Figure 2.

METTL5 catalyzes m⁶A formation in RNA in vitro and in cells. (A) Principle of Methyl-NAIL-MS (Nucleic acid isotope labeling-coupled mass spectrometry) (Reichle et al. 2019). Cells were grown in the presence of CD₃-methionine for metabolic D₃-labeling of methylated nucleosides. Total RNA isolated from these cells was in vitro methylated by recombinant METTL5 with unlabeled SAM. RNA was digested and methylated nucleosides were analyzed by LC-MS/MS. (B) LC-MS/MS elution profiles of indicated modifications in in vitro methylated RNA. In vitro methylated nucleosides (blue) can be distinguished from endogenous methylations (black) by a mass shift of +3 Da. Overlaid MS/MS chromatograms of 6-methyladenosine (m⁶A), 2′-O-methyladenosine (Am), and 5-methylcytosine (m⁵C) are shown. (C, left) Scheme of RNA size exclusion chromatography followed by LC-MS/MS (Chionh et al. 2013). The collected and analyzed fractions are highlighted in gray with sizes of the predominant RNA species per fraction (F1: 28S rRNA; F2: 18S rRNA; F3: 5.8S rRNA and 5S rRNA; F4: 5S rRNA; F5: tRNAs and pri-miRNAs) in the table. (Right) Absolute quantifications of modified nucleosides per respective RNA for 6-methyladenosine (m⁶A), 2′-O-methyladenosine (Am), and 7-methylguanosine (m⁷G) (Borland et al. 2019). Average of three biological replicates and SD are plotted. See Supplemental Figure S2b for additional data. The small decrease in m⁶A abundance in fraction F1 could be due to a minor contamination with 18S rRNA as suggested by model from Piekna-Przybylska et al. (2008) (see also https://people.biochem.umass.edu/fournierlab/3dmodmap/humssu2dframes.php). (E) In vitro RNA MTA with recombinant GFP-METTL5 on 18S rRNA isolated from WT and Mettl5 KO mESC with tritium-labeled SAM. Tritium signal was quantified by liquid scintillation counting (LSC). CPMs for one representative experiment as averages of three technical replicates with SD are plotted. (****) P < 0.0001; (*****) P < 0.00001, calculated with Welch's test (unpaired t-test).