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...

We studied the pathway of 5S ribosomal RNA (rRNA) during oogenesis in Xenopus from its storage in the cytoplasm to incorporation into ribosomes in the nucleus. Ribonucleoprotein particle (RNP) assembly assays reveal striking differences in the behavior of oocyte-type and somatic-type 5S rRNA after microinjection into stage II, III, or IV oocytes or into the cytoplasm of stage V-VI oocytes. Microinjected oocyte-type 5S rRNA predominantly interacts with the 5S rRNA gene-specific transcription factor IIIA (TFIIIA) to form storage 7S RNPs. In contrast, microinjected somatic-type 5S rRNA predominantly interacts with ribosomal protein L5 to form 5S RNPs, which are precursors to ribosome assembly. In addition, a greater amount of somatic-type 5S rRNA accumulates in the nucleus and is assembled into 60S ribosomal subunits. Thus, a slight difference in nucleotide sequence results in differential binding of 5S rRNA to TFIIIA and L5, specializing oocyte-type for storage in the oocyte cytoplasm and somatic-type for rapid mobilization and ribosome assembly. When oocyte-type and somatic-type 5S rRNA molecules were microinjected into the nucleus of stage V-VI oocytes in excess of other ribosomal components, the nucleocytoplasmic distribution of both types of RNA was similar, but the distinctive protein associations were maintained. In contrast, the behavior of oocyte-type and somatic-type 5S rRNA gradually synthesized in situ from microinjected cloned genes was similar, suggesting that nascent RNA is rapidly and directly recruited into ribosomes, thus bypassing an excursion into the cytoplasm prior to ribosome assembly.

Structural requirements of 5S rRNA for nuclear transport and RNA-protein interactions have been studied by analyzing the behavior of oocyte-type 5S rRNA and of 31 different in vitro-generated mutant transcripts after microinjection into the cytoplasm of Xenopus oocytes. Experiments reveal that the sequence and secondary and/or tertiary structure requirements of 5S rRNA for nuclear transport, storage in the cytoplasm as 7S ribonucleoprotein particles, and assembly into 60S ribosomal subunits are complex and nonidentical. Elements of loops A, C, and E, helices II and V, and bulged and hinge nucleotides in the central domain of 5S rRNA carry the essential information for these functional activities. Assembly of microinjected 5S rRNA into 60S ribosomal subunits was shown to occur in the nucleus; thus, the first requirement for subunit assembly is nuclear targeting. The inhibitory effects of ATP depletion, wheat germ agglutinin, and chilling on the nuclear import of 5S rRNA indicate that it crosses the nuclear envelope through the nuclear pore complex by a pathway similar to that used by karyophilic proteins.The orchestration of ribosome biogenesis in eukaryotic cells is a process that requires transfer of macromolecules into and out of the nucleus. In Xenopus oocytes, 5S rRNA is shuttled between the nuclear and cytoplasmic compartments of the oocyte during different stages of oogenesis in a complex pathway involving different protein associations. In previtellogenic oocytes, 5S rRNA is synthesized before other components of ribosomes are available, is exported from the nucleus, and stored in the cytoplasm as 7S ribonucleoprotein particles (RNPs) (5S rRNA complexed with transcription factor IIIA [TFIIIA]) or as 42S RNPs (5S rRNA complexed with other nonribosomal proteins and tRNA). During vitellogenesis, the 5S rRNA is released from storage and a 5S rRNA-ribosomal protein L5 complex, which is a precursor to assembly into the 60S large ribosomal subunit, forms (reference 3 and references therein). We are interested in the mechanisms that govern the subcellular trafficking of 5S rRNA within the oocyte, particularly the requirements for the mobilization of stored 5S rRNA during ribosome assembly.There nuclear RNAs (50), mRNA (16), 40S and 60S ribosomal subunits (6, 38), and 5S rRNA (24) occurs in a manner consistent with a mediated process. Analysis of the nuclear transport of U small nuclear RNAs has multiple defined, kinetically distinct targeting pathways (27,47,48). Nuclear transport of different classes of RNA may thus involve targeting to the pore complex by different cytoplasmic receptors and then translocation into the nucleus by the same pore complex-mediated mechanism. Specific RNA structures have been implicated as requirements for both nuclear import and export (5,22,32,33,67,75). Translocation of RNA molecules across the nuclear envelope may also require interaction with specific proteins (28,31,33,43,62). RNA-protein interactions are important for many regulatory processes. There is growing evidence that R...

In Xenopus laevis oocytes, 5S RNA is stored in the cytoplasm until vitellogenesis, at which time it is imported into the nucleus and targeted to nucleoli for ribosome assembly. This article shows that throughout oogenesis there is a pool of nuclear 5S RNA which is not nucleolar-associated. This distribution reflects that of oocyte-type 5S RNA, which is the major 5S RNA species in oocytes; only small amounts of somatic-type, which differs by six nucleotides, are synthesized. Indeed, 32P-labeled oocyte-type 5S RNA showed a degree of nucleolar localization similar to endogenous 5S RNA (33%) after microinjection. In contrast, 32P-labeled somatic-type 5S RNA showed significantly enhanced localization, whereby 70% of nuclear RNA was associated with nucleoli. A chimeric RNA molecule containing only one somatic-specific nucleotide substitution also showed enhanced localization, in addition to other somatic-specific phenotypes, including enhanced nuclear import and ribosome incorporation. The distribution of 35S-labeled ribosomal protein L5 was similar to that of oocyte-type 5S RNA, even when preassembled with somatic-type 5S RNA. The distribution of a series of 5S RNA mutants was also analyzed. These mutants showed various degrees of localization, suggesting that the efficiency of nucleolar targeting can be influenced by many discrete regions of the 5S RNA molecule.