Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.829
Filtrar
1.
Nat Commun ; 11(1): 1206, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139698

RESUMO

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Compared to Pols II and III, the mechanisms underlying promoter recognition, initiation complex formation and DNA melting by Pol I substantially diverge. Here, we report the high-resolution cryo-EM reconstruction of a Pol I early initiation intermediate assembled on a double-stranded promoter scaffold that prevents the establishment of downstream DNA contacts. Our analyses demonstrate how efficient promoter-backbone interaction is achieved by combined re-arrangements of flexible regions in the 'core factor' subunits Rrn7 and Rrn11. Furthermore, structure-function analysis illustrates how destabilization of the melted DNA region correlates with contraction of the polymerase cleft upon transcription activation, thereby combining promoter recruitment with DNA-melting. This suggests that molecular mechanisms and structural features of Pol I initiation have co-evolved to support the efficient melting, initial transcription and promoter clearance required for high-level rRNA synthesis.


Assuntos
Regiões Promotoras Genéticas , RNA Polimerase I/metabolismo , Saccharomyces cerevisiae/genética , Iniciação da Transcrição Genética , Sequência de Aminoácidos , DNA/química , DNA/ultraestrutura , Modelos Moleculares , Estrutura Secundária de Proteína , Subunidades Proteicas/metabolismo , RNA Polimerase I/química , RNA Polimerase I/ultraestrutura , Fator de Transcrição TFIIB/metabolismo
2.
Nat Commun ; 10(1): 5543, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804486

RESUMO

RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn3 on the rDNA core promoter for transcription initiation. Here, we report cryo-EM structures of closed, intermediate and open Pol I initiation complexes from 2.7 to 3.7 Å resolution to visualize Pol I promoter melting and to structurally and biochemically characterize the recognition mechanism of Pol I promoter DNA. In the closed complex, double-stranded DNA runs outside the DNA-binding cleft. Rotation of CF and upstream DNA with respect to Pol I and Rrn3 results in the spontaneous loading and opening of the promoter followed by cleft closure and positioning of the Pol I A49 tandem winged helix domain (tWH) onto DNA. Conformational rearrangement of A49 tWH leads to a clash with Rrn3 to initiate complex disassembly and promoter escape. Comprehensive insight into the Pol I transcription initiation cycle allows comparisons with promoter opening by Pol II and Pol III.


Assuntos
DNA Fúngico/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase I/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , DNA Fúngico/química , DNA Fúngico/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Polimerase I/química , RNA Polimerase I/metabolismo , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Iniciação da Transcrição Genética
3.
Elife ; 82019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31841110

RESUMO

Ribosomes are universally important in biology and their production is dysregulated by developmental disorders, cancer, and virus infection. Although presumed required for protein synthesis, how ribosome biogenesis impacts virus reproduction and cell-intrinsic immune responses remains untested. Surprisingly, we find that restricting ribosome biogenesis stimulated human cytomegalovirus (HCMV) replication without suppressing translation. Interfering with ribosomal RNA (rRNA) accumulation triggered nucleolar stress and repressed expression of 1392 genes, including High Mobility Group Box 2 (HMGB2), a chromatin-associated protein that facilitates cytoplasmic double-stranded (ds) DNA-sensing by cGAS. Furthermore, it reduced cytoplasmic HMGB2 abundance and impaired induction of interferon beta (IFNB1) mRNA, which encodes a critical anti-proliferative, proinflammatory cytokine, in response to HCMV or dsDNA in uninfected cells. This establishes that rRNA accumulation regulates innate immune responses to dsDNA by controlling HMGB2 abundance. Moreover, it reveals that rRNA accumulation and/or nucleolar activity unexpectedly regulate dsDNA-sensing to restrict virus reproduction and regulate inflammation. (145 words).


