An Rrp6-like protein positively regulates noncoding RNA levels and DNA methylation in Arabidopsis.

Rrp6-mediated nuclear RNA surveillance tunes eukaryotic transcriptomes through noncoding RNA degradation and mRNA quality control, including exosomal RNA decay and transcript retention triggered by defective RNA processing. It is unclear whether Rrp6 can positively regulate noncoding RNAs and whether RNA retention occurs in normal cells. Here we report that AtRRP6L1, an Arabidopsis Rrp6-like protein, controls RNA-directed DNA methylation through positive regulation of noncoding RNAs. Discovered in a forward genetic screen, AtRRP6L1 mutations decrease DNA methylation independently of exosomal RNA degradation. Accumulation of Pol V-transcribed scaffold RNAs requires AtRRP6L1 that binds to RNAs in vitro and in vivo. AtRRP6L1 helps retain Pol V-transcribed RNAs in chromatin to enable their scaffold function. In addition, AtRRP6L1 is required for genome-wide Pol IV-dependent siRNA production that may involve retention of Pol IV transcripts. Our results suggest that AtRRP6L1 functions in epigenetic regulation by helping with the retention of noncoding RNAs in normal cells.

The DEAD-box RNA helicase SHI2 functions in repression of salt-inducible genes and regulation of cold-inducible gene splicing.

Gene regulation is central for growth, development, and adaptation to environmental changes in all living organisms. Many genes are induced by environmental cues, and the expression of these inducible genes is often repressed under normal conditions. Here, we show that the SHINY2 (SHI2) gene is important for repressing salt-inducible genes and also plays a role in cold response. The shi2 mutant displayed hypersensitivity to cold, abscisic acid (ABA), and LiCl. Map-based cloning demonstrates that SHI2 encodes a DEAD- (Asp-Glu-Ala-Asp) box RNA helicase with similarity to a yeast splicing factor. Transcriptomic analysis of the shi2 mutant in response to cold revealed that the shi2 mutation decreased the number of cold-responsive genes and the magnitude of their response, and resulted in the mis-splicing of some cold-responsive genes. Under salt stress, however, the shi2 mutation increased the number of salt-responsive genes but had a negligible effect on mRNA splicing. Our results suggest that SHI2 is a component in a ready-for-transcription repressor complex important for gene repression under normal conditions, and for gene activation and transcription under stress conditions. In addition, SHI2 also serves as a splicing factor required for proper splicing of cold-responsive genes and affects 5' capping and polyadenylation site selection.

An Arabidopsis Nucleoporin NUP85 modulates plant responses to ABA and salt stress.

Several nucleoporins in the nuclear pore complex (NPC) have been reported to be involved in abiotic stress responses in plants. However, the molecular mechanism of how NPC regulates abiotic stress responses, especially the expression of stress responsive genes remains poorly understood. From a forward genetics screen using an abiotic stress-responsive luciferase reporter (RD29A-LUC) in the sickle-1 (sic-1) mutant background, we identified a suppressor caused by a mutation in NUCLEOPORIN 85 (NUP85), which exhibited reduced expression of RD29A-LUC in response to ABA and salt stress. Consistently, the ABA and salinity induced expression of several stress responsive genes such as RD29A, COR15A and COR47 was significantly compromised in nup85 mutants and other nucleoporin mutants such as nup160 and hos1. Subsequently, Immunoprecipitation and mass spectrometry analysis revealed that NUP85 is potentially associated with HOS1 and other nucleoporins within the nup107-160 complex, along with several mediator subunits. We further showed that there is a direct physical interaction between MED18 and NUP85. Similar to NUP85 mutations, MED18 mutation was also found to attenuate expression of stress responsive genes. Taken together, we not only revealed the involvement of NUP85 and other nucleoporins in regulating ABA and salt stress responses, but also uncovered a potential relation between NPC and mediator complex in modulating the gene expression in plants.

The SnRK2 kinases modulate miRNA accumulation in Arabidopsis.

MicroRNAs (miRNAs) regulate gene expression and play critical roles in growth and development as well as stress responses in eukaryotes. miRNA biogenesis in plants requires a processing complex that consists of the core components DICER-LIKE 1 (DCL1), SERRATE (SE) and HYPONASTIC LEAVES (HYL1). Here we show that inactivation of functionally redundant members of the SnRK2 kinases, which are the core components of abscisic acid (ABA) and osmotic stress signaling pathways, leads to reduction in miRNA accumulation under stress conditions. Further analysis revealed that the steady state level of HYL1 protein in plants under osmotic stress is dependent on the SnRK2 kinases. Additionally, our results suggest that the SnRK2 kinases physically associate with the miRNA processing components SE and HYL1 and can phosphorylate these proteins in vitro. These findings reveal an important role for the SnRK2 kinases in the regulation of miRNA accumulation and establish a mechanism by which ABA and osmotic stress signaling is linked to miRNA biogenesis.

Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA-directed DNA methylation.

Argonaute (AGO) family proteins are conserved key components of small RNA-induced silencing pathways. In the RNA-directed DNA methylation (RdDM) pathway in Arabidopsis, AGO6 is generally considered to be redundant with AGO4. In this report, our comprehensive, genomewide analyses of AGO4- and AGO6-dependent DNA methylation revealed that redundancy is unexpectedly negligible in the genetic interactions between AGO4 and AGO6. Immunofluorescence revealed that AGO4 and AGO6 differ in their subnuclear co-localization with RNA polymerases required for RdDM. Pol II and AGO6 are absent from perinucleolar foci, where Pol V and AGO4 are co-localized. In the nucleoplasm, AGO4 displays a strong co-localization with Pol II, whereas AGO6 co-localizes with Pol V. These patterns suggest that RdDM is mediated by distinct, spatially regulated combinations of AGO proteins and RNA polymerases. Consistently, Pol II physically interacts with AGO4 but not AGO6, and the levels of Pol V-dependent scaffold RNAs and Pol V chromatin occupancy are strongly correlated with AGO6 but not AGO4. Our results suggest that AGO4 and AGO6 mainly act sequentially in mediating small RNA-directed DNA methylation.

SAC3B, a central component of the mRNA export complex TREX-2, is required for prevention of epigenetic gene silencing in Arabidopsis.

Epigenetic regulation is important for organismal development and response to the environment. Alteration in epigenetic status has been known mostly from the perspective of enzymatic actions of DNA methylation and/or histone modifications. In a genetic screen for cellular factors involved in preventing epigenetic silencing, we isolated an Arabidopsis mutant defective in SAC3B, a component of the conserved TREX-2 complex that couples mRNA transcription with nuleo-cytoplasmic export. Arabidopsis SAC3B dysfunction causes gene silencing at transgenic and endogenous loci, accompanied by elevation in the repressive histone mark H3K9me2 and by reduction in RNA polymerase Pol II occupancy. SAC3B dysfunction does not alter promoter DNA methylation level of the transgene d35S::LUC, although the DNA demethylase ROS1 is also required for d35S::LUC anti-silencing. THP1 and NUA were identified as SAC3B-associated proteins whose mutations also caused d35S::LUC silencing. RNA-DNA hybrid exists at the repressed loci but is unrelated to gene suppression by the sac3b mutation. Genome-wide analyses demonstrated minor but clear involvement of SAC3B in regulating siRNAs and DNA methylation, particularly at a group of TAS and TAS-like loci. Together our results revealed not only a critical role of mRNA-export factors in transcriptional anti-silencing but also the contribution of SAC3B in shaping plant epigenetic landscapes.

Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling.

The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling.

HOS1 regulates Argonaute1 by promoting transcription of the microRNA gene MIR168b in Arabidopsis.

Proper accumulation and function of miRNAs is essential for plant growth and development. While core components of the miRNA biogenesis pathway and miRNA-induced silencing complex have been well characterized, cellular regulators of miRNAs remain to be fully explored. Here we report that High Expression Of Osmotically Responsive Genes1 (HOS1) is a regulator of an important miRNA, mi168a/b, that targets the Argonaute1 (AGO1) gene in Arabidopsis. HOS1 functions as an ubiquitin E3 ligase to regulate plant cold-stress responses, associates with the nuclear pores to regulate mRNA export, and regulates the circadian clock and flowering time by binding to chromatin of the flowering regulator gene Flowering Locus C (FLC). In a genetic screen for enhancers of sic-1, we isolated a loss-of-function Arabidopsis mutant of HOS1 that is defective in miRNA biogenesis. Like other hos1 mutant alleles, the hos1-7 mutant flowered early and was smaller in stature than the wild-type. Dysfunction in HOS1 reduced the abundance of miR168a/b but not of other miRNAs. In hos1 mutants, pri-MIR168b and pre-MIR168b levels were decreased, and RNA polymerase II occupancy was reduced at the promoter of MIR168b but not that of MIR168a. Chromatin immunoprecipitation assays revealed that HOS1 protein is enriched at the chromatin of the MIR168b promoter. The reduced miR168a/b level in hos1 mutants results in an increase in the mRNA and protein levels of its target gene, AGO1. Our results reveal that HOS1 regulates miR168a/b and AGO1 levels in Arabidopsis by maintaining proper transcription of MIR168b.

The arabidopsis RNA binding protein with K homology motifs, SHINY1, interacts with the C-terminal domain phosphatase-like 1 (CPL1) to repress stress-inducible gene expression.

