Transcription in eukaryotic genomes generates an extensive array of non-protein-coding RNA, the functional significance of which is mostly unknown. We are investigating the link between non-coding RNA and chromatin regulation through analysis of FLC - a regulator of flowering time in Arabidopsis and a target of several chromatin pathways. Here we use an unbiased strategy to characterize non-coding transcripts of FLC and show that sense/antisense transcript levels correlate in a range of mutants and treatments, but change independently in cold-treated plants. Prolonged cold epigenetically silences FLC in a Polycomb-mediated process called vernalization. Our data indicate that upregulation of long non-coding antisense transcripts covering the entire FLC locus may be part of the cold-sensing mechanism. Induction of these antisense transcripts occurs earlier than, and is independent of, other vernalization markers and coincides with a reduction in sense transcription. We show that addition of the FLC antisense promoter sequences to a reporter gene is sufficient to confer cold-induced silencing of the reporter. Our data indicate that cold-induced FLC antisense transcripts have an early role in the epigenetic silencing of FLC, acting to silence FLC transcription transiently. Recruitment of the Polycomb machinery then confers the epigenetic memory. Antisense transcription events originating from 3' ends of genes might be a general mechanism to regulate the corresponding sense transcription in a condition/stage-dependent manner.

Xie and colleagues previously isolated the Arabidopsis COI1 gene that is required for response to jasmonates (JAs), which regulate root growth, pollen fertility, wound healing, and defense against insects and pathogens. In this study, we demonstrate that COI1 associates physically with AtCUL1, AtRbx1, and either of the Arabidopsis Skp1-like proteins ASK1 or ASK2 to assemble ubiquitin-ligase complexes, which we have designated SCF COI1 . COI1 E22A , a single amino acid substitution in the F-box motif of COI1, abolishes the formation of the SCF COI1 complexes and results in loss of the JA response. AtRbx1 double-stranded RNA-mediated genetic interference reduces AtRbx1 expression and affects JAinducible gene expression. Furthermore, we show that the AtCUL1 component of SCF COI1 complexes is modified in planta, where mutations in AXR1 decrease the abundance of the modified AtCUL1 of SCF COI1 and lead to a reduction in JA response. Finally, we demonstrate that the axr1 and coi1 mutations display a synergistic genetic interaction in the double mutant. These results suggest that the COI1 -mediated JA response is dependent on the SCF COI1 complexes in Arabidopsis and that the AXR1 -dependent modification of the AtCUL1 subunit of SCF COI1 complexes is important for JA signaling.

Noncoding RNA is emerging as an important regulator of gene expression in many organisms. We are characterizing RNA-mediated chromatin silencing of the Arabidopsis major floral repressor gene, FLC. Through suppressor mutagenesis, we identify a requirement for CstF64 and CstF77, two conserved RNA 3'-end-processing factors, in FLC silencing. However, FLC sense transcript 3' processing is not affected in the mutants. Instead, CstF64 and CstF77 are required for 3' processing of FLC antisense transcripts. A specific RNA-binding protein directs their activity to a proximal antisense polyadenylation site. This targeted processing triggers localized histone demethylase activity and results in reduced FLC sense transcription. Targeted 3' processing of antisense transcripts may be a common mechanism triggering transcriptional silencing of the corresponding sense gene.

A repressor of the transition to flowering in Arabidopsis is the MADS box protein FLOWERING LOCUS C (FLC). FCA, an RNA-binding protein, and FY, a homolog of the yeast RNA 3' processing factor Pfs2p, downregulate FLC expression and therefore promote flowering. FCA/FY physically interact and alter polyadenylation/3' processing to negatively autoregulate FCA. Here, we show that FCA requires FLOWERING LOCUS D (FLD), a homolog of the human lysine-specific demethylase 1 (LSD1) for FLC downregulation. FCA also partially depends on DICER-LIKE 3, involved in chromatin silencing. fca mutations increased levels of unspliced sense FLC transcript, altered processing of antisense FLC transcripts, and increased H3K4 dimethylation in the central region of FLC. These data support a close association of FCA and FLD in mediating H3K4 demethylation and thus transcriptional silencing of FLC and reveal roles for antisense RNA processing and DCL3 function in this regulation.

SummaryAntisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.

