# Application of Mammalian Nudix Enzymes to Capped RNA Analysis
## 2 Diversity of 5\({}^{\prime}\) RNA Cap Structures--Natural Caps  
Beyond the aforementioned Cap 0 structure, the 5\({}^{\prime}\) mRNA cap can be considered an extended structure where the first transcribed RNA nucleotides (namely N\({}_{1}\) and N\({}_{2}\) in the m\({}^{7}\)GpppN\({}_{1}\)pN\({}_{2}\) structure) are 2\({}^{\prime}\)O-methylated, resulting in the m\({}^{7}\)Gpppm\({}^{2^{\prime}O}\)N\({}_{1}\) (Cap 1) or m\({}^{7}\)Gpppm\({}^{2^{\prime}O}\)N\({}_{1}\)\({}^{2^{\prime}O}\)N\({}_{2}\) (Cap 2) structures [16]. Cap 0 is generally found in lower eukaryotes, whereas Cap 1 and Cap 2 are hallmarks of higher eukaryotes, including humans [8]. The most extensively methylated cap, Cap 4, has been identified in Kinetoplastida (e.g., _Tynanosoma brucei, Trypanosoma cruzi_ and _Leishmana major_), where the first four transcribed nucleotides adjacent to m\({}^{7}\)Guo (m\({}^{7}\)GpppN\({}_{1}\)pN\({}_{2}\)N\({}_{3}\)pN\({}_{4}\)) are heavily methylated (m\({}^{7}\)GpppN\({}_{3}\)\({}^{6,6,2^{\prime}O}\)Apm\({}^{2^{\prime}O}\)Apm\({}^{2^{\prime}O}\)Cpm\({}^{3,2^{\prime}O}\)U) [17]. Additionally, adenosine as the first transcribed nucleotide of Cap 1 can be also methylated at the N\({}^{6}\) position of the purine ring (m\({}^{6}\)A) both in the absence or presence of the 2\({}^{\prime}\)O-methylation. This modification has been shown to have a positive effect on translation efficiency in vitro [18] and on mRNA stability due to increased resistance to Dcp2 [19]. Eukaryotic RNA (e.g., snRNA, snRNA, pn primary miRNA, pre-tRNA) and specific mRNAs (selenoprotein encoding transcripts, or _junD_, _dhx9_ and _tgs1_ mRNA) are modified at their 5\({}^{\prime}\) ends with other cap structures, such as: trimethylguanosine cap (m\({}_{3}\)\({}^{2,2,7}\)GpppN), 5\({}^{\prime}\)-gamma-methyl (tri)phosphate cap (CH\({}_{3}\)pppN), and alfa-dimethyl monophosphate cap [20; 21; 22; 23; 24]. Additional 5\({}^{\prime}\) cap structures have been detected on short RNAs (\(\sim\)20-200 nt), including non-methylated caps (GpppG and GpppA/ApppG), and next to the m\({}^{7}\)Gp or m\({}_{3}\)\({}^{2,2,7}\)Gp moiety, the dimethylguanosine (m\({}_{2}\)\({}^{2,7}\)Gp and m\({}_{2}\)\({}^{2,2}\)Gp), monomethylguanosine  
Figure 1: mRNA 5\({}^{\prime}\) end cap structures. Methylations of m\({}^{7}\)Guo and at 2\({}^{\prime}\)O position of riboses of the first two transcribed nucleotides, which result in Cap 1 and Cap 2 structures, are indicated. Inset the unmethylated cap variant, and m\({}^{6}\)A\({}_{\rm m}\) adenosine containing cap variant (m\({}^{7}\)Gppp m\({}^{6}\)A\({}_{\rm m}\)) are shown. Structures were drawn with ACD/ChemSketch, version 2022.1.2, Advanced Chemistry Development, Inc. (ACD/Labs). (m1 Gp, m2 Gp), and monomethylcitidine (mCp) structures. Moreover, the 2\({}^{\prime}\)O-methylation of the first transcribed nucleotide (N\({}_{1}\)) has been detected together with the aforementioned structures and trimethylated m\({}_{3}\)[2; 2; 7]Gp also with dimethyladenosine m\({}^{6}\)A\({}_{\text{m}}\)[25; 26], Table 1.  
Although initially identified on bacterial RNAs [27], the diversity of naturally occurring 5\({}^{\prime}\) caps present on mRNA and other types of eukaryotic cellular RNAs is further expanded by the detection of structures where the cap is composed of a non-canonical initiating nucleotide (NCIN): nicotinamide adenine dinucleotide NAD+ (NAD-cap) [28; 29], flavin adenine dinucleotide cap (FAD-cap), dephospho-CoA (dpCoA-cap) or UDP-glucose derived caps (uridine diphosphate-glucose UDP-GlcNAc cap and UDP-N-acetyloglucoamie UDP-GlcNAc), so called \"metabolic\" cap structures (Figure 2) [30; 31; 32]. A very recent report showed that adenosine dinucleotide tetrapphosphate (AppppA) can also serve as yet another type of non-canonical RNA cap (Ap\({}_{4}\)A-RNA) in eukaryotic cells (HEK293T, RBL-2H3 cell lines) [33].