Sequence-specific and Shape-selective RNA Recognition by the Human RNA 5-Methylcytosine Methyltransferase NSun6

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. T...

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Bibliographic Details
Published in:The Journal of biological chemistry 2016-11, Vol.291 (46), p.24293-24303
Main Authors: Long, Tao, Li, Jing, Li, Hao, Zhou, Mi, Zhou, Xiao-Long, Liu, Ru-Juan, Wang, En-Duo
Format: Article
Language:English
Subjects:
5-methylcytosine
Humans
Methylation
methyltransferase
NSun family
Protein Domains
Protein Folding
RNA
RNA methylation
RNA methyltransferase
RNA, Transfer, Thr - chemistry
RNA, Transfer, Thr - genetics
RNA, Transfer, Thr - metabolism
S-adenosylmethionine (SAM)
substrate recognition
transfer RNA (tRNA)
tRNA Methyltransferases - chemistry
tRNA Methyltransferases - genetics
tRNA Methyltransferases - metabolism
tRNA modification
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M116.742569