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Results for “RNA, Small Nucleolar”

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At least 19 recordsLinked to original sources

PubMed2000

Precursors to the U3 small nucleolar RNA lack small nucleolar RNP proteins but are stabilized by La binding.

Almost all small eukaryotic RNAs are processed from transiently stabilized 3'-extended forms. A key question is how and why such intermediates are stabilized and how they can then be processed to the mature RNA. Here we report that yeast U3 is also processed from a 3'-extended precursor. The major 3'-extended forms of U3 (U3-3'I and -II) lack the cap trimethylation present in mature U3 and are not associated with small nucleolar RNP (snoRNP) proteins that bind mature U3, i.e., Nop1p, Nop56p, and Nop58p. Depletion of Nop58p leads to the loss of mature U3 but increases the level of U3-3'I and -II, indicating a requirement for the snoRNP proteins for final maturation. Pre-U3 is cleaved by the endonuclease Rnt1p, but U3-3'I and -II do not extend to the Rnt1p cleavage sites. Rather, they terminate at poly(U) tracts, suggesting that they might be bound by Lhp1p (the yeast homologue of La). Immunoprecipitation of Lhp1p fused to Staphylococcus aureus protein A resulted in coprecipitation of both U3-3'I and -II. Deletion of LHP1, which is nonessential, led to the loss of U3-3'I and -II. We conclude that pre-U3 is cleaved by Rnt1p, followed by exonuclease digestion to U3-3'I and -II. These species are stabilized against continued degradation by binding of Lhp1p. Displacement of Lhp1p by binding of the snoRNP proteins allows final maturation, which involves the exosome complex of 3'-->5' exonucleases.

Base Sequence↗

PubMed2001

Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain.

Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB). We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub-nucleolar structure, the nucleolar body (NB). The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures. Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively. Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p. We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis.

Active Transport, Cell Nucleus↗

PubMed2004

Mouse dyskerin mutations affect accumulation of telomerase RNA and small nucleolar RNA, telomerase activity, and ribosomal RNA processing.

Dyskerin is a nucleolar protein present in small nucleolar ribonucleoprotein particles that modify specific uridine residues of rRNA by converting them to pseudouridine. Dyskerin is also a component of the telomerase complex. Point mutations in the human gene encoding dyskerin cause the skin and bone marrow failure syndrome dyskeratosis congenita (DC). To test the extent to which disruption of pseudouridylation or telomerase activity may contribute to the pathogenesis of DC, we introduced two dyskerin mutations into murine embryonic stem cells. The A353V mutation is the most frequent mutation in patients with X-linked DC, whereas the G402E mutation was identified in a single family. The A353V, but not the G402E, mutation led to severe destabilization of telomerase RNA, a reduction in telomerase activity, and a significant continuous loss of telomere length with increasing numbers of cell divisions during in vitro culture. Both mutations caused a defect in overall pseudouridylation and a small but detectable decrease in the rate of pre-rRNA processing. In addition, both mutant embryonic stem cell lines showed a decrease in the accumulation of a subset of H/ACA small nucleolar RNAs, correlating with a significant decrease in site-specific pseudouridylation efficiency. Interestingly, the H/ACA snoRNAs decreased in the G402E mutant cell line differed from those affected in A353V mutant cells. Hence, our findings show that point mutations in dyskerin may affect both the telomerase and pseudouridylation pathways and the extent to which these functions are altered can vary for different mutations.

Animals↗

PubMed1992

Cap structure of U3 small nucleolar RNA in animal and plant cells is different. gamma-Monomethyl phosphate cap structure in plant RNA.

