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George L. Eliceiri - One of the best experts on this subject based on the ideXlab platform.
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The vertebrate E1/U17 Small Nucleolar Ribonucleoprotein particle
Journal of cellular biochemistry, 2006Co-Authors: George L. EliceiriAbstract:Each of the many different box H/ACA Ribonucleoprotein particles (RNPs) present in eukaryotes and archaea consists of four common core proteins and one specific H/ACA Small RNA, which bears the sequence elements H (ANANNA) and ACA. Most of the H/ACA RNPs are Small Nucleolar RNPs (snoRNPs), which are localized in nucleoli, and are one of the two major classes of snoRNPs. Most H/ACA RNPs direct pseudouridine synthesis in pre-rRNA and other RNAs. One H/ACA Small Nucleolar RNA (snoRNA), vertebrate E1/U17 (snR30 in yeast), is required for pre-rRNA cleavage processing that generates mature 18S rRNA. E1 snoRNA is encoded in introns of protein-coding genes, and the evidence suggests that human E1 RNA undergoes uridine insertional RNA editing. The vertebrate E1 RNA consensus secondary structure shows several features that are absent in other box H/ACA snoRNAs. The available UV-induced RNA-protein crosslinking results suggest that the E1 snoRNP is asymmetrical in vertebrate cells, in contrast to other H/ACA snoRNPs. The vertebrate E1 snoRNP in cells is surprisingly complex: (i) E1 RNA contacts directly and specifically several proteins which do not appear to be any of the H/ACA RNP four core proteins; and (ii) multiple E1 RNA sites are needed for E1 snoRNP formation, E1 RNA stability, and E1 RNA–protein direct interactions. J. Cell. Biochem. 98: 486–495, 2006. © 2006 Wiley-Liss, Inc.
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the vertebrate e1 u17 Small Nucleolar Ribonucleoprotein particle
Journal of Cellular Biochemistry, 2006Co-Authors: George L. EliceiriAbstract:Each of the many different box H/ACA Ribonucleoprotein particles (RNPs) present in eukaryotes and archaea consists of four common core proteins and one specific H/ACA Small RNA, which bears the sequence elements H (ANANNA) and ACA. Most of the H/ACA RNPs are Small Nucleolar RNPs (snoRNPs), which are localized in nucleoli, and are one of the two major classes of snoRNPs. Most H/ACA RNPs direct pseudouridine synthesis in pre-rRNA and other RNAs. One H/ACA Small Nucleolar RNA (snoRNA), vertebrate E1/U17 (snR30 in yeast), is required for pre-rRNA cleavage processing that generates mature 18S rRNA. E1 snoRNA is encoded in introns of protein-coding genes, and the evidence suggests that human E1 RNA undergoes uridine insertional RNA editing. The vertebrate E1 RNA consensus secondary structure shows several features that are absent in other box H/ACA snoRNAs. The available UV-induced RNA-protein crosslinking results suggest that the E1 snoRNP is asymmetrical in vertebrate cells, in contrast to other H/ACA snoRNPs. The vertebrate E1 snoRNP in cells is surprisingly complex: (i) E1 RNA contacts directly and specifically several proteins which do not appear to be any of the H/ACA RNP four core proteins; and (ii) multiple E1 RNA sites are needed for E1 snoRNP formation, E1 RNA stability, and E1 RNA–protein direct interactions. J. Cell. Biochem. 98: 486–495, 2006. © 2006 Wiley-Liss, Inc.
Susan J. Baserga - One of the best experts on this subject based on the ideXlab platform.
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An unexpected, conserved element of the U3 snoRNA is required for Mpp10p association.
RNA (New York N.Y.), 2001Co-Authors: Steven Wormsley, Maurille J Fournier, Dmitry Samarsky, Susan J. BasergaAbstract:The U3 Small Nucleolar Ribonucleoprotein (snoRNP) is composed of a Small Nucleolar RNA (snoRNA) and at least 10 proteins. The U3 snoRNA base pairs with the pre-rRNA to carry out the A0, A1, and A2 processing reactions that lead to the release of the 18S rRNA from the nascent pre-rRNA transcript. The yeast U3 snoRNA can be divided into a short 5' domain (nt 1-39) and a larger 3' domain (73 to the 3' end) separated by a stretch of nucleotides called the hinge region (nt 40-72). The sequences required for pre-rRNA base pairing are found in the 5' domain and hinge region whereas the 3' domain is largely covered with proteins. Mpp10p, one of the protein components unique to the U3 snoRNP, plays a role in processing at the A1 and A2 sites. Because of its critical role in U3 snoRNP function, we determined which sequences in the U3 snoRNA are required for Mpp10p association. Unlike fibrillarin and all the previous U3 snoRNP components studied in this manner, sequences in the 3' domain are not sufficient for Mpp10p association. Instead, a conserved sequence element in the U3 snoRNA hinge region is required, placing Mpp10p near the 5' domain that carries out the pre-rRNA base-pairing interactions in the functional center of the U3 snoRNP.
