Structural defects of hypopseudouridylated ribosome observed during IRES-mediated translation initiation.

Abstract

Pseudouridine is an abundant chemical modification found on major functional RNA molecules. Loss of pseudouridine is linked to diseases such as the X-linked dyskeratosis congenita (X-DC). While there is rich biophysical data on pseudouridine in model RNA molecules that show its stabilization role, little is known on its roles in intact functional RNA such as those within ribosome. To fill this gap, we studied structures of yeast ribosome isolated from cells that lack ribosome pseudouridine synthesis capability. We performed cryoEM structural analysis of hypopseudouridylated ribosome during initiation by the Taura syndrome virus (TSV) IRES, as the IRES-mediated translation has been found to be severely impaired in X-DC patient cells or by ribosome isolated from them. We found that though the overall fold of hypopseudouridylated ribosome remains similar to native ribosome, high resolution features reveal significant loss of pseudouridine-mediated interactions through water and long-range base pairings. Strikingly, when bound with TSV IRES and in presence of the elongation factor, eEF2, and GTP hydrolysis, the hypopseudouridylated ribosome favors an unconducive conformation for decoding linked to the loss of heavily pseudouridylated helix H69 and a hypermodified P site uridine, consistent with its reduced rate and fidelity of translation. Our data suggest that loss of pseudouridine in key ribosome sites alters the evolutionarily tuned ribosome motions required for IRES-mediated translation.