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

Analysis of mRNA cap 2′-O-ribose methylation in C. elegans CMTr2 null mutants. (A) Representative western blot from two replicates of human CMTr1 and CMTr2 tagged with FLAGStrep expressed in human HEK293T cells detected with anti-FLAG antibodies. Molecular weight markers in kDa are shown on the left. The asterisk on the right denotes an unspecific band. (B) 2′-O-ribose methylation of a ³²PαGTP capped in vitro transcript starting with AGU by human CMTR1 and CMTr2 expressed in human HEK293T cells, vaccinia CMTr (vCMTr) or extract from untransfected cells (−). 5′ cap structures were separated on a 22% denaturing polyacrylamide gels after digestion with RNase I (lanes 6–8, 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 cap structures are shown on the sides. L: Single nucleotide ladder with nucleotide number indicated in white.