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1.
Mol Cell ; 83(2): 186-202.e11, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36669479

RESUMO

PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes. We show that PGC-1α promotes pausing release in a two-arm mechanism (1) by recruiting the positive transcription elongation factor b (P-TEFb) and (2) by outcompeting the premature transcription termination complex Integrator. Using mice homozygous for five amino acid changes in the CBP80-binding motif (CBM) of PGC-1α that destroy CBM function, we show that efficient differentiation of primary myoblasts to myofibers and timely skeletal muscle regeneration after injury require PGC-1α binding to CBP80. Our findings reveal how PGC-1α activates stress-response gene transcription in a previously unanticipated pre-mRNA quality-control pathway.


Assuntos
Precursores de RNA , Fatores de Transcrição , Animais , Camundongos , Precursores de RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Regiões Promotoras Genéticas , Proteínas de Ligação a DNA/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação ao Cap de RNA/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Músculo Esquelético/metabolismo
2.
Nat Commun ; 14(1): 341, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36670096

RESUMO

The transcriptional response to genotoxic stress involves gene expression arrest, followed by recovery of mRNA synthesis (RRS) after DNA repair. We find that the lack of the EXD2 nuclease impairs RRS and decreases cell survival after UV irradiation, without affecting DNA repair. Overexpression of wild-type, but not nuclease-dead EXD2, restores RRS and cell survival. We observe that UV irradiation triggers the relocation of EXD2 from mitochondria to the nucleus. There, EXD2 is recruited to chromatin where it transiently interacts with RNA Polymerase II (RNAPII) to promote the degradation of nascent mRNAs synthesized at the time of genotoxic attack. Reconstitution of the EXD2-RNAPII partnership on a transcribed DNA template in vitro shows that EXD2 primarily interacts with an elongation-blocked RNAPII and efficiently digests mRNA. Overall, our data highlight a crucial step in the transcriptional response to genotoxic attack in which EXD2 interacts with elongation-stalled RNAPII on chromatin to potentially degrade the associated nascent mRNA, allowing transcription restart after DNA repair.


Assuntos
Dano ao DNA , Reparo do DNA , Cromatina/genética , Transcrição Genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética
3.
Commun Biol ; 6(1): 86, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36690785

RESUMO

Binding of USF1/2 and TFII-I (RBF-2) at conserved sites flanking the HIV-1 LTR enhancer is essential for reactivation from latency in T cells, with TFII-I knockdown rendering the provirus insensitive to T cell signaling. We identified an interaction of TFII-I with the tripartite motif protein TRIM24, and these factors were found to be constitutively associated with the HIV-1 LTR. Similar to the effect of TFII-I depletion, loss of TRIM24 impaired reactivation of HIV-1 in response to T cell signaling. TRIM24 deficiency did not affect recruitment of RNA Pol II to the LTR promoter, but inhibited transcriptional elongation, an effect that was associated with decreased RNA Pol II CTD S2 phosphorylation and impaired recruitment of CDK9. A considerable number of genomic loci are co-occupied by TRIM24/TFII-I, and we found that TRIM24 deletion caused altered T cell immune response, an effect that is facilitated by TFII-I. These results demonstrate a role of TRIM24 for regulation of transcriptional elongation from the HIV-1 promoter, through its interaction with TFII-I, and by recruitment of P-TEFb. Furthermore, these factors co-regulate a significant proportion of genes involved in T cell immune response, consistent with tight coupling of HIV-1 transcriptional activation and T cell signaling.


Assuntos
Infecções por HIV , HIV-1 , Humanos , HIV-1/genética , RNA Polimerase II/metabolismo , Latência Viral , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Transporte
4.
Nat Commun ; 14(1): 166, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36631525

RESUMO

The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3'end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of m6A, the most abundant RNA modification. RBM15 positively regulates m6A levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes.


