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The functional structure of all biologically active molecules is dependent on intra- and inter-molecular interactions. This is especially evident for RNA molecules whose functionality, maturation, and regulation requires formation of correct secondary structure through encoded base-pairing interactions. Unfortunately, intra- and inter-molecular base-pairing information is lacking for most RNAs. Here, we use high-throughput sequencing to interrogate all base-paired RNA in Arabidopsis thaliana, and identify ~200 new small (sm)RNA-producing substrates of RNA-DEPENDENT RNA POLYMERASE 6. Our comprehensive analysis of paired RNAs reveals conserved functionality within introns and both 5’ and 3’ untranslated regions (UTRs) of mRNAs, as well as a novel population of functional RNAs, many of which are the precursors of smRNAs. Finally, we identify intra-molecular base-pairing interactions to produce a genome-wide collection of RNA secondary structure models. These findings highlight the importance of base-paired RNAs in eukaryotes, and present an approach that should be widely applicable for the analysis of this key structural feature of RNA. Double-stranded (dsRNA) specific RNA sequencing (dsRNA-seq) in the unopened flowerbuds of wild-type col-0 plants and rdr6 mutant plants, including 2 samples of col-0 dsRNA (1X- or 2X- Ribominus-treated samples) and 1 sample of rdr6 dsRNA. Corresponding two smRNA libraries (smRNA-seq) of both wild-type col-0 plants and rdr6 mutant plants of the same tissue are also presented.

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