Arabidopsis MED18 Interaction With RNA Pol IV and V Subunit NRPD2a in Transcriptional Regulation of Plant Immune Responses

Mediator is a conserved multiprotein complex important for transcription by RNA polymerase II (Pol II). Arabidopsis Mediator subunit MED18 regulates flowering, hormone signaling and plant immunity. Here we report that Arabidopsis MED18 interacted with NUCLEAR RNA POLYMERASE D2a (NRPD2a), the second largest subunit of the nuclear Pol IV and V, which function in RNA-directed DNA methylation and epigenetic regulation of gene expression. Mutants for both MED18 and NRPD2a were compromised in resistance to necrotrophic fungal pathogen Botrytis cinerea. Mutants for NRPD1a, the largest subunit of Pol IV, were also compromised in resistance to Botrytis, supporting a critical role of Pol IV and V in plant defense against Botrytis. Increased Botrytis susceptibility of both the med18 and nrpd2a mutants were associated with reduced accumulation of reactive oxygen species, which are known to promote resistance to Botrytis. Both the basal and pathogen-induced levels of salicylic acid and jasmonic acid were also significantly altered in the med18 and nrpd2a mutants. Transcriptome profiling found that MED18 and NRPD2a affected both unique and overlapping sets of genes in a broad spectrum of biological processes and pathways that influence plant–pathogen interaction. The genes altered in expression in the med18 and nrpd2a mutants include disease resistance proteins, salicylic acid and jasmonic acid signaling and responses, which are known to affect resistance to necrotrophic pathogens. The novel interaction between subunits of Mediator and plant-specific RNA polymerases provides a new mechanism for epigenetic regulation of resistance and expression of defense-related genes in plant immunity.

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Regulation of RNA Polymerase II Activity is Essential for Terminal Erythroid Maturation

Blood ◽

10.1182/blood.2020009903 ◽

2021 ◽

Author(s):

Zachary Murphy ◽

Kristin Murphy ◽

Jacquelyn A Myers ◽

Michael Roger Getman ◽

Tyler Couch ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Critical Role ◽

Regulation Of Gene Expression ◽

Maturation Stage ◽

Nuclear Condensation ◽

Erythroid Maturation ◽

Specific Regulation

The terminal maturation of human erythroblasts requires significant changes in gene expression in the context of dramatic nuclear condensation. Defects in this process are associated with inherited anemias and myelodysplastic syndromes. The progressively dense appearance of the condensing nucleus in maturing erythroblasts led to the assumption that heterochromatin accumulation underlies this process, but despite extensive study, the precise mechanisms underlying this essential biologic process remain elusive. To delineate the epigenetic changes associated with the terminal maturation of human erythroblasts, we performed mass spectrometry of histone post-translational modifications combined with ChIP-seq, ATAC-seq, and RNA-seq. Our studies revealed that the terminal maturation of human erythroblasts is associated with a dramatic decline in histone marks associated with active transcription elongation, without accumulation of heterochromatin. Chromatin structure and gene expression were instead correlated with dynamic changes in occupancy of elongation competent RNA polymerase II, suggesting that terminal erythroid maturation is controlled largely at the level of transcription. We further demonstrate that RNA Polymerase II "pausing" is highly correlated with transcriptional repression, with elongation competent RNA polymerase II becoming a scare resource in late stage erythroblasts, allocated to erythroid-specific genes. Functional studies confirmed an essential role for maturation stage-specific regulation of RNA polymerase II activity during erythroid maturation, and demonstrate a critical role for HEXIM1 in the regulation of gene expression and RNA polymerase II activity in maturing erythroblasts. Taken together, our findings reveal important insights into the mechanisms that regulate terminal erythroid maturation, and provide a novel paradigm for understanding normal and perturbed erythropoiesis.

