Identification and characterization of genes required for trafficking and biogenesis of small nucleolar RNAs in Saccharomyces cerevisiae
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Small nucleolar RNAs (snoRNAs) are a large family of guide RNAs that orchestrate the processing and modification of the pre-rRNA. Unlike most cellular RNAs, which are exported to the cytoplasm after synthesis in the nucleus, snoRNAs remain within the nucleus and are ultimately targeted to their functional site, the nucleolus. Previous studies on the localization of steady-state snoRNAs indicated that snoRNAs transiently associate with certain nuclear subdomains (Cajal body in vertebrate cells or the nucleolar body in budding yeast cells) prior to their final localization to the nucleolus, implicating compartmentalization of snoRNP biogenesis within these structures. Here, we established an inducible snoRNA expression system to analyze the temporal and spatial trafficking pathway of newly made snoRNAs in the budding yeast Saccharomyces cerevisiae. Our study showed that the newly made snoRNAs temporally associate with the nucleolar body prior to the nucleolus, confirming that the nucleolar body is indeed the intermediate site for snoRNA transport. The cis-acting elements that target snoRNAs to the nucleolus have been identified in previous studies. Although some trans-acting factors have been implicated in snoRNA trafficking, no systematic analysis has been done to identify the protein factors involved in the intranuclear trafficking of snoRNAs. Here, we present new potential trans-acting factors for transport and biogenesis of snoRNAs. We performed fluorescence in situ hybridization (FISH) analysis on a yeast temperature-sensitive mutant collection and a selected part of the yeast knockout mutant collection in order to identify mutants with mislocalization phenotypes of U3 snoRNA. From these two genetic screens, we identified fourteen new potential trans-acting factors that likely play roles at different stages of snoRNA transport including transporting snoRNAs from the nucleoplasm, retaining snoRNAs within the nucleolar body, and targeting snoRNAs to the nucleolus. Some of these potential transport factors are also required for the maturation of U3, indicating that the intranuclear trafficking and biogenesis of snoRNAs are closely interrelated.