U3 small nucleolar RNA (snoRNA) is an abundant small RNA involved in the processing of pre-ribosomal RNA of eukaryotic cells. U3 snoRNA has been previously characterized from several sources, including human, rat, mouse, frog, fruit fly, dinoflagellates, slime mold, and yeast; in all these organisms, U3 snoRNA contains trimethylguanosine cap structure. In all instances where investigated, the trimethylguanosine-capped snRNAs including U3 snoRNA, are synthesized by RNA polymerase II. However, in higher plants, the U3 snoRNA is synthesized by RNA polymerase III and contains a cap structure different from trimethylguanosine (Kiss, T., and Solymosy, F. (1990) Nucleic Acids Res. 18, 1941-1949; Marshallsay, C., Kiss, T., and Filipowicz, W. (1990) Nucleic Acids Res. 18, 3451-3458; Kiss, T., Marshallsay, C., and Filipowicz, W. (1991) Cell 65, 517-526). In this study, we present evidence that cowpea and, most likely, tomato plant U3 snoRNA contains a methyl-pppA cap structure. These data show that the same U3 snoRNA contains different cap structures in different species and suggest that the kind of cap structure that an uridylic acid-rich small nuclear RNA contains is dependent on the RNA polymerase responsible for its synthesis. In vitro synthesized plant U3 snoRNA, with pppA or pppG as its 5' end, was converted to methyl-pppA/G cap structure in vitro when incubated with extracts prepared from wheat germ or HeLa cells. These data show that the capping machinery is conserved in organisms as evolutionarily distant as plants and mammals. Nucleotides 1-45 of tomato U3 snoRNA, which are capable of forming a stem-loop structure, are sufficient to direct the methyl cap formation in vitro.

Animals↗

PubMed1996

Functional base-pairing interaction between highly conserved elements of U3 small nucleolar RNA and the small ribosomal subunit RNA.

The U3 nucleolar RNA has a remarkably wide phyletic distribution extending from the Eukarya to the Archaea. It functions in maturation of the small subunit (SSU) rRNA through a mechanism which is as yet unknown but which involves base-pairing with pre-rRNA. The most conserved part of U3 is within 30 nucleotides of the 5' end, but as yet no function for this domain has been proposed. Elements within this domain are complementary to highly conserved sequences in the SSU rRNA which, in the mature form, fold into a universally conserved pseudoknot. The nature of the complementarity suggests a novel mechanism for U3 function whereby U3 facilitates correct folding of the pseudoknot. Wide phylogenetic comparison provides compelling evidence in support of the interaction in that significant complementary changes have taken place, particularly in the archaeon Sulfolobus, which maintain the base-pairing. Base-substitution mutations in yeast U3 designed to disrupt the base-pairing indicate that the interaction is probably essential. These include cold-sensitivity mutations which exhibit phenotypes similar to U3-depletion, but without impairment of the AO processing step, which occurs within the 5' ETS. These phenotypes are consistent with the destabilization of SSU precursors and partial impairment of the processing steps A1, at the 5' ETS/18 S boundary, and A2, within the ITS1.

Animals↗

PubMed1997

U14 small nucleolar RNA makes multiple contacts with the pre-ribosomal RNA.

The small nucleolar RNA (snoRNA) U14 has two regions of extended primary sequence complementarity to the 18S rRNA. The 3' region (domain B) shows the consensus structure for the methylation guide class of snoRNAs, whereas base-pairing between the 5' region (domain A) and the 18S rRNA sequence is required for the formation of functional ribosomes. Between domains A and B lies another essential region (domain Y). Here we report that yeast U14 can be cross-linked in vivo to the pre-rRNA; cross-linking is detected exclusively with the 35S primary transcript. Many nucleotides in U14 that lie outside of domains A and B are cross-linked to the pre-rRNA; in particular the essential domain Y region is cross-linked at several sites. U14 is, therefore, in far more extensive contact with the pre-rRNA than predicted from simple base-pairing models. Moreover, U14 can be cross-linked to other small RNA species. The functional interactions made by U14 during ribosome synthesis are likely to be very complex.

Base Composition↗

PubMed2002

U87 RNA, a novel C/D box small nucleolar RNA from mammalian cells.

A novel 72 nt small nucleolar RNA (snoRNA) called U87 was found in rat liver cells. This RNA possesses the features of C/D box snoRNA family: boxes C, D', C', D, and 11 nt antisense element complementary to 28S ribosomal RNA (rRNA). The vast majority of C/D box snoRNAs direct site-specific 2'-O-ribose methylation of rRNAs. U87 RNA is suggested to be involved in 2'-O-methylation of a G(3468) residue in 28S rRNA. U87 RNA was detected in different mammalian species with slight length variability. Rat and mouse U87 RNA gene was characterized. Unlike the majority of C/D box snoRNAs U87 RNA lacks the terminal stem required for snoRNA processing. However, U87 gene is flanked by 7 bp inverted repeats potentially able to form a terminal stem in U87 RNA precursor.

Animals↗

PubMed2000

Small evolutionarily conserved RNA, resembling C/D box small nucleolar RNA, is transcribed from PWCR1, a novel imprinted gene in the Prader-Willi deletion region, which Is highly expressed in brain.

