Exploring a unique class of flavoenzymes: Identification and biochemical characterization of ribosomal RNA dihydrouridine synthase

• 成果类型: Article
• 署名作者: Toubdji, Sabrine; Thullier, Quentin; Kilz, Lea - Marie; Marchand, Virginie; Yuan, Yifeng; Sudol, Claudia; Goyenvalle, Catherine; Jean, Olivier Jean-; Rose, Simon; Douthwaite, Stephen; Hardy, Leo; Baharoglu, Zeynep; de Crecy-Lagard, Valerie; Helm, Mark; Motorin, Yuri; Hamdane, Djemel; Bregeon, Damien
• 署名单位: Sorbonne Universite; Centre National de la Recherche Scientifique (CNRS); Sorbonne Universite; Centre National de la Recherche Scientifique (CNRS); Universite PSL; College de France; Institut National de la Sante et de la Recherche Medicale (Inserm); Centre National de la Recherche Scientifique (CNRS); Universite de Lorraine; Universite de Lorraine; Centre National de la Recherche Scientifique (CNRS); Johannes Gutenberg University of Mainz; State University System of Florida; University of Florida; University of Southern Denmark; Centre National de la Recherche Scientifique (CNRS); CNRS - National Institute for Biology (INSB); Pasteur Network; Universite Paris Cite; Institut Pasteur Paris; State University System of Florida; University of Florida
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-12338
• DOI: 10.1073/pnas.2401981121
• 发表日期: 2024-07-30
• 关键词: posttranscriptional modification; messenger-rna; yeast

摘要

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN- dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase- blockage at the rhodamine- labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single- gene deletions, we pinpoint the yhiNgene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives.