U3 snoRNA Research Articles

Comparative analysis of eukaryotic U3 snoRNA

The U3 snoRNA is an exceptional box C/D snoRNA, which is involved in pre-rRNA processing without directing chemical modifications. We report here on a comprehensive computational survey resulting in U3 sequences for morethan 90 additional eukaryotes. This extended data basis is used to improve the secondary structure models. The detailed investigation of the structural variation of U3 snoRNAs turns out to be much more extensive than previously thought. Many fungal U3 genes, in addition, contain introns. U3 promoters are snRNA-like but show substantial variations even between related species.

Mrd1p Is Required for Release of Base-Paired U3 snoRNA within the Preribosomal Complex

In eukaryotes, ribosomes are made from precursor rRNA (pre-rRNA) and ribosomal proteins in a maturation process that requires a large number of snoRNPs and processing factors. A fundamental problem is how the coordinated and productive folding of the pre-rRNA and assembly of successive pre-rRNA-protein complexes is achieved cotranscriptionally. The conserved protein Mrd1p, which contains five RNA binding domains (RBDs), is essential for processing events leading to small ribosomal subunit synthesis. We show that full function of Mrd1p requires all five RBDs and that the RBDs are functionally distinct and needed during different steps in processing. Mrd1p mutations trap U3 snoRNA in pre-rRNP complexes both in base-paired and non-base-paired interactions. A single essential RBD, RBD5, is involved in both types of interactions, but its conserved RNP1 motif is not needed for releasing the base-paired interactions. RBD5 is also required for the late pre-rRNP compaction preceding A(2) cleavage. Our results suggest that Mrd1p modulates successive conformational rearrangements within the pre-rRNP that influence snoRNA-pre-rRNA contacts and couple U3 snoRNA-pre-rRNA remodeling and late steps in pre-rRNP compaction that are essential for cleavage at A(0) to A(2). Mrd1p therefore coordinates key events in biosynthesis of small ribosome subunits.

Evidence that the AAA+Proteins TIP48 and TIP49 Bridge Interactions between 15.5K and the Related NOP56 and NOP58 Proteins during Box C/D snoRNP Biogenesis

The box C/D small nucleolar RNPs (snoRNPs) are essential for the processing and modification of rRNA. TIP48 and TIP49 are two related AAA(+) proteins that are essential for the formation of box C/D snoRNPs. These proteins are key components of the pre-snoRNP complexes, but their exact role in box C/D snoRNP biogenesis is largely uncharacterized. Here we report that TIP48 and TIP49 interact with one another in vitro, and only the TIP48/TIP49 complex, but not the individual proteins, possesses significant ATPase activity. Loss of TIP48 and TIP49 results in a change in pre-snoRNA levels and a loss of U3 snoRNA signal in the Cajal body. We show that TIP48 and TIP49 make multiple interactions with core snoRNP proteins and biogenesis factors and that these interactions are often regulated by the presence of ATP. Furthermore, we demonstrate that TIP48 and TIP49 efficiently bridge interactions between the core box C/D proteins NOP56 or NOP58 and 15.5K. Our data imply that the snoRNP assembly factor NUFIP can regulate the interactions between TIP48 and TIP49 and the core box C/D proteins. We suggest that snoRNP assembly involves an intricate series of interactions that are mediated/regulated by bridging factors and chaperones.

Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs

The U3 small nucleolar ribonucleoprotein (snoRNP) plays an essential role in ribosome biogenesis but, like many RNA-protein complexes, its architecture is poorly understood. To address this problem, binding sites for the snoRNP proteins Nop1, Nop56, Nop58, and Rrp9 were mapped by UV cross-linking and analysis of cDNAs. Cross-linked protein-RNA complexes were purified under highly-denaturing conditions, ensuring that only direct interactions were detected. Recovered RNA fragments were amplified after linker ligation and cDNA synthesis. Cross-linking was successfully performed either in vitro on purified complexes or in vivo in living cells. Cross-linking sites were precisely mapped either by Sanger sequencing of multiple cloned fragments or direct, high-throughput Solexa sequencing. Analysis of RNAs associated with the snoRNP proteins revealed remarkably high signal-to-noise ratios and identified specific binding sites for each of these proteins on the U3 RNA. The results were consistent with previous data, demonstrating the reliability of the method, but also provided insights into the architecture of the U3 snoRNP. The snoRNP proteins were also cross-linked to pre-rRNA fragments, with preferential association at known sites of box C/D snoRNA function. This finding demonstrates that the snoRNP proteins directly contact the pre-rRNA substrate, suggesting roles in snoRNA recruitment. The techniques reported here should be widely applicable to analyses of RNA-protein interactions.

RNA Chaperones Stimulate Formation and Yield of the U3 snoRNA–Pre-rRNA Duplexes Needed for Eukaryotic Ribosome Biogenesis

Short duplexes between the U3 small nucleolar RNA and the precursor ribosomal RNA must form quickly and with high yield to satisfy the high demand for ribosome synthesis in rapidly growing eukaryotic cells. These interactions, designated the U3–ETS (external transcribed spacer) and U3–18S duplexes, are essential to initiate the processing of small subunit ribosomal RNA. Previously, we showed that duplexes corresponding to those in Saccharomyces cerevisiae are only observed in vitro after addition of one of two proteins: Imp3p or Imp4p. Here, we used fluorescence-based and other in vitro assays to determine whether these proteins possess RNA chaperone activities and to assess whether these activities are sufficient to satisfy the duplex yield and rate requirements expected in vivo. Assembly of both proteins with the U3 small nucleolar RNA into a chaperone complex destabilizes a U3 stem structure, apparently to expose its 18S base-pairing site. As a result, the chaperone complex accelerates formation of the U3–18S duplex from an undetectable rate to one comparable with the intrinsic rate observed for hybridizing short duplexes. The chaperone complex also stabilizes the U3–ETS duplex by 2.7 kcal/mol. These chaperone activities provide high U3–ETS duplex yield and rapid U3–18S duplex formation over a broad concentration range to help ensure that the U3–precursor ribosomal RNA interactions limit neither ribosome biogenesis nor rapid cell growth. The thermodynamic and kinetic framework used is general and thus suitable for investigating the mechanism of action of other RNA chaperones.

U3 snoRNA genes are multi-copy and frequently linked to U5 snRNA genes in Euglena gracilis§

BackgroundU3 snoRNA is a box C/D small nucleolar RNA (snoRNA) involved in the processing events that liberate 18S rRNA from the ribosomal RNA precursor (pre-rRNA). Although U3 snoRNA is present in all eukaryotic organisms, most investigations of it have focused on fungi (particularly yeasts), animals and plants. Relatively little is known about U3 snoRNA and its gene(s) in the phylogenetically broad assemblage of protists (mostly unicellular eukaryotes). In the euglenozoon Euglena gracilis, a distant relative of the kinetoplastid protozoa, Southern analysis had previously revealed at least 13 bands hybridizing with U3 snoRNA, suggesting the existence of multiple copies of U3 snoRNA genes.ResultsThrough screening of a λ genomic library and PCR amplification, we recovered 14 U3 snoRNA gene variants, defined by sequence heterogeneities that are mostly located in the U3 3'-stem-loop domain. We identified three different genomic arrangements of Euglena U3 snoRNA genes: i) stand-alone, ii) linked to tRNAArg genes, and iii) linked to a U5 snRNA gene. In arrangement ii), the U3 snoRNA gene is positioned upstream of two identical tRNAArg genes that are convergently transcribed relative to the U3 gene. This scenario is reminiscent of a U3 snoRNA-tRNA gene linkage previously described in trypanosomatids. We document here twelve different U3 snoRNA-U5 snRNA gene arrangements in Euglena; in each case, the U3 gene is linked to a downstream and convergently oriented U5 gene, with the intergenic region differing in length and sequence among the variants.ConclusionThe multiple U3 snoRNA-U5 snRNA gene linkages, which cluster into distinct families based on sequence similarities within the intergenic spacer, presumably arose by genome, chromosome, and/or locus duplications. We discuss possible reasons for the existence of the unusually large number of U3 snoRNA genes in the Euglena genome. Variability in the signal intensities of the multiple Southern hybridization bands raises the possibility that Euglena contains a naturally aneuploid chromosome complement.

Identification, expression and functional analysis of U3 snoRNA genes from Neurospora crassa

By screening of a cDNA library constructed with small RNAs from Neurospora crassa and genome analysis, three U3 snoRNAs of 262, 270 and 275 nt were identified and named as NcU3A, NcU3B and NcU3C, respectively. Expression of the three U3 snoRNA genes was further analyzed by Northern blot hybridization. Meanwhile, two 5′-truncated U3 transcripts with different sizes were also identified, indicating a complex posttranscriptional processing of the U3 snoRNAs. In addition to the known functional elements complementary to 18S rRNA, a novel antisense sequence to 26S rRNA was found in NcU3A. A primer extension assay showed that the antisense sequence did not guide 2′- O-ribose methylation of 26S rRNA but probably functioned as an rRNA chaperon. In the N. crassa genome, three U3 snoRNA genes are independently transcribed and each of them contains a small intron. The same intron-inserting site in the U3 snoRNA genes among N. crassa and yeasts shows the ancient origin of the intron in these organisms. Our results provide new insights into the structural and functional diversity of U3 snoRNA family.

The 5' external transcribed spacer in mouse ribosomal RNA contains two cleavage sites.

The 5' external transcribed spacer (5'ETS) is critical for 18S rRNA formation and is the longest noncoding region in a ribosomal RNA transcript. Here we show that processing in mouse 5'ETS involves two cleavage events. Processing at site A' corresponds to the previously described "primary cleavage," which precedes other processing steps. Processing at the novel site A0 occurs 1 kb downstream from A' yielding two new rRNA precursors: 43S and 29S. The excised 5'-A' and A'-A0 fragments are rapidly degraded under normal conditions. Depletion of the exosome component EXOSC10/PM-Scl100 (ortholog of yeast Rrp6p) results in a strong accumulation of the A'-A0 spacer fragment in mouse cells. We discuss the finding of a second processing site in mammalian 5'ETS in relation to the involvement of the U3 snoRNA in pre-rRNA processing and present a revised map of the mouse 18S rRNA processing pathway.

U3 snoRNA promoter reflects the RNA’s function in ribosome biogenesis

An efficiently expressed "tagged" gene system was used to study promoter sequence elements in genes encoding the U3 snoRNAs of Schizosaccharomyces pombe. Transcription was found dependent on two previously thought, mutually exclusive elements, a TATA-box, as found in other S. pombe snRNA gene promoters, and a Homol D-box, often considered a TATA-box analogue in the promoters of genes that encode ribosomal proteins. The Homol D-box is critical while the TATA-box strongly influences transcription efficiency but is not essential. The results suggest that the U3 snoRNA promoter may represent the fusion of two promoter systems reflecting the special role of this RNA in ribosome biogenesis. Steady state measurements of cellular RNAs support such a coordinated regulation of the U3 snoRNA.

