Messenger RNA Modifications in Translation Regulation

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Over 100 types of chemical modifications have been identified in cellular RNAs. Recent technical advances in high-resolution sequencing have revealed modifications in eukaryotic mRNAs, including N6-methyl adenosine (m6A), 5-methylcytosine (m5C), its oxidation product 5-hydroxymethylcytosine (hm5C), N1-methyladenosine (m1A), N6,2’-O-dimethyladenosine (m6Am) and pseudouridine (Ψ). mRNA modifications may act as epigenetic markers that regulate translation of particular mRNAs, however the exact effect of mRNA modifications on translation in poorly understood. The principle aim of this study is to clarify how m6A, m5C and m1A when present at different locations on mRNA can regulate different steps of translation – initiation, elongation, termination, and ribosome recycling. To this end, we will use array of chemically synthesized mRNAs with modifications at different locations and monitor their translation in a cell-free yeast translation system reconstituted from purified tRNAs, translation factors and ribosomal subunits. The partial reaction of protein synthesis will be monitored in real time utilizing a toolbox of established biochemical and biophysical approaches – including rapid kinetics with quench flow coupled to HPLC and ensemble kinetics monitoring fluorescence and FRET. In addition, in collaboration with the University of Kassel, we aim to study the synergistic effects of the 5-methyloxycarbonylmethyl-2-thiouridine (mcm5s2U34) and 5-methyl-cytosine (m5C) modifications in yeast tRNALys on various stages of translation. Taken together, this project will provide a comprehensive insight into the role of mRNA and tRNA modifications in translation.