Functions of Arabidopsis C-terminal Domain Phosphatase-like 4 in Global Transcriptional Regulation

Phosphoregulation of the carboxyl-terminal domain of RNA polymerase II largest subunit (pol II-CTD) couples transcription and co-transcriptional modification of nascent RNA. Although the molecular mechanisms have been extensively studied in vertebrates, understanding of that in plants is still in its infancy. Through genetics, biochemical and transcriptomic approaches, this dissertation work characterizes functions of a pol II-CTD phosphatase-like protein from Arabidopsis thaliana, CPL4, in the phosphoregulation of pol II-CTD during protein-coding and non-coding RNA transcriptions. CPL4 interacts with and dephosphorylates pol II-CTD both in vitro and in vivo, showing that CPL4 regulates pol II-CTD phosphorylation status in Arabidopsis. An amino acid substitution in the catalytic motif abolished the phosphatase activity of CPL4. The catalytically inactive protein strongly inhibits transcription in transient assays, likely due to a dominant negative effect. Deletion of Breast cancer C-terminal (BRCT) domain alleviates the inhibitory effect of the catalytically inactive CPL4, suggesting that BRCT domain is necessary for CPL4’s function. A suite of xenobiotic stress responsive genes shows constitutive up-regulation in CPL4 knockdown transgenic (CPL4RNAi) lines, indicating that CPL4 negatively regulates the toxic chemical detoxification pathway. The CPL4RNAi plants accumulate aberrant 3’-extended transcripts from many pol II-dependent small nuclear RNA (snRNA) loci. The snRNA 3’-extension gives rise to a snRNA transcript fused with a downstream protein-coding gene (DPG) if present. Such snRNA-DPG fusion transcripts can be found in other plant species. A short, unstable non-coding RNA produced from a protein-coding locus driven by a transposable-embedded snRNA promoter can yield the full-length product in the CPL4RNAi plants. These snRNA-DPGs can be induced in wild type by salt stress, which affects pol II-CTD phosphorylation status. These results indicate a potential stress-inducible conversion of non-coding RNA transcription into protein-coding transcription mediated by pol II-CTD phosphoregulation. CPL4RNAi root explants exhibit enhanced capability of de novo shoot organogenesis due to cytokinin hypersensitivity and earlier induction of shoot apical meristem regulatory genes. A potential involvement of an operon-like cluster of thalianol biosynthesis genes in the CPL4RNAi organogenesis phenotype is implicated. Taken together, Arabidopsis CPL4 is an essential pol II-CTD phosphatase, regulating stress-responsive and organogenesis pathways through protein-coding and non-coding RNA transcriptions

Functions of Arabidopsis C-terminal Domain Phosphatase-like 4 in Global Transcriptional Regulation

Phosphoregulation of the carboxyl-terminal domain of RNA polymerase II largest subunit (pol II-CTD) couples transcription and co-transcriptional modification of nascent RNA. Although the molecular mechanisms have been extensively studied in vertebrates, understanding of that in plants is still in its infancy. Through genetics, biochemical and transcriptomic approaches, this dissertation work characterizes functions of a pol II-CTD phosphatase-like protein from Arabidopsis thaliana, CPL4, in the phosphoregulation of pol II-CTD during protein-coding and non-coding RNA transcriptions. CPL4 interacts with and dephosphorylates pol II-CTD both in vitro and in vivo, showing that CPL4 regulates pol II-CTD phosphorylation status in Arabidopsis. An amino acid substitution in the catalytic motif abolished the phosphatase activity of CPL4. The catalytically inactive protein strongly inhibits transcription in transient assays, likely due to a dominant negative effect. Deletion of Breast cancer C-terminal (BRCT) domain alleviates the inhibitory effect of the catalytically inactive CPL4, suggesting that BRCT domain is necessary for CPL4’s function. A suite of xenobiotic stress responsive genes shows constitutive up-regulation in CPL4 knockdown transgenic (CPL4RNAi) lines, indicating that CPL4 negatively regulates the toxic chemical detoxification pathway. The CPL4RNAi plants accumulate aberrant 3’-extended transcripts from many pol II-dependent small nuclear RNA (snRNA) loci. The snRNA 3’-extension gives rise to a snRNA transcript fused with a downstream protein-coding gene (DPG) if present. Such snRNA-DPG fusion transcripts can be found in other plant species. A short, unstable non-coding RNA produced from a protein-coding locus driven by a transposable-embedded snRNA promoter can yield the full-length product in the CPL4RNAi plants. These snRNA-DPGs can be induced in wild type by salt stress, which affects pol II-CTD phosphorylation status. These results indicate a potential stress-inducible conversion of non-coding RNA transcription into protein-coding transcription mediated by pol II-CTD phosphoregulation. CPL4RNAi root explants exhibit enhanced capability of de novo shoot organogenesis due to cytokinin hypersensitivity and earlier induction of shoot apical meristem regulatory genes. A potential involvement of an operon-like cluster of thalianol biosynthesis genes in the CPL4RNAi organogenesis phenotype is implicated. Taken together, Arabidopsis CPL4 is an essential pol II-CTD phosphatase, regulating stress-responsive and organogenesis pathways through protein-coding and non-coding RNA transcriptions

Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV's 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3' ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs)

Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4

Arabidopsis thaliana Dicer-like 4 (DCL4) produces 21-nt small interfering RNAs from both endogenous and exogenous double-stranded RNAs (dsRNAs), and it interacts with DRB4, a dsRNA-binding protein, in vivo and in vitro. However, the role of DRB4 in DCL4 activity remains unclear because the dsRNA-cleaving activity of DCL4 has not been characterized biochemically. In this study, we biochemically characterize DCL4's Dicer activity and establish that DRB4 is required for this activity in vitro. Crude extracts from Arabidopsis seedlings cleave long dsRNAs into 21-nt small RNAs in a DCL4/DRB4-dependent manner. Immunoaffinity-purified DCL4 complexes produce 21-nt small RNAs from long dsRNA, and these complexes have biochemical properties similar to those of known Dicer family proteins. The DCL4 complexes purified from drb4-1 do not cleave dsRNA, and the addition of recombinant DRB4 to drb4-1 complexes specifically recovers the 21-nt small RNA generation. These results reveal that DCL4 requires DRB4 to cleave long dsRNA into 21-nt small RNAs in vitro. Amino acid substitutions in conserved dsRNA-binding domains (dsRBDs) of DRB4 impair three activities: binding to dsRNA, interacting with DCL4, and facilitating DCL4 activity. These observations indicate that the dsRBDs are critical for DRB4 function. Our biochemical approach and observations clearly show that DRB4 is specifically required for DCL4 activity in vitro

Arabidopsis C-Terminal Domain Phosphatase-Like 1 Functions in miRNA Accumulation and DNA Methylation

<div><p>Arabidopsis CTD-PHOSPHATASE-LIKE 1 (CPL1) is a protein phosphatase that can dephosphorylate RNA polymerase II C-terminal domain (CTD). Unlike typical CTD-phosphatases, CPL1 contains a double-stranded (ds) RNA-binding motif (dsRBM) and has been implicated for gene regulation mediated by dsRNA-dependent pathways. We investigated the role of CPL1 and its dsRBMs in various gene silencing pathways. Genetic interaction analyses revealed that <i>cpl1</i> was able to partially suppress transcriptional gene silencing and DNA hypermethylation phenotype of <i>ros1</i> suggesting CPL1 is involved in the RNA-directed DNA methylation pathway without reducing siRNA production. By contrast, <i>cpl1</i> reduced some miRNA levels at the level of processing. Indeed, CPL1 protein interacted with proteins important for miRNA biogenesis, suggesting that CPL1 regulates miRNA processing. These results suggest that CPL1 regulates DNA methylation via a miRNA-dependent pathway.</p></div

Double-stranded RNA sequencing reveals distinct riboviruses associated with thermoacidophilic bacteria from hot springs in Japan

