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1.
PLoS One ; 16(5): e0247285, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019539

RESUMO

Across eukaryotes, homopolymeric repeats of amino acids are enriched in regulatory proteins such as transcription factors and chromatin remodelers. These domains play important roles in signaling, binding, prion formation, and functional phase separation. Azf1p is a prion-forming yeast transcription factor that contains two homorepeat domains, a polyglutamine and a polyasparagine domain. In this work, we report a new phenotype for Azf1p and identify a large set of genes that are regulated by Azf1p during growth in glucose. We show that the polyasparagine (polyN) domain plays a subtle role in transcription but is dispensable for Azf1p localization and prion formation. Genes upregulated upon deletion of the polyN domain are enriched in functions related to carbon metabolism and storage. This domain may therefore be a useful target for engineering yeast strains for fermentation applications and small molecule production. We also report that both the polyasparagine and polyglutamine domains vary in length across strains of S. cerevisiae and propose a model for how this variation may impact protein function.


Assuntos
Peptídeos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Proteínas Priônicas/metabolismo , Domínios Proteicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética
2.
G3 (Bethesda) ; 10(7): 2543-2551, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32467128

RESUMO

The C-terminal domain (CTD) is an essential domain of the largest subunit of RNA polymerase II, Rpb1p, and is composed of 26 tandem repeats of a seven-amino acid sequence, YSPTSPS. Despite being an essential domain within an essential gene, we have previously demonstrated that the CTD coding region is genetically unstable. Furthermore, yeast with a truncated or mutated CTD sequence are capable of promoting spontaneous genetic expansion or contraction of this coding region to improve fitness. We investigated the mechanism by which the CTD contracts using a tet-off reporter system for RPB1 to monitor genetic instability within the CTD coding region. We report that contractions require the post-replication repair factor Rad5p but, unlike expansions, not the homologous recombination factors Rad51p and Rad52p Sequence analysis of contraction events reveals that deleted regions are flanked by microhomologies. We also find that G-quadruplex forming sequences predicted by the QGRS Mapper are enriched on the noncoding strand of the CTD compared to the body of RPB1 Formation of G-quadruplexes in the CTD coding region could block the replication fork, necessitating post-replication repair. We propose that contractions of the CTD result when microhomologies misalign during Rad5p-dependent template switching via fork reversal.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sequência de Aminoácidos , DNA Helicases , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
FEMS Yeast Res ; 20(3)2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32301985

RESUMO

The pathogenic yeast Candida glabrata is reliant on a suite of cell surface adhesins that play a variety of roles necessary for transmission, establishment and proliferation during infection. One particular adhesin, Epithelial Adhesin 1 [Epa1p], is responsible for binding to host tissue, a process which is essential for fungal propagation. Epa1p structure consists of three domains: an N-terminal intercellular binding domain responsible for epithelial cell binding, a C-terminal GPI anchor for cell wall linkage and a serine/threonine-rich linker domain connecting these terminal domains. The linker domain contains a 40-amino acid tandem repeat region, which we have found to be variable in repeat copy number between isolates from clinical sources. We hypothesized that natural variation in Epa1p repeat copy may modulate protein function. To test this, we recombinantly expressed Epa1p with various repeat copy numbers in S. cerevisiae to determine how differences in repeat copy number affect Epa1p expression, surface display and binding to human epithelial cells. Our data suggest that repeat copy number variation has pleiotropic effects, influencing gene expression, protein surface display and shedding from the cell surface of the Epa1p adhesin. This study serves to demonstrate repeat copy number variation can modulate protein function through a number of mechanisms in order to contribute to pathogenicity of C. glabrata.


Assuntos
Candida glabrata/genética , Adesão Celular/genética , Variações do Número de Cópias de DNA , Células Epiteliais/metabolismo , Proteínas Fúngicas/genética , Sequências de Repetição em Tandem/genética , Candida glabrata/metabolismo , Candidíase/microbiologia , Adesão Celular/fisiologia , Proteínas Fúngicas/metabolismo , Expressão Gênica , Células HeLa , Humanos , Ligação Proteica , Saccharomyces cerevisiae/genética
4.
Elife ; 82019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804179

