SUPPLEMENTAL INFORMATION

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1 SUPPLEMENTAL INFORMATION Identification and validation of circrnas Ribo-minus RNA-seq data sets used in identification of circrnas in Oryza sativa and Arabidopsis thaliana were downloaded from NCBI ( For O. sativa, the data sets used are PRJNA272723, PRJNA272732, PRJNA272733, and PRJNA262032, and for A. thaliana, the data sets used are PRJNA270670, PRJNA241054, PRJNA301350, PRJNA213635, PRJNA304437, PRJNA290457, PRJNA227808, and PRJNA CIRI (Gao, et al., 2015), CIRCexplorer (Zhang et al., 2014; Zhang et al., 2016), PcircRNA_finder (Chen et al., 2016), circrna_finder (Westholm et al., 2014), KNIFE (Szabo et al., 2015) as well as find_circ (Memczak et al., 2013) were used to predict circrnas from the RNA-seq data. Divergent PCR was used to experimentally validate the predicted circrnas following a previously reported protocol (Ye et al. 2015). Analysis of conserved circrnas among the five plant species In our previous study (Ye et al. 2015), we identified 758 conserved circrna pairs between A. thaliana and O. sativa based on their orthologous parent genes and sequence similarity. Based on these 758 circrna pairs, we used BLASTN and BLASTP (Altschul et al., 1997) to determine the conservation of circrnas among the five organisms. Briefly, for each of the 758 circrnas, its two 100-bp sequences flanking the back-splicing junction were extracted from A. thaliana and O. sativa to run BLASTN against each other. Based on the distribution of E-value of the 758 conserved circrnas between A. thaliana and O. sativa, we chose 1e-4 as the threshold E-value because this E-value would include over 80% of the conserved circrna pairs. Furthermore, for the exonic circrna pairs, BLASTP was used to check whether or not their parent genes are orthologous using a threshold E-value <1e-5. In this way, 976 conserved circrna families between A. thaliana and O. sativa were identified (Supplemental Table 2). The circrnas in other three species that showed a high sequence similarity (BLASTN E-value <1e-4) with one of the 976 circrnas were considered to be conserved.

2 Supplementary Table 1. The number of circrnas in PlantcircBase (Release 1) Organism Number of circrnas Full-length sequence of circrnas Acting as circrna-mirnamrna networks* Back-splicing Validated by Sanger mirna Total Total validated sequencing sponges* Tissues References 12, Roots, shoots Ye et al , Leaves Lu et al Oryza sativa 2, Roots Ye et al , Leaves, anthers, pistils, seeds, shoots, roots This study Roots Wang et al , Leaves Ye et al Arabidopsis thaliana NA Sun et al Siliques, seeds Lu et al , Inflorescences, roots, leaves, aerial This study Zea mays Shoots Lu et al Hordeum vulgare Leaves and seeds Darbani et al Solanum lycopersicum Fruit Zuo et al *mirna sponges were predicted using etm_finder (Ye et al. 2014); mirna targets were predicted using psrnatarget (Dai and Zhao 2011).

3 Supplementary Table 2. Number of conserved circrnas in PlantcircBase (Release 1) Organism Oryza sativa Arabidopsis thaliana Oryza sativa Arabidopsis thaliana 976 (4,109/2,767)* --- Zea mays 73 (96/287) 19 (23/58) Hordeum vulgare 11 (12/29) 3 (4/133) Solanum lycopersicum 87 (100/172) 129 (142/226) *Representing a total of 976 conserved circrna families (available at ) between A. thaliana and O. sativa, and 4,109 and 2,767 circrna isoforms of the 976 families in A. thaliana and O. sativa, respectively. Usually, a parent gene or locus generating multiple alternatively spliced circrnas that were grouped as one circrna family.

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5 Supplementary Figure 1 Detail information of a circrna entry (ath_circ_025680). circrna ID: ID of circrnas in PlantcircBase. It is made up of three parts, species_rna type_the serial number of the circrna. For the example of ath_circ_025680, ath is short for Arabidopsis thaliana; circ represents circrna; represents the serial number of the circrna in PlantcircBase. Alias: Name of the circrna used in other papers. Organism: The organism from which the circrna was identified. Position: The exact genomic position of the circrna, including chromosome, start site and end site (1-based). Reference genome: The version of the reference genome used in PlantcircBase. Type: Type of circrna based on its genomic origin, e.g. exonic, intronic and intergenic. Parent gene: The host gene from which the circrna is derived. Parent gene annotation: The annotation of the host gene based on the genome version used in PlantcircBase. Strand: The strand on which the host gene resides. Alternative splicing: In this study, circrnas with overlapping sequences are considered as alternatively spliced isoforms. Splice junction sequence: Sequences showing in upper and lower cases represent the two sides of a back-splicing site. Support reads: The total number of reads that support the junction of the circrna. The supporting reads in different tissues (experiments) are separated by "/". Tissues: Indicating the tissue from which the circrna was identified. Tissues used in different experiments were separated by "/" based on the order of supporting reads showing in the previous item. Exon boundary: It shows whether the splicing sites are on the boundary of exons or not. For example, "Yes-No" represents the donor splicing site is on the exon boundary and the accepter is not on the exon boundary. Splicing signals: The splicing signals of the circrna. For example, AG-GT

6 represents that the donor and the accepter splicing signal is AG and GT, respectively. Evidence 1: Sanger sequencing: Yes represents that Sanger sequencing has been performed to valid the back-splicing site, No represents the opposite, NA represents not available. Evidence 1: PCR primers: PCR primers (including convergent and divergent primers) used in back-splicing validation. circrna sequence: The sequence of a circrna based on assembly using circseq-cup (Ye et al., 2016) or Sanger sequencing. Number of exons covered1: The number of exons that cover the assembly circrna sequence. Evidence 2: Sanger sequencing: "Yes" represents that Sanger sequencing has been performed to valid the full length of circrna, "No" represents the opposite. Evidence 2: PCR primers: PCR primers used in validation of the full length circrna. Genomics sequence: Genomic sequence of the circrna. Number of exons covered2: The number of exons that cover the genomic sequence of the circrna. Conserved circrnas: Conserved circrnas in other plants based on their backsplicing sequence similarity and parent genes. Sponge-miRNA(1-3): mirnas that are predicted to bind the circrna (in other words, the circrna acts as a potential mirna sponge). Only the top three mirnas of a circrna are listed here if more than three were predicted. Sponge-miRNA(1-3)-mRNA: The target mrnas of the mirna listed in the item Sponge-miRNA : Only the top three target mrnas are shown if more than three were predicted. Reference: The study in which the circrna was first reported.

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