Prader-Willi syndrome is a complex neurodevelopmental disorder caused by the inactivation or deletion of imprinted, paternally expressed genes in chromosome band 15q11.2. We report the identification and characterization of PWCR1, a novel imprinted gene within that region, and its mouse orthologue, Pwcr1, which was mapped to the conserved syntenic region on mouse chromosome 7. Expressed only from the paternal allele, both genes require the imprinting-center regulatory element for expression and are transcribed from the same strand. They are intronless and do not appear to encode a protein product. High human/mouse sequence similarity (87% identity) is limited to a 99-bp region called "HMCR" (for "human-mouse conserved region"). The HMCR sequence has features of a C/D box small nucleolar RNA (snoRNA) and is represented in an abundant small transcript in both species. Located in nucleoli, snoRNAs serve as methylation guidance RNAs in the modification of ribosomal RNA and other small nuclear RNAs. In addition to the nonpolyadenylated small RNAs, larger polyadenylated PWCR1 transcripts are found in most human tissues, whereas expression of any Pwcr1 RNAs is limited to mouse brain. Genomic sequence analysis reveals the presence of multiple copies of PWCR1 and Pwcr1 that are organized within local tandem-repeat clusters. On a multispecies Southern blot, hybridization to an HMCR probe encoding the putative snoRNA is limited to mammals.

Alleles↗

PubMed1991

Cooperation between CCAAT and octamer motifs in the distal sequence element of the rat U3 small nucleolar RNA promoter.

Mammalian U3 small nucleolar RNA promoters possess a highly conserved distal sequence element (DSE) consisting of CCAAT and octamer motifs separated by 11-12 base pairs. We show here that both motifs are required for transcription of a rat U3D gene in Xenopus oocytes. Deletion of the CCAAT motif leaves residual DSE activity, while removal of the octamer motif does not. Changing the conserved spacing between the two motifs generally inhibits transcription less than deletion of either motif, but increasing the spacing between the motifs by one helical turn of DNA preserves normal levels of transcription. We also show that the rat U3D DSE is functionally equivalent to the human U2 snRNA DSE, which consists of adjacent GC and octamer motifs, and that elements from the Herpes Simplex Virus thymidine kinase promoter can replace part or all of the U3D DSE. These data are apparently paradoxical; despite high evolutionary conservation, the U3 DSE is relatively insensitive to mutation, and other upstream motifs are also able to drive transcription from the U3 basal promoter. We suggest that the conserved structure of the U3 DSE may be required for regulation rather than efficiency of U3 transcription.

Animals↗

PubMed1999

Box H and box ACA are nucleolar localization elements of U17 small nucleolar RNA.

The nucleolar localization elements (NoLEs) of U17 small nucleolar RNA (snoRNA), which is essential for rRNA processing and belongs to the box H/ACA snoRNA family, were analyzed by fluorescence microscopy. Injection of mutant U17 transcripts into Xenopus laevis oocyte nuclei revealed that deletion of stems 1, 2, and 4 of U17 snoRNA reduced but did not prevent nucleolar localization. The deletion of stem 3 had no adverse effect. Therefore, the hairpins of the hairpin-hinge-hairpin-tail structure formed by these stems are not absolutely critical for nucleolar localization of U17, nor are sequences within stems 1, 3, and 4, which may tether U17 to the rRNA precursor by base pairing. In contrast, box H and box ACA are major NoLEs; their combined substitution or deletion abolished nucleolar localization of U17 snoRNA. Mutation of just box H or just the box ACA region alone did not fully abolish the nucleolar localization of U17. This indicates that the NoLEs of the box H/ACA snoRNA family function differently from the bipartite NoLEs (conserved boxes C and D) of box C/D snoRNAs, where mutation of either box alone prevents nucleolar localization.

Animals↗

PubMed2005

UV-crosslinking of E1 small nucleolar RNA to proteins in frog oocytes.

E1/U17 small nucleolar RNA (snoRNA) is a box H/ACA snoRNA. To detect protein bands that UV-crosslink to E1 RNA primarily at uridines, frog oocytes were injected with [alpha-32P]UTP-labeled E1 RNA and incubated, isolated nuclei were UV irradiated, and nuclear contents were digested with RNase A. Wild-type E1 RNA specifically UV-crosslinked to several protein bands. To identify E1 RNA sites involved in these interactions, we tested 21 E1 RNA mutants, each consisting of substitutions in a conserved sequence or structure. UV-crosslinking of different protein bands to E1 RNA depended on one of the following sets of conserved E1 RNA segments: two 5' end RNA sites; five 5' half RNA sites; two 3' half RNA sites; or 14 sites located throughout E1 RNA. Of these conserved E1 RNA sites, UV-crosslinking apparently depended on sequences at 11 sites, and structures at 2 sites. Gel electrophoresis with and without RNA competition detected protein bands that are not common to all of the box H/ACA snoRNAs.

