U3 snoRNA Research Articles

Assembly and Maturation of the U3 snoRNP in the Nucleoplasm in a Large Dynamic Multiprotein Complex

The assembly and maturation of box C/D snoRNPs, factors essential for ribosome biogenesis, occur in the nucleoplasm. To investigate this process, we have analyzed non-snoRNP factors associated with the nucleoplasmic human U3 snoRNA. We show that both the precursor and mature length nucleoplasmic U3 snoRNAs are present in larger multiprotein complexes that contain the core box C/D proteins as well as many non-snoRNP factors linked to snoRNP assembly (TIP48, TIP49, Nopp140), RNA processing (TGS1, La, LSm4, hRrp46), and subcellular localization (CRM1, PHAX). Using RNAi, we show that most of these factors are essential for box C/D snoRNA accumulation. Furthermore, we demonstrate that the core proteins undergo a restructuring event that stabilizes their binding to the snoRNA. Importantly, restructuring, which may be mediated by the putative remodeling factor TIP49, appears to be linked to nucleolar localization. We believe that the assembly complex coordinates snoRNA processing, snoRNP assembly, restructuring, and localization.

The small-subunit processome is a ribosome assembly intermediate.

The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5' end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps. In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins.

3′-End Polishing of the Kinetoplastid Spliced Leader RNA Is Performed by SNIP, a 3′→5′ Exonuclease with a Motley Assortment of Small RNA Substrates

In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3' extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 3'-->5' exonuclease required for SL RNA 3'-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3' processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 3'-->5' proofreading epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3' extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one- to two- nucleotide 3' extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3'-end formation is a multistep process in which SNIP provides the ultimate 3'-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.

Identification and characterization of coiled body-like structures in pea (Pisum sativum L.).

Coiled bodies (CBs) are nuclear organelles which were considered as "universal" nuclear structures in eukaryotic cells, but the formation and function of CBs, especially in plant cells, remained unclear. In this article we reported that CBs in meristematic cells of pea are oval to round obstacles in nucleus and in adjacent to nucleolus, often have the same electron density with nucleolus. We found that CBs could be stained by the rRNP preference staining method, but no rDNA was detected in the structure. Furthermore, our results of immunoelectron microscopy showed that several processing factors, include fibrillarin, U3 snoRNA and ITS1, were present in CB. It seems probable that CBs is derived structurally from nucleolus and act as transport, storage and processing subnucleolar organelles.

Imp3p and Imp4p mediate formation of essential U3-precursor rRNA (pre-rRNA) duplexes, possibly to recruit the small subunit processome to the pre-rRNA.

In eukaryotes, formation of short duplexes between the U3 small nucleolar RNA (snoRNA) and the precursor rRNA (pre-rRNA) at multiple sites is a prerequisite for three endonucleolytic cleavages that initiate small subunit biogenesis by releasing the 18S rRNA precursor from the pre-rRNA. The most likely role of these RNA duplexes is to guide the U3 snoRNA and its associated proteins, designated the small subunit processome, to the target cleavage sites on the pre-rRNA. Studies by others in Saccharomyces cerevisiae have identified the proteins Mpp10p, Imp3p, and Imp4p as candidates to mediate U3-pre-rRNA interactions. We report here that Imp3p and Imp4p appear to stabilize an otherwise unstable duplex between the U3 snoRNA hinge region and complementary bases in the external transcribed spacer of the pre-rRNA. In addition, Imp4p, but not Imp3p, seems to rearrange the U3 box A stem structure to expose the site that base-pairs with the 5' end of the 18S rRNA, thereby mediating duplex formation at a second site. By mediating formation of both essential U3-pre-rRNA duplexes, Imp3p and Imp4p may help the small subunit processome to dock onto the pre-rRNA, an event indispensable for ribosome biogenesis and hence for cell growth.

