Publications by authors named "Klaus D Grasser"

63 Publications

The Arabidopsis condensin CAP-D subunits arrange interphase chromatin.

New Phytol 2021 05 25;230(3):972-987. Epub 2021 Feb 25.

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstraße 3, D-06466, Seeland, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/nph.17221DOI Listing
May 2021

Multifaceted activities of the plant SAGA complex.

Biochim Biophys Acta Gene Regul Mech 2021 02 31;1864(2):194613. Epub 2020 Jul 31.

Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Ecole Normale Supérieure, CNRS, INSERM, Université PSL, Paris, France. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagrm.2020.194613DOI Listing
February 2021

The transcription and export complex THO/TREX contributes to transcription termination in plants.

PLoS Genet 2020 04 13;16(4):e1008732. Epub 2020 Apr 13.

Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1008732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7179932PMC
April 2020

Critical Role of Transcript Cleavage in Arabidopsis RNA Polymerase II Transcriptional Elongation.

Plant Cell 2020 05 9;32(5):1449-1463. Epub 2020 Mar 9.

Department of Cell Biology & Plant Biochemistry, Biochemistry Centre, University of Regensburg, D-93040 Regensburg, Germany

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 () plants constitutively expressing TFIISmut. Induced, transient expression of TFIISmut in 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1105/tpc.19.00891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203918PMC
May 2020

The FACT Histone Chaperone: Tuning Gene Transcription in the Chromatin Context to Modulate Plant Growth and Development.

Authors:
Klaus D Grasser

Front Plant Sci 2020 19;11:85. Epub 2020 Feb 19.

Cell Biology & Plant Biochemistry, Biochemistry Centre, University of Regensburg, Regensburg, Germany.

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 and 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 RNA polymerase II (RNAPII) as part of the transcript elongation complex and it was identified as repressor of aberrant intragenic transcriptional initiation. 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 and 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 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2020.00085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042381PMC
February 2020

Nucleocytosolic mRNA transport in plants: export factors and their influence on growth and development.

J Exp Bot 2019 08;70(15):3757-3763

Cell Biology & Plant Biochemistry, Biochemistry Centre, University of Regensburg, Universitätsstr. 31, Regensburg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jxb/erz173DOI Listing
August 2019

The SSRP1 subunit of the histone chaperone FACT is required for seed dormancy in Arabidopsis.

J Plant Physiol 2019 May 26;236:105-108. Epub 2019 Mar 26.

Cell Biology & Plant Biochemistry, Biochemistry Centre, University of Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jplph.2019.03.006DOI Listing
May 2019

Histone 2B monoubiquitination complex integrates transcript elongation with RNA processing at circadian clock and flowering regulators.

Proc Natl Acad Sci U S A 2019 04 28;116(16):8060-8069. Epub 2019 Mar 28.

Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium;

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 and had reduced rosette and leaf areas. Strikingly, in mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes () and () that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the and mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the α and β splice forms were altered, and the expression and H2Bub levels were reduced. In the mutant, the delay in flowering time was correlated with an enhanced expression, possibly due to an increased distal versus proximal ratio of its antisense 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 Furthermore, the presence of an intact HUB1 at the is required for SPEN3 function in the formation of the -derived antisense transcripts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1806541116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475378PMC
April 2019

The UAP56-Interacting Export Factors UIEF1 and UIEF2 Function in mRNA Export.

Plant Physiol 2019 04 30;179(4):1525-1536. Epub 2019 Jan 30.

Department of Cell Biology & Plant Biochemistry, Biochemistry Centre, University of Regensburg, D-93053 Regensburg, Germany

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 () 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 , , or both in the double-mutant caused modest growth defects. A cross between the and (defective in the four ALY1-4 mRNA export factors) mutants produced the sextuple mutant , which displayed more severe growth impairment than the plants. Developmental defects including delayed bolting and reduced seed set were observed in the but not the plants. Analysis of the cellular distribution of polyadenylated mRNAs revealed more pronounced nuclear mRNA accumulation in than in and 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1104/pp.18.01476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446781PMC
April 2019

The Arabidopsis Histone Chaperone FACT: Role of the HMG-Box Domain of SSRP1.

J Mol Biol 2018 08 30;430(17):2747-2759. Epub 2018 Jun 30.

