Dissecting structure and function of box H/ACA small nucleolar ribonucleoprotein particles
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The isomerization of up to 100 uridines to pseudouridines in eukaryotic ribosomal RNA (rRNA) is guided by a similar number of box H/ACA small nucleolar RNAs (snoRNAs), each forming a unique small nucleolar ribonucleoprotein particle (snoRNP) with the same four core proteins, NAP57/dyskerin, GAR1, NHP2, and NOP10. These four proteins also associate with the 3' -H/ACA domain of the vertebrate telomerase RNA. Additionally, the nucleolar and Cajal body protein Nopp140 associates with the snoRNPs. Although the composition and function of H/ACA snoRNPs have been established, little is known about their structure and mechanism of action.;To understand the mechanism of snoRNP-mediated pseudouridylation, we established an in vitro assay system. Short site-specifically 32P-labeled rRNA substrates were incubated with subcellular fractions, and the conversion of uridine to pseudouridine was monitored by thin-layer chromatography after digestion to single nucleotides. We demonstrated that immunopurified H/ACA snoRNP core particles consisting of the four core proteins and an H/ACA snoRNA were sufficient for the reaction. SnoRNPs associated quantitatively and reversibly with Nopp140 in a phosphorylation dependent manner. However, pseudouridylation activity was independent of Nopp140, consistent with a chaperoning role for this highly phosphorylated protein. Although snoRNA-rRNA hybridization of up to 14bps was required for the in vitro reaction, rRNA pseudouridylation and release occurred in the absence of ATP and magnesium, suggesting that substrate release took place without RNA helicase activity.;We also studied the molecular interactions of the components of H/ACA snoRNPs. Using in vitro transcription/translation in combination with immunoprecipitation of core proteins, UV-crosslinking, and electrophoretic mobility shift analysis, we demonstrated the following. NOP10 interacted with NAP57 as a prerequisite for NHP2 binding. This core trimer provided the basis for specific binding of H/ACA RNAs. GAR1 associated independently near the active site of NAP57 to form a protein-only H/ACA particle with the core trimer. Point mutation of the catalytic aspartate in the pseudouridine synthase NAP57 had no effect on protein-only particle formation but significantly reduced sno- and telomerase RNA binding. Finally, functional H/ACA snoRNPs assembled in cytosolic extracts were stable and did not exchange their RNA component suggesting that new particle formation requires de novo synthesis.