Assuntos
DNA , Imunidade Inata , Ribossomos/metabolismo , Viroses , Linhagem Celular , Citocinas/metabolismo , Citomegalovirus/imunologia , Fibroblastos , Proteína HMGB2/genética , Humanos , Imunidade Inata/genética , Interferon beta , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , RNA Polimerase I , RNA Mensageiro/metabolismo , RNA Ribossômico , Viroses/genética , Replicação Viral
4.
Genome Res ; 29(12): 1939-1950, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31732535

RESUMO

Transcription factor binding to target sites in vivo is a dynamic process that involves cycles of association and dissociation, with individual proteins differing in their binding dynamics. The dynamics at individual sites on a genomic scale have been investigated in yeast cells, but comparable experiments have not been done in multicellular eukaryotes. Here, we describe a tamoxifen-inducible, time-course ChIP-seq approach to measure transcription factor binding dynamics at target sites throughout the human genome. As observed in yeast cells, the TATA-binding protein (TBP) typically displays rapid turnover at RNA polymerase (Pol) II-transcribed promoters, slow turnover at Pol III promoters, and very slow turnover at the Pol I promoter. Turnover rates vary widely among Pol II promoters in a manner that does not correlate with the level of TBP occupancy. Human Pol II promoters with slow TBP dissociation preferentially contain a TATA consensus motif, support high transcriptional activity of downstream genes, and are linked with specific activators and chromatin remodelers. These properties of human promoters with slow TBP turnover differ from those of yeast promoters with slow turnover. These observations suggest that TBP binding dynamics differentially affect promoter function and gene expression, possibly at the level of transcriptional reinitiation/bursting.


Assuntos
Regulação da Expressão Gênica/fisiologia , Genoma Humano , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Polimerase I/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Humanos
5.
Int J Mol Sci ; 20(19)2019 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-31590335

RESUMO

The stabilization of G-quadruplex DNA structures by small molecules with affinity to oncogene promoters has emerged as a promising anticancer strategy, due to a potential role in gene expression regulation. We explored the ability of BMH-21 (1) and its analogue BA-41 (2) to bind the G-quadruplex structure present in the c-KIT promoter by biophysical methods and molecular modeling. We provide evidence that both compounds interact with the c-KIT 21-mer sequence. The stable monomeric intramolecular parallel G-quadruplex obtained by the mutation of positions 12 and 21 allowed the precise determination of the binding mode by NMR and molecular dynamics studies. Both compounds form a complex characterized by one ligand molecule positioned over the tetrad at the 3'-end, stabilized by an extensive network of π-π interactions. The binding constants (Kb) obtained with fluorescence are similar for both complexes (around 106 M-1). Compound BA-41 (2) showed significant antiproliferative activity against a human lymphoma cell line, SU-DHL4, known to express substantial levels of c-KIT. However, the partial inhibition of c-KIT expression by Western blot analysis suggested that the interaction of compound 2 with the c-KIT promoter is not the primary event and that multiple effects provide a contribution as determinants of biological activity.


Assuntos
Quadruplex G , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , RNA Polimerase I/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-kit/metabolismo
6.
Prostate ; 79(16): 1837-1851, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31524299

RESUMO

BACKGROUND: Advanced prostate cancers depend on protein synthesis for continued survival and accelerated rates of metabolism for growth. RNA polymerase I (Pol I) is the enzyme responsible for ribosomal RNA (rRNA) transcription and a rate-limiting step for ribosome biogenesis. We have shown using a specific and sensitive RNA probe for the 45S rRNA precursor that rRNA synthesis is increased in prostate adenocarcinoma compared to nonmalignant epithelium. We have introduced a first-in-class Pol I inhibitor, BMH-21, that targets cancer cells of multiple origins, and holds potential for clinical translation. METHODS: The effect of BMH-21 was tested in prostate cancer cell lines and in prostate cancer xenograft and mouse genetic models. RESULTS: We show that BMH-21 inhibits Pol I transcription in metastatic, castration-resistant, and enzalutamide treatment-resistant prostate cancer cell lines. The genetic abrogation of Pol I effectively blocks the growth of prostate cancer cells. Silencing of p53, a pathway activated downstream of Pol I, does not diminish this effect. We find that BMH-21 significantly inhibited tumor growth and reduced the Ki67 proliferation index in an enzalutamide-resistant xenograft tumor model. A decrease in 45S rRNA synthesis demonstrated on-target activity. Furthermore, the Pol I inhibitor significantly inhibited tumor growth and pathology in an aggressive genetically modified Hoxb13-MYC|Hoxb13-Cre|Ptenfl/fl (BMPC) mouse prostate cancer model. CONCLUSION: Taken together, BMH-21 is a novel promising molecule for the treatment of castration-resistant prostate cancer.


Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , RNA Polimerase I/antagonistas & inibidores , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Células PC-3 , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Ribossômico/genética , Distribuição Aleatória , Transcrição Genética/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
PLoS Negl Trop Dis ; 13(9): e0007745, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31560682

RESUMO

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus (Flaviviridae), is a causative agent of a severe neuroinfection. Recently, several flaviviruses have been shown to interact with host protein synthesis. In order to determine whether TBEV interacts with this host process in its natural target cells, we analysed de novo protein synthesis in a human cell line derived from cerebellar medulloblastoma (DAOY HTB-186). We observed a significant decrease in the rate of host protein synthesis, including the housekeeping genes HPRT1 and GAPDH and the known interferon-stimulated gene viperin. In addition, TBEV infection resulted in a specific decrease of RNA polymerase I (POLR1) transcripts, 18S and 28S rRNAs and their precursor, 45-47S pre-rRNA, but had no effect on the POLR3 transcribed 5S rRNA levels. To our knowledge, this is the first report of flavivirus-induced decrease of specifically POLR1 rRNA transcripts accompanied by host translational shut-off.


Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Encefalite Transmitida por Carrapatos/virologia , Biossíntese de Proteínas/genética , Animais , Linhagem Celular Tumoral , Encefalite Transmitida por Carrapatos/genética , Encefalite Transmitida por Carrapatos/metabolismo , Humanos , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Precursores de RNA , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Transcrição Genética
8.
Nucleic Acids Res ; 47(19): 10357-10372, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31504794

RESUMO

Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine-binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Histona Acetiltransferases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Acetilação , Carcinogênese/genética , Cromatina/genética , Metilação de DNA/genética , DNA Ribossômico/genética , Histonas/genética , Humanos , RNA Polimerase I/genética , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Transcrição Genética
9.
Mycologia ; 111(5): 857-870, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31414967

RESUMO

In this paper, species of the genus Morchella are investigated in China. Based on morphological characteristics and molecular phylogenetic analyses of the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and the combined data set ITS + nuclear large subunit rDNA (28S) + the translation elongation factor 1-α (TEF1) gene + RNA polymerase II first largest subunit (RPB1) + RNA polymerase II second largest subunit (RPB2), six new phylogenetic species are illustrated and described: M. clivicola, M. confusa, M. odonnellii, M. owneri, M. yangii, and M. yishuica. Furthermore, two new record species, M. dunensis and M. palazonii, which were only known in Europe, are now reported for the first time from Asia. New species of morels will provide additional information on species diversity and genetic resource candidates for improving the cultivation of this economically important fungus.