RESUMO

CAG/CTG trinuncleotide repeats are fragile sequences that when expanded form DNA secondary structures and cause human disease. We evaluated CAG/CTG repeat stability and repair outcomes in histone H2 mutants in S. cerevisiae. Although the two copies of H2A are nearly identical in amino acid sequence, CAG repeat stability depends on H2A copy 1 (H2A.1) but not copy 2 (H2A.2). H2A.1 promotes high-fidelity homologous recombination, sister chromatid recombination (SCR), and break-induced replication whereas H2A.2 does not share these functions. Both decreased SCR and the increase in CAG expansions were due to the unique Thr126 residue in H2A.1 and hta1Δ or hta1-T126A mutants were epistatic to deletion of the Polδ subunit Pol32, suggesting a role for H2A.1 in D-loop extension. We conclude that H2A.1 plays a greater repair-specific role compared to H2A.2 and may be a first step towards evolution of a repair-specific function for H2AX compared to H2A in mammalian cells.


Assuntos
Instabilidade Genômica , Histonas/metabolismo , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Treonina/metabolismo , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/genética
5.
Lab Chip ; 19(20): 3481-3489, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31524206

RESUMO

Fungal adhesion is fundamental to processes ranging from infections to food production to bioengineering. Yet, robust, population-scale quantification methods for yeast surface adhesion are lacking. We developed a microfluidic assay to discriminate and separate genetically-related yeast strains based on adhesion strength, and to quantify effects of ionic strength and substrate hydrophobicity on adhesion. This approach will enable the rapid screening and fractionation of yeast based on adhesive properties for genetic protein engineering, anti-fouling surfaces, and a host of other applications.


Assuntos
Microfluídica/métodos , Saccharomyces cerevisiae/isolamento & purificação , Incrustação Biológica/prevenção & controle , Interações Hidrofóbicas e Hidrofílicas , Mutagênese , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Eletricidade Estática , Propriedades de Superfície , Transativadores/genética , Transativadores/metabolismo
6.
G3 (Bethesda) ; 8(5): 1593-1601, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29523636

RESUMO

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is required to regulate transcription and to integrate it with other essential cellular processes. In the budding yeast Saccharomyces cerevisiae, the CTD of Rpb1p consists of 26 conserved heptad repeats that are post-translationally modified to orchestrate protein factor binding at different stages of the transcription cycle. A long-standing question in the study of the CTD is if there are any functional differences between the 26 repeats. In this study, we present evidence that repeats of identical sequence have different functions based on their position within the CTD. We assembled plasmids expressing Rpb1p with serine to alanine substitutions in three defined regions of the CTD and measured a range of phenotypes for yeast expressing these constructs. Mutations in the beginning and middle regions of the CTD had drastic, and region-specific effects, while mutating the distal region had no observable phenotype. Further mutational analysis determined that Ser5 within the first region of repeats was solely responsible for the observed growth differences and sequencing fast-growing suppressors allowed us to further define the functional regions of the CTD. This mutational analysis is consistent with current structural models for how the RNAPII holoenzyme and the CTD specifically would reside in complex with Mediator and establishes a foundation for studying regioselective binding along the repetitive RNAPII CTD.


Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Saccharomycetales/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Genes Supressores , Inositol/metabolismo , Mutação/genética , Fenótipo , Domínios Proteicos , Sequências Repetitivas de Aminoácidos , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
7.
Biotechniques ; 63(4): 169-173, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29048268

RESUMO

As yeast are starved of nutrients, they enter G0, a quiescent state. Quiescent yeast (Q) cells retain viability for extended periods of time and resume growth following supplementation of missing nutrients. As such, Q cells have become a valuable model for studying longevity and self-renewal of chronologically aged cells. Traditional isolation of Q cells involves a relatively long centrifugation time through a continuous density gradient. Here, we describe a rapid and cost-effective Q-cell isolation technique that uses a single-density, one-step gradient prepared from media containing iodixanol.


Assuntos
Separação Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Fase de Repouso do Ciclo Celular , Saccharomyces cerevisiae/isolamento & purificação , Separação Celular/economia , Centrifugação com Gradiente de Concentração/economia , Análise Custo-Benefício , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Fatores de Tempo , Ácidos Tri-Iodobenzoicos/farmacologia
8.
Cell Rep ; 15(10): 2147-2158, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27239037

RESUMO

Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD) and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7), we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a role for threonine-4 in 3' end processing through control of the transition between cleavage and termination. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3' splice sites through a direct interaction with spliceosomal subcomplex U1. Strikingly, threonine-4 phosphorylation also impacts splicing by serving as a mark of co-transcriptional spliceosome release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes.