Assuntos
RNA Polimerase II , Splicing de RNA , Humanos , Fosfosserina/metabolismo , RNA Polimerase II/metabolismo , Processamento Pós-Transcricional do RNA , RNA/metabolismo , Transcrição Genética , Fosforilação
5.
Cell ; 186(2): 327-345.e28, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36603581

RESUMO

Components of transcriptional machinery are selectively partitioned into specific condensates, often mediated by protein disorder, yet we know little about how this specificity is achieved. Here, we show that condensates composed of the intrinsically disordered region (IDR) of MED1 selectively partition RNA polymerase II together with its positive allosteric regulators while excluding negative regulators. This selective compartmentalization is sufficient to activate transcription and is required for gene activation during a cell-state transition. The IDRs of partitioned proteins are necessary and sufficient for selective compartmentalization and require alternating blocks of charged amino acids. Disrupting this charge pattern prevents partitioning, whereas adding the pattern to proteins promotes partitioning with functional consequences for gene activation. IDRs with similar patterned charge blocks show similar partitioning and function. These findings demonstrate that disorder-mediated interactions can selectively compartmentalize specific functionally related proteins from a complex mixture of biomolecules, leading to regulation of a biochemical pathway.


Assuntos
Proteínas Intrinsicamente Desordenadas , RNA Polimerase II , Transcrição Genética , Proteínas Intrinsicamente Desordenadas/metabolismo , RNA Polimerase II/metabolismo , Ativação Transcricional , Animais , Camundongos
6.
Mol Cell ; 83(2): 203-218.e9, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36626906

RESUMO

Many spliceosomal introns are excised from nascent transcripts emerging from RNA polymerase II (RNA Pol II). The extent of cell-type-specific regulation and possible functions of such co-transcriptional events remain poorly understood. We examined the role of the RNA-binding protein PTBP1 in this process using an acute depletion approach followed by the analysis of chromatin- and RNA Pol II-associated transcripts. We show that PTBP1 activates the co-transcriptional excision of hundreds of introns, a surprising effect given that this protein is known to promote intron retention. Importantly, some co-transcriptionally activated introns fail to complete their splicing without PTBP1. In a striking example, retention of a PTBP1-dependent intron triggers nonsense-mediated decay of transcripts encoding DNA methyltransferase DNMT3B. We provide evidence that this regulation facilitates the natural decline in DNMT3B levels in developing neurons and protects differentiation-specific genes from ectopic methylation. Thus, PTBP1-activated co-transcriptional splicing is a widespread phenomenon mediating epigenetic control of cellular identity.


Assuntos
Células-Tronco Pluripotentes , RNA Polimerase II , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Splicing de RNA/genética , Spliceossomos/metabolismo , Íntrons/genética , Células-Tronco Pluripotentes/metabolismo , Epigênese Genética , Processamento Alternativo
7.
Nucleic Acids Res ; 51(1): 420-433, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36546771

RESUMO

In contrast to the catalytic subunit of telomerase, its RNA subunit (TR) is highly divergent in size, sequence and biogenesis pathways across eukaryotes. Current views on TR evolution assume a common origin of TRs transcribed with RNA polymerase II in Opisthokonta (the supergroup including Animalia and Fungi) and Trypanosomida on one hand, and TRs transcribed with RNA polymerase III under the control of type 3 promoter, found in TSAR and Archaeplastida supergroups (including e.g. ciliates and Viridiplantae taxa, respectively). Here, we focus on unknown TRs in one of the largest Animalia order - Hymenoptera (Arthropoda) with more than 300 available representative genomes. Using a combination of bioinformatic and experimental approaches, we identify their TRs. In contrast to the presumed type of TRs (H/ACA box snoRNAs transcribed with RNA Polymerase II) corresponding to their phylogenetic position, we find here short TRs of the snRNA type, likely transcribed with RNA polymerase III under the control of the type 3 promoter. The newly described insect TRs thus question the hitherto assumed monophyletic origin of TRs across Animalia and point to an evolutionary switch in TR type and biogenesis that was associated with the divergence of Arthropods.


Assuntos
Himenópteros , Telomerase , Animais , Telomerase/genética , Telomerase/metabolismo , Himenópteros/genética , Filogenia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Conformação de Ácido Nucleico , RNA/genética , Plantas/genética , Eucariotos/genética
8.
Proc Natl Acad Sci U S A ; 120(1): e2217476120, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36584296

