Unveil the Molecular Interplay between Aminoglycosides and Pseudouridine in IRES Translation

Eukaryotic ribosomes are enriched with pseudouridine, particularly at the functional centers targeted by antibiotics. Here we investigated the roles of pseudouridine in aminoglycoside-mediated translation inhibition by comparing the structural and functional properties of the wild-type ribosomes and those lacking pseudouridine ( cbf5 -D95A). We showed that the cbf5 -D95A ribosomes have decreased thermostability and high sensitivity to aminoglycosides. When presented with an internal ribosome entry site (IRES) RNA, elongation factor eEF2, GTP, sordarin, hygromycin B preferentially binds to the cbf5 -D95A ribosomes during initiation by blocking eEF2 binding and stalls the ribosomes in a non-rotated conformation, further hindering translocation. Hygromycin B binds to the inter-subunit bridge B2a that is known to be sensitive to pseudouridine, revealing a functional link between pseudouridine and aminoglycoside inhibition. Our results suggest that pseudouridine enhances both thermostability and conformational fitness of the ribosomes, thereby influencing their susceptibility to aminoglycosides. Highlights Loss of pseudouridine increases cell sensitivity to aminoglycosides Pseudouridine enhances ribosome thermostability Hygromycin B competes with eEF2 for the non-rotated ribosome Hygromycin B deforms the codon-anticodon duplex