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All three translation termination codons, or nonsense codons, contain a uridine residue at the first position of the codon1-3. Here, we demonstrate that pseudouridylation (conversion of uridine into pseudouridine (Ψ), ref. 4) of nonsense codons suppresses translation termination both in vitro and in vivo. In vivo targeting of nonsense codons is accomplished by the expression of an H/ACA RNA capable of directing the isomerization of uridine to Ψ within the nonsense codon. Thus, targeted pseudouridylation represents a novel approach for promoting nonsense suppression in vivo. Remarkably, we also show that pseudouridylated nonsense codons code for amino acids with similar properties. Specifically, ΨAA and ΨAG code for serine and threonine, whereas ΨGA codes for tyrosine and phenylalanine, thus suggesting a new mode of decoding. Our results also suggest that RNA modification, as a naturally occurring mechanism, may offer a new way to expand the genetic code.
Ψ, the C5-glycoside isomer of uridine, has many structural and biochemical differences from uridine5 (Supplementary Fig. 1). Thus, it is possible that replacement of the uridine within a nonsense codon with Ψ may affect translation termination. To investigate the possible effect of Ψ on translation termination, we developed an in vitro nonsense suppression assay (Fig. 1a). Briefly, we synthesized an artificial messenger RNA that encoded a 63 histidine (6His) tag at the amino terminus and a Flag tag at the carboxy terminus. In between the 6His tag and Flag tag, a pseudouridylated nonsense codon (ΨAA) was inserted (Fig. 1a). In addition, two control transcripts were created with the same sequence except that the Ψ of the nonsense codon was either changed to uridine (U), thus forming an authentic nonsense codon (UAA), or substituted with a cytidine (C), thus eliminating the nonsense codon (CAA) (Fig. 1a). Anti-6Hisimmunoblot analysis indicated that all three RNAs were equally translated in rabbit reticulocyte lysate (Fig. 1b, top panel). Remarkably, however, according to the anti- Flag blot, the presence of a Ψ within the termination codon resulted in robust nonsense suppression (Fig. 1b, lower panel). Specifically, the ΨAA-containing transcript produced a strong Flag signal which is almost comparable to that produced by the CAA-containing transcript (Fig. 1b). In contrast, only a background level of Flag was produced when the UAA-containing transcript was used (Fig. 1b). Our...