scholarly journals Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis.

1993 ◽  
Vol 13 (4) ◽  
pp. 2469-2477 ◽  
Author(s):  
J P Morrissey ◽  
D Tollervey
Keyword(s):  
18S Rrna ◽  
Progressive Increase ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Small Nucleolar Rna ◽  
Conditional Allele ◽  
Cell Doubling Time ◽  
Associated Proteins ◽  
Nucleolar Rna

Subnuclear fractionation and coprecipitation by antibodies against the nucleolar protein NOP1 demonstrate that the essential Saccharomyces cerevisiae RNA snR30 is localized to the nucleolus. By using aminomethyl trimethyl-psoralen, snR30 can be cross-linked in vivo to 35S pre-rRNA. To determine whether snR30 has a role in rRNA processing, a conditional allele was constructed by replacing the authentic SNR30 promoter with the GAL10 promoter. Repression of snR30 synthesis results in a rapid depletion of snR30 and a progressive increase in cell doubling time. rRNA processing is disrupted during the depletion of snR30; mature 18S rRNA and its 20S precursor underaccumulate, and an aberrant 23S pre-rRNA intermediate can be detected. Initial results indicate that this 23S pre-rRNA is the same as the species detected on depletion of the small nucleolar RNA-associated proteins NOP1 and GAR1 and in an snr10 mutant strain. It was found that the 3' end of 23S pre-rRNA is located in the 3' region of ITS1 between cleavage sites A2 and B1 and not, as previously suggested, at the B1 site, snR30 is the fourth small nucleolar RNA shown to play a role in rRNA processing.

Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis

1993 ◽  
Vol 13 (4) ◽  
pp. 2469-2477
Author(s):  
J P Morrissey ◽  
D Tollervey
Keyword(s):  
18S Rrna ◽  
Progressive Increase ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Small Nucleolar Rna ◽  
Conditional Allele ◽  
Cell Doubling Time ◽  
Associated Proteins ◽  
Nucleolar Rna

Subnuclear fractionation and coprecipitation by antibodies against the nucleolar protein NOP1 demonstrate that the essential Saccharomyces cerevisiae RNA snR30 is localized to the nucleolus. By using aminomethyl trimethyl-psoralen, snR30 can be cross-linked in vivo to 35S pre-rRNA. To determine whether snR30 has a role in rRNA processing, a conditional allele was constructed by replacing the authentic SNR30 promoter with the GAL10 promoter. Repression of snR30 synthesis results in a rapid depletion of snR30 and a progressive increase in cell doubling time. rRNA processing is disrupted during the depletion of snR30; mature 18S rRNA and its 20S precursor underaccumulate, and an aberrant 23S pre-rRNA intermediate can be detected. Initial results indicate that this 23S pre-rRNA is the same as the species detected on depletion of the small nucleolar RNA-associated proteins NOP1 and GAR1 and in an snr10 mutant strain. It was found that the 3' end of 23S pre-rRNA is located in the 3' region of ITS1 between cleavage sites A2 and B1 and not, as previously suggested, at the B1 site, snR30 is the fourth small nucleolar RNA shown to play a role in rRNA processing.

scholarly journals Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing

1999 ◽  
Vol 19 (9) ◽  
pp. 6012-6019 ◽  
Author(s):  
Kishor Sharma ◽  
David Tollervey
Keyword(s):  
18S Rrna ◽  
Homologous Region ◽  
Compensatory Mutation ◽  
Loop Structure ◽  
Rrna Processing ◽  
Base Pairing ◽  
Small Nucleolar Rna ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Nucleolar Rna

ABSTRACT The loop of a stem structure close to the 5′ end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A1, the 5′ end of the 18S rRNA, and at site A2, located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3–pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In theEscherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5′ external transcribed spacer (5′ ETS). Cleavage at site A0in the yeast 5′ ETS strictly requires base pairing between U3 and a sequence within the 5′ ETS. In contrast, the U3-18S interaction is not required for A0 cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A1 cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A1 cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing.

scholarly journals Functional Mapping of the U3 Small Nucleolar RNA from the Yeast Saccharomyces cerevisiae

1998 ◽  
Vol 18 (6) ◽  
pp. 3431-3444 ◽  
Author(s):  
Dmitry A. Samarsky ◽  
Maurille J. Fournier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
18S Rrna ◽  
Glucose Medium ◽  
Small Nucleolar Rna ◽  
Yeast Saccharomyces Cerevisiae ◽  
Tag Effects ◽  
Functional Map ◽  
Rrna Cleavage ◽  
Nucleolar Rna

ABSTRACT 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.

Three new small nucleolar RNAs that are psoralen cross-linked in vivo to unique regions of pre-rRNA

1993 ◽  
Vol 13 (7) ◽  
pp. 4382-4390
Author(s):  
O J Rimoldi ◽  
B Raghu ◽  
M K Nag ◽  
G L Eliceiri
Keyword(s):  
Small Rnas ◽  
18S Rrna ◽  
Rnase H ◽  
Antisense Oligodeoxynucleotide ◽  
28S Rrna ◽  
Small Nucleolar Rnas ◽  
Rrna Sequence ◽  
Nucleolar Rna ◽  
Nucleolar Rnas

We have recently described three novel human small nucleolar RNA species with unique nucleotide sequences, which were named E1, E2, and E3. The present article describes specific psoralen photocross-linking in whole HeLa cells of E1, E2, and E3 RNAs to nucleolar pre-rRNA. These small RNAs were cross-linked to different sections of pre-rRNA. E1 RNA was cross-linked to two segments of nucleolar pre-rRNA; one was within residues 697 to 1163 of the 5' external transcribed spacer, and the other one was between nucleotides 664 and 1021 of the 18S rRNA sequence. E2 RNA was cross-linked to a region within residues 3282 to 3667 of the 28S rRNA sequence. E3 RNA was cross-linked to a sequence between positions 1021 and 1639 of the 18S rRNA sequence. Primer extension analysis located psoralen adducts in E1, E2, and E3 RNAs that were enriched in high-molecular-weight fractions of nucleolar RNA. Some of these psoralen adducts might be cross-links of E1, E2, and E3 RNAs to large nucleolar RNA. Antisense oligodeoxynucleotide-targeted RNase H digestion of nucleolar extracts revealed accessible segments in these three small RNAs. The accessible regions were within nucleotide positions 106 to 130 of E1 RNA, positions 24 to 48 and 42 to 66 of E2 RNA, and positions 7 to 16 and about 116 to 122 of E3 RNA. Some of the molecules of these small nucleolar RNAs sedimented as if associated with larger structures when both nondenatured RNA and a nucleolar extract were analyzed.

scholarly journals U17XS8, a small nucleolar RNA with a 12 nt complementarity to 18S rRNA and coded by a sequence repeated in the six introns ofXenopus laevisribosomal protein S8 gene

1994 ◽  
Vol 22 (5) ◽  
pp. 732-741 ◽  
Author(s):  
Francesco Cecconi ◽  
Paolo Mariottini ◽  
Fabrizio Loreni ◽  
Paola Pierandrei-Amaldi ◽  
Nadia Campioni ◽  
...  
Keyword(s):  
18S Rrna ◽  
Small Nucleolar Rna ◽  
Nucleolar Rna

scholarly journals Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing

1999 ◽  
Vol 19 (8) ◽  
pp. 5441-5452 ◽  
Author(s):  
Sarah J. Lee ◽  
Susan J. Baserga
Keyword(s):  
Ribosomal Proteins ◽  
18S Rrna ◽  
Rna Binding ◽  
Specific Protein ◽  
Rrna Processing ◽  
Binding Domains ◽  
U3 Snorna ◽  
Genes Encoding ◽  
Protein Components

ABSTRACT The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding pre-rRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.

scholarly journals Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs

2003 ◽  
Vol 23 (19) ◽  
pp. 6982-6992 ◽  
Author(s):  
Philip Mitchell ◽  
Elisabeth Petfalski ◽  
Rym Houalla ◽  
Alexandre Podtelejnikov ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Mrna Decay ◽  
Nuclear Protein ◽  
Rrna Processing ◽  
Small Nucleolar Rna ◽  
Whole Cell ◽  
Cell Lysates ◽  
Small Nuclear Rnas ◽  
Nuclear Exosome ◽  
Nucleolar Rna

ABSTRACT 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 Mg2+-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-Δ 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.

scholarly journals Synthesis and Assembly of the Box C+D Small Nucleolar RNPs

2000 ◽  
Vol 20 (8) ◽  
pp. 2650-2659 ◽  
Author(s):  
Denis L. J. Lafontaine ◽  
David Tollervey
Keyword(s):  
18S Rrna ◽  
Protein A ◽  
Small Nucleolar Rnas ◽  
Rrna Processing ◽  
Bona Fide ◽  
Polycistronic Transcripts ◽  
Nucleolar Rnas

