# Application of Mammalian Nudix Enzymes to Capped RNA Analysis
## 7 Discussion  
Among mammalian NUDIX hydrolases with (m)RNA decapping in vitro, Dcp2 and Nudt16 have been used in the analysis of diverse analogues of canonical cap structure. However, mRNA decapping by Dcp2 is a complex process involving multiple regulatory proteins, such as Dcp1, Edc3, and Edc4. The formation of various Dcp2-containing multiprotein complexes ensures precise control over mRNA stability and degradation in vivo, which is crucial for cellular homeostasis and response to environmental changes [145,146]. Moreover, as mentioned earlier, Dcp2 controls stability only of a subset of mRNA in cells [115], exemplified by the recent global profiling study that revealed a number of Dcp2 mRNA substrates, including innate immunity-related transcripts [147,148]. Thus, for the more physiologically relevant results, in vitro decapping with Dcp2 should also be reconsidered in terms of the decapping complex with regulatory proteins and designed gene-specific mRNA (e.g., involved in innate immunity). Available evidence points out to Nudt16 as a decapping enzyme with activity towards the widest range of different cap structures at 5\({}^{\prime}\) end of RNA transcripts: unmethylated (GpppN-), mono-methylated (m7 GppN-), trimethylated (m3\({}^{\text{2,2,7}}\)GpppN-), NAD-, FAD-, and dpCoA-cap. Bearing in mind the broad substrate specificity, Nudt16 can be considered \begin{table}
\begin{tabular}{c c c c c} \hline  & **NAD Cap** & **FAD Cap** & **dpCoA Cap** & **UDP-GlcNAc Cap** \\ \hline Nudt16 & + & + & + & \\ \hline Nudt12 & + & & + & \\ \hline Nudt5 & & & & + \\ \hline Nudt2 & & + & + & \\ \hline \end{tabular}
\end{table} 
Table 3: Activity of selected NUDIX proteins towards metabolite non-canonical RNA caps. as the novel major standard in analysis of modified cap structures at (m)RNA transcripts produced in vitro, next to Dcp2. It already proved to be a potent tool in studies of caps modified in a triphosphate bridge and within 7-methylguanosine (N\({}^{2}\) and 2\({}^{\prime}\)-OH or 3\({}^{\prime}\)-OH of m\({}^{7}\)G). Additionally, it could be used in cap-orientation assay and removing of the reversely capped (with non-ARCA cap analogues) RNA transcripts from IVT reactions (when combined with Xrn1 exoribonuclease). Important advantage of Nudt16 is that it acts effectively as a single subunit, without additional regulatory proteins, what can facilitate its use and optimization. One of the possible limitations is its use in regards to transcripts bearing at their 5\({}^{\prime}\) end dinucleotide cap analogues with adenosine adjacent to the m\({}^{7}\)G (or m\({}_{3}\); 2; 7 G), which are poor substrates for Nudt16 decapping. However, the Nudt12 hydrolase could be used in such case instead. Finally, Nudt2 enzyme is active in vitro towards unmethylated Np\({}_{\text{Pn}}\)N-RNAs (where N = G or A), with lower activity towards those with triphosphate bridge (Np\({}_{3}\)N-RNAs) in comparison to di- or tetraphosphate-containing dinucleotide structures. As such, Nudt2 can be an attractive tool in testing modifications introduced into the polyphosphate chain of synthetic non-methylated dinucleotide analogues (e.g., GppXpG). Those in turn, after incorporation into (m)RNA during IVT reaction, could be subject to methylation by guanine-7-methylfuranferase specific activity (e.g., Vaccina Capping Enzyme, VCE) or introduction, e.g., benzylic moieties with in-vitro-engineered MAT/MTase (methionine adenosylotrasferase/methyltransferase) activities [87]. Nudt2 also showed in vitro activity to m\({}^{7}\)GppN-capped transcripts. Thus, it cannot be excluded from analysis of methylated cap structures. However, further characterization with a wider set of such analogues is needed in that context. Another Nudt2 activity of potentially great interest is its ability to convert triphosphorylated pppN-RNA into monophosphorylated pN-RNA, the latter being a substrate for Xrn1-riboexonuclease-mediated removal from IVT reaction.