An Antisense Circular RNA Regulates Expression of RuBisCO Small Subunit Genes in Arabidopsis.

He Zhang,Xinyu Li,František Baluška,Shuai Liu,Lijuan Yao,Yinglang Wan,Hongyang Wu

doi:10.3389/fpls.2021.665014

Abstract

Circular RNA (circRNA) is a novel class of endogenous long non-coding RNA (lncRNA) and participates in diverse physiological process in plants. From the dataset obtained by high-throughput RNA sequencing, we identified a circRNA encoded by the sense strand of the exon regions spanning two RuBisCO small subunit genes, RBCS2B and RBCS3B, in Arabidopsis thaliana. We further applied the single specific primer-polymerase chain reaction (PCR) and Sanger sequencing techniques to verify this circRNA and named it ag-circRBCS (antisense and across genic-circular RNA RBCS). Using quantitative real-time PCR (qRT-PCR), we found that ag-circRBCS shares a similar rhythmic expression pattern with other RBCS genes. The expression level of ag-circRBCS is 10–40 times lower than the expression levels of RBCS genes in the photosynthetic organs in Arabidopsis, whereas the Arabidopsis root lacked ag-circRBCS expression. Furthermore, we used the delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) to deliver in vitro synthesized ag-circRBCS into Arabidopsis seedlings. Our results indicate that ag-circRBCS could significantly depress the expression of RBCS. Given that ag-circRBCS was expressed at low concentration in vivo, we suggest that ag-circRBCS may represent a fine-tuning mechanism to regulating the expression of RBCS genes and protein content in Arabidopsis.

Highlights

Circular RNA is a class of covalently closed single-stranded RNA molecules that lack 5′ caps and 3′ poly(A) tails that generated by back-splicing events in eukaryotic cells (Wang et al, 2014)
From our previously reported strand-specific RNA sequencing database, we identified a small group of antisense circRNAs encoded by the sense strand of exon regions across RuBisCO small subunit genes in Arabidopsis thaliana, which we named ath_circ_362-366 (Supplementary Figure 1)
In Arabidopsis, the RbcS gene family is further divided into two groups

Summary

Introduction

Circular RNA (circRNA) is a class of covalently closed single-stranded RNA molecules that lack 5′ caps and 3′ poly(A) tails that generated by back-splicing events in eukaryotic cells (Wang et al, 2014). CircSEP3 and circSMARCA5 may form an R-loop with a host DNA locus and regulate gene expression or inhibit the DNA damage repairing (Conn et al, 2017; Xu et al, 2021). Endogenous circRNA containing an internal ribosome entry site (IRES) can be translated in vivo (Wang and Wang, 2015; Zhang et al, 2020), and circRNA–protein complexes have been reported (Schneider et al, 2016). It is still unclear whether the plant and animal circRNAs share similar mechanisms for regulation of gene expression. No plant circRNA with an IRES has been reported to have peptide translation

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Results

Conclusion