Identification, biogenesis, function, and mechanism of action of circular RNAs in plants

Abstract

Circular RNAs (circRNAs) are a class of single-stranded, closed RNA molecules with unique functions that are ubiquitously expressed in all eukaryotes. The biogenesis of circRNAs is regulated by specific cis-acting elements and trans-acting factors in humans and animals. circRNAs mainly exert their biological functions by acting as microRNA sponges, forming R-loops, interacting with RNA-binding proteins, or being translated into polypeptides or proteins in human and animal cells. Genome-wide identification of circRNAs has been performed in multiple plant species, and the results suggest that circRNAs are abundant and ubiquitously expressed in plants. There is emerging compelling evidence to suggest that circRNAs play essential roles during plant growth and development as well as in the responses to biotic and abiotic stress. However, compared with recent advances in human and animal systems, the roles of most circRNAs in plants are unclear at present. Here we review the identification, biogenesis, function, and mechanism of action of plant circRNAs, which will provide a fundamental understanding of the characteristics and complexity of circRNAs in plants.

Keywords: biogenesis; function; identification; mechanism; plant circRNA.

Figure 1

Identification of circRNAs in plants (2014 to May 2022). The numbers represent the total number of circRNAs identified in each study (Bai et al., 2021; Bao et al., 2019; Bian et al., 2021; Chen et al., 2017, 2022; Darbani et al., 2016; Dong et al., 2021; Eisenberg and Levanon, 2018; Fu et al., 2019, 2020; Ghorbani et al., 2018; Han et al., 2021; He et al., 2020; Hong et al., 2020; Jiang et al., 2021; Li et al., 2020a, 2020b, 2021a; Liang et al., 2019; Liu et al., 2017, 2019c, 2020a, 2021a; Luo et al., 2020; Lv et al., 2020; Ma et al., 2021a, 2021b; Medina et al., 2021; Meng et al., 2018; Pan et al., 2018; Qin et al., 2018; Ren et al., 2018; Salih et al., 2021; Shi et al., 2021, 2022; Shu et al., 2021; Sun et al., 2016, 2020, 2021, Tang et al., 2018; Tong et al., 2018; Wang et al., 2017a, 2017b, 2018a, 2018b, 2019c, 2019e, 2020a, 2021a, 2021b, 2021c, 2022a, 2022b, Wu et al., 2020; Xiang et al., 2018; Xu et al., 2019, 2022a; Yang et al., 2020a, 2020b, 2020c, 2022; Yin et al., 2018, 2022; Zeng et al., 2018; Zhan et al., 2021; Zhang et al., 2019a, 2019c, 2020a; Zhao et al., 2017a, 2017c; Zheng et al., 2020; Zhou et al., 2018a, 2018b, 2019, 2020; Zhu et al., 2019; Zuo et al., 2016, 2018, 2019).

Figure 2

Experimental verification methods for studying plant circRNAs. (A) Schematic of probes and primers for northern blotting and PCR, respectively. (B) Identification of circRNAs by northern blotting. For northern blotting, the antisense probe is used as the target probe, and the sense probe serves as the control probe. (C) Identification of circRNAs by PCR. Divergent primers (blue arrows) and convergent primers (yellow arrows) are designed. For PCR products amplified with divergent primers using cDNA as templates, Sanger sequencing is performed to check whether back-splicing sites (BSJs) are included.

Figure 3

Proposed model of circRNA biogenesis in plants. (A) The biogenesis of EcircRNAs in plants may be regulated by reverse complementary sequences present in the flanking introns, RNA binding proteins (RBPs), exon-skipping events, N6-methyladenosine (m6A) modifications, or DNA methylation in the flanking introns. RNA helicases may suppress the base complementary pairing to inhibit EcircRNA biogenesis in plants. (B) ciRNAs are derived from lariat RNAs produced from canonical splicing events. RNA debranching enzyme 1 (DBR1) promotes the degradation of lariat RNAs and inhibits the production of ciRNAs. (C) Mitochondrion-encoded circRNAs (mcircRNAs) may be generated from the degradation of precursor and mature RNAs. Mitochondrial RNAs are degraded by coordination of the endonuclease and 3′–5′ exonuclease, which randomly generate different types of RNA degradation intermediates. These RNA degradation intermediates are circularized by an unknown mechanism to form mcircRNAs.

Figure 4

Confirmed functions of circRNAs in plants. circRNAs from A. thaliana, rice (O. sativa), tomato (Solanum lycopersicum), grapevine (Vitis vinifera), and poplar (Populus tomentosa) play important roles during plant growth and development (Os06circ02797, PDS-circ1, PSY1-circ1, lariat41, and circRNA from SEP3) and the responses to abiotic (circGORK, Vv-circATS1, Circ_0003418, and Os05circ02465) and biotic stress (circR5g05160).

Figure 5

Proposed model for the mechanism of action of circRNAs in plants. (A) EcircRNAs inhibit the expression of host genes or parental genes and promote the production and expression of corresponding exon-skipping AS transcripts by forming R-loop structures with the genomic DNA locus of host genes. (B) circRNAs act as miRNA sponges. circRNAs interact with miRNAs and compete with mRNA to bind to miRNAs, inhibiting the degradation of miRNAs on mRNA and increasing the expression of mRNAs. (C) EcircRNAs or ciRNAs interact with RBPs. (D) ElciRNAs and ciRNAs promote the transcription of their parental genes via interaction with host U1 small nuclear ribonucleoprotein particles and RNA polymerase II or the RNA polymerase II complex, respectively. (E) CircRNAs with open reading frames (ORFs) can be translated into peptides or proteins via internal ribosome entry sites (IRESs) or m6A modifications near the ORFs.