8-oxoguanine riboswitches in bacteria detect and respond to oxidative DNA damage.

Riboswitches rely on structured aptamer domains to selectively sense their target ligands and regulate gene expression. However, some riboswitch aptamers in bacteria carry mutations in their otherwise strictly conserved binding pockets that change ligand specificities. The aptamer domain of a riboswitch class originally found to selectively sense guanine forms a three-stem junction that has since been observed to exploit numerous alterations in its ligand-binding pocket. These rare variants have modified their ligand specificities to sense other purines or purine derivatives, including adenine, 2'-deoxyguanosine (three classes), and xanthine. Herein, we report the characteristics of a rare variant that is narrowly distributed in the Paenibacillaceae family of bacteria. Known representatives are always associated with genes encoding 8-oxoguanine deaminase. As predicted from this gene association, these variant riboswitches tightly bind 8-oxoguanine (8-oxoG), strongly discriminate against other purine derivatives, and function as genetic "ON" switches. Following exposure of cells to certain oxidative stresses, a representative 8-oxoG riboswitch activates gene expression, likely caused by the accumulation of 8-oxoG due to oxidative damage to G nucleobases in DNA, RNA, and the nucleotide pool. Furthermore, an engineered version of the variant aptamer was prepared that exhibits specificity for 8-oxoadenine, further demonstrating that RNA aptamers can acquire mutations that expand their ability to detect and respond to oxidative damage.

Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2'-deoxyguanosine.

The aptamer portions of previously reported riboswitch classes that sense guanine, adenine, or 2'-deoxyguanosine are formed by a highly similar three-stem junction with distinct nucleotide sequences in the regions joining the stems. The nucleotides in these joining regions form the major features of the selective ligand-binding pocket for each aptamer. Previously, we reported the existence of additional, rare variants of the predominant guanine-sensing riboswitch class that carry nucleotide differences in the ligand-binding pocket, suggesting that these RNAs have further diversified their structures and functions. Herein, we report the discovery and analysis of three naturally occurring variants of guanine riboswitches that are narrowly distributed across Firmicutes. These RNAs were identified using comparative sequence analysis methods, which also revealed that some of the gene associations for these variants are atypical for guanine riboswitches or their previously known natural variants. Binding assays demonstrate that the newfound variant riboswitch representatives recognize xanthine, guanine, or 2'-deoxyguanosine, with the guanine class exhibiting greater discrimination against related purines than the more common guanine riboswitch class reported previously. These three additional variant classes, together with the four previously discovered riboswitch classes that employ the same three-stem junction architecture, reveal how a simple structural framework can be diversified to expand the range of purine-based ligands sensed by RNA.

Translation initiation factors GleIF4E2 and GleIF4A can interact directly with the components of the pre-initiation complex to facilitate translation initiation in Giardia lamblia.

Translation initiation factor eIF4F is essential for cap-dependent translation initiation in eukaryotes. eIF4F is a trimeric complex consisting of a scaffold protein eIF4G, cap-binding protein eIF4E and DEAD-box RNA helicase eIF4A. eIF4F binds to the 5' cap structure of the mRNA through eIF4E and facilitates the binding of the preinitiation complex (PIC) via protein-protein interactions of eIF4G with eIF3 in mammals or with eIF5 in yeast. Initiation factor eIF4A is known to unwind the secondary structures of the 5'UTRs encountered by the PIC during its initial binding to the mRNA and while scanning for the initiation codon. In Giardia, homologs for eIF4E (GleIF4E2) and eIF4A (GleIF4A) have been identified but not for eIF4G. To address how PIC is recruited to the 5' end of mRNA in the absence of eIF4G homolog, we have used yeast two-hybrid assays to identify potential interactions of GleIF4E2 with the components of the PIC. The results show that GleIF4E2 can interact with the ß subunit of the initiation factor GleIF2, a component of the PIC. ZDOCK modeling of the GleIF4E2-GleIF2ß complex revealed that the dorsal side of GleIF4E2 is likely involved in binding to GleIF2ß, which mimics the interaction of mammalian eIF4E with eIF4G, and with eIF4E binding proteins. These results suggest that GleIF4E2 can facilitate the recruitment of the PIC to the 5'end of the mRNA by binding directly to the components of the PIC. The role of GleIF4A in translation initiation in Giardia is not clearly understood as the short 5' UTRs of the mRNA are unlikely to form secondary structures. Interestingly, Pateamine A, a specific inhibitor of human eIF4A, inhibited the growth of Giardia in a dose-dependent manner, suggesting that the activity of GleIF4A is probably required for translation. Using yeast two-hybrid assays, we have identified a novel interaction of GleIF4A with i subunit of the initiation factor GleIF3 (GleIF3i), another component of the PIC. These results indicate that the GleIF4A can also interact directly with the components of the PIC. ZDOCK modeling of the GleIF3i-GleIF4A complex suggests that GleIF3i could serve as a stimulator of GleIF4A activity.