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1.
Nucleic Acids Res ; 48(5): 2604-2620, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980825

RESUMO

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA-DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA-DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.


Assuntos
DNA Fúngico/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Moldes Genéticos , Elongação da Transcrição Genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biocatálise , DNA Fúngico/química , Cadeias de Markov , Metiltransferases/química , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , Nucleotídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , RNA Fúngico/biossíntese , Deleção de Sequência , Relação Estrutura-Atividade , Iniciação da Transcrição Genética
2.
Genome Res ; 29(12): 1974-1984, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740578

RESUMO

Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site (TSS) usage, chromatin organization, and posttranscriptional consequences in Saccharomyces cerevisiae We show that TSSs of chromatin-sensitive internal cryptic transcripts retain comparable features of canonical TSSs in terms of DNA sequence, directionality, and chromatin accessibility. We define the 5' and 3' boundaries of cryptic transcripts and show that, contrary to RNA degradation-sensitive ones, they often overlap with the end of the gene, thereby using the canonical polyadenylation site, and associate to polyribosomes. We show that chromatin-sensitive cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Finally, we confirm the presence of the predicted polypeptides by reanalyzing N-terminal proteomic data sets. Our work suggests that a fraction of chromatin-sensitive internal cryptic promoters initiates the transcription of alternative truncated mRNA isoforms. The expression of these chromatin-sensitive isoforms is conserved from yeast to human, expanding the functional consequences of cryptic transcription and proteome complexity.


Assuntos
Cromatina , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sítio de Iniciação de Transcrição , Cromatina/genética , Cromatina/metabolismo , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade de RNA , RNA Fúngico/biossíntese , RNA Fúngico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
J Biol Chem ; 294(33): 12392-12404, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31239353

RESUMO

Genome-wide analyses have revealed that during metal ion starvation, many cells undergo programmed changes in their transcriptome or proteome that lower the levels of abundant metalloproteins, conserving metal ions for more critical functions. Here we investigated how changes in cellular zinc status affect the expression and activity of the zinc-requiring Pho8 alkaline phosphatase from fission yeast (Schizosaccharomyces pombe). In S. pombe, Pho8 is a membrane-tethered and processed glycoprotein that resides in the vacuole. Using alkaline phosphatase activity assays along with various biochemical analyses, we found that Pho8 is active when zinc is plentiful and inactive when zinc is limited. Although Pho8 activity depended on zinc, we also found that higher levels of pho8 mRNAs and Pho8 protein accumulate in zinc-deficient cells. To gain a better understanding of the inverse relationship between pho8 mRNA levels and Pho8 activity, we examined the effects of zinc on the stability and processing of the Pho8 protein. We show that Pho8 is processed regardless of zinc status and that mature Pho8 accumulates under all conditions. We also noted that alkaline phosphatase activity is rapidly restored when zinc is resupplied to cells, even in the presence of the protein synthesis inhibitor cycloheximide. Our results suggest that S. pombe cells maintain inactive pools of Pho8 proteins under low-zinc conditions and that these pools facilitate rapid restoration of Pho8 activity when zinc ions become available.


Assuntos
Fosfatase Alcalina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Zinco/metabolismo , Fosfatase Alcalina/genética , Ativação Enzimática , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
4.
J Biol Chem ; 294(33): 12349-12358, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31235518

RESUMO

In yeast (Saccharomyces cerevisiae), the synthesis of tRNAs by RNA polymerase III (RNAP III) down-regulates the transcription of the nearby RNAP II-transcribed genes by a mechanism that is poorly understood. To clarify the basis of this tRNA gene-mediated (TGM) silencing, here, conducting a bioinformatics analysis of available ChIP-chip and ChIP-sequencing genomic data from yeast, we investigated whether the RNAP III transcriptional machinery can recruit protein factors required for RNAP II transcription. An analysis of 46 genome-wide protein-density profiles revealed that 12 factors normally implicated in RNAP II-mediated gene transcription are more enriched at tRNA than at mRNA loci. These 12 factors typically have RNA-binding properties, participate in the termination stage of the RNAP II transcription, and preferentially localize to the tRNA loci by a mechanism that apparently is based on the RNAP III transcription level. The factors included two kinases of RNAP II (Bur1 and Ctk1), a histone demethylase (Jhd2), and a mutated form of a nucleosome-remodeling factor (Spt6) that have never been reported to be recruited to tRNA loci. Moreover, we show that the expression levels of RNAP II-transcribed genes downstream of tRNA loci correlate with the distance from the tRNA gene by a mechanism that depends on their orientation. These results are consistent with the notion that pre-tRNAs recruit RNAP II-associated factors, thereby reducing the availability of these factors for RNAP II transcription and contributing, at least in part, to the TGM-silencing mechanism.


