50 results on '"Petfalski E"'
Search Results
2. The yeast exosome and human PM-Scl are related complexes of 3 '-> 5 ' exonucleases
- Author
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Allmang, C, Petfalski, E, Podtelejnikov, A, Mann, M, Tollervey, D, and Mitchell, P
- Subjects
RRP4 ,Exoribonucleases ,exosome ,polymyositis-scleroderma - Abstract
We previously identified a complex of 3' --> 5' exoribonucleases, designated the exosome, that is expected to play a major role in diverse RNA processing and degradation pathways. Further biochemical and genetic analyses have revealed six novel components of the complex. Therefore, the complex contains 11 components, 10 of which are predicted to be 3' --> 5' exoribonucleases on the basis of sequence homology. Human homologs were identified for 9 of the 11 yeast exosome components, three of which complement mutations in the respective yeast genes. Two of the newly identified exosome components are homologous to known components of the PM-Scl particle, a multisubunit complex recognized by autoimmune sera of patients suffering from polymyositis-scleroderma overlap syndrome. We demonstrate that the homolog of the Rrp4p exosome subunit is also a component of the PM-Scl complex, thereby providing compelling evidence that the yeast exosome and human PM-Scl complexes are functionally equivalent. The two complexes are similar in size, and biochemical fractionation and indirect immunofluorescence experiments show that, in both yeast and humans, nuclear and cytoplasmic forms of the complex exist that differ only by the presence of the Rrp6p/PM-Scl100 subunit exclusively in the nuclear complex.
- Published
- 1999
3. The Yeast Exosome and Human PM-Scl are Related Complexes of 3'->5' Exonucleases
- Author
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Allmang, C., Petfalski, E., Podtelejnikov, A., Mann, M., Tollervey, D., and Mitchell, P.
- Published
- 1999
4. Recognition of cleavage site A(2) in the yeast pre-rRNA
- Author
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Christine Allmang, Henry Y, Wood H, Jp, Morrissey, Petfalski E, and Tollervey D
- Subjects
Base Sequence ,RNA, Small Nuclear ,Molecular Sequence Data ,Mutation ,RNA Precursors ,Saccharomyces cerevisiae ,Sequence Analysis ,Conserved Sequence ,Research Article - Abstract
Processing of the yeast pre-rRNA at site A(2) internal transcribed spacer 1(ITS1) has been shown to require several small nucleolar ribonucleoprotein particles (snoRNPs) as trans-acting factors. Here we report a detailed mutational analysis of the cid-acting signals required to specify the site of A(2) lie in the 3'-flanking sequence; deletion or substitution of nucleotides in this region strongly inhibits processing, and residual cleavage is inaccurate at the nucleotide level. In contrast, the deletion of the 5'- flanking nucleotides has no detectable effect on processing. An evolutionarily conserved sequence, ACAC, is located at the site of cleavage. Substitution of the 3' AC leads to heterogeneous cleavage, with activation of cleavage at an upstream ACAC sequence, In all mutants that retain an ACAC element, a site of cleavage is detected immediately 5' to this sequence, showing that this element is recognized. An ACAC sequence is, however, not essential for accurate cleavage of site A(2). An additional signal is also present 3' to A(2), in a region that has the potential to form a stem-loop structure that is evolutionarily conserved, but of low stability. As has been found for site A(1) (the 5' end of the yeast 18S rRNA), the identification of the site of processing at A(2) relies on multiple recognition elements.
- Published
- 1996
5. The yeast exosome and human PM-Scl are related complexes of 3' right-arrow 5' exonucleases
- Author
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Allmang, C., primary, Petfalski, E., additional, Podtelejnikov, A., additional, Mann, M., additional, Tollervey, D., additional, and Mitchell, P., additional
- Published
- 1999
- Full Text
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6. The 3' end of yeast 5.8S rRNA is generated by an exonuclease processing mechanism.
- Author
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Mitchell, P, primary, Petfalski, E, additional, and Tollervey, D, additional
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- 1996
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7. Synthetic lethality with fibrillarin identifies NOP77p, a nucleolar protein required for pre-rRNA processing and modification.
- Author
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Bergès, T., primary, Petfalski, E., additional, Tollervey, D., additional, and Hurt, E.C., additional
- Published
- 1994
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8. The POP1 gene encodes a protein component common to the RNase MRP and RNase P ribonucleoproteins.
- Author
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Lygerou, Z, primary, Mitchell, P, additional, Petfalski, E, additional, Séraphin, B, additional, and Tollervey, D, additional
- Published
- 1994
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9. The 5′ end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site.
- Author
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Henry, Y., primary, Wood, H., additional, Morrissey, J.P., additional, Petfalski, E., additional, Kearsey, S., additional, and Tollervey, D., additional
- Published
- 1994
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10. Degradation of ribosomal RNA precursors by the exosome.
- Author
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Allmang, C, Mitchell, P, Petfalski, E, and Tollervey, D
- Abstract
The yeast exosome is a complex of 3'-->5' exonucleases involved in RNA processing and degradation. All 11 known components of the exosome are required during 3' end processing of the 5.8S rRNA. Here we report that depletion of each of the individual components inhibits the early pre-rRNA cleavages at sites A(0), A(1), A(2)and A(3), reducing the levels of the 32S, 20S, 27SA(2)and 27SA(3)pre-rRNAs. The levels of the 27SB pre-rRNAs were also reduced. Consequently, both the 18S and 25S rRNAs were depleted. Since none of these processing steps involves 3'-->5' exonuclease activities, the requirement for the exosome is probably indirect. Correct assembly of trans -acting factors with the pre-ribosomes may be monitored by a quality control system that inhibits pre-rRNA processing. The exosome itself degrades aberrant pre-rRNAs that arise from such inhibition. Exosome mutants stabilize truncated versions of the 23S, 21S and A(2)-C(2)RNAs, none of which are observed in wild-type cells. The putative helicase Dob1p, which functions as a cofactor for the exosome in pre-rRNA processing, also functions in these pre-rRNA degradation activities.
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- 2000
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11. Processing of the yeast pre-rRNA at sites A2and A3is linked
- Author
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Allmang, C., Henry, Y., John Morrissey, Wood, H., Petfalski, E., and Tollervey, D.
12. Recognition of cleavage site A2in the yeast pre-rRNA
- Author
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Allmang, C., Henry, Y., Wood, H., John Morrissey, Petfalski, E., and Tollervey, D.
13. Processing of the yeast pre-rRNA at sites A(2) and A(3) is linked
- Author
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Christine Allmang, Henry Y, Jp, Morrissey, Wood H, Petfalski E, and Tollervey D
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Binding Sites ,Base Sequence ,RNA, Small Nuclear ,Molecular Sequence Data ,Mutation ,RNA Precursors ,Saccharomyces cerevisiae ,Research Article - Abstract
Cleavage of the yeast pre-rRNA at site A(2) in internal transcribed spacer 1 (ITS1) requires multiple snoRNP species, whereas cleavage at site A(3),located 72 nt 3' in ITS1, requires Rnase MRP. Analyses of mutations in the pre- rRNA have revealed an unexpected link between processing at A(2) and A(3). Small substitution mutations in the 3' flanking sequence at A(2) inhibit processing at site A(3), whereas a small deletion at A(3) has been shown to delay processing at site A(2). Moreover, the combination of mutations in cis at both A(2) and A(3) leads to the synthesis of pre-rRNA species with 5' ends within the mature 18S rRNA sequence, at sites between + 482 and + 496. The simultaneous interference with an snoRNP processing complex at site A(2) and an Rnase MPRP complex at site A(3) may activate a pre-rRNA breakdown pathway. The same aberantpre-rRNA species are observed in strains with mutations in the RNA component of Rnase MRP, consistent with interactions between the processing complexes. Furthermore, genetic depletion of the snoRNA, snR30, has been shown to affect the coupling between cleavage by Rnase MRP and subsequent exonuclease digestion.We conclude that an sno-RNP-dependent processing complex that is required for A(2) cleavage and that recognizes the 3' flanking sequence at A(2), interacts with the RNase MRP complex bound to the pre-rRNA around site A(3).