Assuntos
Aminoácidos/metabolismo , Mapas de Interação de Proteínas , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Genoma Fúngico , Modelos Genéticos , Fosforilação , Domínios Proteicos , Proteômica , Splicing de RNA/genética , Spliceossomos/metabolismo , Relação Estrutura-Atividade , Terminação da Transcrição Genética
9.
J Biol Chem ; 291(22): 11540-50, 2016 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-27026700

RESUMO

The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24-26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1 In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length.


Assuntos
Variações do Número de Cópias de DNA/genética , DNA Fúngico/química , RNA Polimerase II/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Western Blotting , Dicroísmo Circular , DNA Fúngico/genética , DNA Fúngico/metabolismo , Evolução Molecular , Dados de Sequência Molecular , Domínios Proteicos , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência do Ácido Nucleico
10.
Mol Cell ; 59(3): 502-11, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26212453

RESUMO

Access to high-quality antibodies is a necessity for the study of histones and their posttranslational modifications (PTMs). Here we debut the Histone Antibody Specificity Database (http://www.histoneantibodies.com), an online and expanding resource cataloging the behavior of widely used, commercially available histone antibodies by peptide microarray. This interactive web portal provides a critical resource to the biological research community that routinely uses these antibodies as detection reagents for a wide range of applications.


Assuntos
Anticorpos/metabolismo , Bases de Dados Genéticas , Histonas/metabolismo , Análise Serial de Proteínas/métodos , Especificidade de Anticorpos , Células HeLa , Humanos , Processamento de Proteína Pós-Traducional
11.
Genes Dev ; 28(13): 1445-60, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24990964

RESUMO

Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. Here we identify chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. Our results reveal an extensive list of known and novel HP1a-interacting proteins, of which we selected three for validation. A strong novel interactor, dADD1 (Drosophila ADD1) (CG8290), is highly enriched in heterochromatin, harbors an ADD domain similar to human ATRX, displays selective binding to H3K9me2 and H3K9me3, and is a classic genetic suppressor of position-effect variegation. Unexpectedly, a second hit, HIPP1 (HP1 and insulator partner protein-1) (CG3680), is strongly connected to CP190-related complexes localized at putative insulator sequences throughout the genome in addition to its colocalization with HP1a in heterochromatin. A third interactor, the histone methyltransferase MES-4, is also enriched in heterochromatin. In addition to these protein-protein interactions, we found that HP1a selectively associated with a broad set of RNAs transcribed from repetitive regions. We propose that this rich network of previously undiscovered interactions will define how HP1a complexes perform their diverse functions in cells and developing organisms.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , RNA/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estágios do Ciclo de Vida/fisiologia , Ligação Proteica , RNA/genética , Análise de Sequência de RNA , Proteína de Ligação a Elemento Regulador de Esterol 1/genética
12.
FEBS Lett ; 587(8): 1247-57, 2013 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-23402885

RESUMO

Post-translational modifications (PTMs) occur on nearly all proteins. Many domains within proteins are modified on multiple amino acid sidechains by diverse enzymes to create a myriad of possible protein species. How these combinations of PTMs lead to distinct biological outcomes is only beginning to be understood. This manuscript highlights several examples of combinatorial PTMs in proteins, and describes recent technological developments, which are driving our ability to understand how PTM patterns may "code" for biological outcomes.


Assuntos
Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/metabolismo , Acetilação , Sítios de Ligação , Humanos , Metilação , Modelos Moleculares , Fosforilação
13.
ACS Chem Biol ; 8(2): 275-82, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23157399

RESUMO

Many proteins composed of tandem repeats (a linear motif, directly repeated within the sequence) are substrates for post-translational modifications (PTMs). Tandem repeats are also dynamic in number, presumably due to instability in the underlying DNA sequence. These observations lead to a hypothesis that cells use a combination of PTMs and variability in repeat number to mediate protein function. Evidence of these processes co-regulating diverse aspects of cellular function can be found in all organisms from bacteria to humans, suggesting a common but poorly described mechanism for regulating and diversifying protein function. This review highlights several examples whereby protein modifications and repetitive protein domains impart diversity. Lastly, it speculates on the possibility of using chemically modified repetitive amino acid sequences to develop peptide-based biomolecules with novel functions.