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Fibrillarin and Other snoRNP Proteins Are Targets of Autoantibodies in Xenobiotic-Induced Autoimmunity
Clinical immunology (Orlando Fla.), 2001Co-Authors: Jian-ming Yang, Susan J. Baserga, Shannon J. Turley, K. Michael PollardAbstract:Exposure of SJL/J mice to mercury induces an anti-Nucleolar autoantibody response. The predominant target is fibrillarin, a 34-kDa component of the Small Nucleolar Ribonucleoprotein particles (snoRNP), but other proteins are also recognized. To characterize these proteins, monoclonal IgG anti-Nucleolar antibodies were produced from HgC12-treated SJL/J mice. One monoclonal, 17C12, recognized fibrillarin, while two others, 7G3 and 6G10, were found to immunoprecipitate snoRNP particles but not fibrillarin. Antibody 6G10 gave a Nucleolar immunofluorescence pattern in human, murine, and amphibian cells, but was negative in immunoblot. The 7G3 monoclone reacted with a 60-kDa protein conserved in human and murine, but not amphibian, cell lines. The 7G3 and 6G10 antigens and fibrillarin colocalized to the nucleolus and Cajal bodies in interphase cells and decorated metaphase chromosomes. These studies suggest that the mercury-induced anti-Nucleolar antibody response targets other protein components of the snoRNP particles in addition to fibrillarin.
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Imp3p and Imp4p, two specific components of the U3 Small Nucleolar Ribonucleoprotein that are essential for pre-18S rRNA processing.
Molecular and cellular biology, 1999Co-Authors: Sarah J. Lee, Susan J. BasergaAbstract:The function of the U3 Small Nucleolar Ribonucleoprotein (snoRNP) is central to the events surrounding pre-rRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.
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M phase phosphoprotein 10 is a human U3 Small Nucleolar Ribonucleoprotein component.
Molecular biology of the cell, 1998Co-Authors: Joanne M. Westendorf, Konstantin N. Konstantinov, Steven Wormsley, Mei-di Shu, Naoko Matsumoto-taniura, Fabienne Pirollet, F. George Klier, Larry Gerace, Susan J. BasergaAbstract:We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential Nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with Nucleolar fibrillarin and other known Nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and preNucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and preNucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 Small Nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 Small Nucleolar Ribonucleoprotein.
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Functional separation of pre-rRNA processing steps revealed by truncation of the U3 Small Nucleolar Ribonucleoprotein component, Mpp10
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Sarah J. Lee, Susan J. BasergaAbstract:The U3 Small Nucleolar Ribonucleoprotein (snoRNP) is required for three cleavage events that generate the mature 18S rRNA from the pre-rRNA. In Saccharomyces cerevisiae, depletion of Mpp10, a U3 snoRNP-specific protein, halts 18S rRNA production and impairs cleavage at the three U3 snoRNP-dependent sites: A0, A1, and A2. We have identified truncation mutations of Mpp10 that affect 18S rRNA synthesis and confer cold-sensitivity and slow growth. However, distinct from yeast cells depleted of Mpp10, the mutants carrying these truncated Mpp10 proteins accumulate a novel precursor, resulting from cleavage at only A0. The Mpp10 truncations do not alter association of Mpp10 with the U3 snoRNA, nor do they affect snoRNA or protein stability. Thus, the role in processing of the U3 snoRNP can be separated into cleavage at the A0 site, which occurs in the presence of truncated Mpp10, and cleavage at the A1/A2 sites, which occurs only with intact Mpp10. These results strongly argue for a role for Mpp10 in processing at the A1/A2 sites.
Reinhard Lührmann - One of the best experts on this subject based on the ideXlab platform.