14. Sen34p depletion blocks tRNA splicing in vivo and delays rRNA processing
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Viviana Volta, David Tollervey, Pier Carlo Marchisio, Patrick Linder, Elisabeth Petfalski, Angela Bachi, Bertrand Emery, Stefano Biffo, Simonetta Piatti, Marcello Ceci, Volta, V, Ceci, M, Emery, B, Bachi, A, Petfalski, E, Tollervey, D, Linder, P, Marchisio, P, Piatti, S, and Biffo, S
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Ribosomal Proteins ,RACK1 ,Saccharomyces cerevisiae Proteins ,Translational efficiency ,RNA Splicing ,Biophysics ,Saccharomyces cerevisiae ,Biology ,Biochemistry ,Intermediate Filament Proteins ,RNA, Transfer ,60S ,Polysome ,eIF6 (alias p27BBP) ,Gene Expression Regulation, Fungal ,Endoribonucleases ,RNA Precursors ,RNA Processing, Post-Transcriptional ,RRNA processing ,Molecular Biology ,Eukaryotic Large Ribosomal Subunit ,Translation (biology) ,Cell Biology ,Phosphoproteins ,Molecular biology ,Cell biology ,Kinetics ,RNA, Ribosomal ,Transfer RNA ,RNA splicing ,Pre-rRNA ,eIF3 ,Eukaryotic Ribosome ,Carrier Proteins ,Gene Deletion - Abstract
Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Ase1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation. (c) 2005 Elsevier Inc. All rights reserved.
- Published
- 2005
15. The conserved RNA-binding protein Seb1 promotes cotranscriptional ribosomal RNA processing by controlling RNA polymerase I progression.
- Author
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Duval M, Yague-Sanz C, Turowski TW, Petfalski E, Tollervey D, and Bachand F
- Subjects
- RNA, Ribosomal genetics, RNA, Ribosomal metabolism, RNA Precursors genetics, RNA Precursors metabolism, Transcription, Genetic, RNA Processing, Post-Transcriptional, DNA, Ribosomal metabolism, RNA Polymerase I genetics, RNA Polymerase I metabolism, Schizosaccharomyces genetics
- Abstract
Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional pre-rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pause-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing., (© 2023. The Author(s).)
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- 2023
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16. Nascent Transcript Folding Plays a Major Role in Determining RNA Polymerase Elongation Rates.
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Turowski TW, Petfalski E, Goddard BD, French SL, Helwak A, and Tollervey D
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- Base Composition, Base Sequence, Binding Sites, Chromatin chemistry, Chromatin metabolism, DNA, Ribosomal genetics, DNA, Ribosomal metabolism, Gene Expression Regulation, Fungal, Protein Binding, RNA Folding, RNA Polymerase I metabolism, RNA Polymerase II metabolism, RNA Polymerase III metabolism, RNA Splice Sites, RNA Splicing, RNA, Fungal genetics, RNA, Fungal metabolism, Saccharomyces cerevisiae metabolism, Schizosaccharomyces genetics, Schizosaccharomyces metabolism, Thermodynamics, RNA Polymerase I genetics, RNA Polymerase II genetics, RNA Polymerase III genetics, RNA, Fungal chemistry, Saccharomyces cerevisiae genetics, Transcription Elongation, Genetic
- Abstract
Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5' enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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17. Defining the RNA interactome by total RNA-associated protein purification.
- Author
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Shchepachev V, Bresson S, Spanos C, Petfalski E, Fischer L, Rappsilber J, and Tollervey D
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- Cross-Linking Reagents chemistry, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins metabolism, RNA chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Thiouracil chemistry, RNA metabolism, RNA-Binding Proteins metabolism, Ribonucleoproteins isolation & purification, Thiouracil analogs & derivatives
- Abstract
The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)-associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA-associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA-protein complexes using silica beads. In a variant approach (PAR-TRAPP), RNAs were labelled with 4-thiouracil prior to 350 nm crosslinking. PAR-TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR-TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA-peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)
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- 2019
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18. RNA Binding by Histone Methyltransferases Set1 and Set2.
- Author
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Sayou C, Millán-Zambrano G, Santos-Rosa H, Petfalski E, Robson S, Houseley J, Kouzarides T, and Tollervey D
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- Chromatin metabolism, Histones metabolism, Methylation, RNA Polymerase II metabolism, Transcription Factors metabolism, DNA-Binding Proteins metabolism, Histone-Lysine N-Methyltransferase metabolism, Methyltransferases metabolism, RNA metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Histone methylation at H3K4 and H3K36 is commonly associated with genes actively transcribed by RNA polymerase II (RNAPII) and is catalyzed by Saccharomyces cerevisiae Set1 and Set2, respectively. Here we report that both methyltransferases can be UV cross-linked to RNA in vivo High-throughput sequencing of the bound RNAs revealed strong Set1 enrichment near the transcription start site, whereas Set2 was distributed along pre-mRNAs. A subset of transcripts showed notably high enrichment for Set1 or Set2 binding relative to RNAPII, suggesting functional posttranscriptional interactions. In particular, Set1 was strongly bound to the SET1 mRNA, Ty1 retrotransposons, and noncoding RNAs from the ribosomal DNA (rDNA) intergenic spacers, consistent with its previously reported silencing roles. Set1 lacking RNA recognition motif 2 (RRM2) showed reduced in vivo cross-linking to RNA and reduced chromatin occupancy. In addition, levels of H3K4 trimethylation were decreased, whereas levels of dimethylation were increased. We conclude that RNA binding by Set1 contributes to both chromatin association and methyltransferase activity., (Copyright © 2017 Sayou et al.)
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- 2017
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19. UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly.
- Author
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Hunziker M, Barandun J, Petfalski E, Tan D, Delan-Forino C, Molloy KR, Kim KH, Dunn-Davies H, Shi Y, Chaker-Margot M, Chait BT, Walz T, Tollervey D, and Klinge S
- Subjects
- Gene Expression Regulation, Fungal, RNA Precursors genetics, RNA Processing, Post-Transcriptional, RNA, Ribosomal, 18S, RNA, Small Nucleolar genetics, RNA, Small Nucleolar physiology, Ribosomal Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Molecular Chaperones physiology, RNA, Fungal metabolism, RNA, Small Nucleolar metabolism, Ribosomes metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes-UtpA and UtpB-interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5' end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5' ETS and U3 snoRNA as well as the 3' boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.
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- 2016
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20. Strand-specific, high-resolution mapping of modified RNA polymerase II.
