Contribution of Nudt12 enzyme to differentially methylated dinucleotides of 5'RNA cap structure

Abstract

Recent findings have substantially broadened our knowledge about the diversity of modifications of the 5'end of RNAs, an issue generally attributed to mRNA cap structure (m⁷GpppN). Nudt12 is one of the recently described new enzymatic activities involved in cap metabolism. However, in contrast to its roles in metabolite-cap turnover (e.g., NAD-cap) and NADH/NAD metabolite hydrolysis, little is known regarding its hydrolytic activity towards dinucleotide cap structures. In order to gain further insight into this Nudt12 activity, comprehensive analysis with a spectrum of cap-like dinucleotides was performed with respect to different nucleotide types adjacent to the (m⁷)G moiety and its methylation status. Among the tested compounds, GpppA, GpppA_m, and Gpppm⁶A_m were identified as novel potent Nudt12 substrates, with K_M values in the same range as that of NADH. Interestingly, substrate inhibition of Nudt12 catalytic activity was detected in the case of the GpppG dinucleotide, a phenomenon not reported to date. Finally, comparison of Nudt12 with DcpS and Nud16, two other enzymes with known activity on dinucleotide cap structures, revealed their overlapping and more specific substrates. Altogether, these findings provide a basis for clarifying the role of Nudt12 in cap-like dinucleotide turnover.

Keywords: DcpS; Differential scanning fluorimetry (DSF); Enzyme kinetics; Nudix family; Nudt12; RNA cap structures; Substrate inhibition.