Abstract
Human NSun6 is an RNA methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to C72 of tRNAThr and tRNACys In the current study, we used mass spectrometry to demonstrate that human NSun6 indeed introduces 5-methylcytosine (m5C) into tRNA, as expected. To further reveal the tRNA recognition mechanism of human NSun6, we measured the methylation activity of human NSun6 and its kinetic parameters for different tRNA substrates and their mutants. We showed that human NSun6 requires a well folded, full-length tRNA as its substrate. In the acceptor region, the CCA terminus, the target site C72, the discriminator base U73, and the second and third base pairs (2:71 and 3:70) of the acceptor stem are all important RNA recognition elements for human NSun6. In addition, two specific base pairs (11:24 and 12:23) in the D-stem of the tRNA substrate are involved in interacting with human NSun6. Together, our findings suggest that human NSun6 relies on a delicate network for RNA recognition, which involves both the primary sequence and tertiary structure of tRNA substrates.
Highlights
Post-transcriptional modifications frequently occur in all types of cellular RNAs
Consistent with results from previous research, our results showed that human NSun6 (hNSun6) independently catalyzed methyl transfer from SAM to tRNAThr(UGU), tRNAThr(AGU), and tRNACys
Recent research has shown that hNSun6 methylates tRNAThr(UGU), tRNAThr(AGU), and
Summary
Human NSun is an RNA methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to C72 of tRNAThr and tRNACys. Our findings suggest that human NSun relies on a delicate network for RNA recognition, which involves both the primary sequence and tertiary structure of tRNA substrates. An m5C modification at position 72 in the acceptor stem of many tRNAs is conserved in higher eukaryotes [1, 2] The function of this m5C72 modification remains elusive, and the enzyme that is thought to establish this modification has only recently been identified [24]. Through cross-linking and deep sequencing methods, the putative human RNA m5C methyltransferase (MTase) NSun (NSun family, member 6) has been found to catalyze the methylation at position 72 in some human tRNAs [24]. The NSun family includes the main RNA m5C MTases in eukaryotes and contains seven members: NSun1–NSun7 [25]. The biological function, modification mechanism, and RNA recognition specificity of NSun all remain elusive. Other cellular RNAs are unlikely to satisfy all of the elements required for hNSun recognition; we conclude that hNSun is a tRNAspecific MTase
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