The phosphorylation state of the C-terminal domain (CTD) of the RNA polymerase II plays crucial roles in transcription and mRNA processing. Previous studies showed that the plant CTD phosphatase-like 1 (CPL1) dephosphorylates Ser-5-specific CTD and regulates abiotic stress response in Arabidopsis. Here, we report the identification of a K-homology domain-containing protein named SHINY1 (SHI1) that interacts with CPL1 to modulate gene expression. The shi1 mutant was isolated from a forward genetic screening for mutants showing elevated expression of the luciferase reporter gene driven by a salt-inducible promoter. The shi1 mutant is more sensitive to cold treatment during vegetative growth and insensitive to abscisic acid in seed germination, resembling the phenotypes of shi4 that is allelic to the cpl1 mutant. Both SHI1 and SHI4/CPL1 are nuclear-localized proteins. SHI1 interacts with SHI4/CPL1 in vitro and in vivo. Loss-of-function mutations in shi1 and shi4 resulted in similar changes in the expression of some stress-inducible genes. Moreover, both shi1 and shi4 mutants display higher mRNA capping efficiency and altered polyadenylation site selection for some of the stress-inducible genes, when compared with wild type. We propose that the SHI1-SHI4/CPL1 complex inhibits transcription by preventing mRNA capping and transition from transcription initiation to elongation.

RNA-binding protein regulates plant DNA methylation by controlling mRNA processing at the intronic heterochromatin-containing gene IBM1.

DNA methylation-dependent heterochromatin formation is a conserved mechanism of epigenetic silencing of transposons and other repeat elements in many higher eukaryotes. Genes adjacent to repetitive elements are often also subjected to this epigenetic silencing. Consequently, plants have evolved antisilencing mechanisms such as active DNA demethylation mediated by the REPRESSOR OF SILENCING 1 (ROS1) family of 5-methylcytosine DNA glycosylases to protect these genes from silencing. Some transposons and other repeat elements have found residence in the introns of genes. It is unclear how these intronic repeat elements-containing genes are regulated. We report here the identification of ANTI-SILENCING 1 (ASI1), a bromo-adjacent homology domain and RNA recognition motif-containing protein, from a forward genetic screen for cellular antisilencing factors in Arabidopsis thaliana. ASI1 is required to prevent promoter DNA hypermethylation and transcriptional silencing of some transgenes. Genome-wide DNA methylation analysis reveals that ASI1 has a similar role to that of the histone H3K9 demethylase INCREASE IN BONSAI METHYLATION 1 (IBM1) in preventing CHG methylation in the bodies of thousands of genes. We found that ASI1 is an RNA-binding protein and ensures the proper expression of IBM1 full-length transcript by associating with an intronic heterochromatic repeat element of IBM1. Through mRNA sequencing, we identified many genes containing intronic transposon elements that require ASI1 for proper expression. Our results suggest that ASI1 associates with intronic heterochromatin and binds the gene transcripts to promote their 3' distal polyadenylation. The study thus reveals a unique mechanism by which higher eukaryotes deal with the collateral effect of silencing intronic repeat elements.

Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis.

MicroRNAs (miRNAs) are important for plant development and stress responses. However, factors regulating miRNA metabolism are not completely understood. SICKLE (SIC), a proline-rich protein critical for development and abiotic stress tolerance of Arabidopsis, was identified in this study. Loss-of-function sic-1 mutant plants exhibited a serrated, sickle-like leaf margin, reduced height, delayed flowering, and abnormal inflorescence phyllotaxy, which are common characteristics of mutants involved in miRNA biogenesis. The sic-1 mutant plants accumulated lower levels of a subset of miRNAs and transacting siRNAs but higher levels of corresponding primary miRNAs than the WT. The SIC protein colocalizes with the miRNA biogenesis component HYL1 in distinct subnuclear bodies. sic-1 mutant plants also accumulated higher levels of introns from hundreds of loci. In addition, sic-1 mutant plants are hypersensitive to chilling and salt stresses. These results suggest that SIC is a unique factor required for the biogenesis of some miRNAs and degradation of some spliced introns and important for plant development and abiotic stress responses.

Regulated AtHKT1 gene expression by a distal enhancer element and DNA methylation in the promoter plays an important role in salt tolerance.