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The roles of nucleolar structure and function in the subcellular location of the HIV-1 Rev protein

Journal of Cell Science ◽

10.1242/jcs.108.8.2811 ◽

1995 ◽

Vol 108 (8) ◽

pp. 2811-2823 ◽

Cited By ~ 3

Author(s):

M. Dundr ◽

G.H. Leno ◽

M.L. Hammarskjold ◽

D. Rekosh ◽

C. Helga-Maria ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Critical Role ◽

Regulation Of Expression ◽

Rna Transcription ◽

Nucleolar Structure ◽

Class 1 ◽

Protein B23 ◽

Hiv 1 ◽

Rev Protein

The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.

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Chitosan Oligosaccharide Induces Resistance to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana by Activating Both Salicylic Acid– and Jasmonic Acid–Mediated Pathways

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-03-18-0071-r ◽

2018 ◽

Vol 31 (12) ◽

pp. 1271-1279 ◽

Cited By ~ 21

Author(s):

Xiaochen Jia ◽

Haihong Zeng ◽

Wenxia Wang ◽

Fuyun Zhang ◽

Heng Yin

Keyword(s):

Salicylic Acid ◽

Jasmonic Acid ◽

Induced Resistance ◽

Pseudomonas Syringae ◽

Plant Immunity ◽

Chitosan Oligosaccharide ◽

Induction Effect ◽

Tomato Dc3000 ◽

Expression Of Genes ◽

Underlying Mechanisms

Chitosan oligosaccharide (COS) is an effective plant immunity elicitor; however, its induction mechanism in plants is complex and needs further investigation. In this study, the Arabidopsis–Pseudomonas syringae pv. tomato DC3000 (hereafter called DC3000) interaction was used to investigate the induction effect and the underlying mechanisms of COS. COS is effective in inducing resistance to DC3000 in Arabidopsis, and our results demonstrate that treatment with COS 3 days before DC3000 inoculation provided the most effective resistance. Disease severity in jar1 (jasmonic acid [JA]-deficient mutant), NahG, and sid2 (salicylic acid [SA]-deficient mutants) suggest both the SA and JA pathways are required for the Arabidopsis response to DC3000. COS pretreatment induced resistance in wild type (WT), jar1, and also, although to a lesser degree, in NahG and sid2 plants, implying that the SA and JA pathways play redundant roles in COS-induced resistance to DC3000. In COS-pretreated plants, expression of genes related to the SA pathway (PR1, PR2, and PR5) and SA content increased in both WT and jar1. Moreover, expression of genes related to the JA pathway (PDF1.2 and VSP2) and JA content both increased in WT and NahG. In conclusion, COS induces resistance to DC3000 in Arabidopsis by activating both SA- and JA-mediated pathways, although SA and JA pathways play redundant roles in this COS-induced resistance.

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The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation

Nucleic Acids Research ◽

10.1093/nar/gkz615 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9024-9036 ◽

Cited By ~ 4

Author(s):

Jered M Wendte ◽

Jeremy R Haag ◽

Olga M Pontes ◽

Jasleen Singh ◽

Sara Metcalf ◽

...

Keyword(s):

Dna Methylation ◽

Catalytic Activity ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cytosine Methylation ◽

Transcriptional Gene Silencing ◽

Rna Dependent Rna Polymerase ◽

Pol Ii ◽

Pol Iv ◽

Non Coding Rnas

Abstract In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV’s largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.

