Significance: Long noncoding RNAs (lncRNAs) play important roles in chromatin regulation in higher eukaryotes. Studies analysing how lncRNAs influence Polycomb silencing suggested they specifically bind and recruit Polycomb Repressive Complex 2 (PRC2) to defined targets. However, the promiscuous binding of PRC2 to RNA has raised questions as to the mechanism by which lncRNA contributes to Polycomb silencing. We investigate the function of a set of cold-induced antisense transcripts in the Polycomb-dependent epigenetic silencing of a floral repressor gene in Arabidopsis . Through analysis of a transgene, in which antisense transcripts are no longer cold-induced, we show that these antisense transcripts are required for coordinated switching of opposing chromatin states. This mechanism links transcriptional shutdown by cold to long-term epigenetic silencing.

Making Antisense of Flowering: The recent discovery of biological roles for long noncoding RNAs raises important questions as to how they are themselves regulated. Sun et al. (p. 619 ) adopted a genetic approach to identify regulators of COOLAIR —a set of antisense transcripts from the locus encoding the major Arabidopsis floral repressor, FLC . Analysis of a mutant misregulating COOLAIR revealed a homeodomain protein that repressed COOLAIR expression via an R-loop covering the COOLAIR promoter. Thus, R-loop stabilization is an integral part of COOLAIR regulation, FLC expression, and flowering time.

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RNA silencing is conserved in a broad range of eukaryotes and includes the phenomena of RNA interference in animals and posttranscriptional gene silencing (PTGS) in plants. In plants, PTGS acts as an antiviral system; a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with positive-strand RNA viruses and provide specificity to this RNA-mediated defense. We present here the results of a survey of virus-specific siRNAs characterized by a sequence analysis of siRNAs from plants infected withCymbidium ringspot tombusvirus(CymRSV). CymRSV siRNA sequences have a nonrandom distribution along the length of the viral genome, suggesting that there are hot spots for virus-derived siRNA generation. CymRSV siRNAs bound to the CymRSV p19 suppressor protein have the same asymmetry in strand polarity as the sequenced siRNAs and are imperfect double-stranded RNA duplexes. Moreover, an analysis of siRNAs derived from two other nonrelated positive-strand RNA viruses showed that they displayed the same asymmetry as CymRSV siRNAs. Finally, we show thatTobacco mosaic virus(TMV) carrying a short inverted repeat of the phytoene desaturase (PDS) gene triggered more accumulation ofPDSsiRNAs than the corresponding antisensePDSsequence. Taken together, these results suggest that virus-derived siRNAs originate predominantly by direct DICER cleavage of imperfect duplexes in the most folded regions of the positive strand of the viral RNA.

RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double-stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein–protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA-induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre-programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high-molecular-weight complex, suggesting the existence of a multi-protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA–RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co-expressed AGO1 in both the presence and absence of P0.