Transcriptome-wide discovery of circular RNAs in Archaea

Abstract

Circular RNA forms had been described in all domains of life. Such RNAs were shown to have diverse biological functions, including roles in the life cycle of viral and viroid genomes, and in maturation of permuted tRNA genes. Despite their potentially important biological roles, discovery of circular RNAs has so far been mostly serendipitous. We have developed circRNA-seq, a combined experimental/computational approach that enriches for circular RNAs and allows profiling their prevalence in a whole-genome, unbiased manner. Application of this approach to the archaeon Sulfolobus solfataricus P2 revealed multiple circular transcripts, a subset of which was further validated independently. The identified circular RNAs included expected forms, such as excised tRNA introns and rRNA processing intermediates, but were also enriched with non-coding RNAs, including C/D box RNAs and RNase P, as well as circular RNAs of unknown function. Many of the identified circles were conserved in Sulfolobus acidocaldarius, further supporting their functional significance. Our results suggest that circular RNAs, and particularly circular non-coding RNAs, are more prevalent in archaea than previously recognized, and might have yet unidentified biological roles. Our study establishes a specific and sensitive approach for identification of circular RNAs using RNA-seq, and can readily be applied to other organisms.

Figure 1.

Identification of circular RNA products in RNA-seq data. (A) An RNA-seq cDNA read that maps to the reference DNA in a non-linear, chiastic manner, is a hallmark of circular RNA. (B) Schematic representation of the Sulfolobus solfataricus tRNA^Trp, which contains a 65 b intron that is cleaved in the process of tRNA maturation and becomes a stable RNA circle. (C) Reads derived from the region around the circularization junction of the excised tRNA^Trp intron show chiastic mapping to the genome of S. solfataricus P2, exemplifying the power of RNA-seq in circular RNA discovery. Multiple different reads spanning the circularization junction confirm that the observed circular junction is not an amplification artifact. Numbers denote position of the S. solfataricus P2 genome.

Figure 2.

Enrichment for circular transcripts in RNA-seq data from RNase R-treated sample. In both panels, top graph presents RNA-seq data from non-treated sample, and bottom graphs present data from RNase R-treated sample, where linear RNAs are depleted. X axis denotes the position on the S. solfataricus P2 genome, with brown arrows denoting genes (right- and left-pointing arrows correspond to genes encoded on the forward and reverse strands, respectively). Red box marks a cRNA region also supported by permuted reads. Y axis denotes RNA-seq coverage of reads fully mapping to the genome. (A) The C/D box RNA sR106 (34) is enriched in the RNase R-treated sample, and its circular formation is independently supported by 2 and 17 permuted reads in total RNA and RNase R-treated samples, respectively. (B) The C/D box RNA sR102 (34) is enriched in the RNase R-treated sample, and its circular formation is independently supported by two and three permuted reads in total RNA and RNase R-treated samples, respectively.

Figure 3.

Functional characteristics of genes encompassing circular transcripts identified in this study.

Figure 4.

Experimental verification of cRNAs by RT-PCR. (A) Left, RT–PCR results of amplification with outward-directed primers, designed to amplify cRNA; right, RT–PCR results of amplification with inward-directed primers, expected to amplify both linear and cRNA form. Single and multiple arrowheads represent single or double/triple size products, respectively. N/S, non-specific amplification (as verified by direct sequencing). RT–PCR with each primer set was performed on total RNA sample, RNase R-treated sample, and DNA sample, all extracted from S. solfataricus grown to stationary phase on organotrophic medium. (B) RT–PCR for verification of circular RNAs. Arrows indicate outward facing primers (top) and inward facing primers (bottom). Purple line/circle denotes the RNA template, pink line denotes expected PCR product. (C) Double and triple sized products can stem from multiple rounds of RT around a circular RNA template, followed by PCR amplification. Arrows mark illustrative PCR primers. (D) Northern blot analyses of two ncRNAs with circular forms: (M) Size marker, (1) ncRNA found in genomic location 1 275 500–1 275 567, (2) ncRNA found in genomic location 442 786–442 854. Circular forms are indicated by ‘C’, and linear forms by ‘L’.