Establishment and maintenance of alternative chromatin states at a multicopy gene locus.

In eukaryotes, each of the more than 100 copies of ribosomal RNA (rRNA) genes exists in either an RNA polymerase I transcribed open chromatin state or a nucleosomal, closed chromatin state. Open rRNA genes guarantee the cell's supply with structural components of the ribosome, whereas closed rRNA genes ensure genomic integrity. We report that the observed balance between open and closed rRNA gene chromatin states in proliferating yeast cells is due to a dynamic equilibrium of transcription-dependent removal and replication-dependent assembly of nucleosomes. Pol I transcription is required for the association of the HMG box protein Hmo1 with open rRNA genes, counteracting replication-independent nucleosome deposition and maintaining the open rRNA gene chromatin state outside of S phase. The findings indicate that the opposing effects of replication and transcription lead to a de novo establishment of chromatin states for rRNA genes during each cell cycle.

RNA polymerase I (Pol I) lobe-binding subunit Rpa12.2 promotes RNA cleavage and proofreading.

Elongating nuclear RNA polymerases (Pols) frequently pause, backtrack, and are then reactivated by endonucleolytic cleavage. Pol backtracking and RNA cleavage are also crucial for proofreading, which contributes to transcription fidelity. RNA polymerase I (Pol I) of the yeast Saccharomyces cerevisiae synthesizes exclusively 35S rRNA, the precursor transcript of mature ribosomal 5.8S, 18S, and 25S rRNA. Pol I contains the specific heterodimeric subunits Rpa34.5/49 and subunit Rpa12.2, which have been implicated in RNA cleavage and elongation activity, respectively. These subunits are associated with the Pol I lobe structure and encompass different structural domains, but the contribution of these domains to RNA elongation is unclear. Here, we used Pol I mutants or reconstituted Pol I enzymes to study the effects of these subunits and/or their distinct domains on RNA cleavage, backtracking, and transcription fidelity in defined in vitro systems. Our findings suggest that the presence of the intact C-terminal domain of Rpa12.2 is sufficient to support the cleavage reaction, but that the N-terminal domains of Rpa12.2 and the heterodimer facilitate backtracking and RNA cleavage. Since both N-terminal and C-terminal domains of Rpa12.2 were also required to faithfully incorporate NTPs in the growing RNA chain, efficient backtracking and RNA cleavage might be a prerequisite for transcription fidelity. We propose that RNA Pols containing efficient RNA cleavage activity are able to add and remove nucleotides until the matching nucleotide supports RNA chain elongation, whereas cleavage-deficient enzymes can escape this proofreading process by incorporating incorrect nucleotides.

Features of yeast RNA polymerase I with special consideration of the lobe binding subunits.

Ribosomal RNAs (rRNAs) are structural components of ribosomes and represent the most abundant cellular RNA fraction. In the yeast Saccharomyces cerevisiae, they account for more than 60 % of the RNA content in a growing cell. The major amount of rRNA is synthesized by RNA polymerase I (Pol I). This enzyme transcribes exclusively the rRNA gene which is tandemly repeated in about 150 copies on chromosome XII. The high number of transcribed rRNA genes, the efficient recruitment of the transcription machinery and the dense packaging of elongating Pol I molecules on the gene ensure that enough rRNA is generated. Specific features of Pol I and of associated factors confer promoter selectivity and both elongation and termination competence. Many excellent reviews exist about the state of research about function and regulation of Pol I and how Pol I initiation complexes are assembled. In this report we focus on the Pol I specific lobe binding subunits which support efficient, error-free, and correctly terminated rRNA synthesis.

The C-terminal region of Net1 is an activator of RNA polymerase I transcription with conserved features from yeast to human.

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA) in all eukaryotes, accounting for the major part of transcriptional activity in proliferating cells. Although basal Pol I transcription factors have been characterized in diverse organisms, the molecular basis of the robust rRNA production in vivo remains largely unknown. In S. cerevisiae, the multifunctional Net1 protein was reported to stimulate Pol I transcription. We found that the Pol I-stimulating function can be attributed to the very C-terminal region (CTR) of Net1. The CTR was required for normal cell growth and Pol I recruitment to rRNA genes in vivo and sufficient to promote Pol I transcription in vitro. Similarity with the acidic tail region of mammalian Pol I transcription factor UBF, which could partly functionally substitute for the CTR, suggests conserved roles for CTR-like domains in Pol I transcription from yeast to human.

RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure.

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I-associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I-specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.

Viewing pre-60S maturation at a minute's timescale.

The formation of ribosomal subunits is a highly dynamic process that is initiated in the nucleus and involves more than 200 trans-acting factors, some of which accompany the pre-ribosomes into the cytoplasm and have to be recycled into the nucleus. The inhibitor diazaborine prevents cytoplasmic release and recycling of shuttling pre-60S maturation factors by inhibiting the AAA-ATPase Drg1. The failure to recycle these proteins results in their depletion in the nucleolus and halts the pathway at an early maturation step. Here, we made use of the fast onset of inhibition by diazaborine to chase the maturation path in real-time from 27SA2 pre-rRNA containing pre-ribosomes localized in the nucleolus up to nearly mature 60S subunits shortly after their export into the cytoplasm. This allows for the first time to put protein assembly and disassembly reactions as well as pre-rRNA processing into a chronological context unraveling temporal and functional linkages during ribosome maturation.

The Reb1-homologue Ydr026c/Nsi1 is required for efficient RNA polymerase I termination in yeast.

Several DNA cis-elements and trans-acting factors were described to be involved in transcription termination and to release the elongating RNA polymerases from their templates. Different models for the molecular mechanism of transcription termination have been suggested for eukaryotic RNA polymerase I (Pol I) from results of in vitro and in vivo experiments. To analyse the molecular requirements for yeast RNA Pol I termination, an in vivo approach was used in which efficient termination resulted in growth inhibition. This led to the identification of a Myb-like protein, Ydr026c, as bona fide termination factor, now designated Nsi1 (NTS1 silencing protein 1), since it was very recently described as silencing factor of ribosomal DNA. Possible Nsi1 functions in regard to the mechanism of transcription termination are discussed.

Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae.

Chromatin is the template for replication and transcription in the eukaryotic nucleus, which needs to be defined in composition and structure before these processes can be fully understood. We report an isolation protocol for the targeted purification of specific genomic regions in their native chromatin context from Saccharomyces cerevisiae. Subdomains of the multicopy ribosomal DNA locus containing transcription units of RNA polymerases I, II or III or an autonomous replication sequence were independently purified in sufficient amounts and purity to analyze protein composition and histone modifications by mass spectrometry. We present and discuss the proteomic data sets obtained for chromatin in different functional states. The native chromatin was further amenable to electron microscopy analysis yielding information about nucleosome occupancy and positioning at the single-molecule level. We also provide evidence that chromatin from virtually every single copy genomic locus of interest can be purified and analyzed by this technique.

Rrp5p, Noc1p and Noc2p form a protein module which is part of early large ribosomal subunit precursors in S. cerevisiae.

Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p-Noc1p-Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity.

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits.

Ribosome biogenesis is a complex multistep process that involves alternating steps of folding and processing of pre-rRNAs in concert with assembly of ribosomal proteins. Recently, there has been increased interest in the roles of ribosomal proteins in eukaryotic ribosome biogenesis in vivo, focusing primarily on their function in pre-rRNA processing. However, much less is known about participation of ribosomal proteins in the formation and rearrangement of preribosomal particles as they mature to functional subunits. We have studied ribosomal proteins L7 and L8, which are required for the same early steps in pre-rRNA processing during assembly of 60S subunits but are located in different domains within ribosomes. Depletion of either leads to defects in processing of 27SA(3) to 27SB pre-rRNA and turnover of pre-rRNAs destined for large ribosomal subunits. A specific subset of proteins is diminished from these residual assembly intermediates: six assembly factors required for processing of 27SA(3) pre-rRNA and four ribosomal proteins bound to domain I of 25S and 5.8S rRNAs surrounding the polypeptide exit tunnel. In addition, specific sets of ribosomal proteins are affected in each mutant: In the absence of L7, proteins bound to domain II, L6, L14, L20, and L33 are greatly diminished, while proteins L13, L15, and L36 that bind to domain I are affected in the absence of L8. Thus, L7 and L8 might establish RNP structures within assembling ribosomes necessary for the stable association and function of the A(3) assembly factors and for proper assembly of the neighborhoods containing domains I and II.

Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro.

Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.

Impact of the yeast S0/uS2-cluster ribosomal protein rpS21/eS21 on rRNA folding and the architecture of small ribosomal subunit precursors.

RpS0/uS2, rpS2/uS5, and rpS21/eS21 form a cluster of ribosomal proteins (S0-cluster) at the head-body junction near the central pseudoknot of eukaryotic small ribosomal subunits (SSU). Previous work in yeast indicated that S0-cluster assembly is required for the stabilisation and maturation of SSU precursors at specific post-nucleolar stages. Here, we analysed the role of S0-cluster formation for rRNA folding. Structures of SSU precursors isolated from yeast S0-cluster expression mutants or control strains were analysed by cryogenic electron microscopy. The obtained resolution was sufficient to detect individual 2'-O-methyl RNA modifications using an unbiased scoring approach. The data show how S0-cluster formation enables the initial recruitment of the pre-rRNA processing factor Nob1 in yeast. Furthermore, they reveal hierarchical effects on the pre-rRNA folding pathway, including the final maturation of the central pseudoknot. Based on these structural insights we discuss how formation of the S0-cluster determines at this early cytoplasmic assembly checkpoint if SSU precursors further mature or are degraded.

Analysis of ribosome biogenesis factor-modules in yeast cells depleted from pre-ribosomes.

Formation of eukaryotic ribosomes requires more than 150 biogenesis factors which transiently interact with the nascent ribosomal subunits. Previously, many pre-ribosomal intermediates could be distinguished by their protein composition and rRNA precursor (pre-rRNA) content. We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means. We compared the protein composition of these largely pre-rRNA free assemblies with the one of analogous pre-ribosomal preparations by semi-quantitative mass spectrometry. The experimental setup minimizes the possibility that the analysed pre-rRNA free protein modules were derived from (partially) disrupted pre-ribosomal particles and provides thereby strong evidence for their pre-ribosome independent existence. In support of the validity of this approach (i) the predicted composition of the analysed protein modules was in agreement with previously described rRNA-free complexes and (ii) in most of the cases we could identify new candidate members of reported protein modules. An unexpected outcome of these analyses was that free large ribosomal subunits are associated with a specific set of ribosome biogenesis factors in cells where neo-production of nascent ribosomes was blocked. The data presented strengthen the idea that assembly of eukaryotic pre-ribosomal particles can result from transient association of distinct building blocks.

TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production.

Ribosome biogenesis is tightly linked to cellular growth. A crucial step in the regulation of ribosomal RNA (rRNA) gene transcription is the formation of the complex between RNA polymerase I (Pol I) and the Pol I-dependent transcription factor Rrn3p. We found that TOR inactivation leads to proteasome-dependent degradation of Rrn3p and a strong reduction in initiation competent Pol I-Rrn3p complexes affecting yeast rRNA gene transcription. Using a mutant expressing non-degradable Rrn3p or a strain in which defined endogenous Rrn3p levels can be adjusted by the Tet-off system, we can demonstrate that Rrn3p levels influence the number of Pol I-Rrn3p complexes and consequently rRNA gene transcription. However, our analysis reveals that the dramatic reduction of rRNA synthesis in the immediate cellular response to impaired TOR signalling cannot be explained by the simple down-regulation of Rrn3p and Pol I-Rrn3p levels.

Preparation of RNA Polymerase Complexes for Their Analysis by Single-Particle Cryo-Electron Microscopy.

Recent technological progress revealed new prospects of high-resolution structure determination of macromolecular complexes using cryo-electron microscopy (cryo-EM) . In the field of RNA polymerase (Pol) I research, a number of cryo-EM studies contributed to understanding the highly specialized mechanisms underlying the transcription of ribosomal RNA genes . Despite a broad applicability of the cryo-EM method itself, preparation of samples for high-resolution data collection can be challenging. Here, we describe strategies for the purification and stabilization of Pol I complexes, exemplarily considering advantages and disadvantages of the methodology. We further provide an easy-to-implement protocol for the coating of EM-grids with self-made carbon support films. In sum, we present an efficient workflow for cryo-grid preparation and optimization, including early stage cryo-EM screening that can be adapted to a wide range of soluble samples for high-resolution structure determination .