Assuntos
Quinases Ciclina-Dependentes , Loci Gênicos , Chaperonas de Histonas , Proteínas Quinases , RNA Polimerase II , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Transcrição Genética/fisiologia , Fatores de Elongação da Transcrição , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
5.
Biochim Biophys Acta Mol Cell Res ; 1866(5): 806-818, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30759361

RESUMO

Mitochondrial tRNAs are processed at their 5'ends by highly divergent but ubiquitous RNase P. In Saccharomyces cerevisiae, Rpm2p is the protein component of RNase P. Here, we identify four novel genes MTA1, MTA2, GEP5 and PET130 of the Saccharomycetaceae family that are necessary for an efficient processing of mitochondrial tRNAs. Null mutants of mta1, mta2 and gep5 have severely reduced levels of mitochondrial tRNAs; in addition, temperature sensitive (ts) mutants of mta1, mta2, pet130 and gep5 accumulated tRNAs precursor transcripts at the restrictive but not at the permissive temperature. The same mitochondrial tRNAs precursors were also identified in rpm2 ts mutants or in the double ts mutants mta1 rpm2 and mta2 rpm2. The genetic and physical association of these four novel genes corroborate the hypothesis that they have their function associated. Different combinations of mta1, mta2, pet130 and gep5 ts alleles display a synthetic respiratory deficient phenotype, an indication of genetic interactions of the genes. Indeed, Mta1p, Mta2p, Pet130p, and Gep5p are associated with the mitochondrial inner membrane and are all extracted and sediment in sucrose gradients as high molecular weight complexes, where they may be present in a common complex with Rpm2p. This is supported by pull-down assays showing co-immunopurification of Rpm2 with Mta1p.


Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , RNA Fúngico/biossíntese , RNA Mitocondrial/biossíntese , RNA de Transferência/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , RNA Fúngico/genética , RNA Mitocondrial/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
6.
Mol Cell ; 72(6): 955-969.e7, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30576657

RESUMO

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.


Assuntos
Regulação Fúngica da Expressão Gênica , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , RNA não Traduzido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Fúngico/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/genética , Fatores de Transcrição/genética , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética
7.
Proc Natl Acad Sci U S A ; 115(29): 7533-7538, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29959206

RESUMO

Despite substantial experimental and computational efforts, mechanistic modeling remains more predictive in engineering than in systems biology. The reason for this discrepancy is not fully understood. One might argue that the randomness and complexity of biological systems are the main barriers to predictive understanding, but these issues are not unique to biology. Instead, we hypothesize that the specific shapes of rare single-molecule event distributions produce substantial yet overlooked challenges for biological models. We demonstrate why modern statistical tools to disentangle complexity and stochasticity, which assume normally distributed fluctuations or enormous datasets, do not apply to the discrete, positive, and nonsymmetric distributions that characterize mRNA fluctuations in single cells. As an example, we integrate single-molecule measurements and advanced computational analyses to explore mitogen-activated protein kinase induction of multiple stress response genes. Through systematic analyses of different metrics to compare the same model to the same data, we elucidate why standard modeling approaches yield nonpredictive models for single-cell gene regulation. We further explain how advanced tools recover precise, reproducible, and predictive understanding of transcription regulation mechanisms, including gene activation, polymerase initiation, elongation, mRNA accumulation, spatial transport, and decay.


Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Modelos Genéticos , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Saccharomyces cerevisiae/metabolismo , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética
8.
Proc Natl Acad Sci U S A ; 115(17): 4429-4434, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29643074

RESUMO

Many fungi are polykaryotic, containing multiple nuclei per cell. In the case of heterokaryons, there are different nuclear types within a single cell. It is unknown what the different nuclear types contribute in terms of mRNA expression levels in fungal heterokaryons. Each cell of the mushroom Agaricus bisporus contains two to 25 nuclei of two nuclear types originating from two parental strains. Using RNA-sequencing data, we assess the differential mRNA contribution of individual nuclear types and its functional impact. We studied differential expression between genes of the two nuclear types, P1 and P2, throughout mushroom development in various tissue types. P1 and P2 produced specific mRNA profiles that changed through mushroom development. Differential regulation occurred at the gene level, rather than at the locus, chromosomal, or nuclear level. P1 dominated mRNA production throughout development, and P2 showed more differentially up-regulated genes in important functional groups. In the vegetative mycelium, P2 up-regulated almost threefold more metabolism genes and carbohydrate active enzymes (cazymes) than P1, suggesting phenotypic differences in growth. We identified widespread transcriptomic variation between the nuclear types of A. bisporus Our method enables studying nucleus-specific expression, which likely influences the phenotype of a fungus in a polykaryotic stage. Our findings have a wider impact to better understand gene regulation in fungi in a heterokaryotic state. This work provides insight into the transcriptomic variation introduced by genomic nuclear separation.


Assuntos
Agaricus/metabolismo , Núcleo Celular/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Regulação para Cima/fisiologia , Agaricus/genética , Núcleo Celular/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Transcriptoma/fisiologia
9.
Mol Cell ; 70(2): 312-326.e7, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29656924

RESUMO

Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other termination factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant termination. We suggest that ncRNA levels and coding gene transcription termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.


Assuntos
DNA Helicases/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Fúngica da Expressão Gênica , RNA Helicases/metabolismo , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , RNA não Traduzido/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Terminação da Transcrição Genética , DNA Helicases/genética , Viabilidade Microbiana , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Helicases/genética , RNA Fúngico/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinação
10.
PLoS One ; 13(3): e0192633, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29561870

RESUMO

The degree of conservation and evolution of cytoplasmic mRNA metabolism pathways across the eukaryotes remains incompletely resolved. In this study, we describe a comprehensive genome and transcriptome-wide analysis of proteins involved in mRNA maturation, translation, and mRNA decay across representative organisms from the six eukaryotic super-groups. We demonstrate that eukaryotes share common pathways for mRNA metabolism that were almost certainly present in the last eukaryotic common ancestor, and show for the first time a correlation between intron density and a selective absence of some Exon Junction Complex (EJC) components in eukaryotes. In addition, we identify pathways that have diversified in individual lineages, with a specific focus on the unique gene gains and losses in members of the Excavata and SAR groups that contribute to their unique gene expression pathways compared to other organisms.


Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Genes Fúngicos/fisiologia , RNA Fúngico , RNA Mensageiro , Saccharomyces cerevisiae , Schizosaccharomyces , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Especificidade da Espécie
11.
PLoS One ; 13(3): e0194438, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29570714

RESUMO

Transcription as the key step in gene expression is a highly regulated process. The speed of transcription elongation depends on the underlying gene sequence and varies on a gene by gene basis. The reason for this sequence dependence is not known in detail. Recently, our group studied the cross talk between the nascent RNA and the transcribing RNA polymerase by screening the Escherichia coli genome for RNA sequences with high affinity to RNA Pol by performing genomic SELEX. This approach led to the identification of RNA polymerase-binding APtamers termed "RAPs". RAPs can have positive and negative effects on gene expression. A subgroup is able to downregulate transcription via the activity of the termination factor Rho. In this study, we used a similar SELEX setup using yeast genomic DNA as source of RNA sequences and highly purified yeast RNA Pol II as bait and obtained almost 1300 yeast-derived RAPs. Yeast RAPs are found throughout the genome within genes and antisense to genes, they are overrepresented in the non-transcribed strand of yeast telomeres and underrepresented in intergenic regions. Genes harbouring a RAP are more likely to show lower mRNA levels. By determining the endogenous expression levels as well as using a reporter system, we show that RAPs located within coding regions can reduce the transcript level downstream of the RAP. Here we demonstrate that RAPs represent a novel type of regulatory RNA signal in Saccharomyces cerevisiae that act in cis and interfere with the elongating transcription machinery to reduce the transcriptional output.


