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Results for “RNA, Small Nucleolar”

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PubMed1995

Ribosomal RNA precursor processing by a eukaryotic U3 small nucleolar RNA-like molecule in an archaeon.

An RNA-containing endonuclease that catalyzes the excision and maturation of the 16S ribosomal RNA (rRNA) from the rRNA primary transcript (pre-rRNA) in the hyperthermophilic archaeon Sulfolobus acidocaldarius has been characterized. The ribonucleoprotein was inactivated by micrococcal nuclease treatment and inactivation was reversed by reconstitution with bulk RNA. A 159-nucleotide RNA with sequence and structural similarity to U3 small nucleolar RNAs of eukaryotes copurified with the endonuclease activity. Oligonucleotide-targeted ribonuclease H inactivation of the U3-like RNA component also abolished processing activity. A motif within the U3 homolog is complementary to the region around the three cleavage sites in the pre-RNA substrate. Thus, U3-mediated processing of pre-rRNA is not specific to eukaryotes; its origin predates the divergence of archaea and eukaryotes.

Base Sequence↗

PubMed2004

Novel non-coding RNAs in Dictyostelium discoideum and their expression during development.

The quest for non-coding RNAs (ncRNAs) in the last few years has revealed a surprisingly large number of small RNAs belonging to previously known as well as entirely novel classes. Computational and experimental approaches have uncovered new ncRNAs in all kingdoms of life. In this work, we used a shotgun cloning approach to construct full-length cDNA libraries of small RNAs from the eukaryotic model organism Dictyostelium discoideum. Interestingly, two entirely novel classes of RNAs were identified of which one is developmentally regulated. The RNAs within each class share conserved 5'- and 3'-termini that can potentially form stem structures. RNAs of both classes show predominantly cytoplasmic localization. In addition, based on conserved structure and/or sequence motifs, several of the identified ncRNAs could be divided into classes known from other organisms, e.g. 18 small nucleolar RNA candidates (17 box C/D, of which a few are developmentally regulated, and one box H/ACA). Two ncRNAs showed a high degree of similarity to the small nuclear U2 RNA and signal recognition particle RNA (SRP RNA), respectively. Furthermore, the majority of the regions upstream of the sequences encoding the isolated RNAs share conserved motifs that may constitute new promoter elements.

Animals↗

PubMed2003

Nucleolar clustering of dispersed tRNA genes.

Early transfer RNA (tRNA) processing events in Saccharomyces cerevisiae are coordinated in the nucleolus, the site normally associated with ribosome biosynthesis. To test whether spatial organization of the tRNA pathway begins with nucleolar clustering of the genes, we have probed the subnuclear location of five different tRNA gene families. The results show that tRNA genes, though dispersed in the linear genome, colocalize with 5S ribosomal DNA and U14 small nucleolar RNA at the nucleolus. Nucleolar localization requires tRNA gene transcription-complex formation, because inactivation of the promoter at a single locus removes its nucleolar association. This organization of tRNA genes must profoundly affect the spatial packaging of the genome and raises the question of whether gene types might be coordinated in three dimensions to regulate transcription.

Cell Nucleolus↗

PubMed2004

The 3' termini of small RNAs in Trypanosoma brucei.

Post-transcriptional 3' end formation is an essential step in the maturation of most small RNAs. Knowledge of the precise 3' ends of mature RNAs is essential for defining 3'-processing activities. We have mapped the mature 3' ends of Spliced Leader RNA, the U1, U2, U4, U5, and U6 small nuclear RNAs, the U3 small nucleolar RNA, and the 5S and 5.8S ribosomal RNAs by ligation-mediated PCR in Trypanosoma brucei. With the exception of U5, two classes of 3' ends were observed: flush with the base of a stem-loop structure, and 1-2 nt extended from a stem-loop. Multiple mechanisms and structural features are likely to influence 3' end maturation of RNAs.

Animals↗

PubMed2003

Rrp47p is an exosome-associated protein required for the 3' processing of stable RNAs.

Related exosome complexes of 3'-->5' exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg(2+)-labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3'-->5' exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3' processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-Delta strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3'-extended nuclear pre-mRNAs or the cytoplasmic 3'-->5' mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.

3' Untranslated Regions↗

PubMed2000

Transient nucleolar localization Of U6 small nuclear RNA in Xenopus Laevis oocytes.

