Direct quantification of 3 ′ terminal 2 ′ -O-methylation of small RNAs by RT-qPCR

Abstract

Modification of nucleotides significantly increases the diversity of functional nucleic acids. As one of the most common modifications of RNAs, methylation of the 2 ′′ -hydroxyl-group of ribonucleotides (2 ′′ -O-methylation) has been found in various RNAs in eukaryotes. However, due to the lack of an efficient method for quantifying small RNA 3 ′′ terminal 2 ′′ -O-methylation, it is difficult to monitor the dynamic change of 3 ′′ terminal 2 ′′ -O-methylation during various biological processes. Capitalizing on the finding that 3 ′′ terminal RNA 2 ′′ -O-methylation can inhibit the activity of poly(A) polymerase, an enzyme that can add the poly(A)-tail to RNA, we develop a method by which the 2 ′′ -O-methylation level of small RNAs, such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), can be directly quantified based on the poly(A)-tailed RT-qPCR technique. With this method, we successfully determine the 2 ′′ -O-methylation level of miRNAs in Arabidopsis thaliana and mouse lung tissue, piRNA in human seminal plasma, and monitor the alteration of miRNA 2 ′′ -O-methylation in Drosophila Schneider 2 cells after knockdown of Drosophila methyltransferase protein Hua enhancer 1 (DmHen-1).