Tethered MNase Structure Probing as Versatile Technique for Analyzing RNPs Using Tagging Cassettes for Homologous Recombination in Saccharomyces cerevisiae.

Micrococcal nuclease (MNase) originating from Staphylococcus aureus is a calcium dependent ribo- and desoxyribonuclease which has endo- and exonucleolytic activity of low sequence preference. MNase is widely used to analyze nucleosome positions in chromatin by probing the enzyme's DNA accessibility in limited digestion reactions. Probing reactions can be performed in a global way by addition of exogenous MNase , or locally by "chromatin endogenous cleavage " (ChEC ) reactions using MNase fusion proteins . The latter approach has recently been adopted for the analysis of local RNA environments of MNase fusion proteins which are incorporated in vivo at specific sites of ribonucleoprotein (RNP ) complexes. In this case, ex vivo activation of MNase by addition of calcium leads to RNA cleavages in proximity to the tethered anchor protein thus providing information about the folding state of its RNA environment.Here, we describe a set of plasmids that can be used as template for PCR-based MNase tagging of genes by homologous recombination in S. cerevisiae . The templates enable both N- and C-terminal tagging with MNase in combination with linker regions of different lengths and properties. In addition, an affinity tag is included in the recombination cassettes which can be used for purification of the particle of interest before or after induction of MNase cleavages in the surrounding RNA or DNA. A step-by-step protocol is provided for tagging of a gene of interest, followed by affinity purification of the resulting fusion protein together with associated RNA and subsequent induction of local MNase cleavages.

Analysis of Yeast RNAP I Transcription of Nucleosomal Templates In Vitro.

Nuclear eukaryotic RNA polymerases (RNAPs) transcribe a chromatin template in vivo. Since the basic unit of chromatin, the nucleosome, renders the DNA largely inaccessible, RNAPs have to overcome the nucleosomal barrier for efficient RNA synthesis. Gaining mechanistical insights in the transcription of chromatin templates will be essential to understand the complex process of eukaryotic gene expression. In this article we describe the use of defined in vitro transcription systems for comparative analysis of highly purified RNAPs I-III from S. cerevisiae (hereafter called yeast) transcribing in vitro reconstituted nucleosomal templates. We also provide a protocol to study promoter-dependent RNAP I transcription of purified native 35S ribosomal RNA (rRNA) gene chromatin.

Specialization of RNA Polymerase I in Comparison to Other Nuclear RNA Polymerases of Saccharomyces cerevisiae.

In archaea and bacteria the major classes of RNAs are synthesized by one DNA-dependent RNA polymerase (RNAP). In contrast, most eukaryotes have three highly specialized RNAPs to transcribe the nuclear genome. RNAP I synthesizes almost exclusively ribosomal (r)RNA, RNAP II synthesizes mRNA as well as many noncoding RNAs involved in RNA processing or RNA silencing pathways and RNAP III synthesizes mainly tRNA and 5S rRNA. This review discusses functional differences of the three nuclear core RNAPs in the yeast S. cerevisiae with a particular focus on RNAP I transcription of nucleolar ribosomal (r)DNA chromatin.

Establishment and Maintenance of Open Ribosomal RNA Gene Chromatin States in Eukaryotes.

In growing eukaryotic cells, nuclear ribosomal (r)RNA synthesis by RNA polymerase (RNAP) I accounts for the vast majority of cellular transcription. This high output is achieved by the presence of multiple copies of rRNA genes in eukaryotic genomes transcribed at a high rate. In contrast to most of the other transcribed genomic loci, actively transcribed rRNA genes are largely devoid of nucleosomes adapting a characteristic "open" chromatin state, whereas a significant fraction of rRNA genes resides in a transcriptionally inactive nucleosomal "closed" chromatin state. Here, we review our current knowledge about the nature of open rRNA gene chromatin and discuss how this state may be established.

Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors.

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.