Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 64
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nucleic Acids Res ; 49(15): 8900-8922, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34370034

RESUMO

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.


Assuntos
Regiões 5' não Traduzidas , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/virologia , Proteínas de Arabidopsis/fisiologia , Proteínas do Capsídeo/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Núcleo Celular/metabolismo , Doenças das Plantas/virologia , RNA Viral/química , DNA Polimerase Dirigida por RNA/metabolismo
2.
New Phytol ; 230(3): 972-987, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33475158

RESUMO

Condensins are best known for their role in shaping chromosomes. Other functions such as organizing interphase chromatin and transcriptional control have been reported in yeasts and animals, but little is known about their function in plants. To elucidate the specific composition of condensin complexes and the expression of CAP-D2 (condensin I) and CAP-D3 (condensin II), we performed biochemical analyses in Arabidopsis. The role of CAP-D3 in interphase chromatin organization and function was evaluated using cytogenetic and transcriptome analysis in cap-d3 T-DNA insertion mutants. CAP-D2 and CAP-D3 are highly expressed in mitotically active tissues. In silico and pull-down experiments indicate that both CAP-D proteins interact with the other condensin I and II subunits. In cap-d3 mutants, an association of heterochromatic sequences occurs, but the nuclear size and the general histone and DNA methylation patterns remain unchanged. Also, CAP-D3 influences the expression of genes affecting the response to water, chemicals, and stress. The expression and composition of the condensin complexes in Arabidopsis are similar to those in other higher eukaryotes. We propose a model for the CAP-D3 function during interphase in which CAP-D3 localizes in euchromatin loops to stiffen them and consequently separates centromeric regions and 45S rDNA repeats.


Assuntos
Arabidopsis , Cromatina , Adenosina Trifosfatases/genética , Animais , Arabidopsis/genética , Proteínas de Ligação a DNA , Interfase , Complexos Multiproteicos
3.
Biochim Biophys Acta Gene Regul Mech ; 1864(2): 194613, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32745625

RESUMO

From yeast to human, the Spt-Ada-GCN5-acetyltransferase (SAGA) gigantic complex modifies chromatin during RNA polymerase II initiation and elongation steps to facilitate transcription. Its enzymatic activity involves a histone acetyltransferase module (HATm) that acetylates multiple lysine residues on the N-terminal tails of histones H2B and H3 and a deubiquitination module (DUBm) that triggers co-transcriptional deubiquitination of histone H2B. With a few notable exceptions described in this review, most SAGA subunits identified in yeast and metazoa are present in plants. Studies from the last 20 years have unveiled that different SAGA subunits are involved in gene expression regulation during the plant life cycle and in response to various types of stress or environmental cues. Their functional analysis in the Arabidopsis thaliana model species is increasingly shedding light on their intrinsic properties and how they can themselves be regulated during plant adaptive responses. Recent biochemical studies have also uncovered multiple associations between plant SAGA and chromatin machineries linked to RNA Pol II transcription. Still, considerably less is known about the molecular links between SAGA or SAGA-like complexes and chromatin dynamics during transcription in Arabidopsis and other plant species. We summarize the emerging knowledge on plant SAGA complex composition and activity, with a particular focus on the best-characterized subunits from its HAT (such as GCN5) and DUB (such as UBP22) modules, and implication of these ensembles in plant development and adaptive responses.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Histona Acetiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , Transativadores/metabolismo , Ubiquitina Tiolesterase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , RNA Polimerase II/metabolismo , Transcrição Genética/fisiologia , Ubiquitinação/fisiologia
4.
PLoS Genet ; 16(4): e1008732, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282821

