Animal Hen1 2′-O-methyltransferases as tools for 3′-terminal functionalization and labelling of single-stranded RNAs

Abstract

S-adenosyl-L-methionine-dependent 2′-O-methylati-on of the 3′-terminal nucleotide plays important roles in biogenesis of eukaryotic small non-coding RNAs, such as siRNAs, miRNAs and Piwi-interacting RNAs (piRNAs). Here we demonstrate that, in contrast to Mg2+/Mn2+-dependent plant and bacterial homologues, the Drosophila DmHen1 and human HsHEN1 piRNA methyltransferases require cobalt cations for their enzymatic activity in vitro. We also show for the first time the capacity of the animal Hen1 to catalyse the transfer of a variety of extended chemical groups from synthetic analogues of the AdoMet cofactor onto a wide range (22–80 nt) of single-stranded RNAs permitting their 3′-terminal functionalization and labelling. Moreover, we provide evidence that deletion of a small C-terminal region of the DmHen1 protein further increases its modification efficiency and abolishes a modest 3′-terminal nucleotide bias observed for the full-length protein. Finally, we show that fluorophore-tagged ssRNA molecules are successfully detected in fluorescence resonance energy transfer assays both individually and in a total RNA mixture. The presented DmHen1-assisted RNA labelling provides a solid basis for developing novel chemo-enzymatic approaches for in vitro studies and in vivo monitoring of single-stranded RNA pools.

Chemo‐enzymatic treatment of RNA to facilitate analyses

“…HEN1 even accepted bulky side chains containing a biotin moiety, allowing for direct and efficient labeling, followed by capture and enrichment of the targeted RNA species—obviating a dedicated chemical functionalization step (Plotnikova, Osipenko, Masevicius, Vilkaitis, & Klimasauskas, ). Recently, similar results have been reported for Drosophila and human HEN1 MTases, which methylate all ssRNAs (Figure c) (Mickutė et al, ). Remarkably, DmHEN1 showed no preference for AdoMet over an azido‐modified AdoMet analog.…”

Covalent labeling of nucleic acids

“…Interestingly, the human Hen1 enzyme, piRNA MTase HsHEN1, requires cobalt ions (Co 2+ or Co 3+ ) for enzymatic activity, whereas Drosophila DmHen1 is also active in the presence of other divalent cations, like Mn 2+ , Mg 2+ and Ca 2+ . 121 However, cobalt ions are cytotoxic, limiting the application of this enzyme to in vitro RNA labeling. 122 Although a one-step procedure appears to be most straightforward to label nucleic acids, there are a few disadvantages that should not be overlooked.…”

“…However, due to the recently optimized conditions for the enzyme, resulting in improved efficiency, this strategy can form the basis for in vitro and in vivo labeling of specific RNAs in single stranded RNA pools, which could become highly relevant for RNA sequencing. 121 Comparable strategies can be applied for tRNA labeling. The MTase Trm1 from Pyrococcus furiosus was used to fluorescently label the N 2 -position of guanosine-26 in tRNA Phe .…”

Repurposing enzymatic transferase reactions for targeted labeling and analysis of DNA and RNA

“…In addition, groups carrying an alkene [15][16][17][18], a norbornene moiety (for bioorthogonal tetrazine ligation [13]), a photocrosslinker [19] and a photoremovable cage [20] have recently become available. An increasingly popular choice is onestep mTAG labeling with fluorophores or biotin [21][22][23][24][25].…”

“…This route affords multi-milligram amounts of cofactor analogs, usually as diastereomeric mixtures [12,27]. Cofactors with large reporter groups such as chromophores or biotin are often obtained by further appending the side chains of functionalized cofactors via suitable conjugation chemistries under mild conditions [24,25,28]. An alternative more recent strategy is based on the natural route of AdoMet production from methionine and ATP catalyzed by AdoMet synthetase (also known as methionine adenosyltransferase, MAT).…”