Systematic identification and characterization of circular RNAs involved in flag leaf senescence of rice

Xiaoping Huang,Xuanzhi Liu,Hongyu Zhang,Weigang Kuang,Yingjin Huang,Qiang Wang,Rong Guo,Haiyan Song,Zhaohai Wang,Jianglin Liao

doi:10.1007/s00425-020-03544-6

Abstract

Main conclusionCircular RNAs (circRNAs) identification, expression profiles, and construction of circRNA-parental gene relationships and circRNA-miRNA-mRNA ceRNA networks indicate that circRNAs are involved in flag leaf senescence of rice.Circular RNAs (circRNAs) are a class of 3′-5′ head-to-tail covalently closed non-coding RNAs which have been proved to play important roles in various biological processes. However, no systematic identification of circRNAs associated with leaf senescence in rice has been studied. In this study, a genome-wide high-throughput sequencing analysis was performed using rice flag leaves developing from normal to senescence. Here, a total of 6612 circRNAs were identified, among which, 113 circRNAs were differentially expressed (DE) during the leaf senescence process. Moreover, 4601 (69.59%) circRNAs were derived from the exons or introns of their parental genes, while 2110 (71%) of the parental genes produced only one circRNA. The sequence alignment analysis showed that hundreds of rice circRNAs were conserved among different plant species. Gene Ontology (GO) enrichment analysis revealed that parental genes of DE circRNAs were enriched in many biological processes closely related to leaf senescence. Through weighted gene co-expression network analysis (WGCNA), six continuously down-expressed circRNAs, 18 continuously up-expressed circRNAs and 15 turn-point high-expressed circRNAs were considered to be highly associated with leaf senescence. Additionally, a total of 17 senescence-associated circRNAs were predicted to have parental genes, in which, regulations of three circRNAs to their parental genes were validated by qRT-PCR. The competing endogenous RNA (ceRNA) networks were also constructed. And a total of 11 senescence-associated circRNAs were predicted to act as miRNA sponges to regulate mRNAs, in which, regulation of two circRNAs to eight mRNAs was validated by qRT-PCR. It is discussed that senescence-associated circRNAs were involved in flag leaf senescence probably through mediating their parental genes and ceRNA networks, to participate in several well-studied senescence-associated processes, mainly including the processes of transcription, translation, and posttranslational modification (especially protein glycosylation), oxidation–reduction process, involvement of senescence-associated genes, hormone signaling pathway, proteolysis, and DNA damage repair. This study not only showed the systematic identification of circRNAs involved in leaf senescence of rice, but also laid a foundation for functional research on candidate circRNAs.

Highlights

With rapid development of high-throughput sequencing technology, a large number of noncoding RNAs, such as long noncoding RNAs and microRNAs, were identified and confirmed as the crucial regulator for gene expression and biological function in different biological processes (Li et al 2015; Shafiq et al 2016)
The transgenic rice lines overexpressing circR5g05160 could lead to enhanced disease resistance to Magnaporthe oryzae (Fan et al 2020). These results showed the essential functions of circRNAs in various biological processes, but not in leaf senescence of rice
To systematically identify cirRNAs involved in leaf senescence of rice, a total of 15 RNA libraries from rice flag leaves at booting stage (FL1, before flag leaf senescence), flowering stage (FL2, before flag leaf senescence), filling stage (FL3, early stage of flag leaf senescence, or turnpoint stage of flag leaf senescence), milking maturity stage (FL4, middle stage of flag leaf senescence) and dough stage (FL5, late stage of flag leaf senescence) were individually constructed, with three biological replicates for each stage

Summary

Introduction

With rapid development of high-throughput sequencing technology, a large number of noncoding RNAs, such as long noncoding RNAs and microRNAs, were identified and confirmed as the crucial regulator for gene expression and biological function in different biological processes (Li et al 2015; Shafiq et al 2016). Different from the traditional linear RNAs terminated with 3′ tails and 5′ caps, circRNAs could form covalently closed ring structures by back-spliced circularization without polyadenylated tails and 5′–3′ polarities (Chen 2016). In the past period of time, circRNAs had been perceived as aberrant splicing and potential functions of circRNAs had not been uncovered (Salzman 2016). Generated mechanism remains largely unclear, it is certain that circRNAs can be classified into exonic, intronic and intergenic circRNAs (Chen 2016)

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Results

Conclusion