Publications by authors named "Ann L Beyer"

37 Publications

The Transcription Factor THO Promotes Transcription Initiation and Elongation by RNA Polymerase I.

J Biol Chem 2016 Feb 9;291(6):3010-8. Epub 2015 Dec 9.

From the Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0024 and

Although ribosomal RNA represents the majority of cellular RNA, and ribosome synthesis is closely connected to cell growth and proliferation rates, a complete understanding of the factors that influence transcription of ribosomal DNA is lacking. Here, we show that the THO complex positively affects transcription by RNA polymerase I (Pol I). We found that THO physically associates with the rDNA repeat and interacts genetically with Pol I transcription initiation factors. Pol I transcription in hpr1 or tho2 null mutants is dramatically reduced to less than 20% of the WT level. Pol I occupancy of the coding region of the rDNA in THO mutants is decreased to ~50% of WT level. Furthermore, although the percentage of active rDNA repeats remains unaffected in the mutant cells, the overall rDNA copy number increases ~2-fold compared with WT. Together, these data show that perturbation of THO function impairs transcription initiation and elongation by Pol I, identifying a new cellular target for the conserved THO complex.
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http://dx.doi.org/10.1074/jbc.M115.673442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742762PMC
February 2016

Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I.

Mol Cell Biol 2015 Jul 27;35(13):2321-31. Epub 2015 Apr 27.

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA

Spt6 (suppressor of Ty6) has many roles in transcription initiation and elongation by RNA polymerase (Pol) II. These effects are mediated through interactions with histones, transcription factors, and the RNA polymerase. Two lines of evidence suggest that Spt6 also plays a role in rRNA synthesis. First, Spt6 physically associates with a Pol I subunit (Rpa43). Second, Spt6 interacts physically and genetically with Spt4/5, which directly affects Pol I transcription. Utilizing a temperature-sensitive allele, spt6-1004, we show that Spt6 is essential for Pol I occupancy of the ribosomal DNA (rDNA) and rRNA synthesis. Our data demonstrate that protein levels of an essential Pol I initiation factor, Rrn3, are reduced when Spt6 is inactivated, leading to low levels of Pol I-Rrn3 complex. Overexpression of RRN3 rescues Pol I-Rrn3 complex formation; however, rRNA synthesis is not restored. These data suggest that Spt6 is involved in either recruiting the Pol I-Rrn3 complex to the rDNA or stabilizing the preinitiation complex. The findings presented here identify an unexpected, essential role for Spt6 in synthesis of rRNA.
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http://dx.doi.org/10.1128/MCB.01499-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456441PMC
July 2015

DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

Mol Cell Biol 2015 Mar 22;35(5):816-30. Epub 2014 Dec 22.

Département des sciences biologiques and Centre de recherche BioMed, Université du Québec à Montréal, Montreal, Quebec, Canada

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.
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http://dx.doi.org/10.1128/MCB.01348-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323488PMC
March 2015

Kinetic analysis demonstrates a requirement for the Rat1 exonuclease in cotranscriptional pre-rRNA cleavage.

PLoS One 2014 3;9(2):e85703. Epub 2014 Feb 3.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, Scotland.

During yeast ribosome synthesis, three early cleavages generate the 20S precursor to the 18S rRNA component of the 40S subunits. These cleavages can occur either on the nascent transcript (nascent transcript cleavage; NTC) or on the 35S pre-rRNA that has been fully transcribed and released from the rDNA (released transcript cleavage; RTC). These alternative pathways cannot be assessed by conventional RNA analyses, since the pre-rRNA products of NTC and RTC are identical. They can, however, be distinguished kinetically by metabolic labeling and quantified by modeling of the kinetic data. The aim of this work was to use these approaches as a practical tool to identify factors that mediate the decision between utilization of NTC and RTC. The maturation pathways of the 40S and 60S ribosomal subunits are largely distinct. However, depletion of some early-acting 60S synthesis factors, including the 5'-exonuclease Rat1, leads to accumulation of the 35S pre-rRNA and delayed 20S pre-rRNA synthesis. We speculated that this might reflect the loss of NTC. Rat1 acts catalytically in 5.8S and 25S rRNA processing but binds to the pre-rRNA prior to these activities. Kinetic data for strains depleted of Rat1 match well with the modeled effects of strongly reduced NTC. This was confirmed by EM visualization of "Miller" chromatin spreads of nascent pre-rRNA transcripts. Modeling further indicates that NTC takes place in a limited time window, when the polymerase has transcribed ∼ 1.5 Kb past the A2 cleavage site. We speculate that assembly of early-acting 60S synthesis factors is monitored as a quality control system prior to NTC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085703PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911906PMC
December 2014

Cohesion promotes nucleolar structure and function.

Mol Biol Cell 2014 Feb 4;25(3):337-46. Epub 2013 Dec 4.