Department of Cell Biology & Plant Biochemistry, Biochemistry Centre, University of Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2018.06.046DOI Listing
August 2018

Targeted Recruitment of the Basal Transcriptional Machinery by LNK Clock Components Controls the Circadian Rhythms of Nascent RNAs in Arabidopsis.

Plant Cell 2018 04 4;30(4):907-924. Epub 2018 Apr 4.

Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra, 08193 Barcelona, Spain

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 The expression of the circadian oscillator genes and 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1105/tpc.18.00052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973845PMC
April 2018

The Adaptor Protein ENY2 Is a Component of the Deubiquitination Module of the Arabidopsis SAGA Transcriptional Co-activator Complex but not of the TREX-2 Complex.

J Mol Biol 2018 05 26;430(10):1479-1494. Epub 2018 Mar 26.

Department of Cell Biology & Plant Biochemistry, Biochemistry Centre, University of Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2018.03.018DOI Listing
May 2018

ALY RNA-Binding Proteins Are Required for Nucleocytosolic mRNA Transport and Modulate Plant Growth and Development.

Plant Physiol 2018 05 14;177(1):226-240. Epub 2018 Mar 14.

Department of Cell Biology and Plant Biochemistry, Biochemistry Centre, University of Regensburg, D-93053 Regensburg, Germany

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 () 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 mutants have a wild-type appearance, disruption of to in 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 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1104/pp.18.00173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933122PMC
May 2018

The Arabidopsis histone chaperone FACT is required for stress-induced expression of anthocyanin biosynthetic genes.

Plant Mol Biol 2018 Mar 13;96(4-5):367-374. Epub 2018 Jan 13.

Cell Biology & Plant Biochemistry, Biochemistry Centre, University of Regensburg, Universitätsstr. 31, 93053, Regensburg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11103-018-0701-5DOI Listing
March 2018

The plant RNA polymerase II elongation complex: A hub coordinating transcript elongation and mRNA processing.

Transcription 2018 4;9(2):117-122. Epub 2017 Oct 4.

a Department of Cell Biology & Plant Biochemistry, Biochemistry Centre , University of Regensburg , Regensburg , Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/21541264.2017.1356902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834219PMC
November 2018

Analysis of In Vivo Chromatin and Protein Interactions of Arabidopsis Transcript Elongation Factors.

Methods Mol Biol 2017 ;1629:105-122

Department of Cell Biology & Plant Biochemistry, Biochemistry Center, University of Regensburg, Universitätsstr. 31, 93053, Regensburg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-7125-1_8DOI Listing
March 2018

The Composition of the Arabidopsis RNA Polymerase II Transcript Elongation Complex Reveals the Interplay between Elongation and mRNA Processing Factors.

Plant Cell 2017 Apr 28;29(4):854-870. Epub 2017 Mar 28.

Department of Cell Biology and Plant Biochemistry, Biochemistry Center, University of Regensburg, D-93053 Regensburg, Germany

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 , 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1105/tpc.16.00735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435424PMC
April 2017

The Arabidopsis THO/TREX component TEX1 functionally interacts with MOS11 and modulates mRNA export and alternative splicing events.

Plant Mol Biol 2017 Feb 21;93(3):283-298. Epub 2016 Dec 21.

Department of Cell Biology and Plant Biochemistry, Biochemistry Centre, University of Regensburg, Universitätsstr. 31, 93053, Regensburg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11103-016-0561-9DOI Listing
February 2017

The zinc-finger protein SPT4 interacts with SPT5L/KTF1 and modulates transcriptional silencing in Arabidopsis.

FEBS Lett 2015 Oct 28;589(21):3254-7. Epub 2015 Sep 28.

Cell Biology & Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2015.09.017DOI Listing
October 2015

Mitotic lifecycle of chromosomal 3xHMG-box proteins and the role of their N-terminal domain in the association with rDNA loci and proteolysis.

New Phytol 2015 Dec 27;208(4):1067-77. Epub 2015 Jul 27.

Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Universitätsstr. 31, D-93053, Regensburg, Germany.