Post-transcriptional Regulation of the U3 Small Nucleolar RNA

A high copy shuttle vector was used to express a "tagged" U3 small nucleolar RNA (snoRNA) gene in Schizosaccharomyces pombe to examine regulatory responses to a high gene dosage. RNA analyses utilizing reverse transcription-PCR amplification and restriction fragment length polymorphism indicated that the tagged gene was both proportionally and highly expressed and that downstream processing and/or termination were critical to U3 snoRNA stability. In contrast, direct measurements of the total cellular U3 snoRNA showed essentially normal levels of mature RNA, although measurements of precursor levels confirmed a highly expressed gene construct. Taken together, the results indicated that the steady state amounts of mature U3 snoRNA were primarily regulated at the post-transcriptional level. This regulatory mechanism prevents over-accumulation of the cellular U3 snoRNA and can efficiently degrade mutant RNA molecules. Together with past studies on other 3' extended RNA precursors, the results support post-transcriptional regulation as a quality control mechanism in which appropriate amounts of functional RNA are stabilized by protein interaction while excess or defective RNA is rapidly degraded. Precursor processing in vitro and mutational analyses were consistent with this model.

Mrd1p binds to pre-rRNA early during transcription independent of U3 snoRNA and is required for compaction of the pre-rRNA into small subunit processomes

In Saccharomyces cerevisiae, synthesis of the small ribosomal subunit requires assembly of the 35S pre-rRNA into a 90S preribosomal complex. SnoRNAs, including U3 snoRNA, and many trans-acting proteins are required for the ordered assembly and function of the 90S preribosomal complex. Here, we show that the conserved protein Mrd1p binds to the pre-rRNA early during transcription and is required for compaction of the pre-18S rRNA into SSU processome particles. We have exploited the fact that an Mrd1p-GFP fusion protein is incorporated into the 90S preribosomal complex, where it acts as a partial loss-of-function mutation. When associated with the pre-rRNA, Mrd1p-GFP functionally interacts with the essential Pwp2, Mpp10 and U3 snoRNP subcomplexes that are functionally interconnected in the 90S preribosomal complex. The fusion protein can partially support 90S preribosome-mediated cleavages at the A0–A2 sites. At the same time, on a substantial fraction of transcripts, the composition and/or structure of the 90S preribosomal complex is perturbed by the fusion protein in such a way that cleavage of the 35S pre-rRNA is either blocked or shifted to aberrant sites. These results show that Mrd1p is required for establishing productive structures within the 90S preribosomal complex.

The HIV Tat protein affects processing of ribosomal RNA precursor

BackgroundInside the cell, the HIV Tat protein is mainly found in the nucleus and nucleolus. The nucleolus, the site of ribosome biogenesis, is a highly organized, non-membrane-bound sub-compartment where proteins with a high affinity for nucleolar components are found. While it is well known that Tat accumulates in the nucleolus via a specific nucleolar targeting sequence, its function in this compartment it still unknown.ResultsTo clarify the significance of the Tat nucleolar localization, we induced the expression of the protein during oogenesis in Drosophila melanogaster strain transgenic for HIV-tat gene. Here we show that Tat localizes in the nucleoli of Drosophila oocyte nurse cells, where it specifically co-localizes with fibrillarin. Tat expression is accompanied by a significant decrease of cytoplasmic ribosomes, which is apparently related to an impairment of ribosomal rRNA precursor processing. Such an event is accounted for by the interaction of Tat with fibrillarin and U3 snoRNA, which are both required for pre-rRNA maturation.ConclusionOur data contribute to understanding the function of Tat in the nucleolus, where ribosomal RNA synthesis and cell cycle control take place. The impairment of nucleolar pre-rRNA maturation through the interaction of Tat with fibrillarin-U3snoRNA complex suggests a process by which the virus modulates host response, thus contributing to apoptosis and protein shut-off in HIV-uninfected cells.

Crystallization and crystallographic analysis of human NUDT16

Human NUDT16, a decapping enzyme belonging to the Nudix superfamily, plays a pivotal role in U8 snoRNA stability. Recombinant NUDT16 expressed in Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. The crystals, which diffracted to 2.10 A resolution, belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.47, b = 79.32, c = 97.20 A. The Matthews coefficient and the solvent content were calculated to be 1.92 A(3) Da(-1) and 35.84%, respectively, for two molecules per asymmetric unit.