Stowers Institute for Medical Research, Kansas City, MO 64110 Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160 Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA 22908.

The cohesin complex contributes to ribosome function, although the molecular mechanisms involved are unclear. Compromised cohesin function is associated with a class of diseases known as cohesinopathies. One cohesinopathy, Roberts syndrome (RBS), occurs when a mutation reduces acetylation of the cohesin Smc3 subunit. Mutation of the cohesin acetyltransferase is associated with impaired rRNA production, ribosome biogenesis, and protein synthesis in yeast and human cells. Cohesin binding to the ribosomal DNA (rDNA) is evolutionarily conserved from bacteria to human cells. We report that the RBS mutation in yeast (eco1-W216G) exhibits a disorganized nucleolus and reduced looping at the rDNA. RNA polymerase I occupancy of the genes remains normal, suggesting that recruitment is not impaired. Impaired rRNA production in the RBS mutant coincides with slower rRNA cleavage. In addition to the RBS mutation, mutations in any subunit of the cohesin ring are associated with defects in ribosome biogenesis. Depletion or artificial destruction of cohesion in a single cell cycle is associated with loss of nucleolar integrity, demonstrating that the defects at the rDNA can be directly attributed to loss of cohesion. Our results strongly suggest that organization of the rDNA provided by cohesion is critical for formation and function of the nucleolus.
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http://dx.doi.org/10.1091/mbc.E13-07-0377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3907274PMC
February 2014

Rrp5 binding at multiple sites coordinates pre-rRNA processing and assembly.

Mol Cell 2013 Dec 14;52(5):707-19. Epub 2013 Nov 14.

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Kings Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland. Electronic address:

In vivo UV crosslinking identified numerous preribosomal RNA (pre-rRNA) binding sites for the large, highly conserved ribosome synthesis factor Rrp5. Intramolecular complementation has shown that the C-terminal domain (CTD) of Rrp5 is required for pre-rRNA cleavage at sites A0-A2 on the pathway of 18S rRNA synthesis, whereas the N-terminal domain (NTD) is required for A3 cleavage on the pathway of 5.8S/25S rRNA synthesis. The CTD was crosslinked to sequences flanking A2 and to the snoRNAs U3, U14, snR30, and snR10, which are required for cleavage at A0-A2. The NTD was crosslinked to sequences flanking A3 and to the RNA component of ribonuclease MRP, which cleaves site A3. Rrp5 could also be directly crosslinked to several large structural proteins and nucleoside triphosphatases. A key role in coordinating preribosomal assembly and processing was confirmed by chromatin spreads. Following depletion of Rrp5, cotranscriptional cleavage was lost and preribosome compaction greatly reduced.
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http://dx.doi.org/10.1016/j.molcel.2013.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3991325PMC
December 2013

Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II.

Cell Rep 2013 Sep 29;4(5):974-84. Epub 2013 Aug 29.

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0024, USA.

Multisubunit RNA polymerases (msRNAPs) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL) in the largest subunit of RNA polymerase I (Pol I) yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss of function in Pol I (Pol II: rpb1- E1103G; Pol I: rpa190-E1224G). Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase activity, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps.
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http://dx.doi.org/10.1016/j.celrep.2013.07.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3801175PMC
September 2013

Rpd3- and spt16-mediated nucleosome assembly and transcriptional regulation on yeast ribosomal DNA genes.

Mol Cell Biol 2013 Jul 20;33(14):2748-59. Epub 2013 May 20.

Department of Biochemistry and Molecular Genetics, University of Virginia, School of Medicine, Charlottesville, Virginia, USA.

Ribosomal DNA (rDNA) genes in eukaryotes are organized into multicopy tandem arrays and transcribed by RNA polymerase I. During cell proliferation, ∼50% of these genes are active and have a relatively open chromatin structure characterized by elevated accessibility to psoralen cross-linking. In Saccharomyces cerevisiae, transcription of rDNA genes becomes repressed and chromatin structure closes when cells enter the diauxic shift and growth dramatically slows. In this study, we found that nucleosomes are massively depleted from the active rDNA genes during log phase and reassembled during the diauxic shift, largely accounting for the differences in psoralen accessibility between active and inactive genes. The Rpd3L histone deacetylase complex was required for diauxic shift-induced H4 and H2B deposition onto rDNA genes, suggesting involvement in assembly or stabilization of the entire nucleosome. The Spt16 subunit of FACT, however, was specifically required for H2B deposition, suggesting specificity for the H2A/H2B dimer. Miller chromatin spreads were used for electron microscopic visualization of rDNA genes in an spt16 mutant, which was found to be deficient in the assembly of normal nucleosomes on inactive genes and the disruption of nucleosomes on active genes, consistent with an inability to fully reactivate polymerase I (Pol I) transcription when cells exit stationary phase.
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http://dx.doi.org/10.1128/MCB.00112-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700123PMC
July 2013

The SWI/SNF chromatin remodeling complex influences transcription by RNA polymerase I in Saccharomyces cerevisiae.

