Conserved Pseudouridines in Helix 69 of the Ribosome are Important for Ribosome Dynamics in Translation

Abstract

The widespread distribution of pseudouridine ({Psi}), an isomer of the canonical uridine base, in RNA indicates its functional importance to the cell. In eukaryotes, it is estimated that around 2% of ribosomal RNA nucleotides are pseudouridines, most of which are located in functional regions of the ribosome. Defects in RNA pseudouridylation induce a range of detrimental effects from compromised cellular protein biosynthesis to disease phenotypes in humans. However, genome-wide changes to mRNA translation profiles by ribosomes lacking specific conserved pseudouridines have not been extensively studied. Here, using a new genomic method called 5P-Seq and in vitro biochemistry, we investigated changes in ribosome dynamics and cellular translation profiles upon loss of {Psi}2258 and {Psi}2260 in helix 69, the two most conserved pseudouridines in the ribosomes. We found that inhibiting the formation of these two pseudouridines challenges ribosomes to maintain the correct open reading frame and causes generally faster ribosome dynamics in translation. Furthermore, mutant ribosomes are more prone to pause while translating a subset of GC-rich codons, especially rare codons such as Arg (CGA) and Arg (CGG). Our data suggest the presence of {Psi}2258 and {Psi}2260 contributes to the dynamics of the H69 RNA stem-loop, and helps to maintain functional interactions with the tRNAs as they move within the ribosome. The optimality of this ribosome-tRNA interaction is likely to be more critical for those limited tRNAs that decode rare codons. Consistent with the changes in ribosome dynamics, our data also show that IRES-mediated translation is compromised in the mutant ribosome. These results explain the importance of {Psi}2258 and {Psi}2260 in H69 to maintain cellular fitness. The strong conservation of {Psi}2258 and {Psi}2260 in the ribosomes from bacteria to humans indicates their functional significance in modulating ribosome functions. We anticipate that the identified functions of these covalent modifications will be conserved in other species.