Recent studies on the 2'-O-methylation and pseudouridylation of U6 small nuclear RNA (snRNA) hypothesize that these posttranscriptional modifications might occur in the nucleolus. In this report, we present direct evidence for the nucleolar localization of U6 snRNA and analyze the kinetics of U6 nucleolar localization after injection of in vitro transcribed fluorescein-labeled transcripts into Xenopus laevis oocytes. In contrast to U3 small nucleolar RNA (snoRNA) which developed strong nucleolar labeling over 4 h and maintained strong nucleolar signals through 24 h, U6 snRNA localized to nucleoli immediately after injection, but nucleolar staining decreased after 4 h. By 24 h after injection of U6 snRNA, only weak nucleolar signals were observed. Unlike the time-dependent profile of strong nucleolar localization of U6 snRNA or U3 snoRNA, injection of fluorescein-labeled U2 snRNA gave weak nucleolar staining at all times throughout a 24-h period; U2 snRNA modifications are believed to occur outside of the nucleolus. The notion that the decrease of U6 signals over time was due to its trafficking out of nucleoli and not to transcript degradation was supported by the demonstration of U6 snRNA stability over time. Therefore, in contrast to snoRNAs like U3, U6 snRNA transiently passes through nucleoli.

Animals↗

PubMed1999

A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3' end.

Simple sequence repeat telomeric DNA is maintained by a specialized reverse transcriptase, telomerase. The integral RNA subunit of telomerase contains a template region that determines the sequence added to chromosome ends. Aside from providing the template, little is known about the role of the telomerase RNA. In addition, no hypotheses have been suggested to account for the striking evolutionary divergence in size and sequence between telomerase RNAs of ciliates, yeasts, and mammals. We show that the two- to threefold increase in size of the mammalian telomerase RNAs relative to ciliate telomerase RNAs is due to the presence of an extra domain resembling a box H/ACA small nucleolar RNA (snoRNA). The human telomerase RNA (hTR) H/ACA domain is essential in vivo for hTR accumulation, hTR 3' end processing, and telomerase activity. By substituting the U64 box H/ACA snoRNA for the hTR H/ACA domain, we demonstrate that a heterologous snoRNA can function to promote chimeric RNA accumulation and 3' end processing but not telomerase activity. In addition, we show that maturation of full-length hTR and its assembly into active telomerase occur from an mRNA promoter-driven RNA polymerase II transcript but not from a U6 snRNA promoter-driven RNA polymerase III transcript. Finally, we show that a small percentage of hTR is associated with nucleoli. These results have implications for the biogenesis and structure of hTR and the human telomerase ribonucleoprotein complex. They also expand the structural and functional diversity of the box H/ACA snoRNA motif.

Animals↗

PubMed2001

Analysis of intergenic spacer transcripts suggests 'read-around' transcription of the extrachromosomal circular rDNA in Euglena gracilis.

We report here the sequence of the 1743 bp intergenic spacer (IGS) that separates the 3'-end of the large subunit ribosomal RNA (rRNA) gene from the 5'-end of the small subunit (SSU) rRNA gene in the circular, extrachromosomal ribosomal DNA (rDNA) of Euglena gracilis. The IGS contains a 277 nt stretch of sequence that is related to a sequence found in ITS 1, an internal transcribed spacer between the SSU and 5.8S rRNA genes. Primer extension analysis of IGS transcripts identified three abundant reverse transcriptase stops that may be analogous to the transcription initiation site (TIS) and two processing sites (A' and A0) that are found in this region in other eukaryotes. Features that could influence processing at these sites include an imperfect palindrome near site A0 and a sequence near site A' that could potentially base pair with U3 small nucleolar RNA. Our identification of the TIS (verified by mung bean nuclease analysis) is considered tentative because we also detected low-abundance transcripts upstream of this site throughout the entire IGS. This result suggests the possibility of 'read-around' transcription, i.e. transcription that proceeds multiple times around the rDNA circle without termination.

Animals↗

PubMed2005

The Trypanosoma brucei La protein is a candidate poly(U) shield that impacts spliced leader RNA maturation and tRNA intron removal.

By virtue of its preferential binding to poly(U) tails on small RNA precursors and nuclear localisation motif, the La protein has been implicated for a role in the stabilisation and nuclear retention of processing intermediates for a variety of small RNAs in eukaryotic cells. As the universal substrate for trans-splicing, the spliced leader RNA is transcribed as a precursor with just such a tail. La protein was targeted for selective knockdown by inducible RNA interference in Trypanosoma brucei. Of three RNA interference strategies employed, a p2T7-177 vector was the most effective in reducing both the La mRNA as well as the protein itself from induced cells. In the relative absence of La protein T. brucei cells were not viable, in contrast to La gene knockouts in yeast. A variety of potential small RNA substrates were examined under induction, including spliced leader RNA, spliced leader associated RNA, the U1, U2, U4, and U6 small nuclear RNAs, 5S ribosomal RNA, U3 small nucleolar RNA, and tRNATyr. None of these molecules showed significant variance in size or abundance in their mature forms, although a discrete subset of intermediates appear for spliced leader RNA and tRNATyr intron splicing under La depletion conditions. 5'-end methylation in the spliced leader RNA and U1 small nuclear RNA was unaffected. The immediate cause of lethality in T. brucei was not apparent, but may represent a cumulative effect of multiple defects including processing of spliced leader RNA, tRNATyr and other unidentified RNA substrates. This study indicates that La protein binding is not essential for maturation of the spliced leader RNA, but does not rule out the presence of an alternative processing pathway that could compensate for the absence of normally-associated La protein.