International audienceMetatranscriptome sequencing expanded the known diversity of the bacterial RNA virome, suggesting that additional riboviruses infecting bacterial hosts remain to be discovered. Here we employed doublestranded RNA sequencing to recover complete genome sequences of two ribovirus groups from acidic hot springs in Japan. One group, denoted hot spring riboviruses (HsRV), consists of viruses with distinct RNA-directed RNA polymerases (RdRPs) that seem to be intermediates between typical ribovirus RdRPs and viral reverse transcriptases. This group forms a distinct phylum, Artimaviricota, or even kingdom within the realm Riboviria. We identified viruses encoding HsRV-like RdRPs in marine water, river sediments and salt marshes, indicating that this group is widespread beyond extreme ecosystems. The second group, denoted hot spring partiti-like viruses (HsPV), forms a distinct branch within the family Partitiviridae. The genome architectures of HsRV and HsPV and their identification in bacteria-dominated habitats suggest that these viruses infect thermoacidophilic bacteria

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand

Regulation of Abiotic Stress Signalling by Arabidopsis C-Terminal Domain Phosphatase-Like 1 Requires Interaction with a K-Homology Domain-Containing Protein

<div><p><i>Arabidopsis thaliana</i> CARBOXYL-TERMINAL DOMAIN (CTD) PHOSPHATASE-LIKE 1 (CPL1) regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds) RNA binding motifs (dsRBMs) at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured <i>Arabidopsis</i> cells and contains multiple K-homology (KH) domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 <i>in vivo</i> function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of <i>rcf3</i> and <i>cpl1</i> mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.</p></div

The <i>cpl1</i> mutation influences DNA methylation.

<p>(A) Bisulfite sequencing results of endogenous <i>RD29A</i> and <i>RD29A-LUC</i> transgene promoters. The ratio of cytosine methylation in percentage was determined at CG, CHG, and CHH sites on endogenous (left) and transgenic (right) <i>RD29A</i> promoters. H represents A, T, or C. (B) PCR-based cytosine methylation assay on RdDM target loci using methylation-sensitive enzymes. The amplifications using undigested DNA templates (-) were used as controls.</p

CPL1 interacts with HYL1-SE complex in nucleus.

<p>(A) Immunoprecipitation with (+) or without (−) anti-HYL1 was performed using a crude extract of calli containing <i>gCPL1-FLAG</i> transgene. CPL1-FLAG was detected by immunoblot using anti-FLAG1 antibody. (B) Immunoprecipitation with (+) or without (−) anti-FLAG was performed using a crude extract of calli containing <i>gCPL1-FLAG</i> transgene. HYL1 was detected by immunoblot using anti-HYL1 antibody. (C) BiFC visualization of CPL1-HYL1 interaction. Epifluorescence (YFP) and bright field images of protoplasts that were transfected with plasmids encoding nYFP-CPL1 and cYFP-HYL1 fusion proteins and NLS-RFP. NLS-RFP was used as a positive control for nuclear localization. Yellow signals on merged images indicate co-localization of YFP and nuclear-localized RFP proteins. Scale bars indicate 10 µm. (D) Luminescence images of <i>N. benthamiana</i> leaves infiltrated with NLUC-HYL1 (top panel) or NLUC-SE (bottom panel) with CLUC-CPL1 fragments. LUC images were obtained 3 days after infiltration. MYB75, CLUC-MYB75 used as a negative control. (E) Yeast two-hybrid assay. Growth of PJ69-4A co-expressing GAL4 DNA binding domain (BD) fused with CPL1 (BD_CPL1) and GAL4 activation domain (AD) fused with HYL1 and SE (AD_HYL1 and AD_SE). Cells were grown on synthetic dropout (SD) media lacking uracil and leucine (-UL) or SD medium lacking uracil, leucine, histidine and adenine (-ULHA). 2×10⁵ cells were used for (1) and diluted 10-fold for (1/10). Photographs were taken after incubation at 28°C for 48 hours. ADv and BDv indicate vector controls.</p