Facile detection of RNA phospho-methylation in cells and tissues.

Abstract

RNAs from various cells and tissues are modified in nearly 200 chemically distinct ways. These modifications can be deposited either on the 5' or 3' ends, or internally on the nucleobases or sugar backbone. 5'-end modifications are crucial for protecting RNAs from untimely degradation/processing, regulating their cellular functions, or discriminating endogenous RNAs from pathogenic RNAs. 5'-end phospho-methylation is a remarkable RNA modification that is enzymatically deposited either on the γ-phosphate of nascent triphosphorylated RNAs by human BCDIN3/MePCE, or on the α-phosphate of processed monophosphorylated RNAs by human BCDIN3D. These 5'-phospho-methyltransferases are part of the BIN3 family of O-methyltransferases conserved from S. pombe to humans and play important cellular and biological roles, many of which await further elucidation. Here, we quickly recapitulate historical methods for the detection of 5'-end phospho-methyl modifications, and focus more specifically on a method that can be used to detect and quantify α-monophosphate methylation from as low as 10-100ng of total RNA from cells or tissues. This method is important for deciphering the roles of BCDIN3D and its homologs across species, as well as serves as starting point for the development of new methods for detection of 5'-end modifications.