5-methylcytosine (m⁵C) modifications of RNA are ubiquitous in nature and play important roles in many biological processes such as protein translational regulation, RNA processing and stress response. Aberrant expressions of RNA:m⁵C methyltransferases are closely associated with various human diseases including cancers. However, no structural information for RNA-bound RNA:m⁵C methyltransferase was available until now, hindering elucidation of the catalytic mechanism behind RNA:m⁵C methylation. Here, we have solved the structures of NSun6, a human tRNA:m⁵C methyltransferase, in the apo form and in complex with a full-length tRNA substrate. These structures show a non-canonical conformation of the bound tRNA, rendering the base moiety of the target cytosine accessible to the enzyme for methylation. Further biochemical assays reveal the critical, but distinct, roles of two conserved cysteine residues for the RNA:m⁵C methylation. Collectively, for the first time, we have solved the complex structure of a RNA:m5C methyltransferase and addressed the catalytic mechanism of the RNA:m⁵C methyltransferase family, which may allow for structure-based drug design toward RNA:m⁵C methyltransferase–related diseases.

Streptococcus pneumoniae causes bacterial pneumonia with high mortality and morbidity. The emergency of multidrug-resistant bacteria threatens the treatment of the disease. Leucyl-tRNA synthetase (LeuRS) plays an essential role in cellular translation and is an attractive drug target for antimicrobial development. Here we report the compound ZCL039, a benzoxaborole-based derivative of AN2690, as a potent anti-pneumococcal agent that inhibits S. pneumoniae LeuRS (SpLeuRS) activity. We show using kinetic, biochemical analyses combined with the crystal structure of ZCL039-AMP in complex with the separated SpLeuRS editing domain, that ZCL039 binds to the LeuRS editing active site which requires the presence of tRNA^Leu, and employs an uncompetitive inhibition mechanism. Further docking models establish that ZCL039 clashes with the eukaryal/archaeal specific insertion I4ae helix within editing domains. These findings demonstrate the potential of benzoxaboroles as effective LeuRS inhibitors for pneumococcus infection therapy, and provide future structure-guided drug design and optimization.

MitoNEET was identified as an outer mitochondrial membrane protein that can potentially bind the anti-diabetes drug pioglitazone. The crystal structure of the cytoplasmic mitoNEET (residues 33-108) is determined in this study. The structure presents a novel protein fold and contains a [2Fe-2S] cluster-binding domain. The [2Fe-2S] cluster is coordinated to the protein by Cys-72, Cys-74, Cys-83, and His-87 residues. This coordination is also novel compared with the traditional [2Fe-2S] cluster coordinated by four cysteines or two cysteines and two histidines. The cytoplasmic mitoNEET forms homodimers in solution and in crystal. The dimerization is mainly mediated by hydrophobic interactions as well as hydrogen bonds coordinated by two water molecules binding at the interface. His-87 residue, which plays an important role in the coordination of the [2Fe-2S] cluster, is exposed to the solvent on the dimer surface. It is proposed that mitoNEET dimer may interact with other proteins via the surface residues in close proximity to the [2Fe-2S] cluster.

TRNA modifications at the anti‐codon loop are critical for accurate decoding. FTSJ1 was hypothesized to be a human tRNA 2′‐O‐methyltransferase. tRNA ^Phe (GAA) from intellectual disability patients with mutations in ftsj1 lacks 2′‐O‐methylation at C32 and G34 (Cm32 and Gm34). However, the catalytic activity, RNA substrates, and pathogenic mechanism of FTSJ1 remain unknown, owing, in part, to the difficulty in reconstituting enzymatic activity in vitro . Here, we identify an interacting protein of FTSJ1, WDR6. For the first time, we reconstitute the 2′‐O‐methylation activity of the FTSJ1‐WDR6 complex in vitro , which occurs at position 34 of specific tRNAs with m ¹ G37 as a prerequisite. We find that modifications at positions 32, 34, and 37 are interdependent and occur in a hierarchical order in vivo . We also show that the translation efficiency of the UUU codon, but not the UUC codon decoded by tRNA ^Phe (GAA), is reduced in ftsj1 knockout cells. Bioinformatics analysis reveals that almost 40% of the high TTT‐biased genes are related to brain/nervous functions. Our data potentially enhance our understanding of the relationship between FTSJ1 and nervous system development.