TOP Promoter Elements Control the Relative Ratio of Intron-encoded snoRNA Versus Spliced mRNA Biosynthesis

In vertebrates almost all snoRNAs are encoded in introns of a specific subclass of polII transcripts: the TOP genes. The majority of these RNAs originate through debranching of the spliced introns, the rest through endonucleolytic cleavage of the precursor that contains them. In both cases it has been suggested that snoRNP factors associate at early steps during transcription and control snoRNA biogenesis. Here, we analyzed the specific case of the U16 snoRNA that was shown to originate mainly through endonucleolytic cleavage. We show that TOP promoter elements determine a specific ratio of snoRNA and mRNA production. Under the control of these sequences the snoRNA is likely to originate from both splicing and cleavage of the pre-mRNA. Conversely, canonical polII promoter elements seem not to be compatible with snoRNA release through the cleavage reaction and produce a lower snoRNA/mRNA ratio. In addition, we show that the proximal part of the TOP promoter is responsible for this peculiar post-transcriptional process that controls the relative ratio between snoRNA and mRNA.

Role of pre-rRNA base pairing and 80S complex formation in subnucleolar localization of the U3 snoRNP.

In the nucleolus the U3 snoRNA is recruited to the 80S pre-rRNA processing complex in the dense fibrillar component (DFC). The U3 snoRNA is found throughout the nucleolus and has been proposed to move with the preribosomes to the granular component (GC). In contrast, the localization of other RNAs, such as the U8 snoRNA, is restricted to the DFC. Here we show that the incorporation of the U3 snoRNA into the 80S processing complex is not dependent on pre-rRNA base pairing sequences but requires the B/C motif, a U3-specific protein-binding element. We also show that the binding of Mpp10 to the 80S U3 complex is dependent on sequences within the U3 snoRNA that base pair with the pre-rRNA adjacent to the initial cleavage site. Furthermore, mutations that inhibit 80S complex formation and/or the association of Mpp10 result in retention of the U3 snoRNA in the DFC. From this we propose that the GC localization of the U3 snoRNA is a direct result of its active involvement in the initial steps of ribosome biogenesis.

Crystals of X29, a Xenopus laevis U8 snoRNA-binding protein with nuclear decapping activity.

Eukaryotic ribosome biosynthesis requires modification (methylation, pseudouridylation) and nucleolytic processing of precursor ribosomal RNAs in the nucleolus. The RNA components of the small nucleolar RNPs (snoRNAs) are essential for many of these events. One snoRNP, called U8, is necessary for maturation of 5.8S and 28S rRNA in vertebrates. In Xenopus laevis, U8 snoRNA was found to bind specifically and with high affinity to a protein called X29. X29 is a Nudix hydrolase, a nucleotide diphosphatase that removes the m(7)G and m(227)G caps from U8 and other RNAs. X29 requires an RNA as substrate and cap analogues are not substrates or inhibitors of cleavage. To study the determinants of X29 activity and its specificity for U8 RNA substrate, X29 was crystallized in an orthorhombic crystal form that diffracts to 2.1 A resolution.

Characterization of a crucifer plant pre-rRNA processing complex.

In cruciferous plants, the primary pre-rRNA cleavage site (P site) is immediately downstream of four similar, highly conserved sequences (A(1), A(2), A(3) and B) located within the 5'-ETS (5'-external transcribed spacer). In the present study, we describe the characterization of a plant NF D (nuclear factor D) that binds and interacts specifically with this A(123)BP cluster in the rDNA sequence. NF D is a high-molecular-mass complex containing nucleolin, fibrillarin and U3 and U14 snoRNAs. Furthermore, we show that NF D binds and cleaves pre-rRNA specifically at the P site. Thus we conclude that NF D is a pre-rRNA processing complex that may first assemble on rDNA and then bind nascent pre-rRNA.

A plant snoRNP complex containing snoRNAs, fibrillarin, and nucleolin-like proteins is competent for both rRNA gene binding and pre-rRNA processing in vitro.