RESUMO

HIV gene expression is modulated by the combinatorial activity of the HIV transcriptional activator, Tat, host transcription factors, and chromatin remodeling complexes. To identify host factors regulating HIV transcription, we used specific single-guide RNAs and endonuclease-deficient Cas9 to perform chromatin affinity purification of the integrated HIV promoter followed by mass spectrometry. The scaffold protein, p32, also called ASF/SF2 splicing factor-associated protein, was identified among the top enriched factors present in actively transcribing HIV promoters but absent in silenced ones. Chromatin immunoprecipitation analysis confirmed the presence of p32 on active HIV promoters and its enhanced recruitment by Tat. HIV uses Tat to efficiently recruit positive transcription elongation factor b (p-TEFb) (CDK9/CCNT1) to TAR, an RNA secondary structure that forms from the first 59 bp of HIV transcripts, to enhance RNAPII transcriptional elongation. The RNA interference of p32 significantly reduced HIV transcription in primary CD4+T cells and in HIV chronically infected cells, independently of either HIV splicing or p32 anti-splicing activity. Conversely, overexpression of p32 specifically increased Tat-dependent HIV transcription. p32 was found to directly interact with Tat's basic domain enhancing Tat stability and half-life. Conversely, p32 associates with Tat via N- and C-terminal domains. Likely due its scaffold properties, p32 also promoted Tat association with TAR, p-TEFb, and RNAPII enhancing Tat-dependent HIV transcription. In sum, we identified p32 as a host factor that interacts with and stabilizes Tat protein, promotes Tat-dependent transcriptional regulation, and may be explored for HIV-targeted transcriptional inhibition.


Assuntos
Infecções por HIV , HIV-1 , Humanos , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , HIV-1/fisiologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Chaperonas Moleculares/metabolismo , Infecções por HIV/genética , Transcrição Genética , Repetição Terminal Longa de HIV/genética
9.
Biochemistry ; 62(1): 95-108, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36525636

RESUMO

Eukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale. We analyzed Saccharomyces cerevisiae Pol II incorporation of ATP or an ATP analog, Sp-ATP-α-S. Here we have measured the rate constants governing individual steps of the Pol II transcription cycle in the presence of ATP or Sp-ATP-α-S. These results suggest that Pol II catalyzes nucleotide incorporation by binding the next cognate nucleotide and immediately catalyzes bond formation and bond formation is either followed by a conformational change or pyrophosphate release. By comparing our previously published RNA polymerase I (Pol I) and Pol I lacking the A12 subunit (Pol I ΔA12) results that we collected under the same conditions with the identical technique, we show that Pol II and Pol I ΔA12 exhibit similar nucleotide addition mechanisms. This observation indicates that removal of the A12 subunit from Pol I results in a Pol II like enzyme. Taken together, these data further our collective understanding of Pol II's nucleotide incorporation mechanism and the evolutionary divergence of RNA polymerases across the three domains of life.


Assuntos
Nucleotídeos , RNA Polimerase II , Nucleotídeos/metabolismo , RNA Polimerase II/metabolismo , Cinética , RNA Polimerase I/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo
10.
Methods Mol Biol ; 2609: 315-328, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36515843

RESUMO

The rate of RNA polymerase II (RNAPII) transcriptional elongation plays a critical role in mRNA biogenesis, from transcription initiation to alternative splicing. As RNAPII moves along the DNA, it must read the DNA sequences wrapped up as chromatin. Thus, the structure of chromatin impedes the movement and speed at which RNAPII moves, presenting a crucial regulation to gene expression. Therefore, factors that bind and regulate the structure of chromatin will impact the rate of RNAPII elongation. We previously showed that PARP1 (poly-ADP-ribose polymerase 1) is one of such factors that bind and alter chromatin dynamics. We also showed that its alteration of chromatin structure modulates RNAPII processivity during transcriptional elongation. Here, we aim to understand how PARP1 alters RNAPII elongation kinetics genome wide.


Assuntos
RNA Polimerase II , Transcrição Genética , RNA Polimerase II/metabolismo , Processamento Alternativo , Cromatina , DNA/metabolismo , Fatores de Elongação da Transcrição/metabolismo
11.
J Mol Biol ; 435(2): 167917, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36502880