Animals↗

PubMed2003

Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin.

Porcine reproductive and respiratory syndrome virus (PRRSV) replicates in the cytoplasm of infected cells, but its nucleocapsid (N) protein localizes specifically to the nucleus and nucleolus. The mechanism of nuclear translocation and whether N associates with particular nucleolar components are unknown. In the present study, we show by confocal microscopy that the PRRSV N protein colocalizes with the small nucleolar RNA (snoRNA)-associated protein fibrillarin. Direct and specific interaction of N with fibrillarin was demonstrated in vivo by the mammalian two-hybrid assay in cells cotransfected with the N and fibrillarin genes and in vitro by the glutathione S-transferase pull-down assay using the expressed fibrillarin protein. Using a series of deletion mutants, the interactive domain of N with fibrillarin was mapped to a region of amino acids 30 to 37. For fibrillarin, the first 80 amino acids, which contain the glycine-arginine-rich region (the GAR domain), was determined to be the domain interactive with N. The N protein was able to bind to the full-length genomic RNA of PRRSV, and the RNA binding domain was identified as the region overlapping with the nuclear localization signal situated at positions 41 to 47. These results suggest that the N protein nuclear transport may be controlled by the binding of RNA to N. The PRRSV N protein was also able to bind to both 28S and 18S ribosomal RNAs. The protein-protein interaction between N and fibrillarin was RNA dependent but independent of N protein phosphorylation. Taken together, our studies demonstrate a specific interaction of the PRRSV nucleocapsid protein with the host cell protein fibrillarin in the nucleolus, and they imply a potential linkage of viral strategies for the modulation of host cell functions, possibly through rRNA precursor processing and ribosome biogenesis.

Animals↗

PubMed2005

mRNA surveillance of expressed pseudogenes in C. elegans.

Messenger RNAs (mRNAs) that contain premature translation termination codons (PTCs) are targeted for rapid degradation in all eukaryotes tested. The mechanisms of nonsense-mediated mRNA decay (NMD) have been described in considerable detail, but the biological roles of NMD in wild-type organisms are poorly understood. mRNAs of wild-type organisms known to be degraded by NMD ("natural targets" of NMD) include by-products of regulated alternative splicing, out-of-frame mRNAs derived from unproductive gene rearrangements, cytoplasmic pre-mRNAs, endogenous retroviral and transposon RNAs, and mRNAs having upstream open reading frames or other unusual sequence features. NMD may function to eliminate aberrant PTC-containing mRNAs in order to protect cells from expression of potentially deleterious truncated proteins. Pseudogenes are nonfunctional genes or gene fragments that accumulate mutations through genetic drift. Such mutations will often introduce shifts of reading frame and/or PTCs, and mRNAs of expressed pseudogenes may thus be substrates of NMD. We demonstrate that mRNAs expressed from C. elegans pseudogenes are degraded by NMD and discuss possible implications for both mRNA surveillance and protein evolution. We describe an expressed pseudogene that encodes a small nucleolar RNA (snoRNA) within an intron and suggest this represents an evolutionary intermediate between snoRNA-encoding host genes that do or do not encode proteins.

Animals↗

PubMed1980

Dictyostelium small nuclear RNA D2 is homologous to rat nucleolar RNA U3 and is encoded by a dispersed multigene family.

We have cloned and partially sequenced a 3.9 kb Bgl II restriction fragment from the genome of the cellular slime mold Dictyostelium discoideum which encodes D2, the most abundant small nuclear RNA (snRNA) in vegetative amoebae. The D2 gene is colinear with the mature snRNA and is surrounded by extremely A + T rich DNA for at least 61 nucleotides 5' to the coding region and 218 nucleotides in the 3' direction. D2 is the only small stable RNA encoded by the cloned DNA fragment. Although D2 RNA is homogeneous in length, a comparison of the RNA and DNA sequencing data indicates that C/U or A/G heterogeneity is present in at least three positions within the D2 RNA sequence. Such minor heterogeneity implies that D2 is encoded by multiple genes, and indeed five different restriction fragments complementary to D2 RNA can be detected when genomic blots are probed with labeled fragments derived from the cloned D2 coding region. Further hybridizaton experiments using the cloned flanking sequences as probes suggest that sequences outside the RNA coding region are not detectably conserved between the five dispersed loci. The D2 RNA sequence is more tha 40% homologous to the small nucleolar RNA U3B from rat Novikoff hepatoma, and the implications of this evolutionary conservation are discussed.