- Author
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Milligan L, Huynh-Thu VA, Delan-Forino C, Tuck A, Petfalski E, Lombraña R, Sanguinetti G, Kudla G, and Tollervey D
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- Binding Sites, Machine Learning, Markov Chains, Phosphorylation, RNA Polymerase II chemistry, RNA, Fungal metabolism, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism, Transcription, Genetic, RNA Polymerase II metabolism, RNA, Messenger metabolism, Saccharomyces cerevisiae genetics
- Abstract
Reversible modification of the RNAPII C-terminal domain links transcription with RNA processing and surveillance activities. To better understand this, we mapped the location of RNAPII carrying the five types of CTD phosphorylation on the RNA transcript, providing strand-specific, nucleotide-resolution information, and we used a machine learning-based approach to define RNAPII states. This revealed enrichment of Ser5P, and depletion of Tyr1P, Ser2P, Thr4P, and Ser7P in the transcription start site (TSS) proximal ~150 nt of most genes, with depletion of all modifications close to the poly(A) site. The TSS region also showed elevated RNAPII relative to regions further 3', with high recruitment of RNA surveillance and termination factors, and correlated with the previously mapped 3' ends of short, unstable ncRNA transcripts. A hidden Markov model identified distinct modification states associated with initiating, early elongating and later elongating RNAPII. The initiation state was enriched near the TSS of protein-coding genes and persisted throughout exon 1 of intron-containing genes. Notably, unstable ncRNAs apparently failed to transition into the elongation states seen on protein-coding genes., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2016
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21. Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits.
- Author
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Turowski TW, Lebaron S, Zhang E, Peil L, Dudnakova T, Petfalski E, Granneman S, Rappsilber J, and Tollervey D
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- Binding Sites, Models, Molecular, Nuclear Proteins metabolism, RNA Cleavage, RNA Precursors metabolism, RNA, Ribosomal metabolism, Ribosomal Proteins chemistry, Ribosomal Proteins metabolism, Ribosome Subunits, Small, Eukaryotic chemistry, Ribosome Subunits, Small, Eukaryotic metabolism, Adenosine Triphosphate metabolism, Protein Serine-Threonine Kinases metabolism, Ribosome Subunits, Small, Eukaryotic enzymology, Saccharomyces cerevisiae Proteins metabolism
- Abstract
During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2014
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22. Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery.
- Author
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Booth DG, Takagi M, Sanchez-Pulido L, Petfalski E, Vargiu G, Samejima K, Imamoto N, Ponting CP, Tollervey D, Earnshaw WC, and Vagnarelli P
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- Cell Nucleolus ultrastructure, Chromosomes, Human ultrastructure, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Ki-67 Antigen genetics, Microscopy, Electron, Molecular Sequence Data, Nuclear Proteins genetics, Nuclear Proteins metabolism, Nucleophosmin, Phosphoproteins genetics, Phosphoproteins metabolism, Phosphorylation, RNA Interference, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Sequence Analysis, Protein, Signal Transduction, Transfection, Nucleolin, Cell Nucleolus metabolism, Chromosomes, Human metabolism, Ki-67 Antigen metabolism, Mitosis, Protein Phosphatase 1 metabolism
- Abstract
When the nucleolus disassembles during open mitosis, many nucleolar proteins and RNAs associate with chromosomes, establishing a perichromosomal compartment coating the chromosome periphery. At present nothing is known about the function of this poorly characterised compartment. In this study, we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells. Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin. Following siRNA depletion of Ki-67, NIFK, B23, nucleolin, and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes. Correlative light and electron microscopy (CLEM) images suggest a near-complete loss of the entire perichromosomal compartment. Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells.DOI: http://dx.doi.org/10.7554/eLife.01641.001., (Copyright © 2014, Booth et al.)
- Published
- 2014
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23. A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease.
- Author
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Granneman S, Petfalski E, and Tollervey D
- Subjects
- Binding Sites, Carrier Proteins genetics, Crystallography, X-Ray, DNA, Ribosomal Spacer, Models, Biological, Multigene Family, Nucleic Acid Conformation, Protein Binding, Ribosomal Proteins genetics, Saccharomyces cerevisiae Proteins genetics, Exonucleases metabolism, Exoribonucleases metabolism, Gene Expression Regulation, Fungal, RNA Precursors genetics, RNA, Ribosomal, 5.8S genetics, Ribosomes metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The 5'-exonuclease Rat1 degrades pre-rRNA spacer fragments and processes the 5'-ends of the 5.8S and 25S rRNAs. UV crosslinking revealed multiple Rat1-binding sites across the pre-rRNA, consistent with its known functions. The major 5.8S 5'-end is generated by Rat1 digestion of the internal transcribed spacer 1 (ITS1) spacer from cleavage site A(3). Processing from A(3) requires the 'A(3)-cluster' proteins, including Cic1, Erb1, Nop7, Nop12 and Nop15, which show interdependent pre-rRNA binding. Surprisingly, A(3)-cluster factors were not crosslinked close to site A(3), but bound sites around the 5.8S 3'- and 25S 5'-regions, which are base paired in mature ribosomes, and in the ITS2 spacer that separates these rRNAs. In contrast, Nop4, a protein required for endonucleolytic cleavage in ITS1, binds the pre-rRNA near the 5'-end of 5.8S. ITS2 was reported to undergo structural remodelling. In vivo chemical probing indicates that A(3)-cluster binding is required for this reorganization, potentially regulating the timing of processing. We predict that Nop4 and the A(3) cluster establish long-range interactions between the 5.8S and 25S rRNAs, which are subsequently maintained by ribosomal protein binding.
- Published
- 2011
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24. Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking.
- Author
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Granneman S, Petfalski E, Swiatkowska A, and Tollervey D
- Subjects
- Base Sequence, Binding Sites, Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, Protein Binding, RNA, Ribosomal, 18S chemistry, RNA, Ribosomal, 18S metabolism, Ribosomal Proteins chemistry, Ribosomal Proteins metabolism, Ribosome Subunits, Small, Eukaryotic chemistry, Saccharomyces cerevisiae chemistry
- Abstract
Understanding of eukaryotic ribosome synthesis has been slowed by a lack of structural data for the pre-ribosomal particles. We report rRNA-binding sites for six late-acting 40S ribosome synthesis factors, three of which cluster around the 3' end of the 18S rRNA in model 3D structures. Enp1 and Ltv1 were previously implicated in 'beak' structure formation during 40S maturation--and their binding sites indicate direct functions. The kinase Rio2, putative GTPase Tsr1 and dimethylase Dim1 bind sequences involved in tRNA interactions and mRNA decoding, indicating that their presence is incompatible with translation. The Dim1- and Tsr1-binding sites overlap with those of homologous Escherichia coli proteins, revealing conservation in assembly pathways. The primary binding sites for the 18S 3'-endonuclease Nob1 are distinct from its cleavage site and were unaltered by mutation of the catalytic PIN domain. Structure probing indicated that at steady state the cleavage site is likely unbound by Nob1 and flexible in the pre-rRNA. Nob1 binds before pre-rRNA cleavage, and we conclude that structural reorganization is needed to bring together the catalytic PIN domain and its target.
- Published
- 2010
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25. Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs.
- Author
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Granneman S, Kudla G, Petfalski E, and Tollervey D
- Subjects
- Base Sequence, Binding Sites, Nucleic Acid Conformation, Protein Binding, RNA Precursors chemistry, RNA, Ribosomal chemistry, RNA, Small Nucleolar chemistry, DNA, Complementary genetics, RNA Precursors metabolism, RNA, Ribosomal metabolism, RNA, Small Nucleolar metabolism, Ultraviolet Rays
- Abstract
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.
- Published
- 2009
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26. Formation and nuclear export of preribosomes are functionally linked to the small-ubiquitin-related modifier pathway.