Through sos3 (salt overly sensitive 3) suppressor screening, two allelic suppressor mutants that are weak alleles of the strong sos3 suppressor sos3hkt1-1 were recovered. Molecular characterization identified T-DNA insertions in the distal promoter region of the Arabidopsis thaliana HKT1 (AtHKT1, At4g10310) in these two weak sos3 suppressors, which results in physical separation of a tandem repeat from the proximal region of the AtHKT1 promoter. The tandem repeat is approximately 3.9 kb upstream of the ATG start codon and functions as an enhancer element to promote reporter gene expression. A putative small RNA target region about 2.6 kb upstream of the ATG start codon is heavily methylated. CHG and CHH methylation but not CG methylation is significantly reduced in the small RNA biogenesis mutant rdr2, indicating that non-CG methylation in this region is mediated by small RNAs. Analysis of AtHKT1 expression in rdr2 suggests that non-CG methylation in the putative small RNA target region represses AtHKT1 expression in shoots. The DNA methylation-deficient mutant met1-3 has nearly complete loss of total cytosine methylation in the putative small RNA target region and is hypersensitive to salt stress. The putative small RNA target region and the tandem repeat are essential for maintaining AtHKT1 expression patterns crucial for salt tolerance.

The role of promoter cis-element, mRNA capping, and ROS in the repression and salt-inducible expression of AtSOT12 in Arabidopsis.

Inducible gene expression is a gene regulatory mechanism central to plant response to environmental cues. The inducible genes are often repressed under normal growth conditions while their expression levels are significantly elevated by conditions such as abiotic stresses. Induction of gene expression requires both cis-acting DNA elements and trans-acting proteins that are modulated through signal transduction pathways. Here we report several molecular events that affect salt induced expression of the Arabidopsis AtSOT12 gene. Promoter deletion analysis revealed that DNA elements residing in the 5' UTR are required for the salt induced expression of AtSOT12. Cytosine methylation in the promoter was low and salt stress slightly increased the DNA methylation level, suggesting that DNA methylation may not contribute to AtSOT12 gene repression. Co-transcriptional processing of AtSOT12 mRNA including capping and polyadenylation site selection was also affected by salt stress. The percentage of capped mRNA increased by salt treatment, and the polyadenylation sites were significantly different before and after exposure to salt stress. The expression level of AtSOT12 under normal growth conditions was markedly higher in the oxi1 mutant defective of reactive oxygen species (ROS) signaling than in the wild type. Moreover, AtSOT12 transcript level was elevated by treatments with DPI and DMTU, two chemicals preventing ROS accumulation. These results suggest that repression of AtSOT12 expression may require physiological level of ROS and ROS signaling.

Protocol: a beginner's guide to the analysis of RNA-directed DNA methylation in plants.

BACKGROUND: DNA methylation is a conserved epigenetic mark that controls genome stability, development and environmental responses in many eukaryotes. DNA methylation can be guided by non-coding RNAs that include small interfering RNAs and scaffold RNAs. Although measurement of DNA methylation and regulatory non-coding RNAs is desirable for many biologists who are interested in exploring epigenetic regulation in their areas, conventional methods have limitations and are technically challenging. For instance, traditional siRNA detection through RNA hybridization requires relatively large amount of small RNAs and involves radioactive isotopes. An alternative approach is RT-qPCR that employs stem loop primers during reverse transcription; however, it requires a prerequisite that the exact sequences of siRNAs should be known. RESULTS: By using the model organism Arabidopsis thaliana, we developed an easy-to-follow, integrative procedure for time-efficient, quantitative measurement of DNA methylation, small interfering RNAs, and scaffold RNAs. Starting with simplified nucleic acid manipulation, we examined DNA methylation levels by using Chop PCR (methylation-sensitive enzyme digestion followed by PCR), which allowed for fast screening for DNA methylation mutants without the need of transgenic reporters. We deployed a simple bioinformatics method for mining published small RNA databases, in order to obtain the nucleotide (nt) sequences of individual 24nt siRNAs within the regions of interest. The protocol of commercial TaqMan Small RNA Assay was subsequently optimized for reliable quantitative detection of individual siRNAs. We used nested qPCR to quantify scaffold RNAs that are of low abundance and without Poly-A tails. In addition, nuclei fraction enables separation of chromatin-associated scaffold RNAs from their cognate non-scaffold transcripts that have been released from chromatin. CONCLUSIONS: We have developed a procedure for quantitative investigations on nucleic acids that are core components of RNA-directed DNA methylation. Our results not only demonstrated the efficacy of this procedure, but also provide lists of methylation-sensitive restriction enzymes, novel DNA methylation marker loci, and related siRNA sequences, all of which can be valuable for future epigenetic studies. Importantly, step-by-step protocols are provided in details such that the approaches can be easily followed by biologists with little experience in epigenetics.

Chemical probes in plant epigenetics studies.

Transcription potential is determined by the accessibility of DNA sequences within the context of chromatin, which is coordinately controlled by various epigenetic modifications. Chemical inhibition of epigenetic regulators provides a quick and effective approach to investigate the roles of epigenetic modifications in controlling many biological processes, especially for species in which genetic information is limited. This mini-review provides a brief overview of epigenetic regulators in the model organism Arabidopsis thaliana and summarizes compounds that have been applied in plant epigenetics studies, with highlights in the applications of these chemical probes in mechanistic and functional investigations.