RESUMO

In addition to the stage of transcriptional initiation, the production of mRNAs is regulated during elongation. Accordingly, the synthesis of mRNAs by RNA polymerase II (RNAPII) in the chromatin context is modulated by various transcript elongation factors. TFIIS is an elongation factor that stimulates the transcript cleavage activity of RNAPII to reactivate stalled elongation complexes at barriers to transcription including nucleosomes. Since Arabidopsis tfIIs mutants grow normally under standard conditions, we have exposed them to heat stress (HS), revealing that tfIIs plants are highly sensitive to elevated temperatures. Transcriptomic analyses demonstrate that particularly HS-induced genes are expressed at lower levels in tfIIs than in wildtype. Mapping the distribution of elongating RNAPII uncovered that in tfIIs plants RNAPII accumulates at the +1 nucleosome of genes that are upregulated upon HS. The promoter-proximal RNAPII accumulation in tfIIs under HS conditions conforms to that observed upon inhibition of the RNAPII transcript cleavage activity. Further analysis of the RNAPII accumulation downstream of transcriptional start sites illustrated that RNAPII stalling occurs at +1 nucleosomes that are depleted in the histone variant H2A.Z upon HS. Therefore, assistance of early transcript elongation by TFIIS is required for reprogramming gene expression to establish plant thermotolerance.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Resposta ao Choque Térmico , Nucleossomos , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição , Arabidopsis/genética , Arabidopsis/metabolismo , Resposta ao Choque Térmico/genética , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo
12.
Nat Commun ; 13(1): 7672, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36539402

RESUMO

Transcription is a dynamic process. To detect the dynamic relationship among protein clusters of RNA polymerase II and coactivators, gene loci, and transcriptional activity, we insert an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site. By optimizing the individual elements, we develop the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNA polymerase II and BRD4 are observed proximal to the transcription start site of Nanog when the gene is transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 tend to be located near the transcription start site, even without transcription activity. Thus, the STREAMING-tag system reveals the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding transcriptional regulation in living cells.


Assuntos
RNA Polimerase II , Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Nucleares/metabolismo , Regulação da Expressão Gênica , Inteínas/genética , Transcrição Genética
13.
Cell Rep ; 41(13): 111865, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36577375

RESUMO

How transcription is regulated as development commences is fundamental to understand how the transcriptionally silent mature gametes are reprogrammed. The embryonic genome is activated for the first time during zygotic genome activation (ZGA). How RNA polymerase II (Pol II) and productive elongation are regulated during this process remains elusive. Here, we generate genome-wide maps of Serine 5 and Serine 2-phosphorylated Pol II during and after ZGA in mouse embryos. We find that both phosphorylated Pol II forms display similar distributions across genes during ZGA, with typical elongation enrichment of Pol II emerging after ZGA. Serine 2-phosphorylated Pol II occurs at genes prior to their activation, suggesting that Serine 2 phosphorylation may prime gene expression. Functional perturbations demonstrate that CDK9 and SPT5 are major ZGA regulators and that SPT5 prevents precocious activation of some genes. Overall, our work sheds molecular insights into transcriptional regulation at the beginning of mammalian development.


Assuntos
RNA Polimerase II , Zigoto , Camundongos , Animais , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Zigoto/metabolismo , Fosforilação , Genoma , Serina/metabolismo , Ativação Transcricional , Regulação da Expressão Gênica no Desenvolvimento , Mamíferos/metabolismo
14.
J Tradit Chin Med ; 42(6): 922-931, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36378050

