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<p>Transfer RNAs (tRNAs) undergo extensive maturation, including modifications essential for translation efficiency and fidelity, as well as for structural stability. The modification cytosine-5 methylation (m<sup>5</sup>C) is catalyzed by NSUN2 at multiple positions within distinct structural regions of a subset of tRNAs, yet the determinants of its substrate specificity remain elusive. In archaea, the protein Archease has been implicated in modulating the cytosine specificity of the NSUN2 ortholog Trm4, whereas mammalian Archease was shown to be involved in pre-tRNA splicing. Here, we investigated whether mammalian Archease influences NSUN2 in a similar way like the archaeal enzyme. Using short hairpin RNA-mediated depletion of Archease in mouse embryonic stem cells and in vitro methylation assays with recombinant proteins, we assessed both the catalytic activity and substrate selectivity of NSUN2 in the presence or the absence of Archease. Our results indicate that neither the efficiency nor positional specificity of NSUN2-mediated tRNA m<sup>5</sup>C methylation is affected by Archease. These findings indicate that, unlike in archaea, Archease does not regulate tRNA methylation by NSUN2 in mammals, highlighting an evolutionary divergence in the functional role of Archease in tRNA maturation pathways.</p>

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The tRNA methylation activity and specificity of NSUN2 are independent of Archease

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月29日

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  • Valentina Stuchlik,
  • Maria Waldl,
  • Anming Huang,
  • Moritz Kleinwächter,
  • Anna Rocchegiani,
  • Fatinah El-Isa,
  • Ivo L. Hofacker,
  • Javier Martinez,
  • Alexandra Lusser,
  • Elisa Vilardo

摘要

Transfer RNAs (tRNAs) undergo extensive maturation, including modifications essential for translation efficiency and fidelity, as well as for structural stability. The modification cytosine-5 methylation (m5C) is catalyzed by NSUN2 at multiple positions within distinct structural regions of a subset of tRNAs, yet the determinants of its substrate specificity remain elusive. In archaea, the protein Archease has been implicated in modulating the cytosine specificity of the NSUN2 ortholog Trm4, whereas mammalian Archease was shown to be involved in pre-tRNA splicing. Here, we investigated whether mammalian Archease influences NSUN2 in a similar way like the archaeal enzyme. Using short hairpin RNA-mediated depletion of Archease in mouse embryonic stem cells and in vitro methylation assays with recombinant proteins, we assessed both the catalytic activity and substrate selectivity of NSUN2 in the presence or the absence of Archease. Our results indicate that neither the efficiency nor positional specificity of NSUN2-mediated tRNA m5C methylation is affected by Archease. These findings indicate that, unlike in archaea, Archease does not regulate tRNA methylation by NSUN2 in mammals, highlighting an evolutionary divergence in the functional role of Archease in tRNA maturation pathways.