Identification of Genes That Function in the Biogenesis and Localization of Small Nucleolar RNAs in Saccharomyces cerevisiae

Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.

Nonpolyadenylated RNA Polymerase II Termination Is Induced by Transcript Cleavage

Although the termination of transcription and 3' RNA processing of the eukaryotic mRNA has been linked to a polyadenylation signal and a transcript cleavage process, much less is known about the termination or processing of nonpolyadenylated RNA polymerase II transcripts. An efficiently expressed plasmid-based expression system was used to study the termination and processing of Schizosaccharomyces pombe U3 small nucleolar RNA (snoRNA) transcripts in vivo. The termination assay was linked to cell transformation, and restriction fragment length polymorphism was used to determine levels of plasmid-derived U3 snoRNA. Mutation analyses in vivo indicate that the maturation of the 3' end is not directly dependent on an external cis-acting sequence or structure; rather, it is dependent on a transcript cleavage that can occur hundreds or even thousands of nucleotides downstream of the mature U3 snoRNA sequence. Similarly, termination is dependent on the same transcript cleavage that is localized in a hairpin structure that normally follows the 3' end of the U3 snoRNA but that also can be moved hundreds or thousands of nucleotides downstream. Both processes, however, can be induced simultaneously and equally efficiently with a single unrelated Pac1 endonuclease-labile structure. The results support a "reversed torpedoes" model in which a single cleavage allows exonucleases and/or other protein factors access to the transcript leading to transcription termination in one direction and RNA maturation in the other direction.

Association of guanine nucleotide-exchange protein BIG1 in HepG2 cell nuclei with nucleolin, U3 snoRNA, and fibrillarin

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, activates class I ADP-ribosylation factors (ARF1-3) by catalyzing the replacement of bound GDP by GTP, an action critical for the regulation of protein transport in eukaryotic cells. Our earlier report [Padilla PI, Pancheco-Rodriguez G, Moss J, Vaughan M (2004) Proc Natl Acad Sci USA 101:2752-2757] that BIG1 concentrated in nucleoli of serum-starved HepG2 cells prompted us to identify molecules associated with BIG1 in dynamic nucleolar structures. Antibodies against BIG1 or nucleolin coprecipitated both proteins from nuclei, which was abolished by the incubation of nuclei with RNase A or DNase, indicating that the interaction depended on nucleic acids. (32)P labeling of RNAs immunoprecipitated with BIG1 or nucleolin from nuclei revealed bands of approximately 210 bases that also hybridized with U3 small nucleolar (sno)RNA-specific oligonucleotides. Clones of U3 snoRNA cDNAs from the material precipitated by antibodies against BIG1 or nucleolin yielded identical nucleotide sequences that also were found in genomic DNA. Later analyses revealed the presence of fibrillarin, nucleoporin p62, and La in BIG1 and nucleolin immunoprecipitates. Our data demonstrate that BIG1, nucleolin, U3, the U3-binding protein fibrillarin, and the RNA-binding protein La may exist together in nuclear complexes, consistent with a potential role for BIG1 in nucleolar processes. Evidence that BIG1 and nucleolin, but not fibrillarin, can be present with p62 at the nuclear envelope confirms the presence of BIG1 and nucleolin in dynamic molecular complexes that change in composition while moving through nuclei. Nuclear functions of BIG1 remain to be determined.