RESUMO

Transcription termination has important regulatory functions, impacting mRNA stability, localization and translation potential. Failure to appropriately terminate transcription can also lead to read-through transcription and the synthesis of antisense RNAs which can have profound impact on gene expression. The Transcription-Export (THO/TREX) protein complex plays an important role in coupling transcription with splicing and export of mRNA. However, little is known about the role of the THO/TREX complex in the control of transcription termination. In this work, we show that two proteins of the THO/TREX complex, namely TREX COMPONENT 1 (TEX1 or THO3) and HYPER RECOMBINATION1 (HPR1 or THO1) contribute to the correct transcription termination at several loci in Arabidopsis thaliana. We first demonstrate this by showing defective termination in tex1 and hpr1 mutants at the nopaline synthase (NOS) terminator present in a T-DNA inserted between exon 1 and 3 of the PHO1 locus in the pho1-7 mutant. Read-through transcription beyond the NOS terminator and splicing-out of the T-DNA resulted in the generation of a near full-length PHO1 mRNA (minus exon 2) in the tex1 pho1-7 and hpr1 pho1-7 double mutants, with enhanced production of a truncated PHO1 protein that retained phosphate export activity. Consequently, the strong reduction of shoot growth associated with the severe phosphate deficiency of the pho1-7 mutant was alleviated in the tex1 pho1-7 and hpr1 pho1-7 double mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants leads to 3'UTR extensions in several endogenous genes. These results demonstrate that THO/TREX complex contributes to the regulation of transcription termination.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Terminação da Transcrição Genética , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas
5.
Front Plant Sci ; 11: 85, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32140163

RESUMO

FACT is a heterodimeric histone chaperone consisting of the SSRP1 and SPT16 proteins and is conserved among eukaryotes. It interacts with the histones H2A-H2B and H3-H4 as well as with DNA. Based on in vitro and in vivo studies mainly in yeast and mammalian cells, FACT can mediate nucleosome disassembly and reassembly and thus facilitates in the chromatin context DNA-dependent processes including transcription, replication and repair. In plants, primarily the role of FACT related to RNA polymerase II transcription has been examined. FACT was found to associate with elongating Arabidopsis RNA polymerase II (RNAPII) as part of the transcript elongation complex and it was identified as repressor of aberrant intragenic transcriptional initiation. Arabidopsis mutants depleted in FACT subunits exhibit various defects in vegetative and reproductive development. Strikingly, FACT modulates important developmental transitions by promoting expression of key repressors of these processes. Thus, FACT facilitates expression of DOG1 and FLC adjusting the switch from seed dormancy to germination and from vegetative to reproductive development, respectively. In the central cell of the female gametophyte, FACT can facilitate DNA demethylation especially within heterochromatin, and thereby contributes to gene imprinting during Arabidopsis reproduction. This review discusses results particularly from the plant perspective about the contribution of FACT to processes that involve reorganisation of nucleosomes with a main focus on RNAPII transcription and its implications for diverse areas of plant biology.

6.
Plant Cell ; 32(5): 1449-1463, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32152189

RESUMO

Transcript elongation factors associate with elongating RNA polymerase II (RNAPII) to control the efficiency of mRNA synthesis and consequently modulate plant growth and development. Encountering obstacles during transcription such as nucleosomes or particular DNA sequences may cause backtracking and transcriptional arrest of RNAPII. The elongation factor TFIIS stimulates the intrinsic transcript cleavage activity of the polymerase, which is required for efficient rescue of backtracked/arrested RNAPII. A TFIIS mutant variant (TFIISmut) lacks the stimulatory activity to promote RNA cleavage, but instead efficiently inhibits unstimulated transcript cleavage by RNAPII. We could not recover viable Arabidopsis (Arabidopsis thaliana) tfIIs plants constitutively expressing TFIISmut. Induced, transient expression of TFIISmut in tfIIs plants provoked severe growth defects, transcriptomic changes and massive, transcription-related redistribution of elongating RNAPII within transcribed regions toward the transcriptional start site. The predominant site of RNAPII accumulation overlapped with the +1 nucleosome, suggesting that upon inhibition of RNA cleavage activity, RNAPII arrest prevalently occurs at this position. In the presence of TFIISmut, the amount of RNAPII was reduced, which could be reverted by inhibiting the proteasome, indicating proteasomal degradation of arrested RNAPII. Our findings suggest that polymerase backtracking/arrest frequently occurs in plant cells, and RNAPII-reactivation is essential for correct transcriptional output and proper growth/development.


Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Transcriptoma/genética
7.
J Plant Physiol ; 236: 105-108, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30947026

RESUMO

SSRP1 is a subunit of the histone chaperone FACT that associates with elongating RNA polymerase II (RNAPII) along the transcribed region of genes. FACT facilitates transcriptional elongation by destabilising nucleosomes in the path of RNAPII, assisting efficient transcription of chromatin templates. In contrast to wild type seeds, freshly harvested seeds of the Arabidopsis ssrp1 mutant germinate efficiently, exhibiting reduced seed dormancy. In line with this phenotype, the ssrp1 seeds have decreased transcript levels of the DOG1 gene, which is a known quantitative trait locus (QTL) for seed dormancy. Analysis of ssrp1 plants harbouring an additional copy of DOG1 show increased levels of DOG1 transcript and consistently more robust seed dormancy. Therefore, our findings indicate that SSRP1 is a novel factor required for the efficient expression of DOG1 and hence a modulator of seed dormancy in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Chaperonas de Histonas/fisiologia , Dormência de Plantas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Germinação , Chaperonas de Histonas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
J Exp Bot ; 70(15): 3757-3763, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30972423

RESUMO

In eukaryotes, the regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step in the expression of protein-coding genes, as it links nuclear mRNA synthesis with cytosolic translation. The pre-mRNAs that are synthesised by RNA polymerase II are processed by 5´-capping, splicing, and 3´-polyadenylation. The multi-subunit THO/TREX complex integrates mRNA biogenesis with their nucleocytosolic transport. Various export factors are recruited to the mRNAs during their maturation, which occurs essentially co-transcriptionally. These RNA-bound export factors ensure efficient transport of the export-competent mRNAs through nuclear pore complexes. In recent years, several factors involved in plant mRNA export have been functionally characterised. Analysis of mutant plants has demonstrated that impaired mRNA export causes defects in growth and development. Moreover, there is accumulating evidence that mRNA export can influence processes such as plant immunity, circadian regulation, and stress responses. Therefore, it is important to learn more details about the mechanism of nucleocytosolic mRNA transport in plants and its physiological significance.


Assuntos
Transporte de RNA/fisiologia , RNA Mensageiro/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Transporte de RNA/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Proc Natl Acad Sci U S A ; 116(16): 8060-8069, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30923114

RESUMO

HISTONE MONOUBIQUITINATION1 (HUB1) and its paralog HUB2 act in a conserved heterotetrameric complex in the chromatin-mediated transcriptional modulation of developmental programs, such as flowering time, dormancy, and the circadian clock. The KHD1 and SPEN3 proteins were identified as interactors of the HUB1 and HUB2 proteins with in vitro RNA-binding activity. Mutants in SPEN3 and KHD1 had reduced rosette and leaf areas. Strikingly, in spen3 mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the hub1-4 mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and FLOWERING LOCUS C (FLC) that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the spen3-1 and hub1-4 mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the CCA1α and CCA1ß splice forms were altered, and the CCA1 expression and H2Bub levels were reduced. In the spen3-1 mutant, the delay in flowering time was correlated with an enhanced FLC expression, possibly due to an increased distal versus proximal ratio of its antisense COOLAIR transcript. Together with transcriptomic and double-mutant analyses, our data revealed that the HUB1 interaction with SPEN3 links H2Bub during transcript elongation with pre-mRNA processing at CCA1 Furthermore, the presence of an intact HUB1 at the FLC is required for SPEN3 function in the formation of the FLC-derived antisense COOLAIR transcripts.


