R-Loop Stabilization Represses Antisense Transcription at the Arabidopsis FLC Locus

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. A homeodomain protein limits expression of antisense transcripts involved in flowering time control. Roles for long noncoding RNAs (lncRNAs) in gene expression are emerging, but regulation of the lncRNA itself is poorly understood. We have identified a homeodomain protein, AtNDX, that regulates COOLAIR, a set of antisense transcripts originating from the 3′ end of Arabidopsis FLOWERING LOCUS C (FLC). AtNDX associates with single-stranded DNA rather than double-stranded DNA non–sequence-specifically in vitro, and localizes to a heterochromatic region in the COOLAIR promoter in vivo. Single-stranded DNA was detected in vivo as part of an RNA-DNA hybrid, or R-loop, that covers the COOLAIR promoter. R-loop stabilization mediated by AtNDX inhibits COOLAIR transcription, which in turn modifies FLC expression. Differential stabilization of R-loops could be a general mechanism influencing gene expression in many organisms.

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