PLoS One 2013 20;8(2):e56793. Epub 2013 Feb 20.

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA.

SWI/SNF is a chromatin remodeling complex that affects transcription initiation and elongation by RNA polymerase II. Here we report that SWI/SNF also plays a role in transcription by RNA polymerase I (Pol I) in Saccharomyces cerevisiae. Deletion of the genes encoding the Snf6p or Snf5p subunits of SWI/SNF was lethal in combination with mutations that impair Pol I transcription initiation and elongation. SWI/SNF physically associated with ribosomal DNA (rDNA) within the coding region, with an apparent peak near the 5' end of the gene. In snf6Δ cells there was a ∼2.5-fold reduction in rRNA synthesis rate compared to WT, but there was no change in average polymerase occupancy per gene, the number of rDNA gene repeats, or the percentage of transcriptionally active rDNA genes. However, both ChIP and EM analyses showed a small but reproducible increase in Pol I density in a region near the 5' end of the gene. Based on these data, we conclude that SWI/SNF plays a positive role in Pol I transcription, potentially by modifying chromatin structure in the rDNA repeats. Our findings demonstrate that SWI/SNF influences the most robust transcription machinery in proliferating cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056793PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3577654PMC
August 2013

Identification of novel proteins associated with yeast snR30 small nucleolar RNA.

Nucleic Acids Res 2011 Dec 5;39(22):9659-70. Epub 2011 Sep 5.

Département des sciences biologiques and Centre de recherche BioMed, Université du Québec à Montréal, Montréal, Québec, H3C 3P8, Canada.

H/ACA small nucleolar RNPs (snoRNPs) that guide pseudouridylation reactions are comprised of one small nucleolar RNA (snoRNA) and four common proteins (Cbf5, Gar1, Nhp2 and Nop10). Unlike other H/ACA snoRNPs, snR30 is essential for the early processing reactions that lead to the production of 18S ribosomal RNA in the yeast Saccharomyces cerevisiae. To determine whether snR30 RNP contains specific proteins that contribute to its unique functional properties, we devised an affinity purification strategy using TAP-tagged Gar1 and an RNA aptamer inserted in snR30 snoRNA to selectively purify the RNP. Northern blotting and pCp labeling experiments showed that S1-tagged snR30 snoRNA can be selectively purified with streptavidin beads. Protein analysis revealed that aptamer-tagged snR30 RNA was associated with the four H/ACA proteins and a number of additional proteins: Nop6, ribosomal proteins S9 and S18 and histones H2B and H4. Using antibodies raised against Nop6 we show that endogenous Nop6 localizes to the nucleolus and that it cosediments with snR30 snoRNA in sucrose density gradients. We demonstrate through primer extension experiments that snR30 snoRNA is required for cleavages at site A0, A1 and A2, and that the absence of Nop6 decreases the efficiency of cleavage at site A2. Finally, electron microscopy analyses of chromatin spreads from cells depleted of snR30 snoRNA show that it is required for SSU processome assembly.
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http://dx.doi.org/10.1093/nar/gkr659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3239182PMC
December 2011

Sch9 regulates ribosome biogenesis via Stb3, Dot6 and Tod6 and the histone deacetylase complex RPD3L.

EMBO J 2011 Jul 5;30(15):3052-64. Epub 2011 Jul 5.

Department of Molecular Biology and National Center for Competence in Research Program 'Frontiers in Genetics', University of Geneva, Geneva, Switzerland.

TORC1 is a conserved multisubunit kinase complex that regulates many aspects of eukaryotic growth including the biosynthesis of ribosomes. The TOR protein kinase resident in TORC1 is responsive to environmental cues and is potently inhibited by the natural product rapamycin. Recent characterization of the rapamycin-sensitive phosphoproteome in yeast has yielded insights into how TORC1 regulates growth. Here, we show that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (Ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of the transcriptional repressors Stb3, Dot6 and Tod6. Deletion of STB3, DOT6 and TOD6 partially bypasses the growth and cell size defects of an sch9 strain and reveals interdependent regulation of both Ribi and RP gene expression, and other aspects of Ribi. Dephosphorylation of Stb3, Dot6 and Tod6 enables recruitment of the RPD3L histone deacetylase complex to repress Ribi/RP gene promoters. Taken together with previous studies, these results suggest that Sch9 is a master regulator of ribosome biogenesis through the control of Ribi, RP, ribosomal RNA and tRNA gene transcription.
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http://dx.doi.org/10.1038/emboj.2011.221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3160192PMC
July 2011

The transcription elongation factor Spt5 influences transcription by RNA polymerase I positively and negatively.

