Published on April 1, 2013 in Biochem J volume None.
PubMed ID: 23621770
Abstract:
The HEN1 methyltransferase from Arabidopsis thaliana modifies the 3'-terminal nucleotides of small regulatory RNAs. Although it is one of the best characterized members of the 2'-O-methyltransferase family, many aspects of its interactions with the cofactor and substrate RNA remained unresolved. To better understand substrate interactions and contributions of individual steps during HEN1 catalysis, we studied binding and methylation kinetics using series of unmethylated, hemimethylated and doubly methylated miRNA and siRNA substrates. Our studies indicate that HEN1 specifically binds double-stranded unmethylated or hemimethylated miR173/miR173* substrates with sub-nanomolar affinity in a cofactor-dependent manner. Kinetic studies under single turnover and pre-steady state conditions in combination with isotope partitioning analysis showed that the binary HEN1*miRNA/miRNA* complex is catalytically competent, however successive methylation of the two strands in a RNA duplex occurs in a non-processive (distributive) manner. We also find that the observed moderate methylation strand preference is largely exerted at the RNA binding step and is fairly independent of the nature of the 3'-terminal nucleobase but shows some dependency on proximal nucleotide mispairs. Our results thus provide novel insights into the mechanism of RNA recognition and modification by a representative small RNA 2'-O-methyltransferase.