Assuntos
Ascomicetos/classificação , Ascomicetos/isolamento & purificação , Filogenia , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , China , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Genes de RNAr , Fator 1 de Elongação de Peptídeos/genética , RNA Polimerase I/genética , RNA Polimerase II/genética , RNA Fúngico/genética , RNA Ribossômico 28S/genética , RNA Ribossômico 5,8S/genética , Análise de Sequência de DNA
10.
Biochim Biophys Acta Gene Regul Mech ; 1862(9): 194408, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31382053

RESUMO

In Saccharomyces cerevisiae, Core Factor (CF) is a key evolutionarily conserved transcription initiation factor that helps recruit RNA polymerase I (Pol I) to the ribosomal DNA (rDNA) promoter. Upregulated Pol I transcription has been linked to many cancers, and targeting Pol I is an attractive and emerging anti-cancer strategy. Using yeast as a model system, we characterized how CF binds to the Pol I promoter by electrophoretic mobility shift assays (EMSA). Synthetic DNA competitors along with anti-tumor drugs and nucleic acid stains that act as DNA groove blockers were used to discover the binding preference of yeast CF. Our results show that CF employs a unique binding mechanism where it prefers the GC-rich minor groove within the rDNA promoter. In addition, we show that yeast CF is able to bind to the human rDNA promoter sequence that is divergent in DNA sequence and demonstrate CF sensitivity to the human specific Pol I inhibitor, CX-5461. Finally, we show that the human Core Promoter Element (CPE) can functionally replace the yeast Core Element (CE) in vivo when aligned by conserved DNA structural features rather than DNA sequence. Together, these findings suggest that the yeast CF and the human ortholog Selectivity Factor 1 (SL1) use an evolutionarily conserved, structure-based mechanism to target DNA. Their shared mechanism may offer a new avenue in using yeast to explore current and future Pol I anti-cancer compounds.


Assuntos
DNA Ribossômico/genética , RNA Polimerase I/genética , Fatores de Transcrição/genética , Transcrição Genética , Benzotiazóis/farmacologia , Sequência Conservada/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Naftiridinas/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Polimerase I/química , Saccharomyces cerevisiae/genética , Fatores de Transcrição/química
11.
Cells ; 8(7)2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261688

RESUMO

The many functions of phosphoinositides in cytosolic signaling were extensively studied; however, their activities in the cell nucleus are much less clear. In this review, we summarize data about their nuclear localization and metabolism, and review the available literature on their involvements in chromatin remodeling, gene transcription, and RNA processing. We discuss the molecular mechanisms via which nuclear phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), modulate nuclear processes. We focus on PI(4,5)P2's role in the modulation of RNA polymerase I activity, and functions of the nuclear lipid islets-recently described nucleoplasmic PI(4,5)P2-rich compartment involved in RNA polymerase II transcription. In conclusion, the high impact of the phosphoinositide-protein complexes on nuclear organization and genome functions is only now emerging and deserves further thorough studies.


Assuntos
Núcleo Celular/metabolismo , Eucariotos/genética , Genoma , Fosfatidilinositol 4,5-Difosfato/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase I/metabolismo , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina , Eucariotos/metabolismo , Ligação Proteica/fisiologia , Processamento Pós-Transcricional do RNA , Transcrição Genética
12.
Nucleic Acids Res ; 47(17): 9180-9197, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350892

RESUMO

Ribonucleotides represent a threat to DNA genome stability and transmission. Two types of Ribonuclease H (RNase H) excise ribonucleotides when they form part of the DNA strand, or hydrolyse RNA when it base-pairs with DNA in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast survives the absence of both enzymes. RNase H1 loss is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. Differential gene expression analysis reveals limited overall changes in RNA levels for RNA Pol II genes after TbRH2A loss, but increased perturbation of nucleotide metabolic genes. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, also leading to altered gene expression. Thus, we demonstrate separation of function between two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion.


