In vivo single-molecule analysis reveals COOLAIR RNA structural diversity

Cellular RNAs are heterogeneous with respect to their alternative processing and secondary structures, but the functional importance of this complexity is still poorly understood. A set of alternatively processed antisense non-coding transcripts, which are collectively called COOLAIR, are generated at the Arabidopsis floral-repressor locus FLOWERING LOCUS C (FLC)(1). Different isoforms of COOLAIR influence FLC transcriptional output in warm and cold conditions(2-7). Here, to further investigate the function of COOLAIR, we developed an RNA structure-profiling method to determine the in vivo structure of single RNA molecules rather than the RNA population average. This revealed that individual isoforms ofthe COOLA/Rtranscript adopt multiple structures with different conformational dynamics. The major distally polyadenylated COOLAIR isoform in warm conditions adopts three predominant structural conformations, the proportions and conformations of which change after cold exposure. An alternatively spliced, strongly cold-upregulated distal COOLAIR isoform(6) shows high structural diversity, in contrast to proximally polyadenylated COOLAIR. A hyper-variable COOLAIR structural element was identified that was complementary to the FLCtranscription start site. Mutations altering the structure of this region changed FLC expression and flowering time, consistent with an important regulatory role of the COOLAIR structure in FLC transcription. Our work demonstrates that isoforms of non-coding RNA transcripts adopt multiple distinct and functionally relevant structural conformations, which change in abundance and shape in response to external conditions.

Automated summary

Cellular RNAs are heterogeneous in terms of their alternative processing and secondary structures, and the functional importance of this complexity is still not well understood. COOLAIR, a group of alternatively processed antisense non-coding transcripts generated at the Arabidopsis FLC locus, influences FLC transcriptional output under different temperature conditions. The study reveals that different isoforms of COOLAIR adopt multiple distinct structural conformations and change in response to external conditions.