MicroRNAs regulate gene expression in numerous biological pathways and are typically methylated at their 3'-termini in plants but not in animals. Here we show that the HEN1 RNA 2'-O-methyltransferase from Arabidopsis thaliana catalyzes the transfer of extended propargylic moieties from synthetic AdoMet cofactor analogs to duplex miRNAs or siRNAs. The presented approach permits selective and efficient covalent labeling of small RNA duplexes with a variety of functional or reporter groups for their enrichment and analysis.

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 the substrate interactions and contributions of individual steps during HEN1 catalysis, we studied the binding and methylation kinetics of the enzyme using a series of unmethylated, hemimethylated and doubly methylated miRNA and siRNA substrates. The present study shows that HEN1 specifically binds double-stranded unmethylated or hemimethylated miR173/miR173* substrates with a subnanomolar 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. The results of the present study thus provide novel insights into the mechanism of RNA recognition and modification by a representative small RNA 2'-O-methyltransferase.

The HEN1 RNA2 '-O-methyltransferase plays important roles in the biogenesis of small non-coding RNAs in plants and proved av aluable tool for selective transfer of functional groups from cofactor analogues onto miRNAa nd siRNAd uplexes in vitro.H erein, we demonstrate the versatile HEN1-mediated methylation and alkylation of small RNA strands in heteroduplexes with ar ange of complementary synthetic DNAoligonucleotides carrying user-defined moieties such as internal or 3'-terminal extensions or chemical reporter groups.The observed DNA-guided covalent functionalization of RNAbroadens our understanding of the substrate specificity of HEN1 and paves the wayf or the development of novel chemo-enzymatic tools with potential applications in miR-Nomics,synthetic biology,and nanomedicine.

The HEN1 RNA 2’-O-methyltransferase plays important roles in biogenesis of small non-coding RNAs in plants and proved a valuable tool for selective transfer of functional groups from cofactor analogues onto miRNA and siRNA duplexes in vitro. Herein, we demonstrate versatile HEN1-mediated methylation and alkylation of small RNA strands in heteroduplexes with a range of complementary synthetic DNA oligonucleotides carrying user-defined moieties such as internal or 3’-terminal extensions or chemical reporter groups. The observed DNA-guided covalent functionalization of RNA broadens our understanding of the substrate specificity of HEN1 and paves the ways for developing novel chemo-enzymatic tools with potential applications in miRNomics, synthetic biology and nanomedicine.

Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.