The high mobility group (HMG)-box is a DNA-binding domain characteristic of various eukaryotic DNA-binding proteins. 3xHMG-box proteins (containing three copies of the HMG-box domain and a unique basic N-terminal domain) are specific for plants and the Arabidopsis genome encodes two versions termed 3xHMG-box1 and 3xHMG-box2, whose expression is cell cycle-dependent, peaking during mitosis. Here, we analysed in detail the spatiotemporal expression, subcellular localisation and chromosome association of the Arabidopsis thaliana 3xHMG-box proteins. Live cell imaging and structured illumination microscopy revealed that the expression of the 3xHMG-box proteins is induced in late G2 phase of the cell cycle and upon nuclear envelope breakdown in prophase they rapidly associate with the chromosomes. 3xHMG-box1 associates preferentially with 45S rDNA loci and the basic N-terminal domain is involved in the targeting of rDNA loci. Shortly after mitosis the 3xHMG-box proteins are degraded and an N-terminal destruction-box mediates the proteolysis. Ectopic expression/localisation of 3xHMG-box1 in interphase nuclei results in reduced plant growth and various developmental defects including early bolting and abnormal flower morphology. The remarkable conservation of 3xHMG-box proteins within the plant kingdom, their characteristic expression during mitosis, and their striking association with chromosomes, suggest that they play a role in the organisation of plant mitotic chromosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/nph.13575DOI Listing
December 2015

Structural insights into the mechanism of negative regulation of single-box high mobility group proteins by the acidic tail domain.

J Biol Chem 2014 Oct 4;289(43):29817-26. Epub 2014 Sep 4.

From the Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom and

The Drosophila and plant (maize) functional counterparts of the abundant vertebrate chromosomal protein HMGB1 (HMG-D and ZmHMGB1, respectively) differ from HMGB1 in having a single HMG box, as well as basic and acidic flanking regions that vary greatly in length and charge. We show that despite these variations, HMG-D and ZmHMGB1 exist in dynamic assemblies in which the basic HMG boxes and linkers associate with their intrinsically disordered, predominantly acidic, tails in a manner analogous to that observed previously for HMGB1. The DNA-binding surfaces of the boxes and linkers are occluded in "auto-inhibited" forms of the protein, which are in equilibrium with transient, more open structures that are "binding-competent." This strongly suggests that the mechanism of auto-inhibition may be a general one. HMG-D and ZmHMGB1 differ from HMGB1 in having phosphorylation sites in their tail and linker regions. In both cases, in vitro phosphorylation of serine residues within the acidic tail stabilizes the assembled form, suggesting another level of regulation for interaction with DNA, chromatin, and other proteins that is not possible for the uniformly acidic (hence unphosphorylatable) tail of HMGB1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.591115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207994PMC
October 2014

Transcript elongation factors: shaping transcriptomes after transcript initiation.

Trends Plant Sci 2014 Nov 11;19(11):717-26. Epub 2014 Aug 11.

Department of Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg (BZR), University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany. Electronic address:

Elongation is a dynamic and highly regulated step of eukaryotic gene transcription. A variety of transcript elongation factors (TEFs), including modulators of RNA polymerase II (RNAPII) activity, histone chaperones, and histone modifiers, have been characterized from plants. These factors control the efficiency of transcript elongation of subsets of genes in the chromatin context and thus contribute to tuning gene expression programs. We review here how genetic and biochemical analyses, primarily in Arabidopsis thaliana, have advanced our understanding of how TEFs adjust plant gene transcription. These studies have revealed that TEFs regulate plant growth and development by modulating diverse processes including hormone signaling, circadian clock, pathogen defense, responses to light, and developmental transitions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tplants.2014.07.002DOI Listing
November 2014

Elongator and SPT4/SPT5 complexes as proxy to study RNA polymerase II transcript elongation control of plant development.

Proteomics 2014 Oct 22;14(19):2109-14. Epub 2014 May 22.

Department of Plant Systems Biology, VIB, Ghent, Belgium; Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.

The elongation phase of the RNA polymerase II (RNAPII) transcription process is dynamic and regulated. Elongator and SUPPRESSOR OF Ty4 (SPT4)/SPT5 are transcript elongation factors that contribute to the regulation of mRNA synthesis by RNA polymerase II in the chromatin context. Recently, the Elongator complex consisting of six subunits and the SPT4/SPT5 heterodimer were isolated from Arabidopsis. Mutant plants affected in the expression of Elongator or SPT4/SPT5 share various auxin-signaling phenotypes. In line with that observation, auxin-related genes are prominent among the genes differentially expressed in these mutants. Exemplified by Elongator and SPT4/SPT5, we discuss here the role that transcript elongation factors may play in the control of plant growth and development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201400024DOI Listing
October 2014

The transcript elongation factor SPT4/SPT5 is involved in auxin-related gene expression in Arabidopsis.