Assuntos
Proteínas de Arabidopsis , Regulação da Expressão Gênica de Plantas , Histonas , RNA de Plantas , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Flores/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Histonas/genética , Histonas/metabolismo , Domínios Proteicos/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologia
10.
Plant Physiol ; 179(4): 1525-1536, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30700540

RESUMO

In eukaryotes, the regulated transport of mRNAs from the nucleus to the cytosol through nuclear pore complexes represents an important step in the expression of protein-coding genes. In plants, the mechanism of nucleocytosolic mRNA transport and the factors involved are poorly understood. The Arabidopsis (Arabidopsis thaliana) genome encodes two likely orthologs of UAP56-interacting factor, which acts as mRNA export factor in mammalian cells. In yeast and plant cells, both proteins interact directly with the mRNA export-related RNA helicase UAP56 and the interaction was mediated by an N-terminal UAP56-binding motif. Accordingly, the two proteins were termed UAP56-INTERACTING EXPORT FACTOR1 and 2 (UIEF1/2). Despite lacking a known RNA-binding motif, recombinant UIEF1 interacted with RNA, and the C-terminal part of UIEF1 mainly contributed to the RNA interaction. Mutation of UIEF1, UIEF2, or both in the double-mutant 2xuief caused modest growth defects. A cross between the 2xuief and 4xaly (defective in the four ALY1-4 mRNA export factors) mutants produced the sextuple mutant 4xaly 2xuief, which displayed more severe growth impairment than the 4xaly plants. Developmental defects including delayed bolting and reduced seed set were observed in the 4xaly but not the 2xuief plants. Analysis of the cellular distribution of polyadenylated mRNAs revealed more pronounced nuclear mRNA accumulation in 4xaly 2xuief than in 2xuief and 4xaly cells. In conclusion, the results indicate that UIEF1 and UIEF2 act as mRNA export factors in plants and that they cooperate with ALY1-ALY4 to mediate efficient nucleocytosolic mRNA transport.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Genoma de Planta , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
11.
J Mol Biol ; 430(17): 2747-2759, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29966609

RESUMO

Histone chaperones play critical roles in regulated structural transitions of chromatin in eukaryotic cells that involve nucleosome disassembly and reassembly. The histone chaperone FACT is a heterodimeric complex consisting in plants and metazoa of SSRP1/SPT16 and is involved in dynamic nucleosome reorganization during various DNA-dependent processes including transcription, replication and repair. The C-terminal HMG-box domain of the SSRP1 subunit mediates interactions with DNA and nucleosomes in vitro, but its relevance in vivo is unclear. Here, we demonstrate that Arabidopsis ssrp1-2 mutant plants express a C-terminally truncated SSRP1 protein. Although the structure of the truncated HMG-box domain is distinctly disturbed, it still exhibits residual DNA-binding activity, but has lost DNA-bending activity. Since ssrp1-2 plants are phenotypically affected but viable, the HMG-box domain may be functionally non-essential. To examine this possibility, SSRP1∆HMG completely lacking the HMG-box domain was studied. SSRP1∆HMG in vitro did not bind to DNA and its interactions with nucleosomes were severely reduced. Nevertheless, the protein showed a nuclear mobility and protein interactions similar to SSRP1. Interestingly, expression of SSRP1∆HMG is almost as efficient as that of full-length SSRP1 in supporting normal growth and development of the otherwise non-viable Arabidopsis ssrp1-1 mutant. SSRP1∆HMG is structurally similar to the fungal ortholog termed Pob3 that shares clear similarity with SSRP1, but it lacks the C-terminal HMG-box. Therefore, our findings indicate that the HMG-box domain conserved among SSRP1 proteins is not critical in Arabidopsis, and thus, the functionality of SSRP1/SPT16 in plants/metazoa and Pob3/Spt16 in fungi is perhaps more similar than anticipated.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Domínios HMG-Box , Chaperonas de Histonas/metabolismo , Nucleossomos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Replicação do DNA , DNA de Plantas/química , DNA de Plantas/genética , DNA de Plantas/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/genética , Nucleossomos/química , Nucleossomos/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Plant Cell ; 30(4): 907-924, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29618629