Assuntos
Antígenos de Protozoários/genética , Instabilidade Genômica/genética , Ribonuclease H/genética , Iniciação da Transcrição Genética , Animais , Antígenos de Protozoários/imunologia , DNA/química , DNA/genética , Dano ao DNA/genética , Replicação do DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Conformação de Ácido Nucleico , RNA/química , RNA/genética , RNA Polimerase I/genética , RNA Polimerase II/genética , Ribonuclease H/química , Ribonuclease H/imunologia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/imunologia , Trypanosoma brucei brucei/patogenicidade
13.
Cells ; 8(6)2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167386

RESUMO

The nucleolus organizes around the sites of transcription by RNA polymerase I (RNA Pol I). rDNA transcription by this enzyme is the key step of ribosome biogenesis and most of the assembly and maturation processes of the ribosome occur co-transcriptionally. Therefore, disturbances in rRNA transcription and processing translate to ribosomal malfunction. Nucleolar malfunction has recently been described in the classical progeria of childhood, Hutchinson-Gilford syndrome (HGPS), which is characterized by severe signs of premature aging, including atherosclerosis, alopecia, and osteoporosis. A deregulated ribosomal biogenesis with enlarged nucleoli is not only characteristic for HGPS patients, but it is also found in the fibroblasts of "normal" aging individuals. Cockayne syndrome (CS) is also characterized by signs of premature aging, including the loss of subcutaneous fat, alopecia, and cataracts. It has been shown that all genes in which a mutation causes CS, are involved in rDNA transcription by RNA Pol I. A disturbed ribosomal biogenesis affects mitochondria and translates into ribosomes with a reduced translational fidelity that causes endoplasmic reticulum (ER) stress and apoptosis. Therefore, it is speculated that disease-causing disturbances in the process of ribosomal biogenesis may be more common than hitherto anticipated.


Assuntos
Nucléolo Celular/metabolismo , Progéria/patologia , Ribossomos/metabolismo , Senilidade Prematura , Nucléolo Celular/genética , Criança , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Estresse do Retículo Endoplasmático , Humanos , Mitocôndrias/metabolismo , Progéria/genética , Progéria/metabolismo , RNA Polimerase I/metabolismo , Ribossomos/genética
14.
Mutat Res ; 815: 20-29, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31063901

RESUMO

Hydroxyurea (HU) is an inhibitor of ribonucleotide reductase that is used as a chemotherapeutic agent to treat a number of chronic diseases. Addition of HU to cell cultures causes reduction of the dNTP cellular pool below levels that are required for DNA replication. This trigger dividing cells to arrest in early S-phase of the cell cycle. Cell division hinges on ribosome biogenesis, which is tightly regulated by rRNA synthesis. Remarkably, HU represses the expression of some genes the products of which are required for rRNA maturation. To gain more information on the cellular response to HU, we employed the yeast Saccharomyces cerevisiae as model organism and analyzed the changing aspects of closed to open forms of rRNA gene chromatin during cell cycle arrest, the arrangement of RNA polymerase-I (RNAPI) on the open genes, the presence of RNAPI transcription-factors, transcription and rRNA maturation. The rRNA gene chromatin structure was analyzed by psoralen crosslinking and the distribution of RNAPI was investigated by chromatin endogenous cleavage. In HU arrested cells nearly all rRNA genes were in the open form of chromatin, but only a portion of them was engaged with RNAPI. Analyses by chromatin immuno-precipitation confirmed that the overall formation of transcription pre-initiation complexes remained unchanged, suggesting that the onset of rRNA gene activation was not significantly affected by HU. Moreover, the in vitro transcription run-on assay indicated that RNAPI retained most of its transcription elongation activity. However, in HU treated cells, we found that: (1) RNAPI accumulated next to the 5'-end of rRNA genes; (2) considerably less rRNA filaments were observed in electron micrographs of rDNA transcription units; and (3) rRNA maturation was compromised. It is established that HU inhibition of ribonucleotide reductase holds back DNA replication. This study indicates a hitherto unexplored cellular response to HU, namely altered rRNA synthesis, which could participate to hamper cell division.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cromatina/genética , Genes de RNAr/genética , Hidroxiureia/farmacologia , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Transcrição Genética/genética , Pontos de Checagem do Ciclo Celular/genética , Divisão Celular/genética , Replicação do DNA/genética , DNA Ribossômico/genética , RNA Polimerase I/genética , RNA Ribossômico/genética , Fase S/efeitos dos fármacos , Fase S/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética
15.
Cell Mol Life Sci ; 76(14): 2789-2797, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31037337

