Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C', box D, and the 3' terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs.

The processing and methylation of precursor rRNA is mediated by the box C/D small nucleolar RNAs (snoRNAs). These snoRNAs differ from most cellular RNAs in that they are not exported to the cytoplasm. Instead, these RNAs are actively retained in the nucleus where they assemble with proteins into mature small nucleolar ribonucleoprotein particles and are targeted to their intranuclear site of action, the nucleolus. In this study, we have identified the cis-acting sequences responsible for the nuclear retention of U3 box C/D snoRNA by analyzing the nucleocytoplasmic distributions of an extensive panel of U3 RNA variants after injection of the RNAs into Xenopus oocyte nuclei. Our data indicate the importance of two conserved sequence motifs in retaining U3 RNA in the nucleus. The first motif is comprised of the conserved box C and box D sequences that characterize the box C/D family. The second motif contains conserved box sequences B and C. Either motif is sufficient for nuclear retention, but disruption of both motifs leads to mislocalization of the RNAs to the cytoplasm. Variant RNAs that are not retained also lack 5 cap hypermethylation and fail to associate with fibrillarin. Furthermore, our results indicate that nuclear retention of U3 RNA does not simply reflect its nucleolar localization. A fragment of U3 containing the box B/C motif is not localized to nucleoli but retained in coiled bodies. Thus, nuclear retention and nucleolar localization are distinct processes with differing sequence requirements.In all eukaryotic cells, there exist a multitude of RNAs which perform diverse functions at distinct subcellular locations. Since RNAs function at sites in the cell which are remote from where they are synthesized, RNA transport and localization are obligatory steps in gene expression for all eukaryotic cells. Despite the fundamental importance of RNA transport and localization, we have a limited understanding of the cellular mechanisms that ensure that individual RNAs are sorted to the intracellular destinations where they function.We are investigating the mechanisms controlling the intracellular trafficking of small nucleolar RNAs (snoRNAs). snoRNAs consist of a family of more than 150 molecules that are known to function in the processing and modification of rRNA within the nucleolus (2,18,47,68,74). The snoRNAs are categorized almost exclusively into two classes based on sequence homology, secondary structure, function, and binding of common proteins. The two major classes of snoRNAs are known as the box C/D snoRNAs and the box H/ACA snoRNAs (3, 16). In contrast to most other cellular RNAs, newly synthesized snoRNAs are not exported from the nucleus to the cytoplasm (52,71,73). Rather, these RNAs remain in the nucleus and are selectively targeted from their sites of synthesis within the nucleoplasm (17, 69) to their intranuclear site of function, the nucleolus. At least some snoRNAs associate with nucleoplasmic structures known as coiled bodies (5,32,53,61,65). Since the association of snoRNAs with coiled ...

U3 small nucleolar RNA (snoRNA) is a member of the Box C/D family of snoRNAs which functions in ribosomal RNA processing. U3-55k is a protein that has been found to interact with U3 but not other members of the Box C/D snoRNA family. We have found that interaction of the U3-55k protein with U3 RNA in vivo is mediated by the conserved Box B/C motif which is unique to U3 snoRNA. Mutation of Box B and Box C, but not of other conserved sequence elements, disrupted interaction of U3-55k with U3 RNA. Furthermore, a fragment of U3 containing only these two conserved elements was bound by U3-55k in vivo. RNA binding assays performed in vitro indicate that Box C may be the primary determinant of the interaction. We have cloned the cDNA encoding the Xenopus laevis U3-55k protein and find strong homology to the human sequence, including six WD repeats. Deletion of WD repeats or sequences near the C-terminus of U3-55k resulted in loss of association with U3 RNA and also loss of localization of U3-55k to the nucleolus, suggesting that protein-protein interactions contribute to the localization and RNA binding of U3-55k in vivo.

The 5'-cap structure of most spliceosomal small nuclear RNAs (snRNAs) and certain small nucleolar RNAs (snoRNAs) undergoes hypermethylation from a 7-methylguanosine to a 2,2, 7-trimethylguanosine structure. 5'-Cap hypermethylation of snRNAs is dependent upon a conserved sequence element known as the Sm site common to most snRNAs. Here we have performed a mutational analysis of U3 and U14 to determine the cis-acting sequences required for 5'-cap hypermethylation of Box C/D snoRNAs. We have found that both the conserved sequence elements Box C (termed C' in U3) and Box D are necessary for cap hypermethylation. Furthermore, the terminal stem structure that is formed by sequences that flank Box C (C' in U3) and Box D is also required. However, mutation of other conserved sequences has no effect on hypermethylation of the cap. Finally, the analysis of fragments of U3 and U14 RNAs indicates that the Box C/D motif, including Box C (C' in U3), Box D and the terminal stem, is capable of directing cap hypermethylation. Thus, the Box C/D motif, which is important for snoRNA processing, stability, nuclear retention, protein binding, nucleolar localization and function, is also necessary and sufficient for cap hypermethylation of these RNAs.

In eukaryotes, many Box C/D small nucleolar RNAs base pair with ribosomal RNA through short complementary guide sequences, thereby marking up to 100 individual nucleotides of ribosomal RNA for 2'-O-methylation. Function of the eukaryotic Box C/D RNAs depends upon interaction with at least six known proteins. Box C/D RNAs are not known to exist in Bacteria but were recently identified in Archaea by biochemical analysis and computational genomic screens and have likely evolved independently in Archaea and Eukarya for more than 2000 million years. We have microinjected Box C/D RNAs from Pyrococcus furiosus, a hyperthermophilic archaeon, into the nuclei of oocytes from the aquatic frog Xenopus laevis. Our results show that Box C/D RNAs derived from this prokaryote are retained in the nucleus, localize to nucleoli, and interact with the X. laevis Box C/D RNA binding proteins fibrillarin, Nop56, and Nop58. Furthermore, we have demonstrated the ability of archaeal Box C/D RNAs to direct site-specific 2'-O-methylation of ribosomal RNA. Our studies have revealed the remarkable ability of archaeal Box C/D RNAs to assemble into functional RNA-protein complexes in the eukaryotic nucleus.