Assuntos
Biblioteca de Peptídeos , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/metabolismo , Sequências de Repetição em Tandem , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas/genética
14.
Nat Struct Mol Biol ; 19(11): 1155-60, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23022729

RESUMO

A fundamental challenge in mammalian biology has been the elucidation of mechanisms linking DNA methylation and histone post-translational modifications. Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has multiple domains that bind chromatin, and it is implicated genetically in the maintenance of DNA methylation. However, molecular mechanisms underlying DNA methylation regulation by UHRF1 are poorly defined. Here we show that UHRF1 association with methylated histone H3 Lys9 (H3K9) is required for DNA methylation maintenance. We further show that UHRF1 association with H3K9 methylation is insensitive to adjacent H3 S10 phosphorylation--a known mitotic 'phospho-methyl switch'. Notably, we demonstrate that UHRF1 mitotic chromatin association is necessary for DNA methylation maintenance through regulation of the stability of DNA methyltransferase-1. Collectively, our results define a previously unknown link between H3K9 methylation and the faithful epigenetic inheritance of DNA methylation, establishing a notable mitotic role for UHRF1 in this process.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , Metilação de DNA/fisiologia , Epigênese Genética/fisiologia , Histonas/metabolismo , Mitose/fisiologia , Sequência de Aminoácidos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Clonagem Molecular , Metilação de DNA/genética , Primers do DNA/genética , Escherichia coli , Polarização de Fluorescência , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Análise em Microsséries , Dados de Sequência Molecular , Fosforilação , Ubiquitina-Proteína Ligases
15.
J Mol Biol ; 424(5): 391-9, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23041298

RESUMO

Antibodies directed against histone posttranslational modifications (PTMs) are critical tools in epigenetics research, particularly in the widely used chromatin immunoprecipitation (ChIP) experiments. However, a lack of quantitative methods for characterizing such antibodies has been a major bottleneck in accurate and reproducible analysis of histone modifications. Here, we report a simple and sensitive method for quantitatively characterizing polyclonal and monoclonal antibodies for histone PTMs in a ChIP-like format. Importantly, it determines the apparent dissociation constants for the interactions of an antibody with peptides harboring cognate or off-target PTMs. Analyses of commercial antibodies revealed large ranges of affinity, specificity and binding capacity as well as substantial lot-to-lot variations, suggesting the importance of quantitatively characterizing each antibody intended to be used in ChIP experiments and optimizing experimental conditions accordingly. Furthermore, using this method, we identified additional factors potentially affecting the interpretation of ChIP experiments.


Assuntos
Anticorpos/imunologia , Afinidade de Anticorpos , Imunoprecipitação da Cromatina/métodos , Histonas/imunologia , Anticorpos/classificação , Sensibilidade e Especificidade
16.
Methods Enzymol ; 512: 107-35, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22910205

RESUMO

Histone posttranslational modifications (PTMs) play a pivotal role in regulating the dynamics and function of chromatin. Supported by an increasing body of literature, histone PTMs such as methylation and acetylation function together in the context of a "histone code," which is read, or interpreted, by effector proteins that then drive a functional output in chromatin (e.g., gene transcription). A growing number of domains that interact with histones and/or their PTMs have been identified. While significant advances have been made in our understanding of how these domains interact with histones, a wide number of putative histone-binding motifs have yet to be characterized, and undoubtedly, novel domains will continue to be discovered. In this chapter, we provide a detailed method for the construction of combinatorially modified histone peptides, microarray fabrication using these peptides, and methods to characterize the interaction of effector proteins, antibodies, and the substrate specificity of histone-modifying enzymes. We discuss these methods in the context of other available technologies and provide a user-friendly approach to enable the exploration of histone-protein-enzyme interactions and function.


