RNA-guided RNA modification in archaea
Youssef, Osama Abdallah
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Post-transcriptional RNA modifications play important roles in all organisms. In eukaryotes and archaea, RNA modifications are brought about by the action of two large families of RNA/protein complexes known as the H/ACA and C/D RNPs. Specifically, H/ACA and C/D RNPs catalyze the conversion of uridine to pseudouridine and 2Õ-O-ribose methylation in cellular RNAs, respectively. The first part of this thesis describes the insights gained into the organization of archaeal H/ACA RNPs. Archaeal H/ACA RNPs consist of a guide RNA and four proteins: Cbf5, Gar1, Nop10, and L7Ae. The guide RNA is required to identify the target RNA nucleotide by base-pairing. We used biochemical assays to detail the RNA/protein and protein/protein interactions that control the assembly and function of these complexes. Our results showed that not only L7Ae but also Cbf5 binds directly to H/ACA guide RNAs. The data presented here revealed that Cbf5 has another important role in addition to its function as the pseudouridine synthase. Cbf5 is required for the recruitment of Gar1 and Nop10 to the guide RNA. We found that all four proteins are required for the efficient catalytic activity of the complex. Unlike the C/D RNPs, H/ACA RNPs do not require L7Ae for the recruitment of the other three proteins to the guide RNA. However, our RNA footprinting data showed that L7Ae plays a key role in the structural organization of H/ACA RNPs. The Sm and Sm-like proteins are a large family of evolutionarily ancient RNA binding proteins that form ring-like structures and mediate important cellular activities. Specific functions for any archaeal Sm-like proteins are unknown. In the second part of this thesis, we report a novel archaeal Sm-like protein (Sm4) from the hyperthermophilic archaeon, Pyrococcus furiosus and its associated RNAs. A 2.8 � X-ray structure indicates that Sm4 can form a stable octamer in the absence of the RNA. We found that Sm4 specifically and directly associates with members of C/D and H/ACA RNAs as well as two uncharacterized non-coding RNAs. We propose that Sm4 is important for the biogenesis or/and function of the modification guide RNAs and thus in the formation of active ribosomes.