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Modulation of RNA Polymerase II Phosphorylation Downstream of Pathogen Perception Orchestrates Plant Immunity

Cell Host & Microbe ◽

10.1016/j.chom.2014.10.018 ◽

2014 ◽

Vol 16 (6) ◽

pp. 748-758 ◽

Cited By ~ 32

Author(s):

Fangjun Li ◽

Cheng Cheng ◽

Fuhao Cui ◽

Marcos V.V. de Oliveira ◽

Xiao Yu ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Plant Immunity

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Novel critical role of a human Mediator complex for basal RNA polymerase II transcription

EMBO Reports ◽

10.1093/embo-reports/kve186 ◽

2001 ◽

Vol 2 (9) ◽

pp. 808-813 ◽

Cited By ~ 93

Author(s):

Gerhard Mittler ◽

Elisabeth Kremmer ◽

H Th. Marc Timmers ◽

Michael Meisterernst

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Critical Role ◽

Mediator Complex ◽

Rna Polymerase Ii Transcription

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Reprogramming and remodeling: transcriptional and epigenetic regulation of salicylic acid-mediated plant defense

Journal of Experimental Botany ◽

10.1093/jxb/eraa072 ◽

2020 ◽

Vol 71 (17) ◽

pp. 5256-5268 ◽

Cited By ~ 1

Author(s):

Jian Chen ◽

Michael Clinton ◽

Guang Qi ◽

Daowen Wang ◽

Fengquan Liu ◽

...

Keyword(s):

Salicylic Acid ◽

Transcription Factors ◽

Plant Defense ◽

Epigenetic Regulation ◽

Current Knowledge ◽

Plant Immunity ◽

Defense Responses ◽

Transcriptional Networks ◽

Transcriptional Reprogramming ◽

Genomic Tools

Abstract As a plant hormone, salicylic acid (SA) plays essential roles in plant defense against biotrophic and hemibiotrophic pathogens. Significant progress has been made in understanding the SA biosynthesis pathways and SA-mediated defense signaling networks in the past two decades. Plant defense responses involve rapid and massive transcriptional reprogramming upon the recognition of pathogens. Plant transcription factors and their co-regulators are critical players in establishing a transcription regulatory network and boosting plant immunity. A multitude of transcription factors and epigenetic regulators have been discovered, and their roles in SA-mediated defense responses have been reported. However, our understanding of plant transcriptional networks is still limited. As such, novel genomic tools and bioinformatic techniques will be necessary if we are to fully understand the mechanisms behind plant immunity. Here, we discuss current knowledge, provide an update on the SA biosynthesis pathway, and describe the transcriptional and epigenetic regulation of SA-mediated plant immune responses.

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An N-Terminal Region of Lassa Virus L Protein Plays a Critical Role in Transcription but Not Replication of the Virus Genome

Journal of Virology ◽

10.1128/jvi.01657-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1934-1944 ◽

Cited By ~ 41

Author(s):

Michaela Lelke ◽

Linda Brunotte ◽

Carola Busch ◽

Stephan Günther

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Large Scale ◽

Critical Role ◽

Terminal Region ◽

Lassa Virus ◽

Mrna Synthesis ◽

L Protein ◽

Mrna Pool ◽

Capping Enzymes

ABSTRACT The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. N- and C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In the present study, we have employed a large-scale mutagenesis approach to map functionally relevant residues in these regions. The main targets were acidic (Asp and Glu) and basic residues (Lys and Arg) known to form catalytic and binding sites of capping enzymes and endoribonucleases. A total of 149 different mutants were generated and tested in the Lassa virus replicon system. Nearly 25% of evolutionarily highly conserved acidic and basic side chains were dispensable for function of L protein in the replicon context. The vast majority of the remaining mutants had defects in both transcription and replication. Seven residues (Asp-89, Glu-102, Asp-119, Lys-122, Asp-129, Glu-180, and Arg-185) were selectively important for mRNA synthesis. The phenotype was particularly pronounced for Asp-89, Glu-102, and Asp-129, which were indispensable for transcription but could be replaced by a variety of amino acid residues without affecting genome replication. Bioinformatics disclosed the remote similarity of this region to type IIs endonucleases. The mutagenesis was complemented by experiments with the RNA polymerase II inhibitor α-amanitin, demonstrating dependence of viral transcription from the cellular mRNA pool. In conclusion, this paper describes an N-terminal region in L protein being important for mRNA, but not genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.

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