Regulatory impact of RNA secondary structure across the Arabidopsis thaliana transcriptome

The secondary structure of an RNA molecule plays an integral role in its maturation, regulation, processing, and functionality. However, the global influence of this feature on plant gene expression is still for the most part unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach in conjunction with transcriptome-wide sequencing of polyA+-selected (RNA-seq), small (smRNA-seq), and ribosome-bound (ribo-seq) RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in Arabidopsis. From this analysis, we find that highly unpaired and paired RNAs are strongly correlated with euchromatic and heterochromatic epigenetic histone modifications, respectively, providing further evidence that secondary structure is necessary for RNA-mediated posttranscriptional regulatory pathways. Additionally, we uncover key structural patterns across protein-coding transcripts that indicate RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in this model plant. We also reveal that RNA folding is significantly anti-correlated with overall transcript abundance, which is likely due to the increased propensity of highly structured mRNAs to be degraded and/or processed into smRNAs. Finally, we find that secondary structure affects mRNA translation, suggesting that this feature regulates plant gene expression at multiple levels. Overall, our findings provide the first global assessment of RNA folding and its significant regulatory effects in a plant transcriptome. Overall design: Single-stranded RNA sequencing (ssRNA-seq) and ribosome-bound RNA sequencing (ribo-seq) in immature buds. A single replicate of each library.