Regulation and function of the DENR/MCT-1 complex

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Regulation of mRNA translation represents a layer of gene expression that is orthogonal to the transcriptional one. One element on mRNAs that influences their translation are short upstream Open Reading Frames (uORFs). When ribosomes translate uORFs in eukaryotes, they 'consume' the ternary complex containing the initiator tRNA, raising the question of how the main ORF further downstream can be translated. Through a long-standing collaboration between the Duncan and Teleman labs, we recently showed that the DENR/MCT-1 complex, consisting of two non-canonical translation factors, promotes translation re-initiation on main ORFs downstream of uORFs. We showed that this complex is active in proliferating cells but not in quiescent cells, likely due to differential phosphorylation. This work raised two important questions which we propose to address in this grant application: 1) How is the DENR/MCT-1 complex regulated by phosphorylation? In Aim 1 we propose to identify phosphorylation sites on the DENR/MCT-1 complex, and the kinases responsible for these phosphorylations (Teleman Lab). 2) How, at the molecular level, does the DENR/MCT-1 complex promote translation reinitiation? In Aim 2 we propose to use translation extracts and semi-reconstituted translation systems to study DENR/MCT-1 function at the molecular level (Duncan Lab). Finally, we will combine these two workstreams in Aim 3 to study how the phosphorylations identified in Aim 1 affect the molecular functions of the DENR/MCT-1 identified in Aim 2. This work will have implications for both basic biology and medicine. It will shed light on the basic and fundamental process of translation re-initiation as well as its regulation. Since DENR and MCT-1 are oncogenes, and many cancer-related genes contain uORFs, this work will also be of cancer relevance.