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Intronic RNAs have been overlooked for a long time: They are functional, but treated as "junk." In this work, we designed a new sequencing strategy to investigate intronic RNAs. By using intron-capture RNA-seq, we systematically analyzed the intronic RNAs in Arabidopsis by zooming into the intronic regions an order of magnitude deeper than in previous work. Our key findings include: (1) Intron-capture RNA-seq is a much more efficient approach to analyze intronic RNAs than total RNA-seq and mRNA-seq. (2) We identified three types of intronic RNAs, and found that the GC pattern differs significantly between the introns with and without intronic RNAs. (3) We detected many hidden elements in introns, including circular RNAs, splice junctions, and transcripts that have previously been overlooked. (4) The expression of these intronic RNAs varies during the time course of pathogen infection, which indicates that an unknown mechanism may exist for these RNAs. (5) We also demonstrated that most of intronic RNAs are detectable in both Arabidopsis and rice, suggesting that these non-coding molecules are conserved. Taken together, this work proposes an efficient strategy to analyze intronic RNAs, and provides an unprecedented view of this essential component in biological pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Circular RNAs (circRNAs) are a group of covalently closed RNAs, and their biological function is largely unknown. In this study, we focused on circRNAs that are generated from exon back-splicing (exonic circRNAs). The linear RNA counterparts encode functional proteins so that we can compare and investigate the relationship between circular and linear RNAs. We compared circRNA expression profiles between untreated and Pseudomonas syringae -infected Arabidopsis and identified and experimentally validated differentially expressed exonic circRNAs by multiple approaches. We found that exonic circRNAs are preferentially enriched in biological processes that associate with biotic and abiotic stress responses. We discovered that circR194 and circR4022 are involved in plant response against P. syringae infection, whereas circR11208 is involved in response against Botrytis cinerea infection. Intriguingly, our results indicate that these exonic circRNAs function synergistically with their corresponding linear RNAs. Furthermore, circR4022 and circR11208 also play substantial roles in Arabidopsis tolerance to salt stress. This study extends our understanding of the molecular functions of plant circRNAs.

Circular RNAs are widely existing in eukaryotes. However, there is as yet no tissue-specific Arabidopsis circular RNA database, which hinders the study of circular RNA in plants. Here, we used 622 Arabidopsis RNA sequencing data sets from 87 independent studies hosted at NCBI SRA and developed AtCircDB to systematically identify, store and retrieve circular RNAs. By analyzing back-splicing sites, we characterized 84 685 circular RNAs, 30 648 tissue-specific circular RNAs and 3486 microRNA-circular RNA interactions. In addition, we used a metric (detection score) to measure the detection ability of the circular RNAs using a big-data approach. By experimental validation, we demonstrate that this metric improves the accuracy of the detection algorithm. We also defined the regions hosting enriched circular RNAs as super circular RNA regions. The results suggest that these regions are highly related to alternative splicing and chloroplast. Finally, we developed a comprehensive tissue-specific database (AtCircDB) to help the community store, retrieve, visualize and download Arabidopsis circular RNAs. This database will greatly expand our understanding of circular RNAs and their related regulatory networks. AtCircDB is freely available at http://genome.sdau.edu.cn/circRNA.

Alternative splicing is an essential biological process to generate proteome diversity and phenotypic complexity. Recent improvements in RNA sequencing accuracy and computational algorithms have provided unprecedented opportunities to examine the expression levels of Arabidopsis (Arabidopsis thaliana) transcripts. In this article, we analyzed 61 RNA sequencing samples from 10 totally independent studies of Arabidopsis and calculated the transcript expression levels in different tissues, treatments, developmental stages, and varieties. These data provide a comprehensive profile of Arabidopsis transcripts with single-base resolution. We quantified the expression levels of 40,745 transcripts annotated in The Arabidopsis Information Resource 10, comprising 73% common transcripts, 15% rare transcripts, and 12% nondetectable transcripts. In addition, we investigated diverse common transcripts in detail, including ubiquitous transcripts, dominant/subordinate transcripts, and switch transcripts, in terms of their expression and transcript ratio. Interestingly, alternative splicing was the highly enriched function for the genes related to dominant/subordinate transcripts and switch transcripts. In addition, motif analysis revealed that TC motifs were enriched in dominant transcripts but not in subordinate transcripts. These motifs were found to have a strong relationship with transcription factor activity. Our results shed light on the complexity of alternative splicing and the diversity of the contributing factors., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

NAGNAG alternative splicing is one type of alternative splicing in mammals and plants. There are two opposite arguments regarding the mechanism of this NAGNAG event, i.e. whether splice variation is controllable by the cell or is just biological noise. In this paper, we systematically investigated NAGNAG acceptors in Arabidopsis thaliana using both cDNA/EST and RNA-Seq data. We identified 9,473 NAGNAG motifs, including 529 cDNA/EST-confirmed NAGNAG acceptors. A nomenclature tree for this type of alternative splicing was defined based on the cDNA/EST validation, location in the exon, sequence and expression level. Low expression of some NAGNAG motifs was observed in various tissues or pathogen-infected samples, indicating the existence of background splicing. Tissue-specific or treatment-specific differences in the dynamic profiles suggest that some NAGNAG acceptors are highly regulated.