Translational control of gene expression in fungi

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Summary Translation of mRNA is a central cellular process, but the mechanisms that control it are not fully understood. Up to 50% of eukaryotic mRNAs contain predicted upstream open reading frames (uORFs), but the roles of the vast majority of these uORFs remain undetermined. In some cases, we know that this additional translational capacity is evolutionarily conserved and serves critical functions in controlling gene expression. A special class of these conserved uORFs encodes peptides that stall protein synthesis in response to the presence of small metabolites. These nascent regulatory peptides act within the ribosome tunnel to arrest translation; by doing so, they control the production of enzymes important in metabolism. However, there remain important gaps in knowledge of the functions of uORFs. First, the mechanisms by which uORF-encoded peptides recognize small molecules and stall eukaryotic ribosomes to control gene expression remain unclear. Second, the extent to which uORFs are translated in cells under different conditions, the extent to which uORFs are evolutionarily conserved, and how and why the translation of particular uORFs controls gene expression, or if their translation serves other functions, is not known. To help bridge these gaps, we will determine the functions of a newly discovered conserved fungal uORF peptide named the inositol regulatory peptide (IRP). Our data indicate that the IRP regulates the expression of the first enzyme necessary for the synthesis of the important molecule inositol. We know that the IRP, while fungal in origin, can regulate reporter genes in mammalian cells as well as the fungus Neurospora crassa, in which we first discovered it. Using fungal and mammalian cell-free translation systems, we obtained direct evidence for translational control by the IRP. We will use both in vivo and in vitro approaches to determine the mechanism of action and physiological consequences of IRP function. In Aim 1, we will perform functional analyses to determine the mechanism of action of the IRP. In Aim 2, we will perform structural analyses to determine the mechanism of action of the IRP. Successful completion of the proposed work will provide mechanistic information that should significantly increase our understanding of translational control mechanisms that are generally important. It will provide new insights into regulatory and metabolic pathways that could be important for developing strategies to manipulate metabolism to improve human health and welfare.