Search

Bud27 is a prefoldin-like, a member of the family of ATP-independent molecular chaperones that associates with RNA polymerases I, II, and III in

RNA molecules and their expression dynamics play essential roles in the establishment of complex cellular phenotypes and/or in the rapid cellular adaption to environmental changes. Accordingly, analyzing RNA expression remains an important step to understand the molecular basis controlling the formation of cellular phenotypes, cellular homeostasis or disease progression. Steady-state RNA levels in the cells are controlled by the sum of highly dynamic molecular processes contributing to RNA expression and can be classified in transcription, maturation and degradation. The main goal of analyzing RNA dynamics is to disentangle the individual contribution of these molecular processes to the life cycle of a given RNA under different physiological conditions. In the recent years, the use of nonradioactive nucleotide/nucleoside analogs and improved chemistry, in combination with time-dependent and high-throughput analysis, have greatly expanded our understanding of RNA metabolism across various cell types, organisms, and growth conditions.In this chapter, we describe a step-by-step protocol allowing pulse labeling of RNA with the nonradioactive nucleotide analog, 4-thiouracil, in the eukaryotic model organism Saccharomyces cerevisiae and the model archaeon Haloferax volcanii.

International audience; Pervasive transcription is widespread in eukaryotes, generating large families of non- coding RNAs. Such pervasive transcription is a key player in the regulatory pathways controlling chromatin state and gene expression. Here, we describe long non-coding RNAs generated from the ribosomal RNA gene promoter called UPStream-initiating transcripts (UPS). In yeast, rDNA genes are organized in tandem repeats in at least two different chromatin states, either transcribed and largely depleted of nucleosomes (open) or assembled in regular arrays of nucleosomes (closed). The production of UPS transcripts by RNA Polymerase II from endogenous rDNA genes was initially documented in mutants defective for rRNA production by RNA polymerase I. We show here that UPS are produced in wild-type cells from closed rDNA genes but are hidden within the enormous production of rRNA. UPS levels are increased when rDNA chromatin states are modified at high temperatures or entering/leaving quiescence. We discuss their role in the regulation of rDNA chromatin states and rRNA production.

15 páginas, 11 figuras. El fichero contiene una rectificación en la página 17, The control of mRNA biogenesis is exerted at several steps. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress by acting during transcriptional initiation and elongation. Here, we genetically show that an intact nuclear pore complex is important for cell survival and maximal expression of stress-responsive genes. The Hog1 SAPK associates with nuclear pore complex components and directly phosphorylates the Nup1, Nup2, and Nup60 components of the inner nuclear basket. Mutation of those factors resulted in a deficient export of stress-responsive genes upon stress. Association of Nup1, Nup2, and Nup60 to stress-responsive promoters occurs upon stress depending on Hog1 activity. Accordingly, STL1 gene territory is maintained at the nuclear periphery upon osmostress in a Hog1-dependent manner. Cells containing non-phosphorylatable mutants in Nup1 or Nup2 display reduced expression of stress-responsive genes. Together, proper mRNA biogenesis of stress-responsive genes requires of the coordinate action of synthesis and export machineries by the Hog1 SAPK, This work was supported by MINECO (Spanish government) Grant BFU2012-33503, the Consolider Ingenio 2010 program (Grant CSD2007-0015), the Fundación Marcelino Botín (to F. P.), and Grant BFU2011-26722 (to E. d. N.). Recipients of an ICREA Acadèmia (Generalitat de Catalunya). Supported by the MINECO Grant BFU2011-23418. Supported by Agence Nationale de la Recherche (Nucleopol and ODynRib-Jeune chercheur program) and Jeune équipe from Fondation pour la Recherche Médicale.

The basal transcription/repair factor II H (TFIIH), found mutated in cancer-prone or premature aging diseases, plays a still unclear role in RNA polymerase I transcription. Furthermore, the impact of this function on TFIIH-related diseases, such as trichothiodystrophy (TTD), remains to be explored. Here, we studied the involvement of TFIIH during the whole process of ribosome biogenesis, from RNAP1 transcription to maturation steps of the ribosomal RNAs. Our results show that TFIIH is recruited to the ribosomal DNA in an active transcription-dependent manner and functions in RNAP1 transcription elongation through ATP hydrolysis of the XPB subunit. Remarkably, we found a TFIIH allele-specific effect, affecting RNAP1 transcription and/or the pre-rRNA maturation process. Interestingly, this effect was observed in mutant TFIIH-TTD cells and also in the brains of TFIIH-TTD mice. Our findings provide evidence that defective ribosome synthesis represents a new faulty mechanism involved in the pathophysiology of TFIIH-related diseases.

