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

Jing Li,Han Dong,Hao Li,Tao Long,Ru-Juan Liu,En-Duo Wang

doi:10.1093/nar/gky1236

Abstract

Human NOL1/NOP2/Sun RNA methyltransferase family member 6 (hNSun6) generates 5-methylcytosine (m5C) 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 m5C72 formation on some specific PhtRNAs in vitro and was thus named as PhNSun6. Remarkably, PhNSun6 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 PhNSun6 is specific to accommodate U73 or G73-containing PhtRNAs. Furthermore, PhNSun6 lacks the eukaryotic NSun6-specific Lys-rich loop, resulting in the non-recognition of D-stem region by PhNSun6. Functionally, the m5C72 modification could slightly promote the thermal stability of PhtRNAs, but did not affect the amino acid accepting activity of PhtRNAs.

Highlights

As linker molecules between ribosomes and growing polypeptide chains, transfer RNAs play a central role in protein synthesis
We found that PH1991 was able to catalyze the methylation of three PhtRNAThr isoacceptors and PhtRNACys(GCA) at 65◦C, similar to Human NOL1/NOP2/Sun RNA methyltransferase family member 6 (hNSun6) at 37◦C (Figure 1B)
The PhtRNAThr(CGU) methylated by PH1991 were analyzed using Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and the modification was confirmed to be member (hNSun6) generates 5methylcytosine (m5C) (Figure 1C), as it was in the methylated PhtRNAThr(GGU), PhtRNAThr(UGU), and PhtRNACys(GCA) (Supplementary Figure S3)

Summary

Introduction

As linker molecules between ribosomes and growing polypeptide chains, transfer RNAs (tRNAs) play a central role in protein synthesis. Further functions of tRNAs have been identified in addition to translation [1,2,3]. Multiple modifications are a characteristic property of tRNAs, and these modifications are crucial for tRNA stability, decoding accuracy, and cellular functions [4,5,6,7,8]. Archaeosine is exclusively found in most of known archaeal tRNAs at position G15 in the D-loop, ensuring the tRNA tertiary structure by tightening the tertiary base pair, G15:C48 [11,12]. An agmatidine modification at the first anticodon position of C34 in archaeal tRNAIle(CAU) is essential for precise decoding [13,14]. Archaea-specific isowyosine and 7-methylwyosine, which are guanosine-37 derivatives, demonstrate the complexity in archaeal wyosine derivatives synthesis [15,16,17,18,19,20]

Methods

Results

Conclusion