Unveil the Molecular Interplay between Aminoglycosides and Pseudouridine in IRES Translation.

UNLABELLED: 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 aminoglycosidesPseudouridine enhances ribosome thermostabilityHygromycin B competes with eEF2 for the non-rotated ribosomeHygromycin B deforms the codon-anticodon duplex.