RESUMO

OBJECTIVE: To investigate the efficacy of Yuzhizi seed extract (FAQSE) on inhibiting the proliferation of hepatocellular carcinoma (HCC) cells in vitro and to explore the anti-HCC action mechanism of FAQSE. METHODS: Human HCC HepG2 and Huh7 cells were used to investigate the anti-HCC effect of FAQSE. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method was used to measure cell viability. Affymetrix microarray was adopted to detect the expression of transcriptome. The differentially expressed genes (DEGs) of each cell line were identified. For co-DEGs of both cell lines, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were enriched using the Database for Annotation, Visualization and Integrated Discovery (DAVID), and the network analysis of protein-protein interaction (PPI) was mapped using the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape software. Some important genes in the PPI network of co-DEGs were selected to verify by quantitative real-time reverse transcription-polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. RESULTS: FAQSE decreased the viability of HepG2 and Huh7 cells. There were 211 co-upregulated and 86 co-downregualted genes in both cell lines after FAQSE treatment. The enriched GO terms of co-upregulated DEGs were primarily involved cell-cell adhesion, viral process, transcription initiation from RNA polymerase II promoter, positive regulation of transcription from RNA polymerase II promoter and actin cytoskeleton organization. The GO terms of co-downregulated DEGs were mainly enriched in the processes of SRP-dependent cotranslational protein targeting to membrane, viral transcription, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, translational initiation and rRNA processing. Main KEGG pathways of co-upregulated DEGs were endocytosis, glutathione metabolism, protein processing in endoplasmic reticulum, synaptic vesicle cycle and lysosome. The major KEGG pathways of co-downregulated DEGs were ribosome, biosynthesis of amino acids, arginine and proline metabolism, systemic lupus erythematosus and complement and coagulation cascades. The top 10 co-DEGs with high hub nodes in STRING analysis were ribosomal protein S27a, transferrin, ribosomal protein S20, ribosomal protein L9, protein phosphatase 2 regulatory subunit B alpha, transthyretin, thioredoxin reductase 1, ribosomal protein L3, ribophorin I and ribosomal protein L24. Alpha-fetoprotein (AFP) was also co-downregulated and contained in the PPI network. The mRNA and protein expression of most verified genes was consistent with the results of co-DEGs analysis. And the AFP level was significantly reduced after FAQSE treatment. CONCLUSIONS: A series of genes and pathways of HepG2 and Huh7 cells were changed after FAQSE treatment, which might be the targets of FAQSE against HCC and worthy of further study. AFP might be important one of them.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Transcriptoma , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Redes Reguladoras de Genes , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular , RNA Mensageiro , Extratos Vegetais/farmacologia , Regulação Neoplásica da Expressão Gênica
15.
Nat Commun ; 13(1): 6871, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36369505

RESUMO

In eukaryotes, small nuclear RNAs (snRNAs) function in many fundamental cellular events such as precursor messenger RNA splicing, gene expression regulation, and ribosomal RNA processing. The snRNA activating protein complex (SNAPc) exclusively recognizes the proximal sequence element (PSE) at snRNA promoters and recruits RNA polymerase II or III to initiate transcription. In view that homozygous gene-knockout of SNAPc core subunits causes mouse embryonic lethality, functions of SNAPc are almost housekeeping. But so far, the structural insight into how SNAPc assembles and regulates snRNA transcription initiation remains unclear. Here we present the cryo-electron microscopy structure of the essential part of human SNAPc in complex with human U6-1 PSE at an overall resolution of 3.49 Å. This structure reveals the three-dimensional features of three conserved subunits (N-terminal domain of SNAP190, SNAP50, and SNAP43) and explains how they are assembled into a stable mini-SNAPc in PSE-binding state with a "wrap-around" mode. We identify three important motifs of SNAP50 that are involved in both major groove and minor groove recognition of PSE, in coordination with the Myb domain of SNAP190. Our findings further elaborate human PSE sequence conservation and compatibility for SNAPc recognition, providing a clear framework of snRNA transcription initiation, especially the U6 system.


Assuntos
RNA Nuclear Pequeno , Fatores de Transcrição , Humanos , Animais , Camundongos , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismo , Microscopia Crioeletrônica , RNA Polimerase II/metabolismo , Transcrição Genética
16.
Cell Mol Life Sci ; 79(12): 597, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36399280

RESUMO

Cervical cancer is the fourth most frequently diagnosed and fatal gynecological cancer. 15-61% of all cases metastasize and develop chemoresistance, reducing the 5-year survival of cervical cancer patients to as low as 17%. Therefore, unraveling the mechanisms contributing to metastasis is critical in developing better-targeted therapies against it. Here, we have identified a novel mechanism where nuclear Caspase-8 directly interacts with and inhibits the activity of CDK9, thereby modulating RNAPII-mediated global transcription, including those of cell-migration- and cell-invasion-associated genes. Crucially, low Caspase-8 expression in cervical cancer patients leads to poor prognosis, higher CDK9 phosphorylation at Thr186, and increased RNAPII activity in cervical cancer cell lines and patient biopsies. Caspase-8 knock-out cells were also more resistant to the small-molecule CDK9 inhibitor BAY1251152 in both 2D- and 3D-culture conditions. Combining BAY1251152 with Cisplatin synergistically overcame chemoresistance of Caspase-8-deficient cervical cancer cells. Therefore, Caspase-8 expression could be a marker in chemoresistant cervical tumors, suggesting CDK9 inhibitor treatment for their sensitization to Cisplatin-based chemotherapy.


