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.DNA methylome | ChIP | gene expression I n higher eukaryotes including plants, DNA methylation is an important epigenetic mark that silences transposons and other repetitive elements. In Arabidopsis thaliana, DOMAINS REARRANGED METHYLASE 2 (DRM2) catalyzes de novo DNA methylation in all cytosine contexts including CG, CHG, and CHH (H represents A, T, or G) (1), through the RNAdirected DNA methylation pathway (RdDM) (2-9). At the same time, preexisting DNA methylation in plants can be pruned by enzymatic excision that is catalyzed by a subfamily of bifunctional DNA glycosylases represented by REPRESSOR OF SI-LENCING 1 (ROS1) and DEMETER (DME) (10)(11)(12)(13)(14). Following the enzymatic removal of 5-methylcytosine, the resultant singlenucleotide gap is filled with an unmodified cytosine through the DNA base excision repair pathway (15,16).Cytosine methylation and demethylation are both tightly linked with histone modifications. Increased DNA methylation was observed in an A. thaliana mutant defective in INCREASED DNA METHYLATION 1 (IDM1), an acetyltransferase that catalyzes acetylation of histone H3 lysine 18 (H3K18) and lysine 23 (H3K23) necessary for subsequent DNA demethylation and prevention of transcriptional silencing (17). In A...

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.cold stress | hydroxyproline-rich glycoprotein | intron decay | mRNA stability M icroRNAs (miRNAs) are a class of endogenous small RNAs that function in gene regulation by guiding mRNA cleavage and translational repression and are critical for plant development and stress responses (1-9). The core components involved in miRNA biogenesis have been identified in plants. RNA polymerase II transcribes MIR genes; a 5′ 7-methyl guanosine cap and a 3′ poly(A) tail are added to produce primary miRNA (primiRNA) transcripts, which form imperfect stem-loop secondary structures by Watson-Crick base pairing between self-complementary foldback regions. In the Arabidopsis nucleus, the stemloop structure of the pri-miRNA is processed by the RNase III enzyme DICER-LIKE1 (DCL1) to produce a pre-miRNA, which is further processed to generate a 21-nt-long miRNA/miRNA* duplex. For accurate dicing, DCL1 requires the help of HYPO-NASTIC LEAVES1 (HYL1, a dsRNA-binding protein) and SERRATE (SE, a C2H2zinc-finger protein) (10). The HUA EN-HANCER 1 (HEN1) methyltransferase catalyzes 2'-O-methylation of the ribose sugar in the 3′ termini of miRNA/miRNA* duplexes (11). HASTY (HST), a homolog of mammalian EXPORTIN 5, helps export methylated miRNA/miRNA* duplexes from the nucleus to the cytosol (12). The mature miRNA is incorporated into ARGONAUTE1 (AGO1), forming an RNA-induced silencing complex, which scans for miRNA-complementary mRNAs and directs the cleavage or translational repression of the target mRNAs (1). miR173 and miRNA390 direct the biogenesis of transacting siRNAs (ta-siRNAs). Noncoding transcripts from TRANS-ACT-ING siRNA genes (TAS) are cleaved by the miRNA-containing AGO1/AGO7 complex (13, 14). The cleaved transcripts are converted into dsRNA by RDR6, and these dsRNAs are processed by DCL4 to yield ∼21-nt ta-siRNAs. Like miRNAs, tasiRNAs negatively regulate gene expression posttranscriptionally (15-18).N...

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.

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.

SUMMARY Rrp6-mediated nuclear RNA surveillance tunes eukaryotic transcriptomes through non-coding 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 non-coding 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 non-coding 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 non-coding RNAs in normal cells.