In eukaryotes the primary cleavage of the precursor rRNA (pre-rRNA) occurs in the 5' external transcribed spacer (5'ETS). In Saccharomyces cerevisiae and animals this cleavage depends on a conserved U3 small nucleolar ribonucleoprotein particle (snoRNP), including fibrillarin, and on other transiently associated proteins such as nucleolin. This large complex can be visualized by electron microscopy bound to the nascent pre-rRNA soon after initiation of transcription. Our group previously described a radish rRNA gene binding activity, NF D, that specifically binds to a cluster of conserved motifs preceding the primary cleavage site in the 5'ETS of crucifer plants including radish, cauliflower, and Arabidopsis thaliana (D. Caparros-Ruiz, S. Lahmy, S. Piersanti, and M. Echeverria, Eur. J. Biochem. 247:981-989, 1997). Here we report the purification and functional characterization of NF D from cauliflower inflorescences. Remarkably NF D also binds to 5'ETS RNA and accurately cleaves it at the primary cleavage site mapped in vivo. NF D is a multiprotein factor of 600 kDa that dissociates into smaller complexes. Two polypeptides of NF D identified by microsequencing are homologues of nucleolin and fibrillarin. The conserved U3 and U14 snoRNAs associated with fibrillarin and required for early pre-rRNA cleavages are also found in NF D. Based on this it is proposed that NF D is a processing complex that assembles on the rDNA prior to its interaction with the nascent pre-rRNA.

Xenopus U3 snoRNA docks on pre-rRNA through a novel base-pairing interaction.

U3 small nucleolar RNA (snoRNA) is essential for rRNA processing to form 18S ribosomal RNA (rRNA). Previously, it has been shown that nucleolin is needed to load U3 snoRNA on pre-rRNA. However, as documented here, this is not sufficient. We present data that base-pairing between the U3 hinges and the external transcribed spacer (ETS) is critical for functional alignment of U3 on its pre-rRNA substrate. Additionally, the interaction between the U3 hinges and the ETS is proposed to serve as an anchor to hold U3 on the pre-rRNA substrate, while box A at the 5' end of U3 snoRNA swivels from ETS contacts to 18S rRNA contacts. Compensatory base changes revealed base-pairing between the 3' hinge of U3 snoRNA and region E1 of the ETS in Xenopus pre-rRNA; this novel interaction is required for 18S rRNA production. In contrast, base-pairing between the 5' hinge of U3 snoRNA and region E2 of the ETS is auxiliary, unlike the case in yeast where it is required. Thus, higher and lower eukaryotes use different interactions for functional association of U3 with pre-rRNA. The U3 hinge sequence varies between species, but covariation in the ETS retains complementarity. This species-specific U3-pre-rRNA interaction offers a potential target for a new class of antibiotics to prevent ribosome biogenesis in eukaryotic pathogens.

Kinetic analysis of ribozyme-substrate complex formation in yeast.

Many RNA-mediated reactions in transcription, translation, RNA processing, and transport require assembly of RNA complexes, yet assembly pathways remain poorly understood. Assembly mechanisms can be difficult to assess in a biological context because many components interact in complex pathways and individual steps are difficult to isolate experimentally. Our previous studies of self-cleaving hairpin ribozymes showed that kinetic and equilibrium parameters measured in yeast agree well with parameters measured in vitro under ionic conditions that mimic the intracellular environment. We now report studies of intermolecular reactions with ribozyme and target sequences expressed in yeast as separate chimeric U3 snoRNAs. In this system, intracellular cleavage rates reflect the kinetics of ribozyme-substrate complex formation through annealing of base-paired helices. Second-order rate constants increased with increasing helix length for in vitro reactions with 2 mM MgCl(2) and 150 mM NaCl and in vivo but not in reactions with 10 mM MgCl(2). Thus, efficient RNA complex formation required a larger extent of complementarity in vivo than in vitro under conditions with high concentrations of divalent cations. The most efficient intracellular cleavage reactions exhibited second-order rate constants that were 15- to 30-fold below rate constants for cleavage of oligonucleotides in vitro. Careful analysis of structural features that influence cleavage efficiency points to substrate binding as the rate-determining step in the intracellular cleavage pathway. Second-order rate constants for intermolecular cleavage agree well with diffusion coefficients reported for U3 snoRNPs in vivo suggesting that complex formation between chimeric ribozyme and substrate snoRNPs in yeast nuclei is diffusion limited.

ESF1 is required for 18S rRNA synthesis in Saccharomyces cerevisiae.

We report that Esf1p (Ydr365cp), an essential, evolutionarily conserved nucleolar protein, is required for the biogenesis of 18S rRNA in Saccharomyces cerevisiae. Depletion of Esf1p resulted in delayed processing of 35S precursor and a striking loss of 18S rRNA. Esf1p physically associated with ribosomal proteins and proteins involved in 18S rRNA biogenesis. Consistent with its role in 18S rRNA biogenesis, Esf1p also physically associated with U3 and U14 snoRNAs, but did not appear to be a core component of the SSU processome. These data indicate that Esf1p plays a direct role in early pre-rRNA processing.

