgenic Drosophila. The 5" UTR sequences of the Antennapedia (Antp), Ultrabithorax (Ubx), and seven in absentia (sina) will be tested for IRES activity using a dicistronic assay system. The ability translation of the distal cistron will be assessed in transgenic Drosophila. Since the dicistronic gene will be driven by a constitutive promoter, the potential translation by the internal initiation mechanism can be assessed for all tissues and stages. Internal deletion experiments will examine whether the putative IRES elements of Antp, Ubx, and sina are necessary for translation in the context of their full 5' UTRs. %%% Initiation of protein synthesis in eukaryotes is preceded by a series of complex reactions primarily involving the ribosome, initiation factors, Net-tRNA, and a mRNA. The cap dependent scanning model was proposed by Kozak in 1989 to explain the order and nature of these reactions. It was first challenged by the discovery that the picornaviruses do not have a terminal cap and are translated by internal ribosome binding. This work will examine the developmental regulation of internal initiation using transgenic Drosophila and should allow insight into the elucidation of alternative initiation mechanisms and also to a possible mode of development gene regulation. ***