Animals↗

PubMed1996

Characterization of the intron-encoded U19 RNA, a new mammalian small nucleolar RNA that is not associated with fibrillarin.

We have characterized a new member (U19) of a group of mammalian small nuclear RNAs that are not precipitable with antibodies against fibrillarin, a conserved nucleolar protein associated with most of the small nucleolar RNAs characterized to date. Human U19 RNA is 200 nucleotides long and possesses 5'-monophosphate and 3'-hydroxyl termini. It lacks functional boxes C and D, sequence motifs required for fibrillarin binding in many other snoRNAs. Human and mouse RNA are 86% homologous and can be folded into similar secondary structures, a finding supported by the results of nuclease probing of the RNA. In the human genome, U19 RNA is encoded in the intron of an as yet not fully characterized gene and could be faithfully processed from a longer precursor RNA in HeLa cell extracts. During fractionation of HeLa cell nucleolar extracts on glycerol gradients, U19 RNA was associated with higher-order structures of approximately 65S, cosedimenting with complexes containing 7-2/MRP RNA, a conserved nucleolar RNA shown to be involved in 5.8S rRNA processing in yeast cells.

Animals↗

PubMed1998

Functional mapping of the U3 small nucleolar RNA from the yeast Saccharomyces cerevisiae.

The U3 small nucleolar RNA participates in early events of eukaryotic pre-rRNA cleavage and is essential for formation of 18S rRNA. Using an in vivo system, we have developed a functional map of the U3 small nucleolar RNA from Saccharomyces cerevisiae. The test strain features a galactose-dependent U3 gene in the chromosome and a plasmid-encoded allele with a unique hybridization tag. Effects of mutations on U3 production were analyzed by evaluating RNA levels in cells grown on galactose medium, and effects on U3 function were assessed by growing cells on glucose medium. The major findings are as follows: (i) boxes C' and D and flanking helices are critical for U3 accumulation; (ii) boxes B and C are not essential for U3 production but are important for function, most likely due to binding of a trans-acting factor(s); (iii) the 5' portion of U3 is required for function but not stability; and, (iv) strikingly, the nonconserved hairpins 2, 3, and 4, which account for 50% of the molecule, are not required for accumulation or function.

Base Sequence↗

PubMed1998

Processing of a dicistronic small nucleolar RNA precursor by the RNA endonuclease Rnt1.

Small nucleolar RNAs (snoRNAs) are intron encoded or expressed from monocistronic independent transcription units, or, in the case of plants, from polycistronic clusters. We show that the snR190 and U14 snoRNAs from the yeast Saccharomyces cerevisiae are co-transcribed as a dicistronic precursor which is processed by the RNA endonuclease Rnt1, the yeast ortholog of bacterial RNase III. RNT1 disruption results in a dramatic decrease in the levels of mature U14 and snR190 and in accumulation of dicistronic snR190-U14 RNAs. Addition of recombinant Rnt1 to yeast extracts made from RNT1 disruptants induces the chase of dicistronic RNAs into mature snoRNAs, showing that dicistronic RNAs correspond to functional precursors stalled in the processing pathway. Rnt1 cleaves a dicistronic transcript in vitro in the absence of other factors, separating snR190 from U14. Thus, one of the functions of eukaryotic RNase III is, as for the bacterial enzyme, to liberate monocistronic RNAs from polycistronic transcripts.

Base Sequence↗

PubMed2002

The hU3-55K protein requires 15.5K binding to the box B/C motif as well as flanking RNA elements for its association with the U3 small nucleolar RNA in Vitro.