- Author
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Panse VG, Kressler D, Pauli A, Petfalski E, Gnädig M, Tollervey D, and Hurt E
- Subjects
- Animals, Cysteine Endopeptidases genetics, Fungal Proteins genetics, Phenotype, Protein Subunits metabolism, RNA Precursors metabolism, RNA, Ribosomal metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Small Ubiquitin-Related Modifier Proteins genetics, Ubiquitin metabolism, Ubiquitin-Conjugating Enzymes genetics, Ubiquitin-Conjugating Enzymes metabolism, Yeasts metabolism, Active Transport, Cell Nucleus physiology, Cysteine Endopeptidases metabolism, Fungal Proteins metabolism, Protein Precursors metabolism, Ribosomes metabolism, Signal Transduction physiology, Small Ubiquitin-Related Modifier Proteins metabolism
- Abstract
Ribosomal precursor particles are initially assembled in the nucleolus prior to their transfer to the nucleoplasm and export to the cytoplasm. In a screen to identify thermosensitive (ts) mutants defective in the export of pre-60S ribosomal subunit, we isolated the rix16-1 mutant. In this strain, nucleolar accumulation of the Rpl25-eGFP reporter was complemented by UBA2 (a subunit of the E1 sumoylation enzyme). Mutations in UBC9 (E2 enzyme), ULP1 [small-ubiquitin-related modifier (SUMO) isopeptidase] and SMT3 (SUMO-1) caused 60S export defects. A directed analysis of the SUMO proteome revealed that many ribosome biogenesis factors are sumoylated. Importantly, preribosomal particles along both the 60S and the 40S synthesis pathways were decorated with SUMO, showing its direct involvement. Consistent with this, early 60S assembly factors were genetically linked to SUMO conjugation. Notably, the SUMO deconjugating enzyme Ulp1, which localizes to the nuclear pore complex (NPC), was functionally linked to the 60S export factor Mtr2. Together our data suggest that sumoylation of preribosomal particles in the nucleus and subsequent desumoylation at the NPC is necessary for efficient ribosome biogenesis and export in eukaryotes.
- Published
- 2006
- Full Text
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27. Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit.
- Author
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Schäfer T, Maco B, Petfalski E, Tollervey D, Böttcher B, Aebi U, and Hurt E
- Subjects
- Models, Molecular, Nuclear Proteins metabolism, Phosphorylation, Protein Conformation, Protein Serine-Threonine Kinases, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, Ribosomal Proteins metabolism, Casein Kinase I chemistry, Casein Kinase I metabolism, Ribosomes chemistry, Ribosomes metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the 'beak'. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.
- Published
- 2006
- Full Text
- View/download PDF
28. Sen34p depletion blocks tRNA splicing in vivo and delays rRNA processing.
- Author
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Volta V, Ceci M, Emery B, Bachi A, Petfalski E, Tollervey D, Linder P, Marchisio PC, Piatti S, and Biffo S
- Subjects
- Carrier Proteins metabolism, Endoribonucleases antagonists & inhibitors, Endoribonucleases genetics, Gene Deletion, Gene Expression Regulation, Fungal, Intermediate Filament Proteins metabolism, Kinetics, Phosphoproteins metabolism, RNA Precursors metabolism, RNA, Transfer biosynthesis, Ribosomal Proteins, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins antagonists & inhibitors, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Endoribonucleases physiology, RNA Processing, Post-Transcriptional, RNA Splicing, RNA, Ribosomal metabolism, RNA, Transfer metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins physiology
- Abstract
Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.
- Published
- 2005
- Full Text
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29. RNA degradation by the exosome is promoted by a nuclear polyadenylation complex.
- Author
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LaCava J, Houseley J, Saveanu C, Petfalski E, Thompson E, Jacquier A, and Tollervey D
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, DEAD-box RNA Helicases, Mass Spectrometry, Oligonucleotide Array Sequence Analysis, Polyadenylation, RNA Polymerase II metabolism, RNA Stability, Saccharomyces cerevisiae metabolism, Carrier Proteins metabolism, Cell Nucleus metabolism, DNA-Directed DNA Polymerase metabolism, Polynucleotide Adenylyltransferase metabolism, RNA metabolism, RNA Helicases metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The exosome complex of 3'-5' exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein complexes in vivo. However, the purified exosome showed weak in vitro activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation complex containing a known exosome cofactor, the RNA helicase Mtr4p; a poly(A) polymerase, Trf4p; and a zinc knuckle protein, Air2p. In vitro, the Trf4p/Air2p/Mtr4p polyadenylation complex (TRAMP) showed distributive RNA polyadenylation activity. The presence of the exosome suppressed poly(A) tail addition, while TRAMP stimulated exosome degradation through structured RNA substrates. In vivo analyses showed that TRAMP is required for polyadenylation and degradation of rRNA and snoRNA precursors that are characterized exosome substrates. Poly(A) tails stimulate RNA degradation in bacteria, suggesting that this is their ancestral function. We speculate that this function was maintained in eukaryotic nuclei, while cytoplasmic mRNA poly(A) tails acquired different roles in translation.
- Published
- 2005
- Full Text
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30. Functional link between ribosome formation and biogenesis of iron-sulfur proteins.
- Author
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Yarunin A, Panse VG, Petfalski E, Dez C, Tollervey D, and Hurt EC
- Subjects
- ATP-Binding Cassette Transporters biosynthesis, ATP-Binding Cassette Transporters genetics, Adenosine Triphosphatases genetics, Adenosine Triphosphatases metabolism, Base Sequence, Biological Transport, Active, DNA, Fungal genetics, Eukaryotic Initiation Factor-3 genetics, Eukaryotic Initiation Factor-3 metabolism, GTP-Binding Proteins genetics, GTP-Binding Proteins metabolism, Genes, Fungal, Iron-Sulfur Proteins genetics, Mutation, RNA Precursors genetics, RNA Precursors metabolism, RNA, Fungal genetics, RNA, Fungal metabolism, RNA, Ribosomal genetics, RNA, Ribosomal metabolism, Ribosomes chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Iron-Sulfur Proteins biosynthesis, Ribosomes metabolism, Saccharomyces cerevisiae Proteins biosynthesis
- Abstract
In genetic screens for ribosomal export mutants, we identified CFD1, NBP35 and NAR1 as factors involved in ribosome biogenesis. Notably, these components were recently reported to function in extramitochondrial iron-sulfur (Fe-S) cluster biosynthesis. In particular, Nar1 was implicated to generate the Fe-S clusters within Rli1, a potential substrate protein of unknown function. We tested whether the Fe-S protein Rli1 functions in ribosome formation. We report that rli1 mutants are impaired in pre-rRNA processing and defective in the export of both ribosomal subunits. In addition, Rli1p is associated with both pre-40S particles and mature 40S subunits, and with the eIF3 translation initiation factor complex. Our data reveal an unexpected link between ribosome biogenesis and the biosynthetic pathway of cytoplasmic Fe-S proteins.
- Published
- 2005
- Full Text
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31. Rea1, a dynein-related nuclear AAA-ATPase, is involved in late rRNA processing and nuclear export of 60 S subunits.
- Author
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Galani K, Nissan TA, Petfalski E, Tollervey D, and Hurt E
- Subjects
- ATP-Binding Cassette Transporters chemistry, ATPases Associated with Diverse Cellular Activities, Alleles, Blotting, Northern, Blotting, Western, Cell Nucleus metabolism, Cytoplasm metabolism, DNA, Ribosomal Spacer, Genes, Reporter, Green Fluorescent Proteins metabolism, Magnesium Chloride pharmacology, Membrane Proteins chemistry, Models, Biological, Mutation, Oligonucleotides chemistry, Plasmids metabolism, Protein Structure, Tertiary, RNA chemistry, RNA, Ribosomal, 5.8S chemistry, Receptors, Steroid, Saccharomyces cerevisiae metabolism, Salts pharmacology, Sodium Dodecyl Sulfate chemistry, Adenosine Triphosphatases chemistry, Adenosine Triphosphatases physiology, RNA, Ribosomal metabolism, Ribosomes chemistry, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins physiology
- Abstract
Rea1, the largest predicted protein in the yeast genome, is a member of the AAA(+) family of ATPases and is associated with pre-60 S ribosomes. Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit. Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex. To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperature-sensitive rea1 alleles. In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA. Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects. Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions.