RESUMO

The heterotrimeric carboxy-terminal domain kinase I (CTDK-I) in yeast is a cyclin-dependent kinase complex that is evolutionally conserved throughout eukaryotes and phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (RNApII) on the second-position serine (Ser2) residue of YSPTSPS heptapeptide repeats. CTDK-I plays indispensable roles in transcription elongation and transcription-coupled processing, such as the 3'-end processing of nascent mRNA transcripts. However, recent studies have revealed additional roles of CTDK-I beyond its primary effect on transcription by RNApII. Here, we describe recent advances in the regulation of genomic stability and rDNA integrity by CTDK-I and highlight the previously underappreciated cellular roles of CTDK-I in rRNA synthesis by RNA polymerase I and translational initiation and elongation. These multiple roles of CTDK-I throughout the cell expand our understanding of how this complex functions to coordinate diverse cellular processes through gene expression and how the human orthologue exerts its roles in diseased states such as tumorigenesis.


Assuntos
Biossíntese de Proteínas , Proteínas Quinases/metabolismo , RNA Polimerase I/metabolismo , RNA Ribossômico/metabolismo , Animais , Humanos
16.
PLoS Genet ; 15(5): e1008138, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31083663

RESUMO

Repetitive DNA sequences within eukaryotic heterochromatin are poorly transcribed and replicate late in S-phase. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). Despite the widespread association between transcription and replication timing, it remains unclear how transcription might impinge on replication, or vice versa. Here we show that, when silencing of an RNA polymerase II (RNA Pol II)-transcribed non-coding RNA at the rDNA is disrupted by SIR2 deletion, RNA polymerase pushes and thereby relocalizes replicative Mcm2-7 helicases away from their loading sites to an adjacent region with low nucleosome occupancy, and this relocalization is associated with increased rDNA origin efficiency. Our results suggest a model in which two of the major defining features of heterochromatin, transcriptional silencing and late replication, are mechanistically linked through suppression of polymerase-mediated displacement of replication initiation complexes.


Assuntos
Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/genética , Sirtuína 2/metabolismo , Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , Replicação do DNA/fisiologia , DNA Ribossômico/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Inativação Gênica , Proteínas de Manutenção de Minicromossomo/genética , RNA Polimerase I/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Genética
17.
PLoS Genet ; 15(5): e1008157, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31136569

RESUMO

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.


Assuntos
Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , RNA Polimerase I/genética , DNA Ribossômico/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Precursores de RNA/genética , RNA Ribossômico , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Genética
18.
Elife ; 82019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30913026

RESUMO

RNA polymerase (Pol) I is a 14-subunit enzyme that solely transcribes pre-ribosomal RNA. Cryo-electron microscopy (EM) structures of Pol I initiation and elongation complexes have given first insights into the molecular mechanisms of Pol I transcription. Here, we present cryo-EM structures of yeast Pol I elongation complexes (ECs) bound to the nucleotide analog GMPCPP at 3.2 to 3.4 Å resolution that provide additional insight into the functional interplay between the Pol I-specific transcription-like factors A49-A34.5 and A12.2. Strikingly, most of the nucleotide-bound ECs lack the A49-A34.5 heterodimer and adopt a Pol II-like conformation, in which the A12.2 C-terminal domain is bound in a previously unobserved position at the A135 surface. Our structural and biochemical data suggest a mechanism where reversible binding of the A49-A34.5 heterodimer could contribute to the regulation of Pol I transcription initiation and elongation.