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cDNA Cloning and Characterization of the Human U3 Small Nucleolar Ribonucleoprotein Complex-Associated 55-Kilodalton Protein
Molecular and cellular biology, 1998Co-Authors: Helma Pluk, Reinhard Lührmann, Jerremy Soffner, Walther J Van VenrooijAbstract:The eukaryotic nucleolus contains a large number of Small RNA molecules (snoRNAs) which, in the form of Small Nucleolar Ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. The most abundant and one of the best-conserved snoRNAs is the U3 RNA. So far, only one human U3 snoRNA-associated protein, fibrillarin, has been characterized. Previously, the U3 snoRNPwas purified from CHO cells, and three proteins of 15, 50, and 55 kDa were found to copurify with the U3 snoRNA (B. Lubben, C. Marshallsay, N. Rottmann, and R. Luhrmann, Nucleic Acids Res. 21:5377-5385, 1993). Here we report the cDNA cloning and characterization of the human U3 snoRNP-associated 55-kDa protein. The isolated cDNA codes for a novel Nucleolar protein which is specifically associated with the U3 snoRNA. This protein, referred to as hU3-55k, is the first characterized U3 snoRNP-specific protein from humans. hU3-55k is a new member of the family of WD-40 repeat proteins and is conserved throughout evolution. It appears that the C-terminal end of hU3-55k is required for Nucleolar localization and U3 snoRNA binding. Eukaryotic cells contain a large number of Small Nucleolar RNAs (snoRNAs) which, in the form of Small Nucleolar ribo- nucleoprotein complexes (snoRNPs), are involved in the var- ious steps of ribosome synthesis (reviewed in references 30 and 46). Several snoRNAs have previously been shown to be re- quired for pre-rRNA processing (12, 30, 46), and a large set of snoRNAs is involved in ribose methylation and pseudouridy- lation of rRNA (8, 15, 24, 33, 34, 49). snoRNAs are heteroge- neous in size, structural elements, and protein association. They are produced by two biosynthetic pathways. Most snoRNAs are encoded within the pre-mRNA introns of ribo- somal or Nucleolar proteins. The processing of such pre- mRNAs via endo- and exonucleolytic cleavages results in the generation of mature noncapped snoRNAs (30). Other
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cdna cloning and characterization of the human u3 Small Nucleolar Ribonucleoprotein complex associated 55 kilodalton protein
Molecular and Cellular Biology, 1998Co-Authors: Helma Pluk, Reinhard Lührmann, Jerremy Soffner, Walther J Van VenrooijAbstract:The eukaryotic nucleolus contains a large number of Small RNA molecules (snoRNAs) which, in the form of Small Nucleolar Ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. The most abundant and one of the best-conserved snoRNAs is the U3 RNA. So far, only one human U3 snoRNA-associated protein, fibrillarin, has been characterized. Previously, the U3 snoRNPwas purified from CHO cells, and three proteins of 15, 50, and 55 kDa were found to copurify with the U3 snoRNA (B. Lubben, C. Marshallsay, N. Rottmann, and R. Luhrmann, Nucleic Acids Res. 21:5377-5385, 1993). Here we report the cDNA cloning and characterization of the human U3 snoRNP-associated 55-kDa protein. The isolated cDNA codes for a novel Nucleolar protein which is specifically associated with the U3 snoRNA. This protein, referred to as hU3-55k, is the first characterized U3 snoRNP-specific protein from humans. hU3-55k is a new member of the family of WD-40 repeat proteins and is conserved throughout evolution. It appears that the C-terminal end of hU3-55k is required for Nucleolar localization and U3 snoRNA binding.
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Isolation and characterization of the Small Nucleolar Ribonucleoprotein particle snR30 from Saccharomyces cerevisiae.
The Journal of biological chemistry, 1995Co-Authors: B Lubben, Patrizia Fabrizio, Berthold Kastner, Reinhard LührmannAbstract:Abstract The nucleolus of the yeast Saccharomyces cerevisiae contains the Small Nucleolar RNA snR30 (snoRNA), that is found associated with at least two proteins, NOP1 and GAR1. All three of these molecules are essential for the cell's viability and have been implicated in pre-rRNA maturation. NOP1 and GAR1 are believed to be general rRNA-processing factors or, alternatively, integral protein components of the Small Nucleolar Ribonucleoprotein particle snR30 (snoRNP). In this paper, we describe procedures for the biochemical isolation of snR30 RNP, and we identify seven snR30 RNP proteins of molecular masses of 10, 23, 25, 38, 46, 48, and 65 kDa, including the previously reported GAR1 protein. Additional proteins, including NOP1, may also be components of snR30 RNP but are lost during our stringent isolation procedure. The 10-, 23-, and 25-kDa (GAR1) and 65-kDa proteins remain tightly associated with the snR30 RNA even after isopycnic sedimentation in cesium sulfate gradients. Electron microscopy of Mono Q-purified snR30 RNPs show a slightly elongated two-domain structure approximately 20 nm long and 14 nm wide.