Assuntos
RNA Polimerase II , Neoplasias do Colo do Útero , Humanos , Feminino , RNA Polimerase II/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Fosforilação , Caspase 8/genética , Caspase 8/metabolismo , Cisplatino/farmacologia , Inibidores de Proteínas Quinases , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo
17.
Genes (Basel) ; 13(11)2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36360188

RESUMO

RPB1, the major and catalytic subunit of human RNA Polymerase II (RNAPII), is specifically degraded by the ubiquitin-proteasome system upon induction of DNA damage by different agents, such as ultraviolet (UV) light. The "last resort" model of RNAPII degradation states that a persistently stalled RNAPII is degraded at the site of the DNA lesion in order to facilitate access to Nucleotide Excision Repair (NER) factors, thereby promoting repair in template strands of active genes. Recent identification and mutation of the lysine residue involved in RPB1 ubiquitylation and degradation unveiled the relevance of RNAPII levels in the control of gene expression. Inhibition of RNAPII degradation after UV light exposure enhanced RNAPII loading onto chromatin, demonstrating that the mere concentration of RNAPII shapes the gene expression response. In this review, we discuss the role of RNAPII ubiquitylation in NER-dependent repair, recent advances in RPB1 degradation mechanisms and its consequences in gene expression under stress, both in normal and repair deficient cells.


Assuntos
Dano ao DNA , RNA Polimerase II , Humanos , Dano ao DNA/genética , Reparo do DNA/genética , Expressão Gênica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Ubiquitinação/genética
18.
G3 (Bethesda) ; 12(12)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36331351

RESUMO

Regulation of RNA polymerase II transcription requires the concerted efforts of several multisubunit coactivator complexes, which interact with the RNA polymerase II preinitiation complex to stimulate transcription. We previously showed that separation of the Mediator core from Mediator's tail module results in modest overactivation of genes annotated as highly dependent on TFIID for expression. However, it is unclear if other coactivators are involved in this phenomenon. Here, we show that the overactivation of certain genes by Mediator core/tail separation is blunted by disruption of the Spt-Ada-Gcn5-Acetyl transferase complex through the removal of its structural Spt20 subunit, though this downregulation does not appear to completely depend on reduced Spt-Ada-Gcn5-Acetyl transferase association with the genome. Consistent with the enrichment of TFIID-dependent genes among genes overactivated by Mediator core/tail separation, depletion of the essential TFIID subunit Taf13 suppressed the overactivation of these genes when Med16 was simultaneously removed. As with Spt-Ada-Gcn5-Acetyl transferase, this effect did not appear to be fully dependent on the reduced genomic association of TFIID. Given that the observed changes in gene expression could not be clearly linked to alterations in Spt-Ada-Gcn5-Acetyl transferase or TFIID occupancy, our data may suggest that the Mediator core/tail connection is important for the modulation of Spt-Ada-Gcn5-Acetyl transferase and/or TFIID conformation and/or function at target genes.


Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regulação Fúngica da Expressão Gênica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transativadores/metabolismo , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Transcrição Genética
19.
Elife ; 112022 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-36421680

RESUMO

Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters - from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.


Assuntos
Nucleotídeos , Poliadenilação , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Poli A/genética , Poli A/metabolismo , Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas , Transcrição Genética
20.
Nat Commun ; 13(1): 7287, 2022 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-36435862

RESUMO

In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, the mechanism by which RNA polymerase II (RNAPII) transcribes the DNA in the chromatosome has remained enigmatic. Here we report the cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I and II), in which RNAPII is paused at the entry linker DNA region of the chromatosome due to H1 binding. In the form I complex, the H1 bound to the nucleosome restricts the linker DNA orientation, and the exit linker DNA is captured by the RNAPII DNA binding cleft. In the form II complex, the RNAPII progresses a few bases ahead by releasing the exit linker DNA from the RNAPII cleft, and directly clashes with the H1 bound to the nucleosome. The transcription elongation factor Spt4/5 masks the RNAPII DNA binding region, and drastically reduces the H1-mediated RNAPII pausing.


Assuntos
Histonas , Nucleossomos , Histonas/metabolismo , RNA Polimerase II/metabolismo , Microscopia Crioeletrônica , DNA/metabolismo
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