Assuntos
Microscopia Crioeletrônica , RNA Polimerase I/ultraestrutura , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/enzimologia
19.
PLoS Genet ; 15(3): e1008055, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30875370

RESUMO

Lethal recessive alleles cause pre- or postnatal death in homozygous affected individuals, reducing fertility. Especially in small size domestic and wild populations, those alleles might be exposed by inbreeding, caused by matings between related parents that inherited the same recessive lethal allele from a common ancestor. In this study we report five relatively common (up to 13.4% carrier frequency) recessive lethal haplotypes in two commercial pig populations. The lethal haplotypes have a large effect on carrier-by-carrier matings, decreasing litter sizes by 15.1 to 21.6%. The causal mutations are of different type including two splice-site variants (affecting POLR1B and TADA2A genes), one frameshift (URB1), and one missense (PNKP) variant, resulting in a complete loss-of-function of these essential genes. The recessive lethal alleles affect up to 2.9% of the litters within a single population and are responsible for the death of 0.52% of the total population of embryos. Moreover, we provide compelling evidence that the identified embryonic lethal alleles contribute to the observed heterosis effect for fertility (i.e. larger litters in crossbred offspring). Together, this work marks specific recessive lethal variation describing its functional consequences at the molecular, phenotypic, and population level, providing a unique model to better understand fertility and heterosis in livestock.


Assuntos
Genes Letais , Mutação com Perda de Função , Sus scrofa/embriologia , Sus scrofa/genética , Sequência de Aminoácidos , Animais , Feminino , Fertilidade/genética , Genes Recessivos , Deriva Genética , Genética Populacional , Haplótipos , Vigor Híbrido/genética , Hibridização Genética/genética , Tamanho da Ninhada de Vivíparos/genética , Masculino , Gravidez , RNA Polimerase I/genética , Análise de Sequência de RNA , Sequenciamento Completo do Genoma
20.
Arthritis Rheumatol ; 71(9): 1571-1579, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30888702

RESUMO

OBJECTIVE: While compelling data suggest a cancer-induced autoimmunity model in scleroderma patients with anti-RNA polymerase III large subunit (anti-RPC155) antibodies, ~85% of these patients do not manifest cancer. This study was undertaken to determine whether additional autoantigens are targeted in anti-RPC155-positive scleroderma patients without detectable cancer. METHODS: The study included 168 scleroderma patients with anti-RPC155 antibodies (80 with a history of cancer and 88 with no cancer diagnosis after >5 years of follow-up). Thirty-five sera (17 from patients with cancer and 18 from patients without cancer) were randomly selected for autoantibody discovery using immunoprecipitation (IP). An ~194-kd band was enriched in the subgroup without cancer; this was identified as RNA polymerase I large subunit (RPA194). RESULTS: RPA194 generated by in vitro transcription/translation was used for IPs performed on the entire cohort to test whether anti-RPA194 was enriched among anti-RPC155-positive patients without cancer. Anti-RPA194 antibodies were significantly more common in the group without cancer (16 [18.2%] of 88) than in the group with cancer (3 [3.8%] of 80) (P = 0.003). Patients with both anti-RPA194 and anti-RPC155 were significantly less likely to have severe gastrointestinal disease than patients with anti-RPC155 only (26.3% versus 51.0%; P = 0.043). CONCLUSION: Anti-RPA194 antibodies are enriched in anti-RPC155-positive scleroderma patients without cancer. Since somatic mutations in the gene encoding RPC155 in cancer in scleroderma patients appears to play a role in immune response initiation against RPC155 in those patients, these data raise the possibility that the development of immune responses to both RPC155 and RPA194 may influence clinical cancer emergence. Further study is required to define whether different autoantibody combinations have utility as tools for cancer risk stratification in scleroderma.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Neoplasias/imunologia , RNA Polimerase III/imunologia , RNA Polimerase I/imunologia , Escleroderma Sistêmico/imunologia , Adulto , Idoso , Autoanticorpos/sangue , Autoantígenos/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Proteção , Escleroderma Sistêmico/sangue
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA