Identification and characterization of small NAD-capped RNAs in yeast

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RNA is an important regulator in biology. Its simple architecture is augmented by diverse chemical modifications. Recently we discovered the ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) to be attached to a specific set of regulatory RNAs in bacteria in a cap-like manner, and to modulate the functions of these RNAs. Furthermore, we discovered the first prokaryotic decapping enzyme, NudC. In the previous funding period we developed new mass-spectrometric and electrophoretic methods for the quantitative analysis of NAD-RNA, as well as preparative methods for their generation. We detected and quantified NAD-RNA in yeast, Arabidopsis, and mouse. NAD captureSeq was applied to four different yeast total RNA samples, and we found that mostly mRNAs are enriched. Another large enriched group are small RNAs. NAD decapping in yeast is catalysed by the NudC homolog Npy1, which is inactive on the canonical m7G cap. The existence of NAD-RNA in prokaryotic as well as eukaryotic organisms points to a more general phenomenon that might be conserved in all kingdoms of life. The aim of this project is to proceed with the identification of NAD-capped-RNA in yeast. Here, we propose to identify, quantify, and characterize small 5’-NAD capped RNAs in the model organism Saccharomyces cerevisiae. To discover such RNAs we will refine the NAD captureSeq to specifically identify NAD-capped RNAs that are smaller than 25 nucleotides. For selected modified RNAs we will unravel the biological significance and biosynthesis. We will address the types and sequences of NAD-capped RNAs, the dependence of the modification on environmental conditions, the biogenesis as well as the decapping in vitro and in vivo. For these studies, we can make use of a range of methods already established in our group.