- Published
- 2004
- Full Text
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32. Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse.
- Author
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Newton K, Petfalski E, Tollervey D, and Cáceres JF
- Subjects
- Amino Acid Sequence, Animals, Apoptosis genetics, Apoptosis physiology, Base Sequence, Chromosomal Proteins, Non-Histone deficiency, Chromosomal Proteins, Non-Histone genetics, Embryonic and Fetal Development genetics, Embryonic and Fetal Development physiology, Female, Gene Expression Regulation, Developmental, Heterozygote, Introns, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Molecular Sequence Data, Mutagenesis, Insertional, Phenotype, Pregnancy, RNA Processing, Post-Transcriptional, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Chromosomal Proteins, Non-Histone metabolism, RNA, Small Nucleolar genetics, RNA, Small Nucleolar metabolism
- Abstract
Fibrillarin, a protein component of C/D box small nucleolar ribonucleoproteins (snoRNPs), directs 2'-O-methylation of rRNA and is also involved in other aspects of rRNA processing. A gene trap screen in embryonic stem (ES) cells resulted in an insertion mutation in the fibrillarin gene. This insertion generated a fusion protein that contained the N-terminal 132 amino acids of fibrillarin fused to a beta-galactosidase-neomycin phosphotransferase reporter. As a result, the N-terminal GAR domain was present in the fusion protein but the methyltransferase-like domain was missing. The ES cell line with the targeted fibrillarin allele was transmitted through the mouse germ line, creating heterozygous animals. Western blot analyses showed a reduction in fibrillarin protein levels in the heterozygous knockout animals. Animals homozygous for the mutation were inviable, and massive apoptosis was observed in early Fibrillarin(-/-) embryos, showing that fibrillarin is essential for development. Fibrillarin(+/-) live-born mice displayed no obvious growth defect, but heterozygous intercrosses revealed a reduced ratio of +/- to +/+ mice, showing that some of the Fibrillarin heterozygous embryos die in utero. Analyses of tissue samples and cultured embryonic fibroblasts showed no discernible alteration in pre-rRNA processing or the level of the U3 snoRNA. However, the level of the intron-encoded box C/D snoRNA U76 was clearly reduced. This suggests a high requirement for snoRNA synthesis during an early stage in development.
- Published
- 2003
- Full Text
- View/download PDF
33. Formation and nuclear export of tRNA, rRNA and mRNA is regulated by the ubiquitin ligase Rsp5p.
- Author
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Neumann S, Petfalski E, Brügger B, Grosshans H, Wieland F, Tollervey D, and Hurt E
- Subjects
- Endosomal Sorting Complexes Required for Transport, Exocytosis, Hybridization, Genetic, Lipids analysis, Lipids biosynthesis, Microscopy, Electron, Mutation, Phenotype, Plasmids, RNA Polymerase II genetics, RNA Polymerase II metabolism, RNA, Messenger genetics, RNA, Ribosomal genetics, RNA, Transfer genetics, Ribosomes chemistry, Ribosomes metabolism, Saccharomyces cerevisiae Proteins analysis, Saccharomyces cerevisiae Proteins genetics, Transcription Factors, Ubiquitin-Protein Ligase Complexes analysis, Ubiquitin-Protein Ligase Complexes genetics, Yeasts genetics, Yeasts ultrastructure, RNA, Messenger metabolism, RNA, Ribosomal metabolism, RNA, Transfer metabolism, Saccharomyces cerevisiae Proteins metabolism, Ubiquitin-Protein Ligase Complexes metabolism, Yeasts metabolism
- Abstract
The yeast ubiquitin-protein ligase Rsp5p regulates processes as diverse as polII transcription and endocytosis. Here, we identify Rsp5p in a screen for tRNA export (tex) mutants. The tex23-1/rsp5-3 mutant, which is complemented by RSP5, not only shows a strong nuclear accumulation of tRNAs at the restrictive temperature, but also is severely impaired in the nuclear export of mRNAs and 60S pre-ribosomal subunits. In contrast, nuclear localization sequence (NLS)-mediated nuclear protein import is unaffected in this mutant. Strikingly, the nuclear RNA export defects seen in the rsp5-3 strain are accompanied by a dramatic inhibition of both rRNA and tRNA processing, a combination of phenotypes that has not been reported for any previously characterized mutation in yeast. These data implicate ubiquitination as a mechanism coordinating the major nuclear RNA biogenesis pathways.
- Published
- 2003
- Full Text
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34. Rrp47p is an exosome-associated protein required for the 3' processing of stable RNAs.
- Author
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Mitchell P, Petfalski E, Houalla R, Podtelejnikov A, Mann M, and Tollervey D
- Subjects
- Alternative Splicing, Cell Nucleus metabolism, Exoribonucleases genetics, Exoribonucleases isolation & purification, Humans, Mutation, RNA Precursors metabolism, RNA, Ribosomal metabolism, RNA, Ribosomal, 5.8S metabolism, RNA, Small Nuclear biosynthesis, RNA, Small Nucleolar biosynthesis, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, 3' Untranslated Regions, Exoribonucleases metabolism, RNA metabolism, RNA Processing, Post-Transcriptional
- 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 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.
- Published
- 2003
- Full Text
- View/download PDF
35. The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes.
- Author
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Schäfer T, Strauss D, Petfalski E, Tollervey D, and Hurt E
- Subjects
- Biological Transport, Cell Nucleolus metabolism, Cytoplasm metabolism, Gene Expression Regulation, Fungal, Green Fluorescent Proteins, Heat-Shock Proteins metabolism, Luminescent Proteins metabolism, Models, Biological, Mutation, RNA Precursors isolation & purification, RNA Precursors metabolism, RNA Processing, Post-Transcriptional, RNA, Fungal genetics, RNA, Fungal metabolism, Ribosomal Proteins genetics, Ribosomal Proteins metabolism, Ribosomes chemistry, Ribosomes metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins isolation & purification, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Recent reports have increased our knowledge of the consecutive steps during 60S ribosome biogenesis substantially, but 40S subunit formation is less well understood. Here, we investigate the maturation of nucleolar 90S pre-ribosomes into cytoplasmic 40S pre-ribosomes. During the transition from 90S to 40S particles, the majority of non-ribosomal proteins (approximately 30 species) dissociate, and significantly fewer factors associate with 40S pre-ribosomes. Notably, some of these components are part of both early 90S and intermediate 40S pre-particles in the nucleolus (e.g. Enp1p, Dim1p and Rrp12p), whereas others (e.g. Rio2p and Nob1p) are found mainly on late cytoplasmic pre-40S subunits. Finally, temperature-sensitive mutants mapping either in earlier (enp1-1) or later (rio2-1) components exhibit defects in the formation and nuclear export of pre-40S subunits. Our data provide an initial biochemical map of the pre-40S ribosomal subunit on its path from the nucleolus to the cytoplasm. This pathway involves fewer changes in composition than seen during 60S biogenesis.