Post‐transcriptional regulation of the U3 small nucleolar RNA

A high copy shuttle vector was used to express a “tagged” U3 snoRNA gene in Schizosaccharomyces pombe to examine regulatory responses to a high gene dosage. RNA analyses utilizing RT‐PCR amplification and restriction fragment length polymorphism indicated that the “tagged” gene was both proportionally and highly expressed and that downstream processing and/or termination were critical to U3 snoRNA stability. In contrast, direct measurements of the total cellular U3 snoRNA showed essentially normal levels of mature RNA although measurements of precursor levels confirmed a highly expressed gene construct. Taken together the results indicated that the steady state amounts of mature U3 snoRNA were primarily regulated at the post‐transcriptional level. This regulatory mechanism prevents an over accumulation of the cellular U3 snoRNA and can efficiently degrade mutant RNA molecules. Together with past studies on other 3’ extended RNA precursors, the results support post‐transcriptional regulation as a quality control mechanism in which appropriate amounts of functional RNA are stabilized by protein interaction while excess or defective RNA is rapidly degraded. Precursor processing in vitro and mutational analyses were consistent with this model. Supported by the Natural Sciences and Engineering Research Council of Canada.

Cwc24p, a novel S. cerevisiae RING‐finger protein, affects U3 snoRNA splicing and rRNA processing

Ribosome biogenesis involve the extensive processing of a precursor rRNA 35S by approximately 150 trans‐acting factors, giving rise to the mature rRNAs. Among these factors are snoRNPs (small nucleolar ribonucleoproteins), that are formed by a guide snoRNA (that base‐pairs with target sites within the pre‐rRNA) and evolutionarily conserved proteins. In this work we present the functional characterization of Cwc24p, a novel S. cerevisiae RING‐finger that interacts with Nop17p (a box C/D snoRNP assembly factor) and with Cef1p (a splicing factor, component of the NTC complex). Consistent with the interaction data, Cwc24p localizes to the cell nucleus. Using a conditional strain (GAL1::CWC24) we observed a small defect in rRNA processing upon depletion of Cwc24p. In order to investigate the involvement of Cwc24p in splicing, a splicing‐specific microarray analysis was conducted and revealed that depletion of Cwc24p leads to the accumulation of both U3 pre‐snoRNAs. U3 snoRNA, which is transcribed from two intron‐containing genes in yeast, is involved in the early cleavages of 35S pre‐rRNA, and the defective splicing of pre‐U3 detected in cells depleted of Cwc24p may be responsible for the accumulation of the 35S precursor rRNA. These results led us to the conclusion that Cwc24p is involved in pre‐U3 snoRNA splicing, indirectly affecting pre‐rRNA processing.Supported by FAPESP and CAPES

Brefeldin A‐inhibited guanine nucleotide‐exchange protein 1 (BIG1) associates with nucleolin and U3 snoRNA in HepG2 cell nuclei

BIG1, a brefeldin A‐inhibited guanine nucleotide‐exchange protein, activates class I ADP‐ribosylation factors (ARF1–3) by catalyzing replacement of bound GDP by GTP, an action critical in secretory transport from endoplasmic reticulum to cis‐Golgi. Our earlier report that BIG1 concentrated in nucleoli of serum‐starved HepG2 cells (Padilla et al., 2004) prompted us to identify molecules associated with BIG1 in the nucleolus. Antibodies against BIG1 co‐precipitated nucleolin (No, an abundant nucleolar protein) from nuclei; likewise, antibodies against No would co‐precipitate BIG1. Incubation of nuclei with RNAse or DNAse abolished co‐immunoprecipitation of BIG1 and No indicating the interaction depended on nucleic acids. Analysis of small nucleolar (sno)RNAs immunoprecipitated with BIG1 and No from nuclei revealed predominant bands of ~210 and ~70 bases; hybridization of the larger with U3‐specific oligonucleotides confirmed its identity. Cloning of U3 snoRNA cDNAs from the material precipitated by antibodies against BIG1 or No yielded identical nucleotide sequences. Continuing characterization of the mechanism and function of BIG1 accumulation in nucleoli of serum‐deprived cells may facilitate their comparison with those of the rapid nuclear accumulation of BIG1 that resulted from protein kinase A‐catalyzed phosphorylation of BIG1 Ser 883. Supported by Intramural Research Program, NHLBI, NIH.

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.