Xenopus U8 snoRNA Binding Protein Is a Conserved Nuclear Decapping Enzyme

U8 snoRNP is required for accumulation of mature 5.8S and 28S rRNA in vertebrates. We are identifying proteins that bind U8 RNA with high specificity to understand how U8 functions in ribosome biogenesis. Here, we characterize a Xenopus 29 kDa protein (X29), which we previously showed binds U8 RNA with high affinity. X29 and putative homologs in other vertebrates contain a NUDIX domain found in MutT and other nucleotide diphosphatases. Recombinant X29 protein has diphosphatase activity that removes m 7G and m 227G caps from U8 and other RNAs in vitro; the putative 29 kDa human homolog also displays this decapping activity. X29 is primarily nucleolar in Xenopus tissue culture cells. We propose that X29 is a member of a conserved family of nuclear decapping proteins that function in regulating the level of U8 snoRNA and other nuclear RNAs with methylated caps.

Two-hybrid Mpp10p interaction-defective Imp4 proteins are not interaction defective in vivo but do confer specific pre-rRNA processing defects in Saccharomyces cerevisiae.

The SSU processome is a large, evolutionarily conserved ribonucleoprotein (RNP), consisting of the U3 snoRNA and at least 28 protein components, that is required for biogenesis of the 18S rRNA. We tested the function of one protein-protein interaction in the SSU processome, Mpp10p-Imp4p, in ribosome biogenesis. Exploiting the reverse two-hybrid system, we screened for mutated Imp4 proteins that were conditionally defective for interaction with Mpp10p. Three different imp4 sequences were isolated that: (i) conferred conditional growth in the two-hybrid strain; (ii) complemented the disrupted imp4; (iii) conferred conditional growth in the context of their normal cellular function; and (iv) resulted in defective pre-rRNA processing at the non-permissive temperatures. Domain swapping revealed that mutations that conferred cold sensitivity resided in the N-terminal coiled-coil domain while mutations in the C-terminus conferred temperature sensitivity. Surprisingly, the mutated Imp4 proteins were not measurably defective for interaction with Mpp10p in the context of the SSU processome. This suggests that other members of the complex may contribute to maintaining the Mpp10p-Imp4p interaction in this large RNP. Since protein-protein interactions are critical for many different aspects of cellular metabolism, our work has implications for the study of other large protein complexes.

Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA.

Pre-mRNA splicing is executed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and numerous proteins. One such protein, 15.5K/Snu13p, is associated with the spliceosomal U4/U6.U5 tri-snRNP and box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which act during preribosomal RNA (rRNA) processing. As such, it is the first splicing factor to be identified in two functionally distinct particles. 15.5K binds to an internal helix-bulge-helix (K-turn) structure in the U4 snRNA and two such structures in the U3 snoRNA. Previous work has concentrated on the structural basis of the interaction of 15.5K with the RNAs and has been carried out in vitro. Here we present a functional analysis of Snu13p in vivo, using a galactose inducible SNU13 strain to investigate the basis of three lethal mutations in Saccharomyces cerevisiae. Two are point mutations that map to the RNA-binding domain, and the third is a C-terminal deletion. These mutations result in accumulation of unspliced pre-mRNA, confirming a role for Snu13p in pre-mRNA splicing. In addition, these mutants also display rRNA processing defects that are variable in nature. Analysis of one mutant in the RNA-binding domain reveals a reduction in the levels of the U4 snRNA, U6 snRNA, and box C/D snoRNAs, but not H/ACA snoRNAs, supporting a role for Snu13p in accumulation and/or maintenance of specific RNAs. The mutations in the RNA-binding domain exhibit differential binding to the U4 snRNA and U3 snoRNA in vitro, suggesting that there are differences in the mode of interaction of Snu13p with these two RNAs.

A complex pathway for 3' processing of the yeast U3 snoRNA.