Assuntos
Histonas/química , Fragmentos de Peptídeos/química , Análise Serial de Proteínas , Processamento de Proteína Pós-Traducional , Acetilação , Sequência de Aminoácidos , Aminoácidos/química , Ensaios Enzimáticos , Enzimas/química , Epigênese Genética , Fluorenos/química , Histonas/metabolismo , Humanos , Metilação , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Biblioteca de Peptídeos , Especificidade por Substrato
17.
J Biol Chem ; 287(5): 3249-56, 2012 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-22157004

RESUMO

Methylation of lysine 36 on histone H3 (H3K36) is catalyzed by the Set2 methyltransferase and is linked to transcriptional regulation. Previous studies have shown that trimethylation of H3K36 by Set2 is directed through its association with the phosphorylated repeats of the RNA polymerase C-terminal domain (RNAPII CTD). Here, we show that disruption of this interaction through the use of yeast mutants defective in CTD phosphorylation at serine 2 results in a destabilization of Set2 protein levels and H3K36 methylation. Consistent with this, we find that Set2 has a short half-life and is co-regulated, with RNAPII CTD phosphorylation levels, during logarithmic growth in yeast. To probe the functional consequence of uncoupling Set2-RNAPII regulation, we expressed a truncated and more stable form of Set2 that is capable of dimethylation but not trimethylation in vivo. Results of high throughput synthetic genetic analyses show that this Set2 variant has distinct genetics from either SET2 or set2Δ and is synthetically sick or lethal with a number of transcription elongation mutants. Collectively, these results provide molecular insight into the regulation of Set2 protein levels that influence H3K36 methylation states.


Assuntos
Histonas/metabolismo , Metiltransferases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Histonas/genética , Metilação , Metiltransferases/genética , Fosforilação/fisiologia , Estabilidade Proteica , Estrutura Terciária de Proteína , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
PLoS One ; 6(3): e18302, 2011 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-21464899

RESUMO

BACKGROUND: The Ccr4-Not complex is a key eukaryotic regulator of gene transcription and cytoplasmic mRNA degradation. Whether this complex also affects aspects of post-transcriptional gene regulation, such as mRNA export, remains largely unexplored. Human Caf1 (hCaf1), a Ccr4-Not complex member, interacts with and regulates the arginine methyltransferase PRMT1, whose targets include RNA binding proteins involved in mRNA export. However, the functional significance of this regulation is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate using co-immunoprecipitation approaches that Ccr4-Not subunits interact with Hmt1, the budding yeast ortholog of PRMT1. Furthermore, using genetic and biochemical approaches, we demonstrate that Ccr4-Not physically and functionally interacts with the heterogenous nuclear ribonucleoproteins (hnRNPs) Nab2 and Hrp1, and that the physical association depends on Hmt1 methyltransferase activity. Using mass spectrometry, co-immunoprecipitation and genetic approaches, we also uncover physical and functional interactions between Ccr4-Not subunits and components of the nuclear pore complex (NPC) and we provide evidence that these interactions impact mRNA export. CONCLUSIONS/SIGNIFICANCE: Taken together, our findings suggest that Ccr4-Not has previously unrealized functional connections to the mRNA processing/export pathway that are likely important for its role in gene expression. These results shed further insight into the biological functions of Ccr4-Not and suggest that this complex is involved in all aspects of mRNA biogenesis, from the regulation of transcription to mRNA export and turnover.


Assuntos
Complexos Multiproteicos/metabolismo , Transporte de RNA , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Proteínas Mutantes/metabolismo , Mutação/genética , Poro Nuclear/metabolismo , Poli A/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleases/isolamento & purificação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
20.
Curr Biol ; 21(1): 53-8, 2011 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-21167713

RESUMO

Increasing evidence suggests that histone posttranslational modifications (PTMs) function in a combinatorial fashion to regulate the diverse activities associated with chromatin. Yet how these patterns of histone PTMs influence the adapter proteins known to bind them is poorly understood. In addition, how histone-specific antibodies are influenced by these same patterns of PTMs is largely unknown. Here we examine the binding properties of histone-specific antibodies and histone-interacting proteins using peptide arrays containing a library of combinatorially modified histone peptides. We find that modification-specific antibodies are more promiscuous in their PTM recognition than expected and are highly influenced by neighboring PTMs. Furthermore, we find that the binding of histone-interaction domains from BPTF, CHD1, and RAG2 to H3 lysine 4 trimethylation is also influenced by combinatorial PTMs. These results provide further support for the histone code hypothesis and raise specific concerns with the quality of the currently available modification-specific histone antibodies.


Assuntos
Anticorpos/metabolismo , Histonas/imunologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína
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