SUMMARY Chemical damage to RNA affects its functional properties and hence may pose a significant hurdle to the translational apparatus; however, the effects of damaged mRNA on the speed and accuracy of the decoding process and their interplay with quality control processes are not known. Here, we systematically explore the effects of oxidative damage on the decoding process using a well-defined bacterial in vitro translation system. We find that the oxidative lesion 8-oxoguanosine reduces the rate of peptide-bond formation by more than three orders of magnitude independent of its position within the codon. Interestingly, 8-oxoguanosine had little effect on the fidelity of the selection process suggesting that the modification stalls the translational machinery. Consistent with these findings, 8-oxoguanosine-mRNAs were observed to accumulate and associate with polyribosomes in yeast strains in which no-go decay is compromised. Our data provide compelling evidence that mRNA-surveillance mechanisms have evolved to cope with damaged mRNA.

Nucleic acids are under constant assault from endogenous and environmental agents that alter their physical and chemical properties. O6-methylation of guanosine (m 6 G) is particularly notable for its high mutagenicity, pairing with T, during DNA replication. Yet, while m 6 G accumulates in both DNA and RNA, little is known about its effects on RNA. Here, we investigate the effects of m 6 G on the decoding process, using a reconstituted bacterial translation system. m 6 G at the first and third position of the codon decreases the accuracy of tRNA selection. The ribosome readily incorporates near-cognate aminoacyltRNAs (aa-tRNAs) by forming m 6 G-uridine codon-anticodon pairs. Surprisingly, the introduction of m 6 G to the second position of the codon does not promote miscoding, but instead slows the observed rates of peptide-bond formation by >1000-fold for cognate aa-tRNAs without altering the rates for near-cognate aa-tRNAs. These in vitro observations were recapitulated in eukaryotic extracts and HEK293 cells. Interestingly, the analogous modification N6-methyladenosine (m 6 A) at the second position has only a minimal effect on tRNA selection, suggesting that the effects on tRNA selection seen with m 6 G are due to altered geometry of the base pair. Given that the m6G:U base pair is predicted to be nearly indistinguishable from a WatsonCrick base pair, our data suggest that the decoding center of the ribosome is extremely sensitive to changes at the second position. Our data, apart from highlighting the deleterious effects that these adducts pose to cellular fitness, shed new insight into decoding and the process by which the ribosome recognizes codon-anticodon pairs.

Nucleotide hydrolysis is essential for many aspects of cellular function. In the case of 3′,5′-bisphosphorylated nucleotides, mammals possess two related 3′-nucleotidases, Golgi-resident 3′-phosphoadenosine 5′-phosphate (PAP) phosphatase (gPAPP) and Bisphosphate 3′-nucleotidase 1 (Bpnt1). gPAPP and Bpnt1 localize to distinct subcellular compartments and are members of a conserved family of metal-dependent lithium-sensitive enzymes. Although recent studies have demonstrated the importance of gPAPP for proper skeletal development in mice and humans, the role of Bpnt1 in mammals remains largely unknown. Here we report that mice deficient for Bpnt1 do not exhibit skeletal defects but instead develop severe liver pathologies, including hypoproteinemia, hepatocellular damage, and in severe cases, frank wholebody edema and death. Accompanying these phenotypes, we observed tissue-specific elevations of the substrate PAP, up to 50-fold in liver, repressed translation, and aberrant nucleolar architecture. Remarkably, the phenotypes of the Bpnt1 knockout are rescued by generating a double mutant mouse deficient for both PAP synthesis and hydrolysis, consistent with a mechanism in which PAP accumulation is toxic to tissue function independent of sulfation. Overall, our study defines a role for Bpnt1 in mammalian physiology and provides mechanistic insights into the importance of sulfur assimilation and cytoplasmic PAP hydrolysis to normal liver function.ribosome biogenesis | nucleolus | phosphoadenosine phosphosulfate | exoribonuclease