Nuclear Retention Elements of U3 Small Nucleolar RNA

Abstract

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 ...

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

“…The procedure for microinjection of Xenopus oocyte nuclei has been described in detail (30,31). In short, a mixture of 32 P-labeled RNAs was resuspended in 20 mg/ml of blue dextran (at a final concentration of 100 fmol/µl U3 snoRNA).…”

“…The Box B/C motif is unique to U3 snoRNA. Both the Box B/C and Box C′/D motifs operate as independent units of U3 RNA nuclear retention in the Xenopus oocyte (30). The Box C′/D motif of U3 also directs the localization of the snoRNA to the nucleolus (31).…”

“…The secondary structure of the 3′ domain positions Boxes B and C, as well as Boxes C′ and D into discrete loops flanked by base paired stems to form the Box B/C and Box C′/D motifs, respectively (23,27,30). The Box C′/D motif of U3 is equivalent to the Box C/D motifs of other members of this snoRNA family in sequence/structure and function (23,27,30). The Box B/C motif is unique to U3 snoRNA.…”

“…Boxes B, C, C′ and D are found in the 3′ domain of U3 and have been characterized as sequences required for protein association (17,19,23,25,29). The secondary structure of the 3′ domain positions Boxes B and C, as well as Boxes C′ and D into discrete loops flanked by base paired stems to form the Box B/C and Box C′/D motifs, respectively (23,27,30). The Box C′/D motif of U3 is equivalent to the Box C/D motifs of other members of this snoRNA family in sequence/structure and function (23,27,30).…”

“…U3-55k belongs to the WD repeat family of proteins (45), characterized by the core sequence [GH]-(X [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41] )- [WD] arranged in repeats of four to eight units (48)(49)(50). β-Transducin (Gβ), a member of the same family, has seven WD repeats which fold into a propeller-like structure with each repeat motif forming a discrete blade of the propeller (51)(52)(53).…”