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Isolation of U3 snoRNP from CHO cells: a novel 55 kDa protein binds to the central part of U3 snoRNA
Nucleic acids research, 1993Co-Authors: Birgit Lübben, Christopher Marshallsay, Norbert Rottmann, Reinhard LührmannAbstract:U3 snoRNP, the most abundant of the Small Nucleolar Ribonucleoprotein particles (snoRNPs), has previously been demonstrated to participate in pre-rRNA maturation. Here we report the purification of U3 snoRNP from CHO cells using anti-m3G-immunoaffinity and mono Q anion-exchange chromatography. Isolated U3 snoRNPs contain three novel proteins, of 15, 50 and 55 kDa respectively. These proteins may represent core U3 snoRNP proteins whose binding mediates the association of other proteins, such as fibrillarin, that are lost during purification. Using a rabbit antiserum raised against the 55 kDa protein, and an in vitro reconstitution assay, we have localised the 55 kDa protein binding site on the U3 snoRNA. Stable binding of the 55 kDa protein requires sequences located between nucleotides 97 and 204 of the human U3 snoRNA, including the evolutionarily conserved B and C sequence motifs.
U. Thomas Meier - One of the best experts on this subject based on the ideXlab platform.
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Genetic Interaction between a Chaperone of Small Nucleolar Ribonucleoprotein Particles and Cytosolic Serine Hydroxymethyltransferase
The Journal of biological chemistry, 2003Co-Authors: Yunfeng Yang, U. Thomas MeierAbstract:Abstract Srp40p is a nonessential yeast Nucleolar protein proposed to function as a chaperone for over 100 Small Nucleolar Ribonucleoprotein particles that are required for rRNA maturation. To verify and expand on its function, genetic screens were performed for the identification of genes that were lethal when mutated in a SRP40 null background (srp40Δ). Unexpectedly, mutation of both cytosolic serine hydroxymethyltransferase (SHM2) and one-carbon tetrahydrofolate synthase (ADE3) was required to achieve synthetic lethality with srp40Δ. Shm2p and Ade3p are cytoplasmic enzymes producing 5,10-methylene tetrahydrofolate in convergent pathways as the primary source for cellular one-carbon groups. Nonetheless, point mutants of Shm2p that were catalytically inactive (i.e. failed to rescue the methionine auxotrophy of a shm2Δ ade3 strain) complemented the synthetic lethal phenotype, thus revealing a novel metabolism-independent function of Shm2p. The same Shm2p mutants exacerbated a giant cell phenotype observed in the shm2Δ ade3 strain suggesting a catalysis-independent role for Shm2p in cell size control, possibly through regulation of ribosome biogenesis via SRP40. Additionally, we show that the Sm-like protein Lsm5p, which as part of Lsm complexes participates in cytosolic and nuclear RNA processing and degradation pathways, is a multicopy suppressor of the synthetic lethality and of the specific depletion of box H/ACA Small Nucleolar RNAs from the srp40Δ shm2 ade3 strain. Finally, rat Nopp140 restored growth and stability of box H/ACA snoRNAs after genetic depletion of SRP40 in the synthetic lethal strain indicating that it is indeed the functional homolog of yeast Srp40p.
