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

Impact of heat stress on CMTr double mutant viability and mRNA cap 2′-O-ribose methylation in Drosophila. (A) Viability of CMTr1^null and CMTr2^null single and double mutant flies at 25°C and 29°C shown as mean ± SE (n = 3, except for CMTr1/2^null at 25°C n = 4, P < 0.001). (B) Recapping of mRNA with ³²P-αGTP from adult Drosophila wild-type and CMTr double mutants at 25°C and adult Drosophila wild-type flies kept at 29°C for 2 d. 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 and 2′-O-ribose methylated with vaccinia CMTr. M2: RNase I digested ³²P-αGTP capped in vitro transcript starting with AGU. Sequences of markers are shown on the left and of cap structures from Drosophila on the right. L: Alkaline hydrolysis of a 5′ ³²P-labeled RNA oligonucleotide with the nucleotide number indicated in white.