東京大学 工学系研究科 教授
Human RNA methyltransferase BCDIN3D is overexpressed in breast cancer cells, and is related to the tumorigenic phenotype and poor prognosis of breast cancer. Here, we show that cytoplasmic tRNAHis is the primary target of BCDIN3D in human cells. Recombinant human BCDIN3D, expressed in Escherichia coli, monomethylates the 5΄-monophosphate of cytoplasmic tRNAHis efficiently in vitro. In BCDN3D-knockout cells, established by CRISPR/Cas9 editing, the methyl moiety at the 5΄-monophosphate of cytoplasmic tRNAHis is lost, and the exogenous expression of BCDIN3D in the knockout cells restores the modification in cytoplasmic tRNAHis. BCIDN3D recognizes the 5΄-guanosine nucleoside at position -1 (G-1) and the eight-nucleotide acceptor helix with the G-1-A73 mis-pair at the top of the acceptor stem of cytoplasmic tRNAHis, which are exceptional structural features among cytoplasmic tRNA species. While the monomethylation of the 5΄-monophosphate of cytoplasmic tRNAHis affects neither the overall aminoacylation process in vitro nor the steady-state level of cytoplasmic tRNAHisin vivo, it protects the cytoplasmic tRNAHis transcript from degradation in vitro. Thus, BCDIN3D acts as a cytoplasmic tRNAHis-specific 5΄-methylphosphate capping enzyme. The present results also suggest the possible involvement of the monomethylation of the 5΄-monophosphate of cytoplasmic tRNAHis and/or cytoplasmic tRNAHis itself in the tumorigenesis of breast cancer cells.
Efficient maturation of transfer RNAs (tRNAs) is required for rapid cell growth. However, the precise timing of tRNA processing in coordination with the order of tRNA modifications has not been thoroughly elucidated. To analyze the modification status of tRNA precursors (pre-tRNAs) during maturation, we isolated pre-tRNAs at various stages from Saccharomyces cerevisiae and subjected them to MS analysis. We detected methylated guanosine cap structures at the 5' termini of pre-tRNAs bearing 5' leader sequences. These capped pre-tRNAs accumulated substantially after inhibition of RNase P activity. Upon depletion of the capping enzyme Ceg1p, the steady state level of capped pre-tRNA was markedly reduced. In addition, a population of capped pre-tRNAs accumulated in strains in which 5' exonucleases were inhibited, indicating that the 5' cap structures protect pre-tRNAs from 5'-exonucleolytic degradation during maturation.
In human mitochondria, the AUA codon encodes methionine via a mitochondrial transfer RNA for methionine (mt-tRNA(Met)) that contains 5-formylcytidine (f(5)C) at the first position of the anticodon (position 34). f(5)C34 is required for deciphering the AUA codon during protein synthesis. Until now, the biogenesis and physiological role of f(5)C34 were unknown. We demonstrate that biogenesis of f(5)C34 is initiated by S-adenosylmethionine (AdoMet)-dependent methylation catalyzed by NSUN3, a putative methyltransferase in mitochondria. NSUN3-knockout cells showed strong reduction in mitochondrial protein synthesis and reduced oxygen consumption, leading to deficient mitochondrial activity. We reconstituted formation of 5-methylcytidine (m(5)C) at position 34 (m(5)C34) on mt-tRNA(Met) with recombinant NSUN3 in the presence of AdoMet, demonstrating that NSUN3-mediated m(5)C34 formation initiates f(5)C34 biogenesis. We also found two disease-associated point mutations in mt-tRNA(Met) that impaired m(5)C34 formation by NSUN3, indicating that a lack of f(5)C34 has pathological consequences.
PIWI-interacting RNAs (piRNAs) play a crucial role in transposon silencing in animal germ cells. In piRNA biogenesis, single-stranded piRNA intermediates are loaded into PIWI-clade proteins and cleaved by Zucchini/MitoPLD, yielding precursor piRNAs (pre-piRNAs). Pre-piRNAs that are longer than the mature piRNA length are then trimmed at their 3′ ends. Although recent studies implicated the Tudor domain protein Papi/Tdrkh in pre-piRNA trimming, the identity of Trimmer and its relationship with Papi/Tdrkh remain unknown. Here, we identified PNLDC1, an uncharacterized 3′-5′ exonuclease, as Trimmer in silkworms. Trimmer is enriched in the mitochondrial fraction and binds to Papi/Tdrkh. Depletion of Trimmer and Papi/Tdrkh additively inhibits trimming, causing accumulation of ∼35–40-nt pre-piRNAs that are impaired for target cleavage and prone to degradation. Our results highlight the cooperative action of Trimmer and Papi/Tdrkh in piRNA maturation.
Ribosome biogenesis requires multiple assembly factors. In Escherichia coli, deletion of RlmE, the methyltransferase responsible for the 2'-O-methyluridine modification at position 2552 (Um2552) in helix 92 of the 23S rRNA, results in slow growth and accumulation of the 45S particle. We demonstrate that the 45S particle that accumulates in ΔrlmE is a genuine precursor that can be assembled into the 50S subunit. Indeed, 50S formation from the 45S precursor could be promoted by RlmE-mediated Um2552 formation in vitro. Ribosomal protein L36 (encoded by rpmJ) was completely absent from the 45S precursor in ΔrlmE, and we observed a strong genetic interaction between rlmE and rpmJ. Structural probing of 23S rRNA and high-salt stripping of 45S components revealed that RlmE-mediated methylation promotes interdomain interactions via the association between helices 92 and 71, stabilized by the single 2'-O-methylation of Um2552, in concert with the incorporation of L36, triggering late steps of 50S subunit assembly.