Assuntos
Proteínas Fúngicas/metabolismo , RNA Polimerase II/metabolismo , RNA Fúngico/biossíntese , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Elongação da Transcrição Genética/fisiologia , Proteínas Fúngicas/genética , RNA Polimerase II/genética , RNA Fúngico/genética , Saccharomyces cerevisiae/genética
12.
Methods Mol Biol ; 1721: 63-72, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29423847

RESUMO

The fission yeast, Schizosaccharomyces pombe, is an important model organism for investigations of gene regulation. Essential to such studies is the ability to quantify the levels of a specific RNA. We describe a protocol for the isolation and quantification of RNA in S. pombe using reverse-transcription followed by quantitative PCR. In this procedure, the cells are lysed using zirconia beads, then total RNA is selectively isolated away from proteins and DNA using the Trizol reagent. Contaminating DNA is then removed from the RNA by using TURBO DNase, which is easily inactivated and requires no subsequent clean-up step. The RNA is then reverse transcribed into cDNA using random nine-mers and oligo dT primers . Quantitative PCR using SYBR green is then performed to quantify RNA levels. This protocol has been tested on several S. pombe genotypes and generates highly reproducible results.


Assuntos
RNA Fúngico/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Schizosaccharomyces/genética , Análise de Sequência de RNA/métodos , RNA Fúngico/biossíntese , Schizosaccharomyces/metabolismo
13.
Methods Mol Biol ; 1721: 73-87, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29423848

RESUMO

The patterns of gene expression in the fission yeast Schizosaccharomyces pombe under various experimental conditions form the basis of any transcriptomic study. We describe a method involving reverse transcription of the mRNA, Polymerase Chain Reaction (PCR), and the subsequent separation of the products onto Urea-Polyacrylamide gel that can be used to study the gene expression patterns in the fission yeast. The method described is cost effective and reproducible with satisfactory resolution of expressed transcripts in the gel. The method has the following essential steps: total RNA isolation and purification, cDNA synthesis from mRNAs, PCR amplification of cDNAs, visualization of PCR products, re-amplification and cloning of the differentially expressed PCR products, sequencing the confirmed clones, and finally cDNA library screening to isolate the genes of interest. The technique is also popularly known as Differential Display Reverse Transcription (DDRT-PCR). After its invention in 1992, a number of modifications have been introduced to optimize the technique and specifically to reduce the major problem of "false positives." Since understanding of specific gene expression patterns that regulate developmental and stress responses is a major concern of biology, DDRT-PCR has become a very popular molecular technique during the past two decades.


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Biblioteca Gênica , RNA Fúngico/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Schizosaccharomyces/genética , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Schizosaccharomyces/metabolismo
14.
RNA ; 24(2): 237-250, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29122971