J Biol Chem 2011 May 5;286(21):18816-24. Epub 2011 Apr 5.

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0024, USA.

Spt5p is a universally conserved transcription factor that plays multiple roles in eukaryotic transcription elongation. Spt5p forms a heterodimer with Spt4p and collaborates with other transcription factors to pause or promote RNA polymerase II transcription elongation. We have shown previously that Spt4p and Spt5p also influence synthesis of ribosomal RNA by RNA polymerase (Pol) I; however, previous studies only characterized defects in Pol I transcription induced by deletion of SPT4. Here we describe two new, partially active mutations in SPT5 and use these mutant strains to characterize the effect of Spt5p on Pol I transcription. Genetic interactions between spt5 and rpa49Δ mutations together with measurements of ribosomal RNA synthesis rates, rDNA copy number, and Pol I occupancy of the rDNA demonstrate that Spt5p plays both positive and negative roles in transcription by Pol I. Electron microscopic analysis of mutant and WT strains confirms these observations and supports the model that Spt4/5 may contribute to pausing of RNA polymerase I early during transcription elongation but promotes transcription elongation downstream of the pause(s). These findings bolster the model that Spt5p and related homologues serve diverse critical roles in the control of transcription.
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http://dx.doi.org/10.1074/jbc.M110.202101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3099698PMC
May 2011

Distinguishing the roles of Topoisomerases I and II in relief of transcription-induced torsional stress in yeast rRNA genes.

Mol Cell Biol 2011 Feb 22;31(3):482-94. Epub 2010 Nov 22.

Department of Microbiology, Box 800734, University of Virginia Health System, Charlottesville, VA 22908-0734, USA.

To better understand the role of topoisomerase activity in relieving transcription-induced supercoiling, yeast genes encoding rRNA were visualized in cells deficient for either or both of the two major topoisomerases. In the absence of both topoisomerase I (Top1) and topoisomerase II (Top2) activity, processivity was severely impaired and polymerases were unable to transcribe through the 6.7-kb gene. Loss of Top1 resulted in increased negative superhelical density (two to six times the normal value) in a significant subset of rRNA genes, as manifested by regions of DNA template melting. The observed DNA bubbles were not R-loops and did not block polymerase movement, since genes with DNA template melting showed no evidence of slowed elongation. Inactivation of Top2, however, resulted in characteristic signs of slowed elongation in rRNA genes, suggesting that Top2 alleviates transcription-induced positive supercoiling. Together, the data indicate that torsion in front of and behind transcribing polymerase I has different consequences and different resolution. Positive torsion in front of the polymerase induces supercoiling (writhe) and is largely resolved by Top2. Negative torsion behind the polymerase induces DNA strand separation and is largely resolved by Top1.
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http://dx.doi.org/10.1128/MCB.00589-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3028620PMC
February 2011

Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis.

Genes Dev 2010 Jul;24(14):1546-58

Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

Pre-rRNA transcription by RNA Polymerase I (Pol I) is very robust on active rDNA repeats. Loss of yeast Topoisomerase I (Top1) generated truncated pre-rRNA fragments, which were stabilized in strains lacking TRAMP (Trf4/Trf5-Air1/Air2-Mtr4 polyadenylation complexes) or exosome degradation activities. Loss of both Top1 and Top2 blocked pre-rRNA synthesis, with pre-rRNAs truncated predominately in the 18S 5' region. Positive supercoils in front of Pol I are predicted to slow elongation, while rDNA opening in its wake might cause R-loop formation. Chromatin immunoprecipitation analysis showed substantial levels of RNA/DNA hybrids in the wild type, particularly over the 18S 5' region. The absence of RNase H1 and H2 in cells depleted of Top1 increased the accumulation of RNA/DNA hybrids and reduced pre-rRNA truncation and pre-rRNA synthesis. Hybrid accumulation over the rDNA was greatly exacerbated when Top1, Top2, and RNase H were all absent. Electron microscopy (EM) analysis revealed Pol I pileups in the wild type, particularly over the 18S. Pileups were longer and more frequent in the absence of Top1, and their frequency was exacerbated when RNase H activity was also lacking. We conclude that the loss of Top1 enhances inherent R-loop formation, particularly over the 5' region of the rDNA, imposing persistent transcription blocks when RNase H is limiting.
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http://dx.doi.org/10.1101/gad.573310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904944PMC
July 2010

Mrd1p is required for release of base-paired U3 snoRNA within the preribosomal complex.

Mol Cell Biol 2009 Nov 24;29(21):5763-74. Epub 2009 Aug 24.

Department of Molecular Biology and Functional Genomics, Stockholm University, SE-106 91 Stockholm, Sweden.

