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 the Tombusviridae family (including genera Tombusvirus, Carmovirus, Necrovirus and others), RT occurs at a UAG stop codon followed by GGR, but enhanced synonymous site conservation was observed for approximately 200 codons 3 0 -adjacent to the UAG (Figure 4) . Some of this conservation, however, may be explained by other conserved elements in the region (see ref. 67 and references therein; see also refs 68, 69) . RNA folding software predicted alphavirus-like 3 0 -adjacent stem-loops in some species and more complex structures in other species, a detailed analysis of which is beyond the scope of this article. The RT site in gammaretroviruses has been studied in depth and our computational analysis supported the known stimulatory spacer sequence and pseudoknot structure but did not reveal further conservation in the vicinity (39) (40) (41) (42) . RT sites in enamoviruses, carrot red leaf luteovirus-associated RNA, Middelburg and Barmah Forest alphaviruses, Providence tetravirus and others, were not analyzed in detail due to lack of sequence data for useful comparative computational analysis (70) (71) (72) . In contrast, to our knowledge, no stimulatory RNA structure has been previously proposed for UGA RT in kelch. However, when we applied our computational analysis to kelch, we found tandem synonymous site conservation peaks 3 0 of the RT codon and the corresponding sequences were predicted to form an RNA stem (268 nt loop in D. melanogaster) that is conserved in all 12 Drosophila species (Figures 3 and 5; Supplementary Data) . The predicted stem has 14 bp with a 1 nt asymmetric bulge near the center of the 5 0 component, and is separated from the RT codon by an 8 nt 'spacer' sequence that is completely conserved in all 12 Drosophila species but, perhaps unusually, the RT codon context is UGA-AUG (UGA-AGC in Anopheles, Culex and Aedes mosquitoes).