Regulation of Eukaryotic Protein Synthesis Initiation

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DESCRIPTION (provided by applicant): The long-term goals of this project are to understand the steps that occur during the recruitment of eukaryotic mRNA to the ribosome and how they are regulated. This process determines both the overall rate of protein synthesis and also the spectrum of mRNAs translated. The four Specific Aims principally concern two interacting initiation factors that are central to this process: eIF4E, the protein that binds the 7-methylguanosine-containing cap of mRNA, and eIF4G, the protein that links together all initiation factors known to be involved in mRNA recruitment. The nematode C. elegans provides unparalleled advantages for studying such a complex biochemical process as initiation of protein synthesis. Surprisingly, it expresses five eIF4E isoforms, termed IFE proteins, which have differing cap-binding properties. In Specific Aim 1, we wi2ll attempt to infer their biological roles by studying their biochemical interactions with m7G- and m3 '2'7G-containing cap analogues and oligonucleotides, with mRNAs, and with other proteins, utilizing MALDI-TOF mass spectrometry in the latter case. In Specific Aim 2, we will determine the effect of human eIF4E phosphorylation on protein synthesis rate. eIF4G binds to both eIF4E and the poly(A)-binding protein, the two C is-acting effectors for mRNA recruitment, yet many isoforms of eIF4G and eIF4G mRNA are observed in mammalian cells. In Specific Aim 3, we will determine the structure, function, and origin of these eIF4G isoforms and characterize the binding of known initiation factors to them. In Specific Aim 4, we will determine the function of eIF4G isoforms in vivo, with special emphasis on the relative importance of eIF4G cleavage in picomavirus-induced host shut-off of protein synthesis. Because overexpression of both eIF4E and eIF4G causes malignant transformation of cells, and because naturally occurring tumors contain greatly elevated levels of eIF4E, these studies have relevance to cancer. They also have relevance for diseases caused by picornaviruses such as poliovirus (polio), rhinovirus (the common cold) and Coxsackievirus (heart failure).