RESUMO

The rhythms of steady-state mRNA expression pervade nearly all circadian systems. However, the mechanisms behind the rhythmic transcriptional synthesis and its correlation with circadian expression remain fully unexplored, particularly in plants. Here, we discovered a multifunctional protein complex that orchestrates the rhythms of transcriptional activity in Arabidopsis thaliana The expression of the circadian oscillator genes TIMING OF CAB EXPRESSION1/PSEUDO-RESPONSE REGULATOR1 and PSEUDO-RESPONSE REGULATOR5 initially relies on the modular function of the clock-related factor REVEILLE8: its MYB domain provides the DNA binding specificity, while its LCL domain recruits the clock components, NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED proteins (LNKs), to target promoters. LNKs, in turn, specifically interact with RNA Polymerase II and the transcript elongation FACT complex to rhythmically co-occupy the target loci. The functional interaction of these components is central for chromatin status, transcript initiation, and elongation as well as for proper rhythms in nascent RNAs. Thus, our findings explain how genome readout of environmental information ultimately results in rhythmic changes of gene expression.


Assuntos
Arabidopsis/genética , Ritmo Circadiano/genética , Complexos Multiproteicos/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Complexos Multiproteicos/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA de Plantas/genética
13.
J Mol Biol ; 430(10): 1479-1494, 2018 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-29588169

RESUMO

The conserved nuclear protein ENY2 (Sus1 in yeast) is involved in coupling transcription and mRNA export in yeast and metazoa, as it is a component both of the transcriptional co-activator complex SAGA and of the mRNA export complex TREX-2. Arabidopsis thaliana ENY2 is widely expressed in the plant and it localizes to the nucleoplasm, but unlike its yeast/metazoan orthologs, it is not enriched in the nuclear envelope. Affinity purification of ENY2 in combination with mass spectrometry revealed that it co-purified with SAGA components, but not with the nuclear pore-associated TREX-2. In addition, further targeted proteomics analyses by reciprocal tagging established the composition of the Arabidopsis SAGA complex consisting of the four modules HATm, SPTm, TAFm and DUBm, and that several SAGA subunits occur in alternative variants. While the HATm, SPTm and TAFm robustly co-purified with each other, the deubiquitination module (DUBm) appears to associate with the other SAGA modules more weakly/dynamically. Consistent with a homology model of the Arabidopsis DUBm, the SGF11 protein interacts directly with ENY2 and UBP22. Plants depleted in the DUBm components, SGF11 or ENY2, are phenotypically only mildly affected, but they contain increased levels of ubiquitinated histone H2B, indicating that the SAGA-DUBm has histone deubiquitination activity in plants. In addition to transcription-related proteins (i.e., transcript elongation factors, Mediator), many splicing factors were found to associate with SAGA, linking the SAGA complex and ongoing transcription with mRNA processing.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Poro Nuclear/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Histonas/metabolismo , Espectrometria de Massas , Poro Nuclear/genética , Proteômica , Transativadores/genética , Ubiquitinação
14.
Plant Physiol ; 177(1): 226-240, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29540591

RESUMO

The regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step linking transcript synthesis and processing with translation. However, in plants, only a few of the factors that act in the mRNA export pathway have been functionally characterized. Flowering plant genomes encode several members of the ALY protein family, which function as mRNA export factors in other organisms. Arabidopsis (Arabidopsis thaliana) ALY1 to ALY4 are commonly detected in root and leaf cells, but they are differentially expressed in reproductive tissue. Moreover, the subnuclear distribution of ALY1/2 differs from that of ALY3/4. ALY1 binds with higher affinity to single-stranded RNA than double-stranded RNA and single-stranded DNA and interacts preferentially with 5-methylcytosine-modified single-stranded RNA. Compared with the full-length protein, the individual RNA recognition motif of ALY1 binds RNA only weakly. ALY proteins interact with the RNA helicase UAP56, indicating a link to the mRNA export machinery. Consistently, ALY1 complements the lethal phenotype of yeast cells lacking the ALY1 ortholog Yra1. Whereas individual aly mutants have a wild-type appearance, disruption of ALY1 to ALY4 in 4xaly plants causes vegetative and reproductive defects, including strongly reduced growth, altered flower morphology, as well as abnormal ovules and female gametophytes, causing reduced seed production. Moreover, polyadenylated mRNAs accumulate in the nuclei of 4xaly cells. Our results highlight the requirement of efficient mRNA nucleocytosolic transport for proper plant growth and development and indicate that ALY1 to ALY4 act partly redundantly in this process; however, differences in expression and subnuclear localization suggest distinct functions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Transporte de RNA , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
15.
Plant Mol Biol ; 96(4-5): 367-374, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29332189