- Published
- 2003
- Full Text
- View/download PDF
36. Lsm Proteins are required for normal processing and stability of ribosomal RNAs.
- Author
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Kufel J, Allmang C, Petfalski E, Beggs J, and Tollervey D
- Subjects
- Base Sequence, Blotting, Northern, Gene Deletion, Genotype, Models, Genetic, Molecular Sequence Data, N-Terminal Acetyltransferase C, Phenotype, Precipitin Tests, RNA metabolism, RNA Cap-Binding Proteins, RNA, Messenger metabolism, RNA, Ribosomal, 18S metabolism, RNA, Ribosomal, 5.8S metabolism, RNA-Binding Proteins genetics, Ribonucleoprotein, U4-U6 Small Nuclear genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, RNA, Ribosomal metabolism, RNA-Binding Proteins physiology, Ribonucleoprotein, U4-U6 Small Nuclear physiology, Saccharomyces cerevisiae Proteins physiology
- Abstract
Depletion of any of the essential Lsm proteins, Lsm2-5p or Lsm8p, delayed pre-rRNA processing and led to the accumulation of many aberrant processing intermediates, indicating that an Lsm complex is required to maintain the normally strict order of processing events. In addition, high levels of degradation products derived from both precursors and mature rRNAs accumulated in Lsm-depleted strains. Depletion of the essential Lsm proteins reduced the apparent processivity of both 5' and 3' exonuclease activities involved in 5.8S rRNA processing, and the degradation intermediates that accumulated were consistent with inefficient 5' and 3' degradation. Many, but not all, pre-rRNA species could be coprecipitated with tagged Lsm3p, but not with tagged Lsm1p or non-tagged control strains, suggesting their direct interaction with an Lsm2-8p complex. We propose that Lsm proteins facilitate RNA protein interactions and structural changes required during ribosomal subunit assembly.
- Published
- 2003
- Full Text
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37. 60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm.
- Author
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Nissan TA, Bassler J, Petfalski E, Tollervey D, and Hurt E
- Subjects
- Biological Transport, Active, Cell Nucleolus metabolism, Cytoplasm metabolism, Fungal Proteins genetics, Fungal Proteins metabolism, Models, Biological, RNA Processing, Post-Transcriptional, RNA, Fungal genetics, RNA, Fungal metabolism, RNA, Ribosomal genetics, RNA, Ribosomal metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Ribosomal Proteins genetics, Ribosomal Proteins metabolism, Ribosomes chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, RNA-Binding Proteins, Ribosomes metabolism, Saccharomyces cerevisiae metabolism
- Abstract
60S ribosomes undergo initial assembly in the nucleolus before export to the cytoplasm and recent analyses have identified several nucleolar pre-60S particles. To unravel the steps in the pathway of ribosome formation, we have purified the pre-60S ribosomes associated with proteins predicted to act at different stages as the pre-ribosomes transit from the nucleolus through the nucleoplasm and are then exported to the cytoplasm for final maturation. About 50 non-ribosomal proteins are associated with the early nucleolar pre-60S ribosomes. During subsequent maturation and transport to the nucleoplasm, many of these factors are removed, while others remain attached and additional factors transiently associate. When the 60S precursor particles are close to exit from the nucleus they associate with at least two export factors, Nmd3 and Mtr2. As the 60S pre-ribosome reaches the cytoplasm, almost all of the factors are dissociated. These data provide an initial biochemical map of 60S ribosomal subunit formation on its path from the nucleolus to the cytoplasm.
- Published
- 2002
- Full Text
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38. 90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors.
- Author
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Grandi P, Rybin V, Bassler J, Petfalski E, Strauss D, Marzioch M, Schäfer T, Kuster B, Tschochner H, Tollervey D, Gavin AC, and Hurt E
- Subjects
- Blotting, Northern, Blotting, Western, Centrifugation, Density Gradient, Electrophoresis, Polyacrylamide Gel, Fungal Proteins chemistry, Fungal Proteins genetics, Fungal Proteins metabolism, Macromolecular Substances, Molecular Weight, Protein Subunits, Ribosomes genetics, RNA Precursors metabolism, RNA, Ribosomal metabolism, Ribonucleoproteins, Small Nucleolar metabolism, Ribosomes chemistry, Ribosomes metabolism, Yeasts cytology, Yeasts genetics
- Abstract
We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.
- Published
- 2002
- Full Text
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39. Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing.
- Author
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Gadal O, Strauss D, Petfalski E, Gleizes PE, Gas N, Tollervey D, and Hurt E
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Cell Nucleolus ultrastructure, Conserved Sequence, Fungal Proteins genetics, Fungal Proteins metabolism, Green Fluorescent Proteins, Luminescent Proteins, Molecular Sequence Data, Mutation, Protein Structure, Tertiary, RNA Precursors chemistry, RNA Precursors genetics, RNA Precursors ultrastructure, RNA Processing, Post-Transcriptional, RNA, Fungal chemistry, RNA, Fungal genetics, RNA, Fungal metabolism, RNA, Ribosomal chemistry, RNA, Ribosomal genetics, RNA, Ribosomal ultrastructure, Sequence Alignment, Temperature, Yeasts growth & development, Cell Nucleolus metabolism, RNA Precursors metabolism, RNA, Ribosomal metabolism, Ribosomal Proteins genetics, Ribosomal Proteins metabolism, Yeasts genetics
- Abstract
Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.
- Published
- 2002
- Full Text
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40. Identification of a 60S preribosomal particle that is closely linked to nuclear export.
- Author
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Bassler J, Grandi P, Gadal O, Lessmann T, Petfalski E, Tollervey D, Lechner J, and Hurt E
- Subjects
- Amino Acid Sequence, Animals, Blotting, Northern, Centrifugation, Density Gradient, Fungal Proteins genetics, GTP Phosphohydrolases chemistry, GTP Phosphohydrolases genetics, Genes, Reporter genetics, Humans, Molecular Sequence Data, Nuclear Proteins genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Ribosomal Proteins genetics, Ribosomal Proteins metabolism, Ribosomes chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Sequence Alignment, Temperature, Transformation, Genetic, Active Transport, Cell Nucleus physiology, Cell Nucleus metabolism, Fungal Proteins metabolism, GTP Phosphohydrolases metabolism, Nuclear Proteins metabolism, Ribosomes metabolism, Saccharomyces cerevisiae Proteins
- Abstract
A nuclear GTPase, Nug1p, was identified in a genetic screen for components linked to 60S ribosomal subunit export. Nug1p cosedimented with nuclear 60S preribosomes and was required for subunit export to the cytoplasm. Tagged Nug1p coprecipitated with proteins of the 60S subunit, late precursors to the 25S and 5.8S rRNAs, and at least 21 nonribosomal proteins. These included a homologous nuclear GTPase, Nug2p, the Noc2p/Noc3p heterodimer, Rix1p, and Rlp7p, each of which was implicated in 60S subunit export. Other known ribosome synthesis factors and proteins of previously unknown function, including the 559 kDa protein Ylr106p, also copurified. Eight of these proteins were copurified with nuclear pore complexes, suggesting that this complex represents the transport intermediate for 60S subunit export.
- Published
- 2001
- Full Text
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41. A nuclear AAA-type ATPase (Rix7p) is required for biogenesis and nuclear export of 60S ribosomal subunits.
- Author
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Gadal O, Strauss D, Braspenning J, Hoepfner D, Petfalski E, Philippsen P, Tollervey D, and Hurt E
- Subjects
- Adenosine Triphosphatases genetics, Base Sequence, Biological Transport, Cell Nucleolus metabolism, Cytoplasm metabolism, DNA Primers, Green Fluorescent Proteins, Luminescent Proteins metabolism, Mutation, Nuclear Proteins, RNA Processing, Post-Transcriptional genetics, RNA, Ribosomal metabolism, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Adenosine Triphosphatases metabolism, Cell Nucleus enzymology, Ribosomes metabolism, Saccharomyces cerevisiae Proteins
- Abstract
Ribosomal precursor particles are assembled in the nucleolus before export into the cytoplasm. Using a visual assay for nuclear accumulation of 60S subunits, we have isolated several conditional-lethal strains with defects in ribosomal export (rix mutants). Here we report the characterization of a mutation in an essential gene, RIX7, which encodes a novel member of the AAA ATPase superfamily. The rix7-1 temperature-sensitive allele carries a point mutation that causes defects in pre-rRNA processing, biogenesis of 60S ribosomal subunits, and their subsequent export into the cytoplasm. Rix7p, which associates with 60S ribosomal precursor particles, localizes throughout the nucleus in exponentially growing cells, but concentrates in the nucleolus in stationary phase cells. When cells resume growth upon shift to fresh medium, Rix7p-green fluorescent protein exhibits a transient perinuclear location. We propose that a nuclear AAA ATPase is required for restructuring nucleoplasmic 60S pre-ribosomal particles to make them competent for nuclear export.