ABSTRACT Two core small nucleolar RNP (snoRNP) proteins, Nop1p (fibrillarin in vertebrates) and Nop58p (also known as Nop5p) have previously been reported to be specifically associated with the box C+D class of small nucleolar RNAs (snoRNAs). Here we report that Nop56p, a protein related in sequence to Nop58p, is a bona fide box C+D snoRNP component; all tested box C+D snoRNAs were coprecipitated with protein A-tagged Nop56p. Analysis of in vivo snoRNP assembly indicated that Nop56p was stably associated with the snoRNAs only in the presence of Nop1p. In contrast, Nop58p and Nop1p associate independently with the snoRNAs. Genetic depletion of Nop56p resulted in inhibition of early pre-rRNA processing events at sites A0, A1, and A2 and mild depletion of 18S rRNA. However, Nop56p depletion did not lead to codepletion of the box C+D snoRNAs. This is in contrast to Nop58p, which was required for the accumulation of all tested box C+D snoRNAs. Unexpectedly, we found that Nop1p was specifically required for the synthesis and accumulation of box C+D snoRNAs processed from pre-mRNA introns and polycistronic transcripts.

scholarly journals Dhr1p, a Putative DEAH-Box RNA Helicase, Is Associated with the Box C+D snoRNP U3

2000 ◽  
Vol 20 (19) ◽  
pp. 7238-7246 ◽  
Author(s):  
Alan Colley ◽  
Jean D. Beggs ◽  
David Tollervey ◽  
Denis L. J. Lafontaine
Keyword(s):  
18S Rrna ◽  
Mrna Splicing ◽  
Rrna Synthesis ◽  
Detectable Effect ◽  
Rna Helicases ◽  
Structural Reorganization ◽  
Rrna Cleavage ◽  
Processing Steps

ABSTRACT Putative RNA helicases are involved in most aspects of gene expression. All previously characterized members of the DEAH-box family of putative RNA helicases are involved in pre-mRNA splicing. Here we report the analysis of two novel DEAH-box RNA helicases, Dhr1p and Dhr2p, that were found to be predominantly nucleolar. Both genes are essential for viability, and MET-regulated alleles were therefore created. Depletion of Dhr1p or Dhr2p had no detectable effect on pre-mRNA splicing in vivo or in vitro. Both Dhr1p and Dhr2p were, however, required for 18S rRNA synthesis. Depletion of Dhr2p inhibited pre-rRNA cleavage at sites A0, A1, and A2, while Dhr1p depletion inhibited cleavage at sites A1 and A2. No coprecipitation of snoRNAs was detected with ProtA-Dhr2p, but Dhr1p-ProtA was stably associated with the U3 snoRNA. Depletion of Dhr1p inhibited processing steps that require base pairing of U3 to the 5′ end of the 18S rRNA. We speculate that Dhr1p is targeted to the preribosomal particles by the U3-18S rRNA interaction and is required for the structural reorganization of the rRNA during formation of the central pseudoknot.

scholarly journals Yeast 18S rRNA Dimethylase Dim1p: a Quality Control Mechanism in Ribosome Synthesis?

1998 ◽  
Vol 18 (4) ◽  
pp. 2360-2370 ◽  
Author(s):  
Denis L. J. Lafontaine ◽  
Thomas Preiss ◽  
David Tollervey
Keyword(s):  
18S Rrna ◽  
Small Subunit ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rrna Processing ◽  
Small Subunit Rrna ◽  
Enzymatic Function ◽  
Processing Steps

ABSTRACT One of the few rRNA modifications conserved between bacteria and eukaryotes is the base dimethylation present at the 3′ end of the small subunit rRNA. In the yeast Saccharomyces cerevisiae, this modification is carried out by Dim1p. We previously reported that genetic depletion of Dim1p not only blocked this modification but also strongly inhibited the pre-rRNA processing steps that lead to the synthesis of 18S rRNA. This prevented the formation of mature but unmodified 18S rRNA. The processing steps inhibited were nucleolar, and consistent with this, Dim1p was shown to localize mostly to this cellular compartment. dim1-2 was isolated from a library of conditionally lethal alleles of DIM1. In dim1-2strains, pre-rRNA processing was not affected at the permissive temperature for growth, but dimethylation was blocked, leading to strong accumulation of nondimethylated 18S rRNA. This demonstrates that the enzymatic function of Dim1p in dimethylation can be separated from its involvement in pre-rRNA processing. The growth rate ofdim1-2 strains was not affected, showing the dimethylation to be dispensable in vivo. Extracts of dim1-2 strains, however, were incompetent for translation in vitro. This suggests that dimethylation is required under the suboptimal in vitro conditions but only fine-tunes ribosomal function in vivo. Unexpectedly, when transcription of pre-rRNA was driven by a polymerase II PGKpromoter, its processing became insensitive to temperature-sensitive mutations in DIM1 or to depletion of Dim1p. This observation, which demonstrates that Dim1p is not directly required for pre-rRNA processing reactions, is consistent with the inhibition of pre-rRNA processing by an active repression system in the absence of Dim1p.

Sign in / Sign up

Export Citation Format

Share Document