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

J Biol Chem. 2016 Nov 11;291(46):24293-24303. doi: 10.1074/jbc.M116.742569. Epub 2016 Oct 4.


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.

Keywords: 5-methylcytosine; NSun family; RNA methylation; RNA methyltransferase; S-adenosylmethionine (SAM); methyltransferase; substrate recognition; tRNA modification; transfer RNA (tRNA).

MeSH terms

  • Humans
  • Methylation
  • Protein Domains
  • Protein Folding*
  • RNA, Transfer, Thr / chemistry*
  • RNA, Transfer, Thr / genetics
  • RNA, Transfer, Thr / metabolism
  • tRNA Methyltransferases / chemistry*
  • tRNA Methyltransferases / genetics
  • tRNA Methyltransferases / metabolism


  • RNA, Transfer, Thr
  • NSUN6 protein, human
  • tRNA Methyltransferases

Associated data

  • PDB/1B23