- Published
- 2001
- Full Text
- View/download PDF
42. Maturation and intranuclear transport of pre-ribosomes requires Noc proteins.
- Author
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Milkereit P, Gadal O, Podtelejnikov A, Trumtel S, Gas N, Petfalski E, Tollervey D, Mann M, Hurt E, and Tschochner H
- Subjects
- Active Transport, Cell Nucleus physiology, Carrier Proteins genetics, Carrier Proteins metabolism, Cell Compartmentation physiology, Cytoplasm metabolism, Gene Expression Regulation, Fungal, Genotype, Green Fluorescent Proteins, Indicators and Reagents pharmacokinetics, Luminescent Proteins pharmacokinetics, Molecular Sequence Data, Nuclear Proteins metabolism, RNA Precursors metabolism, RNA-Binding Proteins, Ribosomal Proteins biosynthesis, Ribosomal Proteins metabolism, Saccharomyces cerevisiae, Schizosaccharomyces, Schizosaccharomyces pombe Proteins metabolism, Sequence Homology, Amino Acid, Cell Nucleolus metabolism, Heat-Shock Proteins metabolism, Intermediate Filament Proteins metabolism, Nuclear Proteins genetics, Nucleocytoplasmic Transport Proteins, Ribosomes metabolism, Saccharomyces cerevisiae Proteins, Schizosaccharomyces pombe Proteins genetics
- Abstract
How pre-ribosomes temporally and spatially mature during intranuclear biogenesis is not known. Here, we report three nucleolar proteins, Noc1p to Noc3p, that are required for ribosome maturation and transport. They can be isolated in two distinct complexes: Noc1p/Noc2p associates with 90S and 66S pre-ribosomes and is enriched in the nucleolus, and Noc2p/Noc3p associates with 66S pre-ribosomes and is mainly nucleoplasmic. Mutation of each Noc protein impairs intranuclear transport of 60S subunits at different stages and inhibits pre-rRNA processing. Overexpression of a conserved domain common to Noc1p and Noc3p is dominant-negative for cell growth, with a defect in nuclear 60S subunit transport, but no inhibition of pre-rRNA processing. We propose that the dynamic interaction of Noc proteins is crucial for intranuclear movement of ribosomal precursor particles, and, thereby represent a prerequisite for proper maturation.
- Published
- 2001
- Full Text
- View/download PDF
43. Three novel components of the human exosome.
- Author
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Brouwer R, Allmang C, Raijmakers R, van Aarssen Y, Egberts WV, Petfalski E, van Venrooij WJ, Tollervey D, and Pruijn GJ
- Subjects
- Animals, Base Sequence, Cell Nucleus chemistry, Cloning, Molecular, Cytoplasm chemistry, Exoribonucleases genetics, Exosome Multienzyme Ribonuclease Complex, HeLa Cells, Humans, Molecular Sequence Data, Molecular Weight, Precipitin Tests, RNA-Binding Proteins, Rabbits, Exoribonucleases analysis
- Abstract
The yeast exosome is a complex of 3' --> 5' exoribonucleases. Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were expressed and used to raise rabbit antisera. In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes. All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm. Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3' --> 5' exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex.
- Published
- 2001
- Full Text
- View/download PDF
44. Precursors to the U3 small nucleolar RNA lack small nucleolar RNP proteins but are stabilized by La binding.
- Author
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Kufel J, Allmang C, Chanfreau G, Petfalski E, Lafontaine DL, and Tollervey D
- Subjects
- Base Sequence, Endoribonucleases metabolism, Exosome Multienzyme Ribonuclease Complex, Fungal Proteins genetics, Molecular Sequence Data, Multienzyme Complexes genetics, Multienzyme Complexes metabolism, Nuclear Proteins genetics, Nuclear Proteins metabolism, Precipitin Tests, RNA Processing, Post-Transcriptional, RNA Stability, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Recombinant Fusion Proteins metabolism, Ribonuclease III, Ribonucleoproteins, Small Nucleolar genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Staphylococcal Protein A genetics, Staphylococcal Protein A immunology, Staphylococcal Protein A metabolism, Exoribonucleases, Fungal Proteins metabolism, RNA Precursors metabolism, RNA, Small Nucleolar metabolism, RNA-Binding Proteins metabolism, Ribonucleoproteins, Small Nucleolar metabolism, Saccharomyces cerevisiae Proteins
- Abstract
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.
- Published
- 2000
- Full Text
- View/download PDF
45. Functions of the exosome in rRNA, snoRNA and snRNA synthesis.
- Author
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Allmang C, Kufel J, Chanfreau G, Mitchell P, Petfalski E, and Tollervey D
- Subjects
- Base Sequence, Exodeoxyribonuclease V, Nucleic Acid Conformation, RNA Processing, Post-Transcriptional, RNA, Ribosomal chemistry, RNA, Ribosomal metabolism, RNA, Small Nuclear chemistry, RNA, Small Nuclear metabolism, RNA, Small Nucleolar chemistry, RNA, Small Nucleolar metabolism, Saccharomyces cerevisiae genetics, Exodeoxyribonucleases metabolism, RNA, Ribosomal biosynthesis, RNA, Small Nuclear biosynthesis, RNA, Small Nucleolar biosynthesis, Saccharomyces cerevisiae enzymology
- Abstract
The yeast nuclear exosome contains multiple 3'-->5' exoribonucleases, raising the question of why so many activities are present in the complex. All components are required during the 3' processing of the 5.8S rRNA, together with the putative RNA helicase Dob1p/Mtr4p. During this processing three distinct steps can be resolved, and hand-over between different exonucleases appears to occur at least twice. 3' processing of snoRNAs (small nucleolar RNAs) that are excised from polycistronic precursors or from mRNA introns is also a multi-step process that involves the exosome, with final trimming specifically dependent on the Rrp6p component. The spliceosomal U4 snRNA (small nuclear RNA) is synthesized from a 3' extended precursor that is cleaved by Rnt1p at sites 135 and 169 nt downstream of the mature 3' end. This cleavage is followed by 3'-->5' processing of the pre-snRNA involving the exosome complex and Dob1p. The exosome, together with Rnt1p, also participates in the 3' processing of the U1 and U5 snRNAs. We conclude that the exosome is involved in the processing of many RNA substrates and that different components can have distinct functions.
- Published
- 1999
- Full Text
- View/download PDF
46. The yeast exosome and human PM-Scl are related complexes of 3' --> 5' exonucleases.
- Author
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Allmang C, Petfalski E, Podtelejnikov A, Mann M, Tollervey D, and Mitchell P
- Subjects
- Exodeoxyribonucleases, Exosome Multienzyme Ribonuclease Complex, Fungal Proteins metabolism, Humans, Autoantigens metabolism, Exoribonucleases metabolism, Saccharomyces cerevisiae metabolism
- Abstract
We previously identified a complex of 3' --> 5' exoribonucleases, designated the exosome, that is expected to play a major role in diverse RNA processing and degradation pathways. Further biochemical and genetic analyses have revealed six novel components of the complex. Therefore, the complex contains 11 components, 10 of which are predicted to be 3' --> 5' exoribonucleases on the basis of sequence homology. Human homologs were identified for 9 of the 11 yeast exosome components, three of which complement mutations in the respective yeast genes. Two of the newly identified exosome components are homologous to known components of the PM-Scl particle, a multisubunit complex recognized by autoimmune sera of patients suffering from polymyositis-scleroderma overlap syndrome. We demonstrate that the homolog of the Rrp4p exosome subunit is also a component of the PM-Scl complex, thereby providing compelling evidence that the yeast exosome and human PM-Scl complexes are functionally equivalent. The two complexes are similar in size, and biochemical fractionation and indirect immunofluorescence experiments show that, in both yeast and humans, nuclear and cytoplasmic forms of the complex exist that differ only by the presence of the Rrp6p/PM-Scl100 subunit exclusively in the nuclear complex.