The 15.5K protein directly binds to the 5' stem-loop of the U4 small nuclear RNA, the small nucleolar (sno) RNA box C/D motif, and the U3 snoRNA-specific box B/C motif. The box B/C motif has also been shown to be essential for the association of the U3 small nucleolar ribonucleoprotein-specific protein hU3-55K. We therefore set out to determine how 15.5K and hU3-55K recognize the box B/C motif. By using an in vitro assembly assay, we show that hU3-55K effectively binds a sub-fragment of the U3 snoRNA surrounding the B/C motif that we have named the U3BC RNA. The association of hU3-55K with the U3BC RNA is dependent on the binding of 15.5K to the box B/C motif. The association of hU3-55K with the U3BC RNA was found to be also dependent on a conserved RNA structure that flanks the box B/C motif. Furthermore, we show that hU3-55K, a WD 40 repeat containing protein, directly cross-links to the U3BC RNA. Our data support a new structural model of the box B/C region of the U3 snoRNA in which the box B/C motif is base-paired to form a structure highly similar to that of both the U4 5' stem-loop and the box C/D motif.

Amino Acid Sequence↗

PubMed1986

Isolation and characterization of a human U3 small nucleolar RNA gene.

U3 RNA is an abundant, capped, small nucleolar RNA, implicated in the processing of preribosomal RNA. In this study, a DNA clone coding for U3 RNA (clone U3-1) was isolated from a human genomic library and characterized. The DNA sequence was identical to that of human U3 RNA isolated from HeLa cells. The flanking regions showed homology to the enhancer, promoter, and 3'-processing signal found in U1 and U2 snRNA genes. Further, the recently identified "U3 box" (GATTGGCTGCN10TATGTTAATTATGG) of rat U3 genes (Stroke and Weiner, (1985) J. Mol. Biol. 184, 183-193), was also found in the human U3 gene. This gene was transcribed in Xenopus oocytes; it is the first cloned true human U3 gene.

Base Sequence↗

PubMed1994

Processing of U14 small nucleolar RNA from three different introns of the mouse 70-kDa-cognate-heat-shock-protein pre-messenger RNA.

U14 is a small nucleolar RNA required for the processing of eukaryotic rRNA precursors. The U14 genes of mouse as well as rat, hamster, human, Xenopus and trout are encoded within introns of the constitutively expressed 70-kDa-cognate-heat-shock protein gene (hsc70). We demonstrate here that U14.6 and U14.8 snRNAs, in addition to the previously characterized U14.5, are processed from their respective introns when hsc70 pre-mRNA transcripts containing these intronic snRNAs are injected into Xenopus oocyte nuclei. Identical intermediates are observed in the processing of all three mouse U14 snRNAs indicating similar processing pathways. The production of U14 snRNA processing intermediates possessing either mature 5' or 3' termini demonstrated that processing can occur at either end independent of maturation at the other terminus. Processing of U14.6 from hsc70 intron 6 is not dependent upon the base pairing of intron sequences flanking the 5' and 3' termini of the encoded U14 snRNA molecule. Therefore, excision of an intronic snRNA does not require extending the 5',3' terminal helix of U14 snRNA secondary structure into flanking intron regions as originally suggested. Microinjection of the plasmid vector containing the mouse hsc70/U14.5 snRNA coding region revealed that undetermined plasmid sequences can serve as non-specific promoters to generate spurious RNA transcripts. The processing of these transcripts and examination of the plasmid-initiated transcriptional-start sites indicated that a U14-specific promoter is not present in or around the intron-encoded U14.5 gene. These results strongly suggest that biosynthesis of mouse U14 snRNA results from an intron-processing pathway.

Amino Acid Sequence↗

PubMed2000

Multiple conserved segments of E1 small nucleolar RNA are involved in the formation of a ribonucleoprotein particle in frog oocytes.

E1/U17 small nucleolar RNA (snoRNA) is a box H/ACA snoRNA. To identify E1 RNA elements required for its assembly into a ribonucleoprotein (RNP) particle, we have made substitution mutations in evolutionarily conserved sequences and structures of frog E1 RNA. After E1 RNA was injected into the nucleus of frog oocytes, assembly of this exogenous RNA into an RNP was monitored by non-denaturing gel electrophoresis. Unexpectedly, nucleotide substitutions in many phylogenetically conserved segments of E1 RNA produced RNPs with abnormal gel-electrophoresis patterns. These RNA segments were at least nine conserved sequences and an apparently conserved structure. In another region needed for RNP formation, the requirement may be sequence(s) and/or structure. Base substitutions in each of these and in one additional conserved E1 RNA segment reduced the stability of this snoRNA in frog oocytes. Nucleolar localization was assayed by fluorescence microscopy after injection of fluorescein-labelled RNA. The H box (ANANNA) and the ACA box are both needed for efficient nucleolar localization of frog E1 RNA.

Animals↗