Mature U3 snoRNA in yeast is generated from the 3'-extended precursors by endonucleolytic cleavage followed by exonucleolytic trimming. These precursors terminate in poly(U) tracts and are normally stabilised by binding of the yeast La homologue, Lhp1p. We report that normal 3' processing of U3 requires the nuclear Lsm proteins. On depletion of any of the five essential proteins, Lsm2-5p or Lsm8p, the normal 3'-extended precursors to the U3 snoRNA were lost. Truncated fragments of both mature and pre-U3 accumulated in the Lsm-depleted strains, consistent with substantial RNA degradation. Pre-U3 species were co-precipitated with TAP-tagged Lsm3p, but the association with spliced pre-U3 was lost in strains lacking Lhp1p. The association of Lhp1p with pre-U3 was also reduced on depletion of Lsm3p or Lsm5p, indicating that binding of Lhp1p and the Lsm proteins is interdependent. In contrast, a tagged Sm-protein detectably co-precipitated spliced pre-U3 species only in strains lacking Lhp1p. We propose that the Lsm2-8p complex functions as a chaperone in conjunction with Lhp1p to stabilise pre-U3 RNA species during 3' processing. The Sm complex may function as a back-up to stabilise 3' ends that are not protected by Lhp1p.

Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse.

Fibrillarin, a protein component of C/D box small nucleolar ribonucleoproteins (snoRNPs), directs 2'-O-methylation of rRNA and is also involved in other aspects of rRNA processing. A gene trap screen in embryonic stem (ES) cells resulted in an insertion mutation in the fibrillarin gene. This insertion generated a fusion protein that contained the N-terminal 132 amino acids of fibrillarin fused to a beta-galactosidase-neomycin phosphotransferase reporter. As a result, the N-terminal GAR domain was present in the fusion protein but the methyltransferase-like domain was missing. The ES cell line with the targeted fibrillarin allele was transmitted through the mouse germ line, creating heterozygous animals. Western blot analyses showed a reduction in fibrillarin protein levels in the heterozygous knockout animals. Animals homozygous for the mutation were inviable, and massive apoptosis was observed in early Fibrillarin(-/-) embryos, showing that fibrillarin is essential for development. Fibrillarin(+/-) live-born mice displayed no obvious growth defect, but heterozygous intercrosses revealed a reduced ratio of +/- to +/+ mice, showing that some of the Fibrillarin heterozygous embryos die in utero. Analyses of tissue samples and cultured embryonic fibroblasts showed no discernible alteration in pre-rRNA processing or the level of the U3 snoRNA. However, the level of the intron-encoded box C/D snoRNA U76 was clearly reduced. This suggests a high requirement for snoRNA synthesis during an early stage in development.

Components of U3 snoRNA-containing complexes shuttle between nuclei and the cytoplasm and differentially localize in nucleoli: implications for assembly and function.

U3 small nucleolar RNA (snoRNA) and associated proteins are required for the processing of preribosomal RNA (pre-rRNA) and assembly of preribosomes. There are two major U3 snoRNA-containing complexes. The monoparticle contains U3 snoRNA and the core Box C/D snoRNA-associated proteins and an early preribosome-associated complex contains the monoparticle and additional factors that we refer to as preribosome-associated proteins. To address how and where the U3 snoRNA-containing preribosome assembles and how these processes are temporally and spatially regulated, we have examined the dynamics and distribution of human U3 complex-associated components in cells with active or inactive transcription of rDNA. We found that U3 complex-associated proteins shuttle between the nucleus and the cytoplasm independent of the synthesis and export of preribosomal particles, suggesting that the shuttling of these proteins may either provide opportunities for their regulation, or contribute to or modulate ribosome export. In addition, monoparticle and preribosome associated components predominantly localize to different nucleolar substructures, fibrillar components, and granular components, respectively, in active nucleoli, and partition separately into the two components during nucleolar segregation induced by inhibition of pol I transcription. Although the predominant localizations of these two sets of factors differ, there are significant areas of overlap that may represent the sites where they reside as a single complex. These results are consistent with a model in which U3 monoparticles associate with the fibrillar components of nucleoli and bind pre-rRNA during transcription, triggering recruitment of preribosome-associated proteins to assemble the complex necessary for pre-rRNA processing.

Inhibition of HIV-1 infection by lentiviral vectors expressing pol III-promoted anti-HIV RNAs

A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96–98% in 293 cells to 15–20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34+-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.