Wang Wei-Syuan, Chen Yu-Hsiang, Liou Gunn-Guang, Murashko Oleg N, Lin-Chao Sue
Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan.
Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Science, National Defense Medical Center, Taipei 11490, Taiwan.
PNAS Nexus. 2025 Aug 18;4(9):pgaf269. doi: 10.1093/pnasnexus/pgaf269. eCollection 2025 Sep.
endoribonuclease E (RNase E), encoded by the essential gene and conserved across γ-Proteobacteria, plays a central role in RNA processing and decay. We show here that -null strain, -null strain complemented with catalytic-null RNase E mutant, and C-terminal-truncated strain (Rned500) all lack flagellar biogenesis and motility under both aerobic and anaerobic conditions, which are restored by wild-type RNase E complementation. The Rned500 displays dysregulated expression of the three-tiered flagellar transcriptional cascade, increased stability of flagellar mRNAs, and reduced flagellar protein levels through sRNA-dependent translational inhibition. However, ectopic expression of flagellar master regulators or flagellar proteins fails to restore flagellar biogenesis and motility. To investigate the underlying defect, we examined the cellular localization of the early flagellar structural protein FliF and found it mislocalized in Rned500, indicating a disruption of early flagellar assembly. This defect is further supported by the impaired secretion of the flagellar anti-sigma factor FlgM in Rned500, a process that requires a functional flagellar basal body. Complementation with wild-type RNase E in Rned500 fully restores expression of the flagellar cascade, proper membrane localization of FliF, flagella formation, and motility. Wild-type RNase E-expressing strains, but not Rned500, activate Toll-like receptor 5 (TLR5)-dependent nuclear factor-kappa B signaling in THP-1 human monocytic cells through flagellin. This response, confirmed by a TLR5 dual-luciferase reporter assay in transfected HEK293T human embryonic kidney cells, highlights RNase E's role in enabling flagellar expression required for cellular immune activation. Collectively, these results identify RNase E as a key flagellar biogenesis regulator, revealing novel posttranscriptional control mechanisms.
核糖核酸内切酶E（RNase E）由必需基因编码，在γ-变形菌中保守，在RNA加工和降解中起核心作用。我们在此表明，缺失该基因的菌株、用催化失活的RNase E突变体互补的缺失该基因的菌株以及C端截短的菌株（Rned500）在有氧和无氧条件下均缺乏鞭毛生物合成和运动能力，而野生型RNase E互补可恢复这些能力。Rned500表现出三层鞭毛转录级联反应的表达失调、鞭毛mRNA稳定性增加以及通过sRNA依赖性翻译抑制导致鞭毛蛋白水平降低。然而，鞭毛主调节因子或鞭毛蛋白的异位表达未能恢复鞭毛生物合成和运动能力。为了研究潜在缺陷，我们检查了早期鞭毛结构蛋白FliF的细胞定位，发现它在Rned500中定位错误，表明早期鞭毛组装受到破坏。Rned500中鞭毛抗σ因子FlgM的分泌受损进一步支持了这一缺陷，这一过程需要功能性鞭毛基体。用野生型RNase E对Rned500进行互补可完全恢复鞭毛级联反应的表达、FliF的正确膜定位、鞭毛形成和运动能力。表达野生型RNase E的菌株而非Rned500通过鞭毛蛋白在THP-1人单核细胞中激活Toll样受体5（TLR5）依赖性核因子κB信号传导。在转染的HEK293T人胚肾细胞中通过TLR5双荧光素酶报告基因测定法证实的这一反应突出了RNase E在实现细胞免疫激活所需的鞭毛表达中的作用。总的来说，这些结果确定RNase E是关键的鞭毛生物合成调节因子，揭示了新的转录后控制机制。
