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

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-1μM-1 on mRNA cap analog, down to MettL16 which has the lowest rate of ∼3 h-1μM-1 acting on an RNA hairpin. Both PCIF1 and MettL5 have a binding affinity (Km) of ∼1 μM or lesser for both substrates of SAM and RNA, whereas MettL16 has significantly lower binding affinities for both (Km >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 200-500 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.