Distinct characteristics of Arabidopsis thaliana circular RNAs

In all living organisms, regulation of gene expression is fundamental for survival and adaptation. Gene expression can be modulated at various steps, including at the level of RNA processing. During the last few years, the importance of alternative splicing of mRNAs in controlling plant development and stress responses were emerged and highlighted its importance. Recently, an other type of alternative splicing has been reported which leads to the generation of circular RNAs (circRNAs), a novel class on endogenous noncoding RNAs. Several functions of circular RNAs have been proven or proposed, including functioning as microRNA or RNA-binding protein decoys, playing regulatory roles in gene expression or affecting transcriptional control via special RNA-RNA interactions. Despite the widening knowledge of circRNAs and their functional aspects in the animal kingdom, relatively little is known about circRNAs in plants. In order to detect and classify circRNAs in Arabidopsis thaliana, we created a workflow that includes generation of Illumina libraries enriched for circRNAs and a comparison of biocomputational tools developed for detecting endogenous circular RNAs in other species. With the power of high-throughput sequencing and evaluation of algorithms, high-fidelity candidates were subjected for an analysis of their functional role in plant development and stress-related responses, especially regarding the role of splicing, including alternative splicing events, splice site preference and strength variances and transcript composition and to comprehend the role of RNA processing in stress response. Here we present an approach combining bioinformatic tools and molecular techniques to investigate the adaptability of detection methods of circRNAs from other species to plant circular RNAs, and based on our high-fidelity results identify and understand the characteristics of circRNAs in Arabidopsis thaliana. Overall design: In order to identify and analyze circRNAs in plants, Arabidopsis thaliana liquid culture seedlings were subjected for NGS RNA library preparation. Plant material was treated with with the hormones salicylic acid and jasmonic acid, which mimic biotic stress conditions. Rnase R treatment, ribosomal and poly-A depletion were applied to enhance the signal. Libraries were done in triplicates.

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