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) Genomic organization of the C. elegans CMTr2 locus depicting the exon intron structure is shown on the left. The methyltransferase domain including key amino acid residues involved in catalysis (K117, D235, and K275 in yellow) is shown in dark blue and the catalytically inactive methyltransferase-like domain in light blue. The deletion in the CMTr2 null allele (tm4453) leads to a frameshift and is validated by PCR products separated on an agarose gel on the right. M: 100 bp ladder. (B) Recapping of mRNA with ³²P-αGTP from adult C. elegans wild-type and CMTr2 null mutants. 5′ cap structures were separated on a 22% denaturing polyacrylamide gels after digestion with RNase I (lanes 4 and 5, 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 C. elegans on the right. L: Alkaline hydrolysis of a 5′ ³²P-labeled RNA oligonucleotide with the nucleotide number indicated in white.