Identification and Functional Characterization of Tomato CircRNAs Derived from Genes Involved in Fruit Pigment Accumulation

Abstract

1CircRNAs, a class of widespread circular RNAs produced from precursor mRNA back-splicing, have been implicated in regulation of gene expression in eukaryotes, but their biological functions in plants have not yet been elucidated. By deep sequencing of rRNA-removed and RNase R-digested RNA samples we have identified several thousands of putative back-splicing sites in tomato fruit (Solanum lycopersicum) and show that the abundance of some of these circRNAs derived from fruit pigment biosynthesis genes are regulated by fruit ripening. Herein, we overexpressed a circRNA derived from Phytoene Synthase 1 (PSY1) in tomato 'Ailsa Craig' and microTom. The PSY1 mRNA abundance, the lycopene and β-carotene accumulation were decreased significantly in the transgenic tomato fruits, likely due to the continuous highly expressed circRNAs and/or the low abundant linear RNAs generated from the overexpression vector. Besides, overexpression of a circRNA derived from Phytoene Desaturase (PDS) showed similar results. Our results provide biological insights into plant circRNAs.CircRNAs, a class of circular RNAs in eukaryotes, are derived from precursor mRNA back-splicing 1 . Although circRNAs have been identified more than 20 years ago [2][3][4] , they had been considered to be produced from aberrant splicing and their existence and functional potential were both underestimated. Nowadays, with the development of next-generation sequencing and bioinformatics, circRNAs have been identified in various eukaryotic species [5][6][7][8][9] . Most of the identified circRNAs are expressed at low levels, indicating the possibility that the majority of circRNAs might be splicing byproducts with little functional potential 8,[10][11][12] . However, many circRNAs are more abundant than their linear counterparts [5][6][7][8]13 , suggesting the potential functional significance of these circular RNA molecules. Recent studies revealed that circRNAs are more stable than linear mRNAs, and most of them are cytoplasmic 6,7,13 . In addition, the circularization of circRNAs are conserved among species, and the expression of circRNAs are often cell, tissue and developmental stage-specific 7,8,[13][14][15] . Recent studies have revealed that circRNAs may play roles in gene expression regulation, although the function of most circRNAs remain largely unknown [16][17][18] . The biogenesis of circRNAs is considered to be regulated by both cis-elements and trans-acting factors 16,18 . Complementary sequences or inverted repeats in the introns flanking the back-splice site could promote exon circularization by pairing to form hairpin structures 10,13,19,20 , and multiple circRNAs may be produced from one single gene due to different sequence pairing, which is referred as to alternative circularization 10 . However, there are also circRNAs produced from exons without being bracketed by complementary sequences [21][22][23] , indicating that other cis-elements may account for the circularization, such as sequences recognized by RNA-binding proteins (RBPs) [24][25]...

Identification and characterization of circular RNAs during the sea buckthorn fruit development

“…Another one circRNA (circZDS) was transcripted from ζ-carotene desaturase (ZDS) gene, which was important for carotenoid biosynthesis [35][36][37][38]. Also, PSY1-circ1 and PDS1-circ1, respectively derived from PSY1 and PDS gene, can change fruit pigment by affect the lycopene and β-carotene accumulation in tomato [11]. All these results suggested that circRNAs could have important biological functions in the metabolite synthesis process during sea buckthorn fruit development.…”

“…A lot of fruit ripening related circRNAs were also discovered in tomato [7]. Moreover, two fruit pigment related circRNAs were verified in tomato [11]. As a medicinal and edible plant, sea buckthorn has been considered as good plant for studying the process of fruit ripening because of its continuous maturation process, abundant nourishments and availability of genome information.…”

“…In addition, the sequences of circRNAs are often conserved in various species, which suggests that they are an evolutionarily conserved feature. And many conserved circRNAs were derived from important gene loci implying their potentially important roles [6,10,11].…”

Systematic identification and analysis of heat‐stress‐responsive lncRNAs, circRNAs and miRNAs with associated co‐expression and ceRNA networks in cucumber (Cucumis sativus L.)

“…The results showed that 102 target mRNAs of 39 circRNAs were involved in the pathways involved in ethylene synthesis and signal transduction (Wang et al ). The research showed that circRNA is involved in the regulation of pigment accumulation during tomato fruit ripening and overexpression of circRNA related to pigment synthesis can significantly reduce the expression level of the parental gene and the color of the response (Tan et al ). The research has found that circular RNA might play an important role in development of moso bamboo by regulating the splicing of several rapid‐growth related genes (Wang et al ).…”

“…Similar research found that circRNAs play an important role in the regulation and control of tomato fruits (Yin et al ). CircRNA is widely distributed in plants and has been identified in several plants such as Arabidopsis, rice, tomato and soybean using deep RNA‐seq and bioinformatic tools (Lu et al , Chen et al , Conn et al , Tan et al , Zhao et al , Zhou et al ). However, at present, comprehensive studies of circRNA in plants are lacking, and the functions of circRNA have not yet been clarified.…”