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|Title: ||Evolutionary Conservation of Eukaryotic and Archaeal Box C/D Ribonucleoprotein Complex Structure|
|Authors: ||Kuhn, Jeffrey Francis|
|Advisors: ||E. Stuart Maxwell, Committee Chair|
Dennis Brown, Committee Member
Paul Wollenzien, Committee Member
James Brown, Committee Member
|Issue Date: ||9-Oct-2002|
|Abstract: ||Ribosome biogenesis requires a large population of small nucleolar RNAs (snoRNAs) for pre-rRNA processing and nucleotide modification. These snoRNAs can be classified into two major families based on sequence and structural elements: the box C/D snoRNAs and the box H/ACA snoRNAs. The box C/D snoRNAs possess conserved nucleotide boxes C and D contained within a folded structural element defined as the box C/D core motif that is essential for snoRNA biogenesis, nucleolar transport, and nucleotide modification. This dissertation describes the purification, identification and characterization of box C/D snoRNA-associated proteins. These proteins are conserved throughout evolution and homologs exist in Eukaryota and Archaea. Homologs of the box C/D snoRNAs exist in Archaea as well, termed sRNAs. This work also examines the structure of the box C/D core motif RNA and investigates the organization of the box C/D RNP particle.
Eukaryotic snoRNA-associated proteins were initially identified by affinity chromatography using the box C/D core motif RNA as the selection agent. The four proteins isolated consist of two protein pairs, with members of each pair being highly related in sequence. One pair of proteins, Nop56p and Nop58p/Nop5p, are essential nucleolar proteins associated with box C/D snoRNAs, consistent with their designation as "core" snoRNP proteins. The second pair of proteins, termed p50 and p55, are essential nucleoplasmic proteins and have been designated "accessory" proteins. Immunoprecipitation experiments suggest that the eukaryotic proteins p50 and p55 are transiently associated with the box C/D snoRNP complex in the nucleoplasm, consistent with a role in snoRNA biogenesis and/or snoRNA transport events. These results have led to the designation of p50 and p55 as accessory proteins. Sequence analysis reveals that these snoRNA-associated proteins are conserved throughout evolution and homologs exist in archaeal genomes, where a single homolog for each of the Nop56p/Nop58p and p50/p55 protein pairs exists.
Thermal denaturation analysis of the box C/D core motif RNA demonstrates that it possesses a well-defined structure in the absence of snoRNA binding proteins. Together with a mutational analysis, the thermal denaturation experiments reveal that the box C/D core motif has an ordered structure which is stable in solution and requires a set of critical G-A residues. These results are consistent with the previous suggestion that the box C/D core motif forms a "kink-turn" (K-turn) motif, a novel RNA fold defined by a highly kinked phosphodiester backbone and two base paired stems flanking an asymmetric bulge region which contains tandem sheared G-A base pairs critical to RNA folding.
Sequence analysis reveals that the archaeal homolog of the eukaryotic core protein termed 15.5kD is the archaeal ribosomal protein L7. The 15.5kD protein serves a dual function in eukaryotes by binding both the spliceosomal U4 snRNA and the box C/D snoRNAs. Interestingly, archaeal L7 also serves a dual function by binding the 23S rRNA as well as the box C/D sRNAs. Our binding analyses reveal that L7 binds the box C/D core motif with high affinity. In addition, we have cloned the remaining archaeal homologs of the box C/D-associated proteins and binding analyses demonstrate that these proteins, together with the sRNAs, are able to form sRNP complexes which closely resemble eukaryotic snoRNPs. Nuclease mapping experiments have begun to establish the overall organization of the archaeal box C/D sRNP particle. The striking similarity of the box C/D sRNP/snoRNP complex to the L7:KT15 RNP of the 50S ribosomal subunit suggests that the archaeal sRNPs and eukaryotic snoRNPs could have their evolutionary origins in primordial ribosomes of the ancient RNA world.|
|Appears in Collections:||Dissertations|
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