Determination of 2′-O-methyl groups in 32P-labeled RNAs

Abstract

A two-dimensional method for the detection of ribose methyl groups in32P-labeled RNAs is described which is suitable for both qualitative and quantitative purposes. The method involves fractionation of an alkaline hydrolysate of the RNA by two-dimensional electrophoresis of the digest on DEAE-cellulose paper; prior to the second electrophoresis the products are dephosphorylated on the paper by alkaline phosphatase. After visualization of the ribose-methylated (i.e., alkali-stable) oligonucleotides by radioautography, quantitation of the number of ribose methyl groups is possible since each ribose methyl group corresponds to one phosphate group. When applied to a bacterial 23S rRNA the method proved to be accurate enough for determination of the low level (0.1%) of ribose-methylated nucleosides in this RNA. It could also be demonstrated by this sensitive method that the RNA from the small ribosomal subunit of yeast mitochondria is devoid of ribose methyl groups.