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Report COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures Graphical Abstract Highlights d Long non-coding (lnc)RNA, COOLAIR, has a complex structure with multi-helix junctions d The distally polyadenylated lncRNA contains two right-hand turn (r-turn) motifs d A phenotypically signi?cant lncRNA isoform has an altered secondary structure d The COOLAIR secondary structure has been evolutionarily conserved across species Authors Emily J.

Hawkes, Scott P. Hennelly, Irina V. Novikova, Judith A. Irwin, Caroline Dean, Karissa Y. Sanbonmatsu Correspondence [email protected] In Brief Hawkes et al. chemically probed the Arabidopsis thaliana long non-coding RNA, COOLAIR, to reveal its complex secondary structure. The evolutionary conservation of this secondary structure across Brassicaceae species, despite low sequence similarity, indicates a functional role. This is supported by variation in secondary structure in a phenotypically signi?cant COOLAIR splicing isoform. Hawkes et al., 2016, Cell Reports 16, 3087C3096 September 20,

2016 http://dx.doi.org/10.1016/j.celrep.2016.08.045 Cell Reports Report COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures Emily J. Hawkes,1,5 Scott P. Hennelly,2,3,5 Irina V. Novikova,2,4 Judith A. Irwin,1 Caroline Dean,1 and Karissa Y. Sanbonmatsu2,3,6,7,* 1John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK 2Los Alamos National Laboratory, Los Alamos, NM 87545, USA 3New Mexico Consortium, Los Alamos, NM 87544, USA 4Paci?c Northwest National Laboratory, Environmental Molecular Sciences Laboratory, Richland, WA 99354, USA 5Co-?rst author 6Lead Contact 7Twitter: @karissascience *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2016.08.045 SUMMARY There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. We investigated anti- sense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their sec- ondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally pol- yadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP de?ning a functionally distinct A. thaliana FLC haplo- type. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and clon- ing. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junc- tions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription. INTRODUCTION Long non-coding RNAs (lncRNAs) have emerged as potentially important players in the epigenetic regulation of development and disease in many organisms. These RNAs are typically 1C10 kb in length, polyadenylated, capped, and alternatively spliced (Guttman and Rinn, 2012;

Ulitsky and Bartel, 2013). They can be cis- or trans-acting and have been associated with gene regulation in mechanisms including chromatin scaf- folding, Polycomb complex (PRC2) recruitment to chromatin, mRNA decay, and decoys for proteins and microRNAs (miRNAs). Speci?c functional studies have shown lncRNAs to be essential for Xist regulation, paraspeckle formation, lineage commitment, stem cell development, cancer-associated ef- fects, coactivation of hormone response, and brain development (Klattenhoff et al., 2013;