- Published
- 1999
- Full Text
- View/download PDF
47. Processing of the precursors to small nucleolar RNAs and rRNAs requires common components.
- Author
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Petfalski E, Dandekar T, Henry Y, and Tollervey D
- Subjects
- Exoribonucleases genetics, Exoribonucleases metabolism, Introns, Mutagenesis, RNA Precursors metabolism, RNA Processing, Post-Transcriptional, RNA, Fungal metabolism, RNA, Ribosomal metabolism, RNA, Small Nuclear metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins
- Abstract
The genes encoding the small nucleolar RNA (snoRNA) species snR190 and U14 are located close together in the genome of Saccharomyces cerevisiae. Here we report that these two snoRNAs are synthesized by processing of a larger common transcript. In strains mutant for two 5'-->3' exonucleases, Xrn1p and Rat1p, families of 5'-extended forms of snR190 and U14 accumulate; these have 5' extensions of up to 42 and 55 nucleotides, respectively. We conclude that the 5' ends of both snR190 and U14 are generated by exonuclease digestion from upstream processing sites. In contrast to snR190 and U14, the snoRNAs U18 and U24 are excised from the introns of pre-mRNAs which encode proteins in their exonic sequences. Analysis of RNA extracted from a dbr1-delta strain, which lacks intron lariat-debranching activity, shows that U24 can be synthesized only from the debranched lariat. In contrast, a substantial level of U18 can be synthesized in the absence of debranching activity. The 5' ends of these snoRNAs are also generated by Xrn1p and Rat1p. The same exonucleases are responsible for the degradation of several excised fragments of the pre-rRNA spacer regions, in addition to generating the 5' end of the 5.8S rRNA. Processing of the pre-rRNA and both intronic and polycistronic snoRNAs therefore involves common components.
- Published
- 1998
- Full Text
- View/download PDF
48. The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'-->5' exoribonucleases.
- Author
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Mitchell P, Petfalski E, Shevchenko A, Mann M, and Tollervey D
- Subjects
- Amino Acid Sequence, Exoribonucleases chemistry, Exoribonucleases genetics, Exoribonucleases isolation & purification, Fungal Proteins chemistry, Fungal Proteins genetics, Fungal Proteins isolation & purification, Genetic Complementation Test, HeLa Cells, Humans, Molecular Sequence Data, Molecular Weight, Mutation, RNA, Ribosomal, 5.8S metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Exoribonucleases metabolism, Fungal Proteins metabolism, Multienzyme Complexes metabolism, RNA Processing, Post-Transcriptional physiology, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins
- Abstract
We identified a complex in S. cerevisiae, the "exosome," consisting of the five essential proteins Rrp4p, Rrp41p, Rrp42p, Rrp43p, and Rrp44p (Dis3p). Remarkably, four of these proteins are homologous to characterized bacterial 3'-->5' exoribonucleases; Rrp44p is homologous to RNase II, while Rrp41p, Rrp42p, and Rrp43p are related to RNase PH. Recombinant Rrp4p, Rrp44p, and Rrp41p are 3'-->5' exoribonucleases in vitro that have distributive, processive, and phosphorolytic activities, respectively. All components of the exosome are required for 3' processing of the 5.8S rRNA. Human Rrp4p is found in a comparably sized complex, and expression of the hRRP4 gene in yeast complements the rrp4-1 mutation. We conclude that the exosome constitutes a highly conserved eukaryotic RNA processing complex.
- Published
- 1997
- Full Text
- View/download PDF
49. Processing of the yeast pre-rRNA at sites A(2) and A(3) is linked.
- Author
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Allmang C, Henry Y, Morrissey JP, Wood H, Petfalski E, and Tollervey D
- Subjects
- Base Sequence, Binding Sites, Molecular Sequence Data, Mutation, RNA, Small Nuclear genetics, RNA Precursors genetics, Saccharomyces cerevisiae genetics
- Abstract
Cleavage of the yeast pre-rRNA at site A(2) in internal transcribed spacer 1 (ITS1) requires multiple snoRNP species, whereas cleavage at site A(3),located 72 nt 3' in ITS1, requires Rnase MRP. Analyses of mutations in the pre- rRNA have revealed an unexpected link between processing at A(2) and A(3). Small substitution mutations in the 3' flanking sequence at A(2) inhibit processing at site A(3), whereas a small deletion at A(3) has been shown to delay processing at site A(2). Moreover, the combination of mutations in cis at both A(2) and A(3) leads to the synthesis of pre-rRNA species with 5' ends within the mature 18S rRNA sequence, at sites between + 482 and + 496. The simultaneous interference with an snoRNP processing complex at site A(2) and an Rnase MPRP complex at site A(3) may activate a pre-rRNA breakdown pathway. The same aberantpre-rRNA species are observed in strains with mutations in the RNA component of Rnase MRP, consistent with interactions between the processing complexes. Furthermore, genetic depletion of the snoRNA, snR30, has been shown to affect the coupling between cleavage by Rnase MRP and subsequent exonuclease digestion.We conclude that an sno-RNP-dependent processing complex that is required for A(2) cleavage and that recognizes the 3' flanking sequence at A(2), interacts with the RNase MRP complex bound to the pre-rRNA around site A(3).
- Published
- 1996
50. Recognition of cleavage site A(2) in the yeast pre-rRNA.
- Author
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Allmang C, Henry Y, Wood H, Morrissey JP, Petfalski E, and Tollervey D
- Subjects
- Base Sequence, Conserved Sequence, Molecular Sequence Data, Mutation, RNA Precursors metabolism, RNA, Small Nuclear genetics, Sequence Analysis, RNA Precursors genetics, RNA, Small Nuclear metabolism, Saccharomyces cerevisiae genetics
- Abstract
Processing of the yeast pre-rRNA at site A(2) internal transcribed spacer 1(ITS1) has been shown to require several small nucleolar ribonucleoprotein particles (snoRNPs) as trans-acting factors. Here we report a detailed mutational analysis of the cid-acting signals required to specify the site of A(2) lie in the 3'-flanking sequence; deletion or substitution of nucleotides in this region strongly inhibits processing, and residual cleavage is inaccurate at the nucleotide level. In contrast, the deletion of the 5'- flanking nucleotides has no detectable effect on processing. An evolutionarily conserved sequence, ACAC, is located at the site of cleavage. Substitution of the 3' AC leads to heterogeneous cleavage, with activation of cleavage at an upstream ACAC sequence, In all mutants that retain an ACAC element, a site of cleavage is detected immediately 5' to this sequence, showing that this element is recognized. An ACAC sequence is, however, not essential for accurate cleavage of site A(2). An additional signal is also present 3' to A(2), in a region that has the potential to form a stem-loop structure that is evolutionarily conserved, but of low stability. As has been found for site A(1) (the 5' end of the yeast 18S rRNA), the identification of the site of processing at A(2) relies on multiple recognition elements.
- Published
- 1996
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