An important component of cellular biochemistry is the concentration of proteins and nucleic acids in non-membranous compartments1,2. These biomolecular condensates are formed from processes including liquid-liquid phase separation (LLPS). The multivalent interactions necessary for LLPS have been studied extensively in vitro1,3. However, what regulates LLPS in vivo is still poorly understood. Here, we identify an in vivo regulator of LLPS through a genetic suppressor screen for loss of function of the Arabidopsis RNA-binding protein FCA. FCA contains prion-like domains that phase-separate in vitro, and exhibits behavior in vivo consistent with phase separation. The mutant screen identified a functional requirement for a coiled coil protein, FLL2, in FCA nuclear body formation. FCA reduces transcriptional read-through by promoting proximal polyadenylation at many sites in the Arabidopsis genome3,4. FLL2 was required to promote this proximal polyadenylation, but not binding of FCA to target RNA. Ectopic expression of FLL2 increased the size and number of FCA nuclear bodies. Crosslinking with formaldehyde captured in vivo interactions between FLL2, FCA and the polymerase and nuclease modules of the RNA 3’ end processing machinery. These 3’ RNA processing components were found to colocalize with FCA in the nuclear bodies in vivo. We conclude that FLL2 promotes liquid-liquid phase separation, important for dynamics of polyadenylation complexes at specific poly A sites. Our findings show that coiled coil proteins can promote LLPS, expanding our understanding of the principles governing the in vivo dynamics of liquid-like bodies.

The Arabidopsis thaliana CORONATINE INSENSITIVE1 (COI1) gene encodes an F-box protein to assemble SCF COI1 complexes essential for response to jasmonates (JAs), which are a family of plant signaling molecules required for many essential functions, including plant defense and reproduction. To better understand the molecular basis of JA action, we screened for suppressors of coi1 and isolated a coi1 suppressor1 (cos1) mutant. The cos1 mutation restores the coi1-related phenotypes, including defects in JA sensitivity, senescence, and plant defense responses. The COS1 gene was cloned through a map-based approach and found to encode lumazine synthase, a key component in the riboflavin pathway that is essential for diverse yet critical cellular processes. We demonstrated a novel function for the riboflavin pathway that acts downstream of COI1 in the JA signaling pathway and is required for suppression of the COI1-mediated root growth, senescence, and plant defense.

Small RNA-mediated chromatin silencing is well characterized for repeated sequences and transposons, but its role in regulating single-copy endogenous genes is unclear. We have identified two small RNAs (30 and 24 nucleotides) corresponding to the reverse strand 3 to the canonical poly(A) site of FLOWERING LOCUS C (FLC), an Arabidopsis gene encoding a repressor of flowering. Genome searches suggest that these RNAs originate from the FLC locus in a genomic region lacking repeats. The 24-nt small RNA, which is most abundant in developing fruits, is absent in mutants defective in RNA polymerase IVa, RNA-DEPENDENT RNA POLYMERASE 2, and DICER-LIKE 3, components required for RNAi-mediated chromatin silencing. The corresponding genomic region shows histone 3 lysine 9 dimethylation, which was reduced in a dcl2,3,4 triple mutant. Investigations into the origins of the small RNAs revealed a polymerase IVa-dependent spliced, antisense transcript covering the 3 FLC region. Mutation of this genomic region by T-DNA insertion led to FLC misexpression and delayed flowering, suggesting that RNAi-mediated chromatin modification is an important component of endogenous pathways that function to suppress FLC expression.flowering ͉ small interfering RNA ͉ antisense transcription T he role of the RNAi machinery in the epigenetic silencing of repeated sequences and transposons is well documented (1). Double-stranded RNA, generated by a variety of mechanisms including antisense transcription, transcription through inverted repeats, or RDR activity, is cleaved by DICER(s) (DCL) to produce short interfering (si)RNAs, which direct chromatinmodifying machinery to regions of the chromatin containing complementary DNA sequences. The siRNAs and chromatinmodification complexes are tethered to the silenced loci, maintaining continued production of siRNAs and the silenced state.In Arabidopsis, the production and function of siRNAs mediating the epigenetic silencing of repeated sequences require RDR2, DCL3, HUA ENHANCER 1 (HEN1), ARGONAUTE (AGO) 4 (2-4), and two functionally distinct forms of a plantspecific RNA polymerase IV complex, containing either polymerase IV largest subunit (NRPD) 1a or NRPD1b with NRPD2 (5-8). Several observations suggest that RNAi-mediated chromatin silencing could also be playing a major role in regulation of single-copy sequences. siRNAs complementary to both genic and intergenic sequences have been identified in Arabidopsis (9) and associated with AGO4 (10). In mammals, DICER has been shown to play a role in the turnover of intergenic transcripts and the corresponding chromatin modification in the human ␤-globin cluster (11), and antisense/intergenic transcripts have been found to be extensive in the Arabidopsis, Drosophila, and mammalian genomes (12, 13). Cis-antisense transcripts have also been shown to trigger posttranscriptional silencing, conferring salt tolerance and plant immunity in Arabidopsis (14, 15).Considerable activity has focused on dissecting the epigenetic basis of the regulation of FLC, a MADS box tr...