Expression Landscape of circRNAs in Arabidopsis thaliana Seedlings and Adult Tissues

Abstract

RNA-seq is currently the only method that can provide a comprehensive landscape of circular RNA (circRNAs) in the whole organism and its particular organs. Recent years have brought an increasing number of RNA-seq-based reports on plant circRNAs. Notably, the picture they revealed is questionable and depends on the applied circRNA identification and quantification techniques. In consequence, little is known about the biogenesis and functions of circRNAs in plants. In this work, we tested two experimental and six bioinformatics procedures of circRNA analysis to determine the optimal approach for studying the profiles of circRNAs in Arabidopsis thaliana. Then using the optimized strategy, we determined the accumulation of circular and corresponding linear transcripts in plant seedlings and organs. We observed that only a small fraction of circRNAs was reproducibly generated. Among them, two groups of circRNAs were discovered: ubiquitous and organ-specific. The highest number of circRNAs with significantly increased accumulation in comparison to other organs/seedlings was found in roots. The circRNAs in seedlings, leaves and flowers originated mainly from genes involved in photosynthesis and the response to stimulus. The levels of circular and linear transcripts were not correlated. Although RNase R treatment enriches the analyzed RNA samples in circular transcripts, it may also have a negative impact on the stability of some of the circRNAs. We also showed that the normalization of NGS data by the library size is not proper for circRNAs quantification. Alternatively, we proposed four other normalization types whose accuracy was confirmed by ddPCR. Moreover, we provided a comprehensive characterization of circRNAs in A. thaliana organs and in seedlings. Our analyses revealed that plant circRNAs are formed in both stochastic and controlled processes. The latter are less frequent and likely engage circRNA-specific mechanisms. Only a few circRNAs were organ-specific. The lack of correlation between the accumulation of linear and circular transcripts indicated that their biogenesis depends on different mechanisms.

Comparative analysis of circular RNA enrichment methods

“…This might be explained by the fact that polyA+RNase R+ rRNA – treatment method can more efficient at removing linear RNAs, thus leading to an increased the resolution of the analysis. This result has also been confirmed in Philips A, et al [ 36 ] study. Nevertheless, the number of identified circRNA candidates by using polyA+RNase R and rRNA – +polyA+RNase R enrichment methods were not difference with compared to rRNA depletion group.…”

Deregulation of ncRNA in Neurodegenerative Disease: Focus on circRNA, lncRNA and miRNA in Amyotrophic Lateral Sclerosis

“…RNA sequencing technology is currently the only method capable of providing a complete landscape of circRNAs throughout the body and in specific tissue areas ( Philips et al, 2020 ). In particular, ribo-minus RNA-seq, has allowed identifying new change in circRNAs expression and also investigate the roles of these circRNAs in the condition of interest ( Cooper et al, 2018 ).…”

At-C-RNA database, a one-stop source for information on circRNAs inArabidopsis thalianain a unified format

“…The following information is available for each circRNA: (i) the study in which the datasets were generated, (ii) plant line and organ in which circRNAs were identified, (iii) the average score, computed for all circRNAs according to the same procedure, (iv) the individual component scores and (v) information regarding whether a circRNA was confirmed with RNase R experiments. It is worth mentioning that we introduced a novel ‘reproducibility’ criterion in At-C-RNA, as in our previous studies, and we showed that most circRNAs in A. thaliana are produced spontaneously and thus possibly carry no biological function ( 11 ). It is important to highlight the reproducible circRNAs, which may have functional potential and thus are especially important to the wide range of studies.…”

“…Moreover, we compared the sources used by other plant circRNA databases and we chose those that met our criteria. In total, we utilized third-party data from eight studies (100 SRA files) and two datasets (110 SRA files) from our previous studies ( 11 , 12 ). All analyzed datasets are presented in Table 1 .…”

“…Given the above, comprehensive comparative analyses of circRNAs in this model species have been significantly hampered. To change this situation, we reanalyzed RNA-seq raw data available in the public domain with our protocol for circRNA identification in plants ( 11 ) and developed At-C-RNA database to integrate and standardize the available circRNA data (see Figure 1 ).…”