Nucleolar Localization Elements ofXenopus laevisU3 Small Nucleolar RNA

Abstract

The Nucleolar Localization Elements (NoLEs) of Xenopus laevis U3 small nucleolar RNA (snoRNA) have been defined. Fluorescein-labeled wild-type U3 snoRNA injected into Xenopus oocyte nuclei localized specifically to nucleoli as shown by fluorescence microscopy. Injection of mutated U3 snoRNA revealed that the 5Ј region containing Boxes A and AЈ, known to be important for rRNA processing, is not essential for nucleolar localization. Nucleolar localization of U3 snoRNA was independent of the presence and nature of the 5Ј cap and the terminal stem. In contrast, Boxes C and D, common to the Box C/D snoRNA family, are critical elements for U3 localization. Mutation of the hinge region, Box B, or Box CЈ led to reduced U3 nucleolar localization. Results of competition experiments suggested that Boxes C and D act in a cooperative manner. It is proposed that Box B facilitates U3 snoRNA nucleolar localization by the primary NoLEs (Boxes C and D), with the hinge region of U3 subsequently base pairing to the external transcribed spacer of pre-rRNA, thus positioning U3 snoRNA for its roles in rRNA processing. INTRODUCTIONMany aspects of how macromolecules are targeted to their correct subcellular destination still need to be defined. Although principles governing RNA export to the cytoplasm and import into the nucleus are beginning to be understood, very little is known about signals that direct RNA within the nucleus to the nucleolus. The nucleolus contains a vast array of different RNA species involved in ribosome biogenesis: ribosomal RNA (rRNA) and its precursors and ϳ200 small nucleolar RNAs (snoRNAs). Unlike rRNA whose genes are located within the nucleolus, the other transcripts found in the nucleolus must travel there from their sites of synthesis in the nucleoplasm. What are the "zip codes" for targeting snoRNA to the nucleolus?To address this question, we have studied U3 snoRNA because it is the most abundant snoRNA in the nucleolus, has been sequenced from a large number of animals and plants (Gu and Reddy, 1997), and is well characterized in terms of secondary structure and function in rRNA processing. U3 snoRNA is synthesized in the nucleoplasm in the proximity of coiled bodies (Gao et al., 1997), and formation of its trimethylguanosine cap can occur in the nucleoplasm (Terns and Dahlberg, 1994;Terns et al., 1995), unlike spliceosomal snoRNAs that are exported to the cytoplasm for cap trimethylation (Mattaj, 1986). Once U3 snoRNA is transported to the nucleolus, it is found highly concentrated in the dense fibrillar component (Matera et al., 1994) where initial rRNA processing events are believed to occur, but up to half of the U3 snoRNA is also detected by electron microscopy to be diffuse in the granular component Puvion-Dutilleul et al., 1991Azum-Gélade et al., 1994) where later rRNA processing cleavages take place. On the basis of the localization of U3 snoRNA after various actinomycin D treatments (Puvion-Dutilleul et al., 1992;Rivera-Leó n and Gerbi, 1997), a model has been proposed in which U3 snoRNA trave...

Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

“…The box CϩD sequences, together with a stem structure that normally brings them together in the snoRNA secondary structure are the only elements essential for the synthesis and stability of this class of snoRNA, and their presence is the only feature that is clearly conserved among the box CϩD snoRNAs (Baserga et al+, 1991;Huang et al+, 1992;Caffarelli et al+, 1996;Watkins et al+, 1996;Xia et al+, 1997)+ A simple model for the involvement of Nop58p in snoRNA stability is through protection from the exonucleolytic activities that normally generate the mature ends+ If so, the alteration in the 39 end of U24 on depletion of Nop58p suggests that this interaction might be via binding to the conserved box D at the 39 end of the snoRNAs+ Curiously, inspection of the 39 end of U24 (CUGAUGAAU OH ) (Qu et al+, 1995) reveals the presence of a consensus box C motif (UGAUGA), partially overlapping the box D element (CUGA)+ We speculate that on depletion of Nop58p, the putative box C binding protein(s), which normally binds to the 59 end of the snoRNA, also binds to the cryptic 39 box C element+ This might confer extra protection to U24 and explain why this species is more resistant to Nop58p depletion+ During depletion of Nop58p, Nop1p was reported to be delocalized to the nucleoplasm and cytoplasm (Wu et al+, 1998)+ In the conditions used (up to 12 h in glucose medium) the snoRNAs were presumably strongly depleted+ This indicates that Nop1p does not localize to the nucleolus on its own, but rather is localized to and/or anchored to the nucleolus in association with snoRNPs+ The association of the box CϩD snoRNAs with the nucleolus does not require complementarity to the pre-rRNA (Lange et al+, 1998b)+ Instead, both snoRNA stability and nucleolar targeting require the conserved box CϩD elements and the terminal stem (Lange et al+, 1998a(Lange et al+, , 1998cSamarsky et al+, 1998)+ It seems probable that Nop58p stabilizes the snoRNAs via binding to one or both of these sequence elements and is, therefore, a good candidate to provide the nucleolar targeting signals+ Testing of this hypothesis will require the separation of the role of Nop58p in snoRNA stability from its putative targeting function+…”

Interaction of the U3-55k protein with U3 snoRNA is mediated by the Box B/C motif of U3 and the WD repeats of U3-55k

“…It is now clear that U3-55k is also localized in the nucleoli of multiple cell types (Figs 2B and C and 6D; 45). The sequence elements involved in the localization of U3 RNA to nucleoli have been investigated (31,69,70). We have found that while the Box B/C motif of U3 is involved in the retention of the RNA in the nucleus, it is neither required nor sufficient for nucleolar localization (30,31).…”

U87 RNA, a novel C/D box small nucleolar RNA from mammalian cells

“…Several laboratories have demonstrated that snoRNAs with substitution mutations disrupting the terminal stem cannot reach nucleolus and are accumulated in so-called coiled bodies (Narayanan et al, 1999); according to alternative data such RNAs are able to reach the nucleolus (Lange et al, 1998). Naturally occurring snoRNAs lacking the terminal stem in mature state are probably transferred into nucleolus as a longer IR-containing precursors.…”