RESUMO

Expression of fission yeast glycerophosphate transporter Tgp1 is repressed in phosphate-rich medium and induced during phosphate starvation. Repression is enforced by transcription of the nc-tgp1 locus upstream of tgp1 to produce a long noncoding (lnc) RNA. Here we identify two essential elements of the nc-tgp1 promoter: a TATA box -30TATATATA-23 and a HomolD box -64CAGTCACA-57, mutations of which inactivate the nc-tgp1 promoter and de-repress the downstream tgp1 promoter under phosphate-replete conditions. The nc-tgp1 lncRNA poly(A) site maps to nucleotide +1636 of the transcription unit, which coincides with the binding site for Pho7 (1632TCGGACATTCAA1643), the transcription factor that drives tgp1 expression. Overlap between the lncRNA template and the tgp1 promoter points to transcriptional interference as the simplest basis for lncRNA repression. We identify a shorter RNA derived from the nc-tgp1 locus, polyadenylated at position +508, well upstream of the tgp1 promoter. Mutating the nc-tgp1-short RNA polyadenylation signal abolishes de-repression of the downstream tgp1 promoter elicited by Pol2 CTD Ser5Ala phospho-site mutation. Ser5 mutation favors utilization of the short RNA poly(A) site, thereby diminishing transcription of the lncRNA that interferes with the tgp1 promoter. Mutating the nc-tgp1-short RNA polyadenylation signal attenuates induction of the tgp1 promoter during phosphate starvation. Polyadenylation site choice governed by CTD Ser5 status adds a new level of lncRNA control of gene expression and reveals a new feature of the fission yeast CTD code.


Assuntos
Proteínas de Membrana Transportadoras/genética , RNA Polimerase II/genética , RNA Longo não Codificante/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/biossíntese , Mutação , Fosfatos/fisiologia , Poliadenilação , Regiões Promotoras Genéticas , RNA Fúngico/biossíntese , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Proteínas de Schizosaccharomyces pombe/biossíntese , Serina/genética , TATA Box , Sítio de Iniciação de Transcrição
15.
Biochim Biophys Acta Gene Regul Mech ; 1861(4): 310-319, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29127063

RESUMO

RNA polymerase III (RNAPIII) transcribes tRNA genes, 5S RNA as well as a number of other non-coding RNAs. Because transcription by RNAPIII is an energy-demanding process, its activity is tightly linked to the stress levels and nutrient status of the cell. Multiple signaling pathways control RNAPIII activity in response to environmental cues, but exactly how these pathways regulate RNAPIII is still poorly understood. One major target of these pathways is the transcriptional repressor Maf1, which inhibits RNAPIII activity under conditions that are detrimental to cell growth. However, recent studies have found that the cell can also directly regulate the RNAPIII machinery through phosphorylation and sumoylation of RNAPIII subunits. In this review we summarize post-translational modifications of RNAPIII subunits that mainly have been identified in large-scale proteomics studies, and we highlight several examples to discuss their relevance for regulation of RNAPIII.


Assuntos
Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , RNA Polimerase III/metabolismo , RNA de Transferência/biossíntese , Animais , Caseína Quinases/genética , Proteína Coatomer/genética , Regulação Fúngica da Expressão Gênica , Fosforilação , Subunidades Proteicas , RNA Polimerase III/química , RNA Polimerase III/genética , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA de Transferência/genética , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Sumoilação , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIIIB/genética , Fatores de Transcrição/genética
16.
Cell Rep ; 19(12): 2477-2489, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28636937

RESUMO

RNAi factors and their catalytic activities are essential for heterochromatin assembly in S. pombe. This has led to the idea that siRNAs can promote H3K9 methylation by recruiting the cryptic loci regulator complex (CLRC), also known as recombination in K complex (RIKC), to the nucleation site. The conserved RNA-binding protein Rct1 (AtCyp59/SIG-7) interacts with splicing factors and RNA polymerase II. Here we show that Rct1 promotes processing of pericentromeric transcripts into siRNAs via the RNA recognition motif. Surprisingly, loss of siRNA in rct1 mutants has no effect on H3K9 di- or tri-methylation, resembling other splicing mutants, suggesting that post-transcriptional gene silencing per se is not required to maintain heterochromatin. Splicing of the Argonaute gene is also defective in rct1 mutants and contributes to loss of silencing but not to loss of siRNA. Our results suggest that Rct1 guides transcripts to the RNAi machinery by promoting splicing of elongating non-coding transcripts.