Nucleic Acids Res 2014 Apr 4;42(7):4332-47. Epub 2014 Feb 4.

Department of Cell Biology & Plant Biochemistry, Biochemie-Zentrum Regensburg (BZR), University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466 Gatersleben, Germany and Institute for Biochemistry I, Biochemie-Zentrum Regensburg (BZR), University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.

The heterodimeric complex SPT4/SPT5 is a transcript elongation factor (TEF) that directly interacts with RNA polymerase II (RNAPII) to regulate messenger RNA synthesis in the chromatin context. We provide biochemical evidence that in Arabidopsis, SPT4 occurs in a complex with SPT5, demonstrating that the SPT4/SPT5 complex is conserved in plants. Each subunit is encoded by two genes SPT4-1/2 and SPT5-1/2. A mutant affected in the tissue-specifically expressed SPT5-1 is viable, whereas inactivation of the generally expressed SPT5-2 is homozygous lethal. RNAi-mediated downregulation of SPT4 decreases cell proliferation and causes growth reduction and developmental defects. These plants display especially auxin signalling phenotypes. Consistently, auxin-related genes, most strikingly AUX/IAA genes, are downregulated in SPT4-RNAi plants that exhibit an enhanced auxin response. In Arabidopsis nuclei, SPT5 clearly localizes to the transcriptionally active euchromatin, and essentially co-localizes with transcribing RNAPII. Typical for TEFs, SPT5 is found over the entire transcription unit of RNAPII-transcribed genes. In SPT4-RNAi plants, elevated levels of RNAPII and SPT5 are detected within transcribed regions (including those of downregulated genes), indicating transcript elongation defects in these plants. Therefore, SPT4/SPT5 acts as a TEF in Arabidopsis, regulating transcription during the elongation stage with particular impact on the expression of certain auxin-related genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gku096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985667PMC
April 2014

The seed dormancy defect of Arabidopsis mutants lacking the transcript elongation factor TFIIS is caused by reduced expression of the DOG1 gene.

FEBS Lett 2014 Jan 16;588(1):47-51. Epub 2013 Nov 16.

Cell Biology & Plant Biochemistry, Biochemie-Zentrum Regensburg, Regensburg University, Universitätsstr. 31, D-93053 Regensburg, Germany; Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark. Electronic address:

TFIIS is a transcript elongation factor that facilitates transcription by RNA polymerase II, as it assists the enzyme to bypass blocks to mRNA synthesis. Previously, we have reported that Arabidopsis plants lacking TFIIS exhibit reduced seed dormancy. Among the genes differentially expressed in tfIIs seeds, the DOG1 gene was identified that is a known QTL for seed dormancy. Here we have analysed plants that overexpress TFIIS in wild type background, or that harbour an additional copy of DOG1 in tfIIs mutant background. These experiments demonstrate that the down-regulation of DOG1 expression causes the seed dormancy phenotype of tfIIs mutants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2013.10.047DOI Listing
January 2014

Arabidopsis DEAD-box RNA helicase UAP56 interacts with both RNA and DNA as well as with mRNA export factors.

PLoS One 2013 26;8(3):e60644. Epub 2013 Mar 26.

Department of Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, Regensburg, Germany.

The DEAD-box protein UAP56 (U2AF65-associcated protein) is an RNA helicase that in yeast and metazoa is critically involved in mRNA splicing and export. In Arabidopsis, two adjacent genes code for an identical UAP56 protein, and both genes are expressed. In case one of the genes is inactivated by a T-DNA insertion, wild type transcript level is maintained by the other intact gene. In contrast to other organisms that are severely affected by elevated UAP56 levels, Arabidopsis plants that overexpress UAP56 have wild type appearance. UAP56 localises predominantly to euchromatic regions of Arabidopsis nuclei, and associates with genes transcribed by RNA polymerase II independently from the presence of introns, while it is not detected at non-transcribed loci. Biochemical characterisation revealed that in addition to ssRNA and dsRNA, UAP56 interacts with dsDNA, but not with ssDNA. Moreover, the enzyme displays ATPase activity that is stimulated by RNA and dsDNA and it has ATP-dependent RNA helicase activity unwinding dsRNA, whereas it does not unwind dsDNA. Protein interaction studies showed that UAP56 directly interacts with the mRNA export factors ALY2 and MOS11, suggesting that it is involved in mRNA export from plant cell nuclei.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060644PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608606PMC
September 2013

Plant proteins containing high mobility group box DNA-binding domains modulate different nuclear processes.

