UV irradiation responses in Giardia intestinalis.

The response to ultraviolet light (UV) radiation, a natural stressor to the intestinal protozoan parasite Giardia intestinalis, was studied to deepen the understanding of how the surrounding environment affects the parasite during transmission. UV radiation at 10 mJ/cm(2) kills Giardia cysts effectively whereas trophozoites and encysting parasites can recover from UV treatment at 100 mJ/cm(2) and 50 mJ/cm(2) respectively. Staining for phosphorylated histone H2A showed that UV treatment induces double-stranded DNA breaks and flow cytometry analyses revealed that UV treatment of trophozoites induces DNA replication arrest. Active DNA replication coupled to DNA repair could be an explanation to why UV light does not kill trophozoites and encysting cells as efficiently as the non-replicating cysts. We also examined UV-induced gene expression responses in both trophozoites and cysts using RNA sequencing (RNA seq). UV radiation induces small overall changes in gene expression in Giardia but cysts show a stronger response than trophozoites. Heat shock proteins, kinesins and Nek kinases are up-regulated, whereas alpha-giardins and histones are down-regulated in UV treated trophozoites. Expression of variable surface proteins (VSPs) is changed in both trophozoites and cysts. Our data show that Giardia cysts have limited ability to repair UV-induced damage and this may have implications for drinking- and waste-water treatment when setting criteria for the use of UV disinfection to ensure safe water.

RNA editing in Trypanosoma brucei: characterization of gRNA U-tail interactions with partially edited mRNA substrates.

Guide RNAs (gRNAs), key components of the RNA editing reaction in Trypanosoma brucei, direct the insertion and deletion of uridylate (U) residues. Analyses of gRNAs reveal three functional elements. The 5'-end of the gRNA contains the anchor, which is responsible for selection and binding to the pre-edited mRNA. The second element (the guiding region) provides the information required for editing. At the 3'-end of the gRNA is a non-encoded U-tail, whose function remains unclear. However, the cleavage-ligation model for editing proposes that the U-tail binds to purine-rich regions upstream of editing sites, thereby strengthening the interaction and holding onto the 5' cleavage product. Our previous studies demonstrated that the U-tail interacts with upstream sequences and may play roles in both stabilization and tethering. These studies also indicated that the U-tail interactions involved mRNA regions that were to be subsequently edited. This raised the question of what happens to the mRNA-U-tail interaction as editing proceeds in the 3'-->5' direction. We examined gCYb-558 and its U-tail interaction with 5'CYbUT and two partially edited 5'CYb substrates. Our results indicate that the 3'-end of the U-tail interacts with the same sequence in all three mRNAs. Predicted secondary structures using crosslinking data suggest that a similar structure is maintained as editing proceeds. These results indicate that the role of the U-tail may also involve maintenance of important secondary structure motifs.