RESUMO

KEY MESSAGE: The histone chaperone FACT is involved in the expression of genes encoding anthocyanin biosynthetic enzymes also upon induction by moderate high-light and therefore contributes to the stress-induced plant pigmentation. The histone chaperone FACT consists of the SSRP1 and SPT16 proteins and associates with transcribing RNAPII (RNAPII) along the transcribed region of genes. FACT can promote transcriptional elongation by destabilising nucleosomes in the path of RNA polymerase II, thereby facilitating efficient transcription of chromatin templates. Transcript profiling of Arabidopsis plants depleted in SSRP1 or SPT16 demonstrates that only a small subset of genes is differentially expressed relative to wild type. The majority of these genes is either up- or down-regulated in both the ssrp1 and spt16 plants. Among the down-regulated genes, those encoding enzymes of the biosynthetic pathway of the plant secondary metabolites termed anthocyanins (but not regulators of the pathway) are overrepresented. Upon exposure to moderate high-light stress several of these genes are up-regulated to a lesser extent in ssrp1/spt16 compared to wild type plants, and accordingly the mutant plants accumulate lower amounts of anthocyanin pigments. Moreover, the expression of SSRP1 and SPT16 is induced under these conditions. Therefore, our findings indicate that FACT is a novel factor required for the accumulation of anthocyanins in response to light-induction.


Assuntos
Antocianinas/biossíntese , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Vias Biossintéticas/genética , Genes de Plantas , Chaperonas de Histonas/metabolismo , Estresse Fisiológico/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos da radiação , Regulação para Cima/efeitos da radiação
16.
Transcription ; 9(2): 117-122, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28886274

RESUMO

Characterisation of the Arabidopsis RNA polymerase II (RNAPII) elongation complex revealed an assembly of a conserved set of transcript elongation factors associated with chromatin remodellers, histone modifiers as well as with various pre-mRNA splicing and polyadenylation factors. Therefore, transcribing RNAPII streamlines the processes of mRNA synthesis and processing in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética , Elongação da Transcrição Genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Fatores de Alongamento de Peptídeos/genética , Poliadenilação , RNA Polimerase II/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo
17.
Methods Mol Biol ; 1629: 105-122, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28623582

RESUMO

A central step to elucidate the function of proteins commonly comprises the analysis of their molecular interactions in vivo. For nuclear regulatory proteins this involves determining protein-protein interactions as well as mapping of chromatin binding sites. Here, we present two protocols to identify protein-protein and chromatin interactions of transcript elongation factors (TEFs) in Arabidopsis. The first protocol (Subheading 3.1) describes protein affinity-purification coupled to mass spectrometry (AP-MS) that utilizes suspension cultured cells as experimental system. This approach provides an unbiased view of proteins interacting with epitope-tagged TEFs. The second protocol (Subheading 3.2) depicts details about a chromatin immunoprecipitation (ChIP) procedure to characterize genomic binding sites of TEFs. These methods should be valuable tools for the analysis of a broad variety of nuclear proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Transcrição Genética , Proteínas de Arabidopsis/isolamento & purificação , Sítios de Ligação , Imunoprecipitação da Cromatina , Cromatografia de Afinidade , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Ligação Proteica , Estatística como Assunto , Espectrometria de Massas em Tandem
18.
Plant Cell ; 29(4): 854-870, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28351991