Plant Physiol 2012 Jul 14;159(3):875-83. Epub 2012 May 14.

Department of Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, D-93053 Regensburg, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1104/pp.112.198283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3387713PMC
July 2012

The plant-specific family of DNA-binding proteins containing three HMG-box domains interacts with mitotic and meiotic chromosomes.

New Phytol 2011 Nov 22;192(3):577-89. Epub 2011 Jul 22.

Cell Biology and Plant Biochemistry, Regensburg University, Regensburg, Germany.

• The high mobility group (HMG)-box represents a DNA-binding domain that is found in various eukaryotic DNA-interacting proteins. Proteins that contain three copies of the HMG-box domain, termed 3 × HMG-box proteins, appear to be specific to plants. The Arabidopsis genome encodes two 3 × HMG-box proteins that were studied here. • DNA interactions were examined using electrophoretic mobility shift assays, whereas expression, subcellular localization and chromosome association were mainly analysed by different types of fluorescence microscopy. • The 3 × HMG-box proteins bind structure specifically to DNA, display DNA bending activity and, in addition to the three HMG-box domains, the basic N-terminal domain contributes to DNA binding. The expression of the two Arabidopsis genes encoding 3 × HMG-box proteins is linked to cell proliferation. In synchronized cells, expression is cell cycle dependent and peaks in cells undergoing mitosis. 3 × HMG-box proteins are excluded from the nuclei of interphase cells and localize to the cytosol, but, during mitosis, they associate with condensed chromosomes. The 3 × HMG-box2 protein generally associates with mitotic chromosomes, while 3 × HMG-box1 is detected specifically at 45S rDNA loci. • In addition to mitotic chromosomes the 3 × HMG-box proteins associate with meiotic chromosomes, suggesting that they are involved in a general process of chromosome function related to cell division, such as chromosome condensation and/or segregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1469-8137.2011.03828.xDOI Listing
November 2011

Reduced expression of the DOG1 gene in Arabidopsis mutant seeds lacking the transcript elongation factor TFIIS.

FEBS Lett 2011 Jun 8;585(12):1929-33. Epub 2011 May 8.

Cell Biology and Plant Biochemistry, Regensburg University, Regensburg, Germany.

TFIIS is a transcript elongation factor that facilitates transcription by RNA polymerase II through blocks to elongation. Arabidopsis plants lacking TFIIS are affected in seed dormancy, which represents a block to complete germination under favourable conditions. We have comparatively profiled the transcript levels of seeds of tfIIs mutants and control plants. Among the differentially expressed genes, the DOG1 gene was identified that is a QTL for seed dormancy. The reduced expression of DOG1 in tfIIs seeds was confirmed by quantitative RT-PCR and Northern analyses, suggesting that down-regulation of DOG1 expression is involved in the seed dormancy phenotype of tfIIs mutants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2011.04.077DOI Listing
June 2011

Unexpected mobility of plant chromatin-associated HMGB proteins.

Plant Signal Behav 2011 Jun 1;6(6):878-80. Epub 2011 Jun 1.

Faculty of Biology & Institute for Genome Research and Systems Biology, Bielefeld University, Bielefeld, Germany.

High mobility group (HMG) proteins of the HMGB family containing a highly conserved HMG box are chromatin-associated proteins that interact with DNA and nucleosomes and catalyze changes in DNA topology, thereby facilitating important DNA-dependent processes. The genome of Arabidopsis thaliana encodes 15 different HMG-box proteins that are further subdivided into four groups: HMGB-type proteins, ARID-HMG proteins, 3xHMG proteins that contain three HMG boxes and the structure-specific recognition protein 1 (SSRP1). Typically, HMGB proteins are localized exclusively to the nucleus, like Arabidopsis HMGB1 and B5. However, these Arabidopsis HMGB proteins showed a very high mobility within the nuclear compartment. Recent studies revealed that Arabidopsis HMGB2/3 and B4 proteins are predominantly nuclear but also exist in the cytoplasm, suggesting an as yet unknown cytoplasmic function of these chromosomal HMG proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3218493PMC
http://dx.doi.org/10.4161/psb.6.6.15255DOI Listing
June 2011