The small GTPase Ran is the central regulator of macromolecular transport between the cytoplasm and the nucleus. Recent work has suggested that RanGTP also plays an important role in regulating some intra-nuclear processes. In this study, we have investigated whether RanGTP is required for the intra-nuclear transport of RNAs. Specifically, we directly analyzed the nucleolar localization of Box C/D and Box H/ACA small nucleolar RNAs (snoRNAs)in mammalian (tsBN2) cells, Saccharomyces cerevisiae and Xenopus oocytes under conditions that deplete nuclear RanGTP and prevent RNA export to the cytoplasm. Our data suggest that depletion of nuclear RanGTP does not significantly alter the nucleolar localization of U3 snoRNA in tsBN2 cells. Complementary studies in the budding yeast S. cerevisiae using conditional Ran mutants as well as mutants in Ran regulatory proteins also indicate that disruption of the Ran gradient or of Ran itself does not detectably affect the nucleolar localization of snoRNAs. Finally, microinjection into Xenopus oocytes was used to clearly demonstrate that a specific pool of snoRNAs could still be efficiently targeted to the nucleolus even when the RanGTP gradient was disrupted by microinjection of mutant Ran proteins. Taken together, our data from three phylogenetically distinct experimental systems suggest that nuclear RanGTP,which is essential for trafficking of RNAs between the nuclear and cytoplasmic compartments, is not required for nuclear retention or nucleolar localization of snoRNAs.

Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.After synthesis in the nucleus, all RNA species undergo a series of maturation steps and transport from the site of synthesis to the site of action, and it is increasingly clear that RNA biogenesis and trafficking are closely linked (54). Whether they are destined to function in the cytoplasm (e.g., rRNA, tRNA, mRNA) or the nucleus (e.g., snRNAs or snoRNAs), all RNAs undergo intranuclear trafficking. It is well established that interactions between trans-acting factors and specific features of RNA substrates can govern which transport pathways will be utilized (38,59,73). However, little is known about the intranuclear trafficking factors and how they affect the molecular movement of RNAs within the nucleus.Small nucleolar RNAs (snoRNAs) provide excellent models to study intranuclear RNA transport. snoRNAs remain within the nucleus, where they undergo biogenesis including covalent alterations (e.g., 5Ј cap hypermethylation and 3Ј-end processing), as well as assembly with specific proteins into stable and functional RNP complexes (snoRNPs) (40,54,70,84). Ultimately, snoRNAs are targeted to nucleoli, where they function in rRNA maturation.The numerous snoRNA species fall into two structurally and functionally distinct classes called the box C/D and box H/ACA snoRNAs (40,54,70,84). Species of both RNA classes interact with pre-rRNA (via base pairing), mainly to guide site-specific nucleotide modification. The majority of box C/D snoRNAs orchestrate 2Ј-O-methylation (12,41,65,89), while the bulk of the box H/ACA RNA species guide pseudouridylation (21, 64). A few RNAs of both classes (e.g., box C/D snoRNA U3 and box H/ACA snoRNA U17 [vertebrates]/snR30 [yeast]) are critical for promoting specific endonucleolytic cleavages of pre-rRNA transcripts ...