RESUMO

Transcript elongation factors (TEFs) are a heterogeneous group of proteins that control the efficiency of transcript elongation of subsets of genes by RNA polymerase II (RNAPII) in the chromatin context. Using reciprocal tagging in combination with affinity purification and mass spectrometry, we demonstrate that in Arabidopsis thaliana, the TEFs SPT4/SPT5, SPT6, FACT, PAF1-C, and TFIIS copurified with each other and with elongating RNAPII, while P-TEFb was not among the interactors. Additionally, NAP1 histone chaperones, ATP-dependent chromatin remodeling factors, and some histone-modifying enzymes including Elongator were repeatedly found associated with TEFs. Analysis of double mutant plants defective in different combinations of TEFs revealed genetic interactions between genes encoding subunits of PAF1-C, FACT, and TFIIS, resulting in synergistic/epistatic effects on plant growth/development. Analysis of subnuclear localization, gene expression, and chromatin association did not provide evidence for an involvement of the TEFs in transcription by RNAPI (or RNAPIII). Proteomics analyses also revealed multiple interactions between the transcript elongation complex and factors involved in mRNA splicing and polyadenylation, including an association of PAF1-C with the polyadenylation factor CstF. Therefore, the RNAPII transcript elongation complex represents a platform for interactions among different TEFs, as well as for coordinating ongoing transcription with mRNA processing.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Proteômica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
19.
Plant Mol Biol ; 93(3): 283-298, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28004241

RESUMO

KEY MESSAGE: We identify proteins that associate with the THO core complex, and show that the TEX1 and MOS11 components functionally interact, affecting mRNA export and splicing as well as plant development. TREX (TRanscription-EXport) is a multiprotein complex that plays a central role in the coordination of synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicase UAP56 and the mRNA export factors ALY2-4 and MOS11 we detected interactions with the mRNA export complex TREX-2 and multiple spliceosomal components. Plants defective in the THO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 double-mutant plants show marked defects in vegetative and reproductive development. In tex1 plants, the levels of tasiRNAs are reduced, while miR173 levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed, while no mRNA export defect was detected with tex1 cells. Nevertheless, in tex1 mos11 double-mutants, the mRNA export defect was clearly enhanced relative to mos11. The subnuclear distribution of TEX1 substantially overlaps with that of splicing-related SR proteins and in tex1 plants the ratio of certain alternative splicing events is altered. Our results demonstrate that Arabidopsis TEX1 and MOS11 are involved in distinct steps of the biogenesis of mRNAs and small RNAs, and that they interact regarding some aspects, but act independently in others.


Assuntos
Processamento Alternativo/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Transporte de RNA , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Inflorescência/metabolismo , Inflorescência/ultraestrutura , Modelos Biológicos , Fenótipo , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/genética
20.
FEBS Lett ; 589(21): 3254-7, 2015 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-26424658

RESUMO

The Arabidopsis multidomain protein SPT5L/KTF1 (which has similarity to the transcript elongation factor SPT5) associates with RNA polymerase V (RNAPV) and is an accessory factor in RNA-directed DNA methylation. The zinc-finger protein SPT4 was found to interact with SPT5L (and SPT5) both in vivo and in vitro. Here, we show that plants depleted of SPT4 relative to wild type display reduced DNA methylation and the locus specificity is shared with SPT5L, suggesting a cooperation of SPT4 and SPT5L. Unlike observed for SPT5, no reduced protein level of SPT5L is determined in SPT4-deficient plants. These experiments demonstrate that in addition to the RNA polymerase II-associated SPT4/SPT5 that is generally conserved in eukaryotes, flowering plants have SPT4/SPT5L that is involved in RNAPV-mediated transcriptional silencing.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Inativação Gênica , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Metilação de DNA , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/química , Dedos de Zinco
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...