Eukaryotic transcription of ribosomal RNAs (rRNAs) by RNA polymerase I can account for more than half of the total cellular transcripts depending on organism and growth condition. To support this level of expression, eukaryotic rRNA genes are present in multiple copies. Interestingly, these genes co-exist in different chromatin states that may differ significantly in their nucleosome content and generally correlate well with transcriptional activity. Here we review how these chromatin states have been discovered and characterized focusing particularly on their structural protein components. The establishment and maintenance of rRNA gene chromatin states and their impact on rRNA synthesis are discussed. This article is part of a Special Issue entitled: Transcription by Odd Pols.

The native template of all eukaryotic nuclear RNA polymerases is chromatin. To understand how transcription occurs in vivo, it is important to define the chromatin environment of transcribing RNA Pols. Here, we describe a method used to characterize the distribution and the protein environment of RNA Pol I on ribosomal DNA during transcription in the yeast S. cerevisiae. The method is based on conventional chromatin immunoprecipitation and we propose quality control analyses at different steps of the procedure. Finally, the obtained samples are a useful source for downstream analyses by semiquantitative mass spectrometry or quantitative PCR.

The native template of all eukaryotic nuclear RNA polymerases is chromatin. To understand how transcription occurs in vivo, it is important to define the chromatin environment of transcribing RNA Pols. Here, we describe a method used to characterize the distribution and the protein environment of RNA Pol I on ribosomal DNA during transcription in the yeast S. cerevisiae. The method is based on conventional chromatin immunoprecipitation and we propose quality control analyses at different steps of the procedure. Finally, the obtained samples are a useful source for downstream analyses by semiquantitative mass spectrometry or quantitative PCR.

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.

Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35–47S) can be achieved by up to 150 RNA polymerase I (Pol I) enzymes simultaneously transcribing each rRNA gene. In this paper, we present recent advances made in understanding the regulatory mechanisms that control elongation. Built-in Pol I elongation factors, such as Rpa34/Rpa49 in budding yeast and PAF53/CAST in humans, are instrumental to the extremely high rate of rRNA production per gene. rRNA elongation mechanisms are intrinsically linked to chromatin structure and to the higher-order organization of the rRNA genes (rDNA). Factors such as Hmo1 in yeast and UBF1 in humans are key players in rDNA chromatin structurein vivo. Finally, elongation factors known to regulate messengers RNA production by RNA polymerase II are also involved in rRNA production and work cooperatively with Rpa49in vivo.

17 páginas, 8 figuras.-- This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License., The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. Recently, it has been shown that the formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. However, the mode of assembly of other 90S pre-ribosome components remains obscure as yet. In this study, we have investigated the assembly of three proteins (Utp20, Imp4 and Bms1) previously regarded as potential nucleating factors of the 90S particle. Here, we demonstrate that the loading of those three proteins onto the pre-rRNA takes place independently of Rrp5/UTP-C and, instead, occurs downstream of the tUTP and U3/UTP-B subcomplexes. We also demonstrate that Bms1 and Utp20 are required for the recruitment of a subset of proteins to nascent pre-ribosomes. Finally, we show that proteins associated through secondary steps condition the stability of the two assembly branches in partially assembled pre-ribosomes. These results provide new information about the functional relationships among 90S particle components and the events that are required for their stepwise incorporation onto the primary pre-rRNA. © 2011 The Author(s)., Spanish Ministry of Science and Innovation (MICINN) (BFU-2008-02729 and RD06/0020/0001); Castilla-León Autonomous Government (GR95); Samuel Solórzano Barruso Foundation (FS/2-2009). MICINN funding is co-sponsored by the European Union FEDER Program. Funding for open access charge: Spanish Ministry of Science and Innovation (MICINN) (BFU-2008-02729).

The interactions among the yeast stalk components (P0, P1alpha, P1beta, P2alpha and P2beta) and with EF-2 have been explored using immunoprecipitation, affinity chromatography and the two-hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1alpha and P1beta were found to interact with P2beta and P2alpha respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C-terminal P0 fragments. Immunoprecipitation shows the association of EF-2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two-hybrid assay. This interaction improves using the 100-amino-acid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1alpha with P2beta and of P1beta with P2alpha rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.

Different models have been proposed explaining how eukaryotic gene transcription is terminated. Recently, Nsi1, a factor involved in silencing of ribosomal DNA (rDNA), was shown to be required for efficient termination of rDNA transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. Nsi1 contains Myb-like DNA binding domains and associates in vivo near the 3′ end of rRNA genes to rDNA, but information about which and how DNA sequences might influence Nsi1-dependent termination is lacking. Here, we show that binding of Nsi1 to a stretch of 11 nucleotides in the correct orientation was sufficient to pause elongating Pol I shortly upstream of the Nsi1 binding site and to release the transcripts in vitro. The same minimal DNA element triggered Nsi1-dependent termination of pre-rRNA synthesis using an in vivo reporter assay. Termination efficiency in the in vivo system could be enhanced by inclusion of specific DNA sequences downstream of the Nsi1 binding site. These data and the finding that Nsi1 blocks efficiently only Pol I-dependent RNA synthesis in an in vitro transcription system improve our understanding of a unique mechanism of transcription termination.

