As an RNA-guided RNA modification enzyme, the box H/ACA RNP recruits the guide RNA to recognize the substrate RNA, whereas the protein partners carry out the catalysis. Most intriguingly, box H/ACA RNPs share the same four conserved proteins, Cbf5, Nop10, L7Ae and Gar1, and are able to utilize more than 100 guide RNAs to guide posttranscriptional modifications in most stable RNAs. With respect to structural studies, archaeal box H/ACA ribonucleoproteins (RNPs) are probably one of the most extensively characterized ribonucleoprotein particles to this day. The dissertation presented here describes this work's contribution to the understanding of the nature of the RNA-guided RNA modification enzyme and of the most complex pseudouridine synthases. In chapter 2, we explored the structural basis of an archaeal box H/ACA protein complex comprised of three of the four essential proteins, Cbf5, Nop10 and Gar1. It was the first time we obtained molecular insights into these three proteins and the implications of a severe disease called dyskeratosis congenita (DC). We have also identified a DC mutation cluster site within a modeled dyskerin (Cbf5 homolog in humans) structure. In chapter 3, we further characterized the three-dimensional structure of a catalytically deficient archaeal box H/ACA RNP complex, including the guide RNA, the substrate RNA, Cbf5, Nop10 and Gar1. We devised a non-intrusive 2-aminopurine (2-AP) fluorescence assay which allowed us to determine the precise placement of the target uridine at the active site requires a conformational change of the guide-substrate RNA duplex by L7Ae. In chapter 4, we further examined the structural basis for accurate placement of substrate by accessory proteins using the 2-AP fluorescence assay. Our results revealed that each of the three accessory proteins, Nop10, L7Ae and Gar1, as well as an active site residue, have distinct effects on substrate conformations, suggesting the cooperative network of box H/ACA RNP. In chapter 5, we described a substrate-bound functional archaeal box H/ACA RNP that revealed detailed information about the active site. The substrate RNA containing 5-fluoruridine at the modification position is fully docked and rearranged in a manner similar to those of stand-alone pseudouridine synthases. The complementary biochemical studies further revealed the importance of a conserved protein loop and a guide-substrate RNA pocket in the binding to the substrate. With further comparison of available structures of stand-alone pseudouridine synthases-RNA complexes, we are able to summarize the common mechanism among all pseudouridine synthases, perhaps also a theme in other widespread RNA-guided enzymes. The accomplishments of this work greatly enhance our understanding of the enzymatic architecture of box H/ACA RNPs, unravel the many intriguing features of the most complex pseudouridine synthases, and shed light on the nature of the RNA-guided RNA modification and the assembly architecture of the telomerase holoenzyme.