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Immunopurified Small Nucleolar Ribonucleoprotein Particles Pseudouridylate rRNA Independently of Their Association with Phosphorylated Nopp140
Molecular and Cellular Biology, 2002Co-Authors: C. C. Query, U. Thomas MeierAbstract:The isomerization of up to 100 uridines to pseudouridines (Ψs) in eukaryotic rRNA is guided by a similar number of box H/ACA Small Nucleolar RNAs (snoRNAs), each forming a unique Small Nucleolar ribonucleo-protein particle (snoRNP) with the same four core proteins, NAP57 (also known as dyskerin or Cbf5p), GAR1, NHP2, and NOP10. Additionally, the Nucleolar and Cajal body protein Nopp140 (Srp40p) associates with the snoRNPs. To understand the role of these factors in pseudouridylation, we established an in vitro assay system. Short site-specifically 32 P-labeled rRNA substrates were incubated with subcellular fractions, and the conversion of uridine to Ψ was monitored by thin-layer chromatography after digestion to single nucleotides. Immunopurified box H/ACA core particles were sufficient for the reaction. SnoRNPs associated quantitatively and reversibly with Nopp140. However, pseudouridylation activity was independent of Nopp140, consistent with a chaperoning role for this highly phosphorylated protein. Although up to 14 bp between the snoRNA and rRNA were required for the in vitro reaction, rRNA pseudouridylation and release occurred in the absence of ATP and magnesium. These data suggest that substrate release takes place without RNA helicase activity but may be aided by the snoRNP core proteins.
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Conserved composition of mammalian box H/ACA and box C/D Small Nucleolar Ribonucleoprotein particles and their interaction with the common factor Nopp140.
Molecular biology of the cell, 2000Co-Authors: Yunfeng Yang, Cynthia Isaac, Chen Wang, François Dragon, Vanda Pogac̆ić, U. Thomas MeierAbstract:Small Nucleolar Ribonucleoprotein particles (snoRNPs) mainly catalyze the modification of rRNA. The two major classes of snoRNPs, box H/ACA and box C/D, function in the pseudouridylation and 29-O-methylation, respectively, of specific nucleotides. The emerging view based on studies in yeast is that each class of snoRNPs is composed of a unique set of proteins. Here we present a characterization of mammalian snoRNPs. We show that the previously characterized NAP57 is specific for box H/ACA snoRNPs, whereas the newly identified NAP65, the rat homologue of yeast Nop5/58p, is a component of the box C/D class. Using coimmunoprecipitation experiments, we show that the Nucleolar and coiled-body protein Nopp140 interacts with both classes of snoRNPs. This interaction is corroborated in vivo by the exclusive depletion of snoRNP proteins from nucleoli in cells transfected with a dominant negative Nopp140 construct. Interestingly, RNA polymerase I transcription is arrested in nucleoli depleted of snoRNPs, raising the possibility of a feedback mechanism between rRNA modification and transcription. Moreover, the Nopp140snoRNP interaction appears to be conserved in yeast, because depletion of Srp40p, the yeast Nopp140 homologue, in a conditional lethal strain induces the loss of box H/ACA Small Nucleolar RNAs. We propose that Nopp140 functions as a chaperone of snoRNPs in yeast and vertebrate cells.
Michael P. Terns - One of the best experts on this subject based on the ideXlab platform.
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Determinants of the interaction of the spinal muscular atrophy disease protein SMN with the dimethylarginine-modified box H/ACA Small Nucleolar Ribonucleoprotein GAR1.
The Journal of biological chemistry, 2002Co-Authors: Sarah E. Whitehead, Kevin W. Jones, Xing Zhang, Xiaodong Cheng, Rebecca M. Terns, Michael P. TernsAbstract:Deletion or mutation of the SMN1 (survival of motor neurons) gene causes the common, fatal neuromuscular disease spinal muscular atrophy. The SMN protein is important in Small nuclear Ribonucleoprotein (snRNP) assembly and interacts with snRNP proteins via arginine/glycine-rich domains. Recently, SMN was also found to interact with core protein components of the two major families of Small Nucleolar RNPs, fibrillarin and GAR1, suggesting that SMN may also function in the assembly of Small Nucleolar RNPs. Here we present results that indicate that the interaction of SMN with GAR1 is mediated by the Tudor domain of SMN. Single point mutations within the Tudor domain, including a spinal muscular atrophy patient mutation, impair the interaction of SMN with GAR1. Furthermore, we find that either of the two arginine/glycine-rich domains of GAR1 can provide for interaction with SMN, but removal of both results in loss of the interaction. Finally, we have found that unlike the interaction of SMN with the Sm snRNP proteins, interaction with GAR1 and fibrillarin is not enhanced by arginine dimethylation. Our results argue against post-translational arginine dimethylation as a general requirement for SMN recognition of proteins bearing arginine/glycine-rich domains.
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Nuclear Retention Elements of U3 Small Nucleolar RNA
Molecular and cellular biology, 1999Co-Authors: Wayne A. Speckmann, Rebecca M. Terns, Aarthi Narayanan, Michael P. TernsAbstract: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.