Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 291, Issue 46, Pages 24293-24303Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M116.742569
Keywords
5-methylcytosine; RNA methylation; RNA methyltransferase; S-adenosylmethionine (SAM); transfer RNA (tRNA); NSun family; methyltransferase; substrate recognition; tRNA modification
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Funding
- National Natural Science Foundation of China [91440204, 31270775, 81471113]
- National Key Basic Research Foundation of China [2012CB911000]
- Youth Innovation Promotion Association (Chinese Academy of Sciences)
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Human NSun6 is an RNA methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to C72 of tRNA(Thr) and tRNA(Cys). In the current study, we used mass spectrometry to demonstrate that human NSun6 indeed introduces 5-methylcytosine (m(5)C) 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.
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