The stalk of the large ribosomal subunit is involved in the interaction of the elongation factors with the ribosome, as biochemical data have clearly indicated and as has been directly confirmed by electron microscopy. The eukaryotic ribosomal-stalk elements are functionally equivalent to the bacterial components, but while some of them are highly conserved, others have evolved notably. The highly probable presence of the acidic proteins in the ribosome as monomers, together with the existence of one specific binding site for each 12-kDa protein in the particles, favors the homogeneous rather than the heterogeneous ribosomal-stalk model in yeast. The data available on the assembly of the yeast stalk are not abundant. An analysis of two Saccharomyces cerevisiae strains carrying a disruption of the genes encoding the two proteins of the same type, either P1 or P2, has shown that their ribosomes do not carry any 12-kDa acidic protein. The experimental evidence indicates that the in vitro assembly of the yeast ribosomal stalk involves a number of consecutive binding steps which are apparently initiated by the interaction of the P1 proteins and is followed by the binding of the P2 polypeptides.

Most methods currently available for the analysis of chromatin in vivo rely on a priori knowledge of putative chromatin components or their posttranslational modification state. The isolation of defined native chromosomal regions provides an attractive alternative to obtain a largely unbiased molecular description of chromatin. Here, we describe a strategy combining site-specific recombination at the chromosome with an efficient tandem affinity purification protocol to isolate a single-copy gene locus from the yeast Saccharomyces cerevisiae. The method allows robust enrichment of a targeted chromatin domain, making it amenable to compositional, structural, and biochemical analyses. This technique appears to be suitable to obtain a detailed description of chromatin composition and specific posttranslational histone modification state at virtually any genomic locus in yeast.

Most methods currently available for the analysis of chromatin in vivo rely on a priori knowledge of putative chromatin components or their posttranslational modification state. The isolation of defined native chromosomal regions provides an attractive alternative to obtain a largely unbiased molecular description of chromatin. Here, we describe a strategy combining site-specific recombination at the chromosome with an efficient tandem affinity purification protocol to isolate a single-copy gene locus from the yeast Saccharomyces cerevisiae. The method allows robust enrichment of a targeted chromatin domain, making it amenable to compositional, structural, and biochemical analyses. This technique appears to be suitable to obtain a detailed description of chromatin composition and specific posttranslational histone modification state at virtually any genomic locus in yeast.

Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of similar to 70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Delta. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.

The synthesis of ribosomal RNA (rRNA) precursor molecules by RNA polymerase I (Pol I) terminates with the dissociation of the protein-DNA-RNA ternary complex. Based on in vitro results the mechanism of Pol I termination appeared initially to be rather conserved and simple until this process was more thoroughly re-investigated in vivo. A picture emerged that Pol I termination seems to be connected to co-transcriptional processing, re-initiation of transcription and, possibly, other processes downstream of Pol I transcription units. In this article, our current understanding of the mechanism of Pol I termination and how this process might be implicated in other biological processes in yeast and mammals is summarized and discussed. This article is part of a Special Issue entitled: Transcription by Odd Pols.

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.

The Pol I subunits Rpa34 and Rpa49, required for elongation and 35S rRNA production, promote clustering of neighboring Pol I complexes to enhance the rRNA gene transcription cycle., RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.

The ribosomal stalk is essential for translation; however, its overall structure is poorly understood. Characterization of the region involved in the interactions between protein P0 and the 12 kDa acidic proteins P1 and P2 is fundamental to understand the assembly and function of this structure in the eukaryotic ribosome. The acidic protein content is important for the ribosome efficiency and affects the translation of specific mRNAs. By usage of a series of progressively truncated fragments of protein P0 in the two-hybrid test, a region between positions 213 and 250 was identified as the minimal protein part able to interact with the acidic proteins. Extensions at either end affect the binding capacity of the fragment either positively or negatively depending on the number of added amino acids. Deletions inside the binding region confirm its in vivo relevance since they drastically reduce the P0 interacting capacity with the 12 kDa acidic proteins, which are severely reduced in the ribosome when the truncated protein is expressed in the cell. Moreover, recombinant His-tagged P0 fragments containing the binding site and bound to Ni(2+)-NTA columns can form a complex with the P1 and P2 proteins, which is able to bind elongation factor EF2. The results indicate the existence of a region in P0 that specifically interacts with the acidic proteins. These interactions are, however, hindered by the presence of neighbor protein domains, suggesting the need for conformational changes in the complete P0 to allow the assembly of the ribosomal stalk.