In eukaryotes, ribosomes are made from precursor rRNA (pre-rRNA) and ribosomal proteins in a maturation process that requires a large number of snoRNPs and processing factors. A fundamental problem is how the coordinated and productive folding of the pre-rRNA and assembly of successive pre-rRNA-protein complexes is achieved cotranscriptionally. The conserved protein Mrd1p, which contains five RNA binding domains (RBDs), is essential for processing events leading to small ribosomal subunit synthesis. We show that full function of Mrd1p requires all five RBDs and that the RBDs are functionally distinct and needed during different steps in processing. Mrd1p mutations trap U3 snoRNA in pre-rRNP complexes both in base-paired and non-base-paired interactions. A single essential RBD, RBD5, is involved in both types of interactions, but its conserved RNP1 motif is not needed for releasing the base-paired interactions. RBD5 is also required for the late pre-rRNP compaction preceding A(2) cleavage. Our results suggest that Mrd1p modulates successive conformational rearrangements within the pre-rRNP that influence snoRNA-pre-rRNA contacts and couple U3 snoRNA-pre-rRNA remodeling and late steps in pre-rRNP compaction that are essential for cleavage at A(0) to A(2). Mrd1p therefore coordinates key events in biosynthesis of small ribosome subunits.
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http://dx.doi.org/10.1128/MCB.00428-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2772733PMC
November 2009

The Paf1 complex is required for efficient transcription elongation by RNA polymerase I.

Proc Natl Acad Sci U S A 2009 Feb 22;106(7):2153-8. Epub 2009 Jan 22.

Department of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, AL 35294-0024, USA.

Regulation of RNA polymerase I (Pol I) transcription is critical for controlling ribosome synthesis. Most previous investigations into Pol I transcription regulation have focused on transcription initiation. To date, the factors involved in the control of Pol I transcription elongation are poorly understood. The Paf1 complex (Paf1C) is a well-defined factor that influences polymerase II (Pol II) transcription elongation. We found that Paf1C associates with rDNA. Deletion of genes for Paf1C subunits (CDC73, CTR9, or PAF1) reduces the rRNA synthesis rate; however, there is no significant alteration of rDNA copy number or Pol I occupancy of the rDNA. Furthermore, EM analysis revealed a substantial increase in the frequency of large gaps between transcribing polymerases in ctr9Delta mutant cells compared with WT. Together, these data indicate that Paf1C promotes Pol I transcription through the rDNA by increasing the net rate of elongation. Thus, the multifunctional, conserved transcription factor Paf1C plays an important role in transcription elongation by Pol I in vivo.
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http://dx.doi.org/10.1073/pnas.0812939106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650124PMC
February 2009

Electron microscope visualization of RNA transcription and processing in Saccharomyces cerevisiae by Miller chromatin spreading.

Methods Mol Biol 2009 ;464:55-69

Department of Microbiology, University of Virginia Health System, Charlottesville, VA, USA.

The Miller chromatin spreading technique for electron microscopic visualization of gently dispersed interphase chromatin has proven extremely valuable for analysis of genetic activities in vivo. It provides a unique view of transcription and RNA processing at the level of individual active genes. The budding yeast Saccharomyces cerevisiae has also been an invaluable model system for geneticists and molecular biologists. In this chapter, we describe methods for applying the Miller chromatin-spreading method to Saccharomyces cerevisiae. This allows one to use electron microscopic visualization of a gene of interest to study effects of specific mutations on gene activity. We are applying the method to study transcription and processing of ribosomal RNA.
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http://dx.doi.org/10.1007/978-1-60327-461-6_4DOI Listing
September 2010

Transcription of multiple yeast ribosomal DNA genes requires targeting of UAF to the promoter by Uaf30.

Mol Cell Biol 2008 Nov 2;28(21):6709-19. Epub 2008 Sep 2.

Department of Biochemistry and Molecular Genetics, University of Virginia Health System, School of Medicine, Charlottesville, VA 22908, USA.

Upstream activating factor (UAF) is a multisubunit complex that functions in the activation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I). Cells lacking the Uaf30 subunit of UAF reduce the rRNA synthesis rate by approximately 70% compared to wild-type cells and produce rRNA using both Pol I and Pol II. Miller chromatin spreads demonstrated that even though there is an overall reduction in rRNA synthesis in uaf30 mutants, the active rDNA genes in such strains are overloaded with polymerases. This phenotype was specific to defects in Uaf30, as mutations in other UAF subunits resulted in a complete absence of rDNA genes with high or even modest Pol densities. The lack of Uaf30 prevented UAF from efficiently binding to the rDNA promoter in vivo, leading to an inability to activate a large number of rDNA genes. The relatively few genes that did become activated were highly transcribed, apparently to compensate for the reduced rRNA synthesis capacity. The results show that Uaf30p is a key targeting factor for the UAF complex that facilitates activation of a large proportion of rDNA genes in the tandem array.
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http://dx.doi.org/10.1128/MCB.00703-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2573240PMC
November 2008

TOR regulates the subcellular distribution of DIM2, a KH domain protein required for cotranscriptional ribosome assembly and pre-40S ribosome export.

