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1.
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
2.
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
3.
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 Gênica , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Transcriptoma/genética
4.
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 Gênica , 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
5.
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 Gênica/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
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