Animals↗

PubMed1999

Gene organization and sequence of the region containing the ribosomal protein genes RPL13A and RPS11 in the human genome and conserved features in the mouse genome.

We have determined the organization and sequence of the region containing two ribosomal protein (rp) genes in the human and mouse genomes. The two genes, human RPL13A and RPS11, and mouse Rpl13a and Rps11, are tandemly located in both genomes with an interval of only 4.6kb in the case of the human genes and 1.6kb in the case of the mouse genes. The human RPL13A and RPS11 are 4236bp and 3254bp in length and comprise eight and five exons respectively, whereas the mouse Rps11 is 1951bp long and has five exons. Structural comparison of these genes, including previously reported mouse Rpl13a, revealed a significant conservation of sequences in the promoter regions. Although most rp genes are dispersed throughout the human genome, the conserved features and adjacent localization indicate possible coordinate transcription of the two genes. Furthermore, we have found that four small nucleolar RNA (snoRNA) genes are located in the introns of the two rp genes, both human and mouse. U32, U33, and U34 snoRNAs are encoded in introns 2, 4, and 5 of RPL13A respectively, and U35 in the sixth intron of RPL13A and the third intron of RPS11. The same organization of these snoRNA genes was also observed in the case of the mouse genes.

Animals↗

PubMed2003

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.

Biological Transport↗

PubMed2005

The snoRNA HBII-52 regulates alternative splicing of the serotonin receptor 2C.

The Prader-Willi syndrome is a congenital disease that is caused by the loss of paternal gene expression from a maternally imprinted region on chromosome 15. This region contains a small nucleolar RNA (snoRNA), HBII-52, that exhibits sequence complementarity to the alternatively spliced exon Vb of the serotonin receptor 5-HT(2C)R. We found that HBII-52 regulates alternative splicing of 5-HT(2C)R by binding to a silencing element in exon Vb. Prader-Willi syndrome patients do not express HBII-52. They have different 5-HT(2C)R messenger RNA (mRNA) isoforms than healthy individuals. Our results show that a snoRNA regulates the processing of an mRNA expressed from a gene located on a different chromosome, and the results indicate that a defect in pre-mRNA processing contributes to the Prader-Willi syndrome.

Alternative Splicing↗

PubMed2004

The small subunit processome is required for cell cycle progression at G1.

Without ribosome biogenesis, translation of mRNA into protein ceases and cellular growth stops. We asked whether ribosome biogenesis is cell cycle regulated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and we determined that it is not regulated in the same manner as in metazoan cells. We therefore turned our attention to cellular sensors that relay cell size information via ribosome biogenesis. Our results indicate that the small subunit (SSU) processome, a complex consisting of 40 proteins and the U3 small nucleolar RNA necessary for ribosome biogenesis, is not mitotically regulated. Furthermore, Nan1/Utp17, an SSU processome protein, does not provide a link between ribosome biogenesis and cell growth. However, when individual SSU processome proteins are depleted, cells arrest in the G1 phase of the cell cycle. This arrest was further supported by the lack of staining for proteins expressed in post-G1. Similarly, synchronized cells depleted of SSU processome proteins did not enter G2. This suggests that when ribosomes are no longer made, the cells stall in the G1. Therefore, yeast cells must grow to a critical size, which is dependent upon having a sufficient number of ribosomes during the G1 phase of the cell cycle, before cell division can occur.

Cell Cycle↗

PubMed2001

Analysis of 41 kb of the DNA sequence from the right arm of chromosome II of Schizosaccharomyces pombe.

We report the complete sequence of cosmid c18A7 (41 046 bp insert), located on the right arm of chromosome II of the Schizosaccharomyces pombe genome. The sequence, which partially overlaps with cosmids SPBC4F6 and SPBC336, contains 16 open reading frames (ORFs) capable of coding for proteins of at least 100 amino acid residues in length (one partial) and one small nucleolar RNA (snoRNA). Four known genes were found: swi10 (encoding a mating-type switching protein also involved in nucleotide excision repair); dim1 (encoding a dimethyladenosine transferase); arf1 (encoding ADP-ribosylation factor 1); and pol3 (cdc6) the partial fragment, encoding the 125 kDa catalytic subunit of the DNA polymerase type B. Six ORFs similar to known proteins were found. They include a transporter of the major facilitator superfamily class, a vacuolar sorting protein, an asparagine synthase, a nuclear protein, a reticulum oxidoreductin and a heat shock protein. Each protein product of the other six ORFs has conserved domains and can be assigned a molecular, but not a biological, function. The sequence has been submitted to the EMBL database under Accession No. AL080287.

Amino Acid Sequence↗

PubMed2000

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.

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.

Amino Acid Motifs↗