Two new requirements for producing normal ribosomes: (I) A novel helicase is needed for snoRNP biogenesis, and; (II) Pseudoridine modifications in the reaction center are important for ribosome function
Eukaryotic cells contain scores of small nucleolar RNAs (snoRNAs) that are complexed with proteins in particles known as snoRNPs. SnoRNPs function in processing of ribosomal RNA (rRNA) and nucleotide modification of ribosomal and other RNAs that pass through the nucleolus. The modifications involved are: (1) isomerization of uridine to pseudouridine (Ψ), and; (2) methylation of the 2′-hydroxyl of ribose moieties (Nm). The Ψ and Nm modified nucleotides are the most abundant in rRNA.
The purpose of the thesis work is to obtain a better understanding of the synthesis of snoRNPs and the function(s) of the modifications they form. To this end, three lines of research were carried out. Two focus on different snoRNA-associated proteins. The third examines the effect of snoRNP-mediated pseudouridylation of rRNA on ribosome structure and function.
One of the snoRNA-associated proteins studied is a conserved helicase previously isolated from a mouse nuclear extract on the basis of association with a conserved snoRNA structure known as the box C/D motif. Here, it was shown that the yeast ortholog of this protein (Rvb2p) is required for production of snoRNAs (both major families) and for localization of snoRNP proteins. The findings are consistent with a role for Rvb2p in the assembly or trafficking of snoRNPs.
A second project was aimed at determining if a particular H/ACA snoRNP protein is the enzyme that forms Ψ in rRNA. The protein, Cbf5p, was previously shown to have limited sequence homology with known Ψ synthases (96). Here, in collaboration with the John Carbon laboratory, it was determined that point mutations in Cbf5p in this region of homology can abolish in vivo pseudouridylation of rRNA, arguing that Cbf5p is indeed the global Ψ synthase.
Finally, the function of Ψ in the peptidyl transferase center (PTC) was investigated by depleting cells of snoRNPs that modify this domain, and evaluating the under-modified ribosomes. Interestingly, cells lacking Ψs in the PTC are impaired in growth and protein synthesis. Chemical probing revealed changes in the structure of the mutant ribosomes. Enhanced reactivity with dimethylsulfate was observed for nucleotides predicted to influence binding of elongation and initiation factors. The results demonstrate that the structure and activity of yeast ribosomes depend on pseudouridylation of the PTC. The possibility that guide snoRNPs have function(s) other than modification is discussed.