CMTr mediated 2′-O-ribose methylation status of cap-adjacent nucleotides across animals

Analysis of mRNA cap 2′-O-ribose methylation in various species. (A) Recapping of mRNA with ³²P-αGTP from Trypanosomes (T. brucei), adult C. elegans, adult Drosophila wild-type and CMTr1/2 double flies, worker honey bees, zebrafish inner organs and brain, mouse brain, and human HEK293T cells. 5′ cap structures were separated on a 22% denaturing polyacrylamide gels after digestion with RNase I (lanes 4–11, right). Markers—M1: RNase I digested ³²P-αGTP capped in vitro transcript starting with AGU. M2: RNase I digested ³²P-αGTP capped in vitro transcript starting with AGU and 2′-O-ribose methylated with vaccinia CMTr. Sequences of markers are shown on the left and of cap structures from different species on the right, except for the sequence from Trypanososmes, which is shown on the left. L: Alkaline hydrolysis of a 5′ ³²P-labeled RNA oligonucleotide with the nucleotide number indicated in white. (B) Schematic diagram of a 2D thin layer chromatography (TLC) depicting standard and 2′-O-ribose methylated nucleotides. For orientation, pA is indicated with an asterisk and pAm with an arrow. (C–J) TLCs showing modifications of the first cap-adjacent nucleotides of T. brucei (C), C. elegans (D), Drosophila (E), honey bees (F), zebrafish embryos and inner organs (G,H), mouse brain (I), and human HCT116 cells (J).