NSUN6 is a human RNA methyltransferase that catalyzes formation of m⁵C72 in specific tRNAs

Abstract

Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m 5 C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m 5 C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNA Cys and tRNA Thr as RNA substrates of NSUN6 and identified the cytosine C72 at the 3 ′ end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3 ′ -CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis.

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

“…The constructs were transfected into HEK293 Flp‐In T‐Rex cells according to the manufacturer's instructions and as described (Sloan et al , 2015). UV and 5‐AzaC cross‐linking and analysis of cDNA (CRAC) experiments were carried out as previously described (Bohnsack et al , 2012; Haag et al , 2015a; see also Appendix Supplementary Methods). Detection of co‐immunoprecipitated tRNA by Northern blot was performed as previously described (Haag et al , 2015a).…”

“…UV and 5‐AzaC cross‐linking and analysis of cDNA (CRAC) experiments were carried out as previously described (Bohnsack et al , 2012; Haag et al , 2015a; see also Appendix Supplementary Methods). Detection of co‐immunoprecipitated tRNA by Northern blot was performed as previously described (Haag et al , 2015a). In brief, after cross‐linking and immunoprecipitation of protein–RNA complexes the RNA was eluted by proteinase K digestion for 16 h, precipitated and resuspended in loading dye (95% formamide, 5 mM EDTA, bromophenol blue).…”

“…After washing with PBS, cells were mounted on coverslips using Vectashield ® (Vector labs) for confocal microscopy and localisation analysis. Alternatively, immunofluorescence using an antibody against ABH1 (see Appendix Table S2) was performed as previously described (Haag et al , 2015a). Isolation of mitochondria, analysis of submitochondrial localisation and protease protection assays were performed as described using the antibodies listed in Appendix Table S2 (Dennerlein et al , 2015).…”

“…m 5 C modifications can be installed by any of the seven proteins of the Nol1/Nop2/SUN domain (NSUN) family and by an enzyme named DNA methyltransferase 2 (DNMT2). DNMT2 mainly catalyses the m 5 C modification in position 38 of tRNA Asp in human cells (Goll et al , 2006), while the so far characterised NSUN proteins show specificity for tRNAs (NSUN2, NSUN6; Schaefer et al , 2010; Tuorto et al , 2012; Blanco et al , 2014; Haag et al , 2015a) or rRNA (NSUN1/NOP2, NSUN5; Sloan et al , 2013; Tafforeau et al , 2013; Schosserer et al , 2015). NSUN2 can also modify vault RNAs and mRNAs (Hussain et al , 2013), and NSUN4 was described to localise to mitochondria where it was shown to methylate the mitochondrial 12S rRNA in mice (Cámara et al , 2011; Metodiev et al , 2014).…”

How RNA modification allows non-conventional decoding in mitochondria

“…1,2,3 This enzyme is a member of the Nol1/Nop2/SUN domain (NSUN) family, which also contains the RNA methyltransferases NSUN2 and NSUN6 that modify cytoplasmic tRNAs. 4,5 In contrast, NSUN3 localizes to the mitochondrial matrix where it specifically recognizes the anticodon stem loop (ASL) of mt-tRNA Met . Interestingly, mutations that compromise basepairing in the ASL, including a pathogenic mutation, reduce C34 methylation by NSUN3, implying that lack of this modification in mt-tRNA Met can lead to disease.…”