RNA 2008 Oct 28;14(10):2061-73. Epub 2008 Aug 28.

Fonds de la Recherche Scientifique (FRS-FNRS), Académie Wallonie-Bruxelles, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, Charleroi-Gosselies, B-6041, Belgium.

Eukaryotic ribosome synthesis is a highly dynamic process that involves the transient association of scores of trans-acting factors to nascent pre-ribosomes. Many ribosome synthesis factors are nucleocytoplasmic shuttling proteins that engage the assembly pathway at early nucleolar stages and escort pre-ribosomes to the nucleoplasm and/or the cytoplasm. Here, we report that two 40S ribosome synthesis factors, the KH-domain protein DIM2 and the HEAT-repeats/Armadillo-domain and export factor RRP12, are nucleolar restricted upon nutritional, osmotic, and oxidative stress. Nucleolar entrapment of DIM2 and RRP12 was triggered by rapamycin treatment and was under the strict control of the target of rapamycin (TOR) signaling cascade. DIM2 binds pre-rRNAs directly through its KH domain at the 5'-end of ITS1 (D-A(2) segment) and, consistent with its requirements in early nucleolar pre-rRNA processing, is required for efficient cotranscriptional ribosome assembly. The substitution of a single and highly conserved amino acid (G207A) within the KH motif is sufficient to inhibit pre-rRNA processing in a fashion similar to genetic depletion of DIM2. DIM2 carries an evolutionarily conserved putative nuclear export sequence (NES) at its carboxyl-terminal end that is required for efficient pre-40S ribosome export. Strikingly, DIM2 and RRP12 are both involved in the nucleocytoplasmic translocation of pre-ribosomes, suggesting that this step in the ribosome assembly pathway has been selected as a regulatory target for the TOR pathway.
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http://dx.doi.org/10.1261/rna.1176708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553727PMC
October 2008

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

Nucleic Acids Res 2008 Aug 27;36(13):4364-80. Epub 2008 Jun 27.

Department of Molecular Biology and Functional Genomics, Stockholm University, SE-106 91, Stockholm, Sweden.

In Saccharomyces cerevisiae, synthesis of the small ribosomal subunit requires assembly of the 35S pre-rRNA into a 90S preribosomal complex. SnoRNAs, including U3 snoRNA, and many trans-acting proteins are required for the ordered assembly and function of the 90S preribosomal complex. Here, we show that the conserved protein Mrd1p binds to the pre-rRNA early during transcription and is required for compaction of the pre-18S rRNA into SSU processome particles. We have exploited the fact that an Mrd1p-GFP fusion protein is incorporated into the 90S preribosomal complex, where it acts as a partial loss-of-function mutation. When associated with the pre-rRNA, Mrd1p-GFP functionally interacts with the essential Pwp2, Mpp10 and U3 snoRNP subcomplexes that are functionally interconnected in the 90S preribosomal complex. The fusion protein can partially support 90S preribosome-mediated cleavages at the A(0)-A(2) sites. At the same time, on a substantial fraction of transcripts, the composition and/or structure of the 90S preribosomal complex is perturbed by the fusion protein in such a way that cleavage of the 35S pre-rRNA is either blocked or shifted to aberrant sites. These results show that Mrd1p is required for establishing productive structures within the 90S preribosomal complex.
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http://dx.doi.org/10.1093/nar/gkn384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2490760PMC
August 2008

Visual analysis of the yeast 5S rRNA gene transcriptome: regulation and role of La protein.

Mol Cell Biol 2008 Jul 12;28(14):4576-87. Epub 2008 May 12.

Department of Microbiology, University of Virginia Health System, Charlottesville, Virginia 22908-0734, USA.

5S rRNA genes from Saccharomyces cerevisiae were examined by Miller chromatin spreading, representing the first quantitative analysis of RNA polymerase III genes in situ by electron microscopy. These very short genes, approximately 132 nucleotides (nt), were engaged by one to three RNA polymerases. Analysis in different growth conditions and in strains with a fourfold range in gene copy number revealed regulation at two levels: number of active genes and polymerase loading per gene. Repressive growth conditions (presence of rapamycin or postexponential growth) led first to fewer active genes, followed by lower polymerase loading per active gene. The polymerase III elongation rate was estimated to be in the range of 60 to 75 nt/s, with a reinitiation interval of approximately 1.2 s. The yeast La protein, Lhp1, was associated with 5S genes. Its absence had no discernible effect on the amount or size of 5S RNA produced yet resulted in more polymerases per gene on average, consistent with a non-rate-limiting role for Lhp1 in a process such as polymerase release/recycling upon transcription termination.
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http://dx.doi.org/10.1128/MCB.00127-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2447126PMC
July 2008

Transcription elongation by RNA polymerase I is linked to efficient rRNA processing and ribosome assembly.

