Archaeal NSUN6 catalyzes m5C72 modification on a wide-range of specific tRNAs

Abstract

Human NOL1/NOP2/Sun RNA methyltransferase family member 6 (hNSun6) generates 5-methylcytosine (m 5 C) at C72 of four specific tRNAs, and its homologs are present only in higher eukaryotes and hyperthermophilic archaea. Archaeal NSun6 homologs possess conserved catalytic residues, but have distinct differences in their RNA recognition motifs from eukaryotic NSun6s. Until now, the biochemical properties and functions of archaeal NSun6 homologs were unknown. In archaeon Pyrococcus horikoshii OT3, the gene encoding the NSun6 homolog is PH1991 . We demonstrated that the PH1991 protein could catalyze m 5 C72 formation on some specific Ph tRNAs in vitro and was thus named as Ph NSun6. Remarkably, Ph NSun6 has a much wider range of tRNA substrates than hNSun6, which was attributed to its tRNA substrate specificity. The mechanism was further elucidated using biochemical and crystallographic experiments. Structurally, the binding pocket for nucleotide 73 in Ph NSun6 is specific to accommodate U73 or G73-containing Ph tRNAs. Furthermore, Ph NSun6 lacks the eukaryotic NSun6-specific Lys-rich loop, resulting in the non-recognition of D-stem region by Ph NSun6. Functionally, the m 5 C72 modification could slightly promote the thermal stability of Ph tRNAs, but did not affect the amino acid accepting activity of Ph tRNAs.

CIGAR-seq, a CRISPR/Cas-based method for unbiased screening of novel mRNA modification regulators

“…As shown in Figure 2B, NSUN6-dependent m 5 C sites were embedded in slightly GC-rich environments with a strongly enriched TCCA motif at 3' of m 5 C sites. Previously, a similar 3' TCCA motif was also found at NSUN6 target sites in tRNAs (Li et al, 2019), and has also been proposed as sequence motif around NSUN2-independent sites in another study (Huang et al, 2019). In comparison, the sequence feature around NSUN2-dependent m 5 C sites is distinct, which is enrich for 3' NGGG motif (Huang et al, 2019, Yang, Yang et al, 2017b).…”

“…As shown in Fig 1D, it turned out that NSUN6, a member of NOL1/NOP2/sun domain (NSUN) family, was identified as the best hit ( Fig 1D). Interestingly, NSUN6 was previously reported to introduce the m 5 C in tRNA (Li, Li et al, 2019). However, two previous studies did not show significant m 5 C changes in mRNA after NSUN6 perturbation in HeLa cells (Huang et al, 2019, Yang et al, 2017a.…”

CIGAR‐seq, a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators

“…As shown in Fig 1D, it turned out that NSUN6, a member of NOL1/NOP2/sun domain (NSUN) family, was identified as the best hit with all six gRNAs decreasing the m 5 C level effectively (Fig 1D and Dataset EV2). Interestingly, NSUN6 was previously reported to introduce the m 5 C in tRNA (Li et al , 2019). However, two previous studies did not show significant m 5 C changes in mRNA after NSUN6 perturbation in HeLa cells (Yang et al , 2017; Huang et al , 2019), which might be due to the incomplete gene silencing mediated by siRNA.…”

Sequence- and structure-selective mRNA m5C methylation by NSUN6 in animals

“…For example, of the 503 Type II sites found in at least one of the 10 human tissue and cell samples ( Figure 1A), only 61 sites had a methylation level > 0.4 in one or more samples. Interestingly, it is known that archaeal NSUN6 has a much wider range of tRNA substrates [32], but both human and fly NSUN6 proteins only methylated 4 tRNAs at C72. Moreover, miCLIP data revealed that about 30% of human NSUN6 miCLIP reads were mapped to mRNAs.…”