Assuntos
Ciclofilinas/fisiologia , Heterocromatina/genética , RNA Fúngico/genética , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/genética , Montagem e Desmontagem da Cromatina , Ciclofilinas/química , Exossomos/metabolismo , Regulação Fúngica da Expressão Gênica , Heterocromatina/metabolismo , Histonas/metabolismo , Metilação , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Interferência de RNA , RNA Polimerase II/metabolismo , Processamento de RNA , RNA Fúngico/biossíntese , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química
17.
Cold Spring Harb Protoc ; 2017(5)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28461655

RESUMO

Traditionally, the half-lives of mRNAs were measured after inhibition of transcription to allow decay of the preexisting population. The protocol presented here is a more recently developed strategy in which mRNA turnover is analyzed by measuring the decline in levels of newly synthesized RNA labeled with 4-thiouridine (4sU) during a brief pulse. After RNA extraction, the 4sU is biotinylated and the labeled species are purified using streptavidin beads. DNA microarrays can then be used to compare this population with total RNA, allowing half-lives to be calculated.


Assuntos
Marcadores de Afinidade , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Schizosaccharomyces/metabolismo , Tiouridina , Antimetabólitos , Biotinilação , Meia-Vida , Indicadores e Reagentes , Análise de Sequência com Séries de Oligonucleotídeos , RNA Fúngico/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Estreptavidina
18.
J Cell Biol ; 216(7): 1907-1914, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28539386

RESUMO

Eukaryotic genomes are replicated in a reproducible temporal order; however, the physiological significance is poorly understood. We compared replication timing in divergent yeast species and identified genomic features with conserved replication times. Histone genes were among the earliest replicating loci in all species. We specifically delayed the replication of HTA1-HTB1 and discovered that this halved the expression of these histone genes. Finally, we showed that histone and cell cycle genes in general are exempt from Rtt109-dependent dosage compensation, suggesting the existence of pathways excluding specific loci from dosage compensation mechanisms. Thus, we have uncovered one of the first physiological requirements for regulated replication time and demonstrated a direct link between replication timing and gene expression.


Assuntos
Proteínas de Ciclo Celular/genética , Período de Replicação do DNA , DNA Fúngico/biossíntese , Regulação Fúngica da Expressão Gênica , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/biossíntese , DNA Fúngico/genética , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/biossíntese , Mutação , Filogenia , RNA Fúngico/biossíntese , RNA Fúngico/genética , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Transcrição Genética
19.
Mol Cell ; 66(1): 77-88.e5, 2017 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-28366642

RESUMO

Spt5 is an essential and conserved factor that functions in transcription and co-transcriptional processes. However, many aspects of the requirement for Spt5 in transcription are poorly understood. We have analyzed the consequences of Spt5 depletion in Schizosaccharomyces pombe using four genome-wide approaches. Our results demonstrate that Spt5 is crucial for a normal rate of RNA synthesis and distribution of RNAPII over transcription units. In the absence of Spt5, RNAPII localization changes dramatically, with reduced levels and a relative accumulation over the first ∼500 bp, suggesting that Spt5 is required for transcription past a barrier. Spt5 depletion also results in widespread antisense transcription initiating within this barrier region. Deletions of this region alter the distribution of RNAPII on the sense strand, suggesting that the barrier observed after Spt5 depletion is normally a site at which Spt5 stimulates elongation. Our results reveal a global requirement for Spt5 in transcription elongation.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , RNA Antissenso/biossíntese , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , Proteínas Cromossômicas não Histona/genética , Biologia Computacional , Bases de Dados Genéticas , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Genótipo , Mutação , Fenótipo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Processamento de RNA , RNA Antissenso/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Tempo , Fatores de Elongação da Transcrição/genética
20.
Mol Cell ; 66(1): 38-49.e6, 2017 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-28318822

RESUMO

At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3'-processing of the pre-mRNA and transcription termination. Here, we present a genome-wide analysis of the 3'-transition in budding yeast. We find that the 3'-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the polyadenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and requires the "torpedo" exonuclease Rat1 (XRN2 in human). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3'-transition can result in increased transcription at downstream genes.


Assuntos
DNA Fúngico/metabolismo , Processamento de Terminações 3' de RNA , RNA Polimerase II/metabolismo , Precursores de RNA/biossíntese , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Modelos Genéticos , Ligação Proteica , RNA Polimerase II/genética , Precursores de RNA/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
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