Mol Cell 2007 Apr;26(2):217-29

Department of Biological Chemistry, University of California, Irvine, 240-D Medical Sciences I, Irvine, CA 92697-1700, USA.

The synthesis of ribosomes in eukaryotic cells is a complex process involving many nonribosomal protein factors and snoRNAs. In general, the processes of rRNA transcription and ribosome assembly are treated as temporally or spatially distinct. Here, we describe the identification of a point mutation in the second largest subunit of RNA polymerase I near the active center of the enzyme that results in an elongation-defective enzyme in the yeast Saccharomyces cerevisiae. In vivo, this mutant shows significant defects in rRNA processing and ribosome assembly. Taken together, these data suggest that transcription of rRNA by RNA polymerase I is linked to rRNA processing and maturation. Thus, RNA polymerase I, elongation factors, and rRNA sequence elements appear to function together to optimize transcription elongation, coordinating cotranscriptional interactions of many factors/snoRNAs with pre-rRNA for correct rRNA processing and ribosome assembly.
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http://dx.doi.org/10.1016/j.molcel.2007.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1927085PMC
April 2007

Regulation of rRNA synthesis by TATA-binding protein-associated factor Mot1.

Mol Cell Biol 2007 Apr 12;27(8):2886-96. Epub 2007 Feb 12.

Department of Biochemistry and Molecular Genetics, University of Virginia Health System, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908-0733, USA.

Mot1 is an essential, conserved, TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae with well-established roles in the global control of RNA polymerase II (Pol II) transcription. Previous results have suggested that Mot1 functions exclusively in Pol II transcription, but here we report a novel role for Mot1 in regulating transcription by RNA polymerase I (Pol I). In vivo, Mot1 is associated with the ribosomal DNA, and loss of Mot1 results in decreased rRNA synthesis. Consistent with a direct role for Mot1 in Pol I transcription, Mot1 also associates with the Pol I promoter in vitro in a reaction that depends on components of the Pol I general transcription machinery. Remarkably, in addition to Mot1's role in initiation, rRNA processing is delayed in mot1 cells. Taken together, these results support a model in which Mot1 affects the rate and efficiency of rRNA synthesis by both direct and indirect mechanisms, with resulting effects on transcription activation and the coupling of rRNA synthesis to processing.
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http://dx.doi.org/10.1128/MCB.00054-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1899949PMC
April 2007

Transcription and translation are coupled in Archaea.

Mol Biol Evol 2007 Apr 20;24(4):893-5. Epub 2007 Jan 20.

Polysomes have been visualized by electron microscopy attached directly to dispersed strands of genomic DNA extruded from lysed cells of the hyperthermophilic archaeon Thermococcus kodakaraensis. These complexes are consistent with transcription and translation being coupled in this Archaeon, with translation of transcripts being initiated before the transcript is complete.
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http://dx.doi.org/10.1093/molbev/msm007DOI Listing
April 2007

The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation.

Proc Natl Acad Sci U S A 2006 Jun 12;103(25):9464-9. Epub 2006 Jun 12.

Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA.

Ribosome biogenesis is a complex process that requires >150 transacting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and previously undescribed components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single-point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results suggest that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A(1) and/or A(2.).
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http://dx.doi.org/10.1073/pnas.0603673103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1480430PMC
June 2006

Role of histone deacetylase Rpd3 in regulating rRNA gene transcription and nucleolar structure in yeast.

Mol Cell Biol 2006 May;26(10):3889-901

Department of Biological Chemistry, University of California--Irvine, 240D Medical Sciences I, Irvine, California 92697-1700, USA.

The 35S rRNA genes at the RDN1 locus in Saccharomyces cerevisiae can be transcribed by RNA polymerase (Pol) II in addition to Pol I, but Pol II transcription is usually silenced. The deletion of RRN9 encoding an essential subunit of the Pol I transcription factor, upstream activation factor, is known to abolish Pol I transcription and derepress Pol II transcription of rRNA genes, giving rise to polymerase switched (PSW) variants. We found that deletion of histone deacetylase gene RPD3 inhibits the appearance of PSW variants in rrn9 deletion mutants. This inhibition can be explained by the observed specific inhibition of Pol II transcription of rRNA genes by the rpd3Delta mutation. We propose that Rpd3 plays a role in the maintenance of an rRNA gene chromatin structure(s) that allows Pol II transcription of rRNA genes, which may explain the apparently paradoxical previous observation that rpd3 mutations increase, rather than decrease, silencing of reporter Pol II genes inserted in rRNA genes. We have additionally demonstrated that Rpd3 is not required for inhibition of Pol I transcription by rapamycin, supporting the model that Tor-dependent repression of the active form of rRNA genes during entry into stationary phase is Rpd3 independent.
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http://dx.doi.org/10.1128/MCB.26.10.3889-3901.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489006PMC
May 2006

Histones are required for transcription of yeast rRNA genes by RNA polymerase I.

