Enzymatic characterization of three human RNA adenosine methyltransferases reveals diverse substrate affinities and reaction optima

Abstract

RNA methylations of varied RNA species (mRNA, tRNA, rRNA, non-coding RNA) generate a range of modified nucleotides, including N6-methyladenosine. Here we study the enzymology of three human RNA methyltransferases that methylate the adenosine amino group in diverse contexts, when it is: the first transcribed nucleotide after the mRNA cap (PCIF1), at position 1832 of 18S rRNA (MettL5-Trm112 complex), and within a hairpin in the 3′ UTR of the S-adenosyl- l -methionine synthetase (MettL16). Among these three enzymes, the catalytic efficiency ranges from PCIF1, with the fastest turnover rate of >230 h −1 μM −1 on mRNA cap analog, down to MettL16, which has the lowest rate of ∼3 h −1 μM −1 acting on an RNA hairpin. Both PCIF1 and MettL5 have a binding affinity ( K m ) of ∼1 μM or less for both substrates of SAM and RNA, whereas MettL16 has significantly lower binding affinities for both ( K m >0.4 mM for SAM and ∼10 μM for RNA). The three enzymes are active over a wide pH range (∼5.4–9.4) and have different preferences for ionic strength. Sodium chloride at 200 mM markedly diminished methylation activity of MettL5-Trm112 complex, whereas MettL16 had higher activity in the range of 200 to 500 mM NaCl. Zinc ion inhibited activities of all three enzymes. Together, these results illustrate the diversity of RNA adenosine methyltransferases in their enzymatic mechanisms and substrate specificities and underline the need for assay optimization in their study.

Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs

“…Both oligonucleotides contain additional internal Ade residues. Under the conditions established for the capped substrate at pH 8.0 (23), we observed PCIF1 methylation of both uncapped substrates, with higher activity on oligo R2 (Figure 2A). Replacement of the 5' A and 3' A in oligo R2 (with U) resulted in only low residual activity (Figure 2B).…”

“…We next measured the PCIF1 kinetic parameters on oligo R2, with and without 2'O methylation, at three different pH values (5.4, 8.0 and 9.4). This pH range was used because we previously characterized PCIF1 activity on a cap analog and observed increasing catalytic efficiency from lower to higher pH that was independent of the buffering agent used (23). By varying concentrations of the RNA substrates, we determined kinetic parameters for reaction rate (kcat), RNA binding affinity (KM), and catalytic efficiency (kcat/KM) (Figure 4).…”

“…The highly purified recombinant enzymes used in this study were recently characterized in our laboratory: human PCIF1 (pXC2055) (23), human MettL3-14 (49,50), human MettL5-Trm112 (pXC2062-pXC2076), and human MettL16 (pXC2210) (23). The example of purified PCIF1 and its activity is shown in Figure S1A-C.…”

“…Methylation assays of PCIF1 on different RNA oligonucleotides were conducted in the reaction buffer containing 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM DTT, 20 µM or 100 µM SAM, with various PCIF1 and substrate concentrations. Methylation assays of MettL3-MettL14, MettL5-Trm112 and MettL16 were conducted under the optimal conditions for each enzyme as previously characterized in our laboratory (23,49,50).…”

Structure, dynamics, and inhibition of Staphylococcus aureus m1A22-tRNA methyltransferase

“…The MTase-Glo™ Methyltransferase Assay, which detects luminescence generated in a luciferase-coupled reaction driven by ATP, which in turn is synthesized enzymatically from the SAH produced in the methyltransferase reaction (21), was used to quantify the methylation of S. aureus tRNA Leu as catalysed by SaTrmK. This is a robust and sensitive assay used to characterise RNA methyltransferase activity (22) and to screen tRNA methyltransferase inhibitors (23), as it has been shown to result in lower false-positive rates than other methods (24). In the absence of tRNA, SaTrmK contributes no background luminescence, as expected since the enzyme is not purified bound to SAH.…”