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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.

In order to avoid nuclease contamination two-step purification scheme was applied. As a first step affinity chromatography was used as soluble filtered lysate was loaded onto equilibrated 5 ml GSTrap HP column (GE Healthcare; the used Equilibration buffer (pH 7.3) contained 140 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4 and 1 mM EDTA). GST tagged proteins were eluted with 50 mM Tris-HCl (pH 8.0), 10 mM GSH and 1 mM DTT. Then fractions that were enriched in the proteins of interest were further loaded on the 5 ml anion exchange column HiTrap Q HP (GE Healthcare) washed with 50 mM Tris-HCl (pH 8.0). Hen1 proteins were eluted with 1 M NaCl and dialyzed against 25 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT and 0.1% Triton X-100 at first without and later with 50% glycerol, collected and stored at -20°C.

Preparation of templates for in vitro transcription
For in vitro transcription of 43 nt and longer RNA substrates PCR-amplified DNA was used as a template, which itself was amplified from pUC19-T7II-siR43/60 or 80-HDV plasmid with T7II-Fw and HDV-Rv primers.
These plasmids were constructed using pTZ19-siR43/60/80. pTZ19-siR43 was a result of ligation of siR-DNA-43-1/2 with PstI and HindIII (Thermo Scientific) digested vector. pTZ19-siR60 and siR80 was generated after siR-DNA-60-1/2 and siR-DNA-80-1/2, respectively, were ligated with MunI and HindIII (Thermo Scientific) restricted pTZ19-siR43. As initial in vitro transcription experiments resulted in heterogeneous transcripts, T7 class III promoter was changed to class II promoter and HDV ribozyme at the 3' end of each sequence was added. This was done through three steps of PCR amplification: first, sequences of T7 II promoter, respective RNA and 5' of HDV ribozyme were amplified using T7II-Fw and siR-HDV-Rv primers and respective plasmid as a template, second, HDV ribozyme was obtained after PCR from partially complementary HDV-Fw and HDV-Rv primers (1), third, PCR products from first and second steps were mixed and amplified with terminal primers, namely T7II-Fw and HDV-Rv. DNA fragments generated in the last step were ligated with pUC19/SmaI (Thermo Scientific) resulting in pUC19-T7II-siR43/60 or 80-HDV.
All primers used for preparation of longer RNA substrates are listed in Supplementary Table S4.
For polyacrylamide (PAA) gel analysis RNA was mixed with 2 x RNA loading buffer (Fisher Scientific), heated for 5 min at 85°C and resolved on 10-13% denaturing PAA gel with 7 M urea. Phosphor imaging plates (Fujifilm) were exposed to radioactive gels and later scanned with FLA-5100 Image Reader (Fujifilm). Results were analysed using MultiGauge v.3.0 software (Fujifilm).

HPLC-MS analysis of modified nucleosides
1 µM of methyltransferase was used to modify 2 µM of miR173 in the presence of 0.1 mM of AdoMet 1/Ado-6-amine 2/Ado-6-azide 3 or 0.05 mM Ado-13-biotin 4 in the Reaction buffer or 6 µM of Ado-14-Cy3 5 in the Cy3 reaction buffer. After 1.5 h of incubation at 37˚C, RNA was extracted with chloroform, precipitated and resuspended in P1 buffer (10 mM NaOAc, 1mM Zn(OAc)2, pH 6.5) for degradation to nucleotides with 0.01 u/µl of P1 nuclease (Sigma-Aldrich). After 2 h of incubation at 50°C, followed the overnight dephosphorylation to nucleosides with 0.02 u/µl of FastAP phosphatase at 37°C. Enzymes were inactivated by heating at 70°C for 15 min and precipitated by centrifugation at 20,000 x g for 40 min at 4 °C. The collected supernatant was loaded on Discovery SH C18 column (Supelco) for analyses using 1290 Infinity HPLC System (Agilent Technologies) and eluted with linear gradient from 20 mM ammonium formate (pH 3.5) to 80% methanol. Mass spectra of modification products were acquired on an Agilent Q-TOF 6520 mass analyser in a positive ionization mode and assessed with Agilent MassHunter Workstation software.

Electrophoretic mobility shift assay (EMSA)
0.2 μM of 32 P-labelled miR173 was incubated with no protein (control) or with 1 μM of DmHen1 or HsHEN1 for 30 min at 37°C in Binding buffer (10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.1 mg/ml BSA and 5% glycerol) containing 0.1 mM of AdoHcy without or with 1, 5 or 10 mM metal cofactor in the form of chloride salt. Potential RNA-protein complexes were analysed on native 8% PAA gel in 0.5 x TB buffer (pH 7.5). The gel was dried and exposed to phosphor imaging plate. After scanning with FLA-5100 Image Reader (Fujifilm) results were analysed using MultiGauge v.3.0 software (Fujifilm).

Synthesis of Ado-14-Cy3 cofactor
Reaction was monitored performing HPLC/MS experiments, HRMS data were obtained using Agilent 6230 TOF mass spectrometer (ESI). The purification of target cofactor was performed with preparative reversed- Ado-14-Cy3 cofactor 5 Cy3 alkyne 6 After that, water was added to the reaction mixture. The resulting solution was washed with CH2Cl2 (5 

FRET analysis on polyacrylamide gel
For visualization of siR23-Cy3/DNA-2Cy5 FRET pair in PAA gel, siR23 was modified with Cy3 as described above and siR23-CH3 was prepared as a control. Three pairs of RNA/DNA hybrids were annealed: siR23- Calculated kchem values, which were acquired by using first order kinetics and fitting single-exponential equation to experimental data from at least two replicates, are listed in Table 1.
Supplementary Figure S5. DmHen1 modifies RNA substrates of different length with identical 3' terminal sequences at similar rate in the presence of AdoMet 1 or its synthetic analogues. Reactions were performed as described in Supplementary Figure S4 with the exception that 0.05 mM of Ado-13-biotin 4 was used instead.
Results are mean of two experiments. kchem values are listed in Table 2.
Supplementary Figure S6. Scheme of attachment of required fluorophore, Cy5 or Cy5.5, using two-step labelling approach for RNA functionalization. Experimental results are presented in Figure 5B.
Supplementary Figure S7. One-step labelling of long ssRNAs with bulky side chains of synthetic cofactor analogues -Ado-13-biotin 4 (A) or Ado-14-Cy3 5 (B and C using DmHen1 or DmHen1ΔC, respectively). While the portion of modified 32 P-RNA is seen by the lower mobility of respective bands (A, B and C, left), Cy3 labelling is also detected using 532 nm laser (B and C right). Modification reactions were performed with 1 µM of DmHen1 or DmHen1ΔC, 0.1 µM of 32 P-labeled RNA and 50 µM of Ado-13-biotin 4, 6 µM of Ado-14-Cy3 5 or AdoMet 1 as a control for 30 min at 37°C. In the presence of DNA-5Cy5 higher FRET signal is registered, even though 5th position for Cy5 attachment holds no apparent advantage as can be seen from siR23/DNA heteroduplex structure modelled using 3D-Nus Web server (2). Green sphere -2'-O atom of ribose, to which Cy3 moiety is transferred by DmHen1; red spheresmethyl groups of thymine, which are replaced by Cy5 in DNA-2Cy5 and DNA-5Cy5 oligonucleotides at second and at fifth position, respectively.