Proc Natl Acad Sci U S A 2005 Jul 7;102(29):10129-34. Epub 2005 Jul 7.

Department of Biological Chemistry, University of California, Irvine, CA 92697-1700, USA.

Nucleosomes and their histone components have generally been recognized to act negatively on transcription. However, purified upstream activating factor (UAF), a transcription initiation factor required for RNA polymerase (Pol) I transcription in Saccharomyces cerevisiae, contains histones H3 and H4 and four nonhistone protein subunits. Other studies have shown that histones H3 and H4 are associated with actively transcribed rRNA genes. To examine their functional role in Pol I transcription, we constructed yeast strains in which synthesis of H3 is achieved from the glucose-repressible GAL10 promoter. We found that partial depletion of H3 (approximately 50% depletion) resulted in a strong inhibition (>80%) of Pol I transcription. A combination of biochemical analysis and electron microscopic (EM) analysis of Miller chromatin spreads indicated that initiation and elongation steps and rRNA processing were compromised upon histone depletion. A clear decrease in relative amounts of UAF, presumably caused by reduced stability, was also observed under the conditions of H3 depletion. Therefore, the observed inhibition of initiation can be explained, in part, by the decrease in UAF concentration. In addition, the EM results suggested that the defects in rRNA transcript elongation and processing may be a result of loss of histones from rRNA genes rather than (or in addition to) an indirect consequence of effects of histone depletion on expression of other genes. Thus, these results show functional importance of histones associated with actively transcribed rRNA genes.
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http://dx.doi.org/10.1073/pnas.0504563102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1177414PMC
July 2005

Esf2p, a U3-associated factor required for small-subunit processome assembly and compaction.

Mol Cell Biol 2005 Jul;25(13):5523-34

Fonds National de la Recherche Scientifique, Université Libre de Bruxelles, Institut de Biologie et de Médecine Moléculaires, Charleroi-Gosselies, Belgium.

Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A0 through A2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5' external transcribed spacer (ETS) and the box C+D snoRNA U3, as well as additional box C+D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5' ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.
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http://dx.doi.org/10.1128/MCB.25.13.5523-5534.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1156982PMC
July 2005

Pre-18S ribosomal RNA is structurally compacted into the SSU processome prior to being cleaved from nascent transcripts in Saccharomyces cerevisiae.

Mol Cell 2004 Dec;16(6):943-54

Department of Microbiology, University of Virginia Health System, Charlottesville, VA 22908, USA.

Recent studies have revealed multiple dynamic complexes that are precursors to eukaryotic ribosomes. EM visualization of nascent rRNA transcripts provides in vivo temporal and structural context for these events. In exponentially growing S. cerevisiae, pre-18S rRNA is dramatically compacted into a large particle (SSU processome) within seconds of completion of its transcription and is released cotranscriptionally by cleavage in ITS1. After cleavage, a new terminal knob is formed on the nascent large subunit rRNA, compacting it progressively in a 5'-3' direction. Depletion of individual components shows that cotranscriptional SSU processome formation is a sensitive indicator of the occurrence or timing of the early A0-A2 cleavages and depends on factors not isolated in preribosome complexes, as well as on favorable growth conditions. The results show that the approximately 40 components of the SSU processome/90S preribosome can complete their tasks within approximately 85 s in optimal conditions.
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http://dx.doi.org/10.1016/j.molcel.2004.11.031DOI Listing
December 2004

RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components.

Genes Dev 2004 Oct;18(20):2506-17

Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8024, USA.

Sequential events in macromolecular biosynthesis are often elegantly coordinated. The small ribosomal subunit (SSU) processome is a large ribonucleoprotein (RNP) required for processing of precursors to the small subunit RNA, the 18S, of the ribosome. We have found that a subcomplex of SSU processome proteins, the t-Utps, is also required for optimal rRNA transcription in vivo in the yeast Saccharomyces cerevisiae. The t-Utps are ribosomal chromatin (r-chromatin)-associated, and they exist in a complex in the absence of the U3 snoRNA. Transcription is required neither for the formation of the subcomplex nor for its r-chromatin association. The t-Utps are associated with the pre-18S rRNAs independent of the presence of the U3 snoRNA. This association may thus represent an early step in the formation of the SSU processome. Our results indicate that rRNA transcription and pre-rRNA processing are coordinated via specific components of the SSU processome.
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http://dx.doi.org/10.1101/gad.1226604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC529538PMC
October 2004