Stimulation of stop codon readthrough: frequent presence of an extended 3′ RNA structural element

Firth, Andrew E.; Wills, Norma M.; Gesteland, Raymond F.; Atkins, John F.

In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3′-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼150 nt 3′-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem–loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem–loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3′ RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.