Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification

Abstract

Circular RNAs (circRNAs) are newly discovered incipient non-coding RNAs with potential roles in disease progression in living organisms. Significant reports, since their inception, highlight the abundance and putative functional roles of circRNAs in every organism checked for, like O. sativa, Arabidopsis, human, and mouse. CircRNA expression is generally less than their linear mRNA counterparts which fairly explains the competitive edge of canonical splicing over non-canonical splicing. However, existing methods may not be sensitive enough for the discovery of low-level expressed circRNAs. By combining template-dependent multiple displacement amplification (tdMDA), Illumina sequencing, and bioinformatics tools, we have developed an experimental protocol that is able to detect 1,875 novel and known circRNAs from O. sativa. The same method also revealed 9,242 putative circRNAs in less than 40 million reads for the first time from the Nicotiana benthamiana whose genome has not been fully annotated. Supported by the PCR-based validation and Sanger sequencing of selective circRNAs, our method represents a valuable tool in profiling circRNAs from the organisms with or without genome annotation.

Circular RNAs—The Road Less Traveled

“…Similarly, the expression of circMBNL1 was reported in human and Drosophila heads (Ashwal-Fluss et al, 2014). More than 700 common exonic circRNAs, shared between O. sativa and A. thaliana (Ye et al, 2015), were reported, and a similar observation was also observed between O. sativa Indica and other plants (Guria et al, 2019). These examples depict the conservation of circRNA that originated from the genomic locus, which evolved with a paralogous or orthologous nature.…”

“…Japonica, Oryza sativa ssp. Indica, Nicotiana benthamiana (Guria et al, 2019), and in 12 other plant species (Chu et al, 2017). However, their identification and characterization reveal that the biogenesis of circRNAs originates through the act of backsplicing and not through mis-splicing, as was reported previously.…”

“…On the other hand, it has also been validated that any sequence can be circularized if the last three nucleotides in the 5 ′ and 3 ′ spliceosomal recognition sites remain unchanged (Starke et al, 2015). Conversely, most of the plant circRNAs are joined by non-canonical splice sites (Ye et al, 2017;Chu et al, 2018a,b;Guria et al, 2019); the probable reason for this could be the flexibility in binding of the spliceosome machinery. Due to high complementarity, the microRNA (miRNA)-mediated cleavage of circRNAs is possibly another striking reason for the lower number of circRNAs in plants, as shown in Vitis vinifera L. (Gao et al, 2019).…”

“…Recently, we have also reported that ∼20% of circRNAs are highly expressed than linear RNAs, which are spliced out from the same locus in O. sativa ssp. Indica (Guria et al, 2019).…”

“…Recently, we reported extremely lengthy circRNAs from O. sativa ssp. Indica that encompass sequences for different miRNAs (Guria et al, 2019). Hence, the processivity of these mature miRNAs may be linked to circRNA sequences.…”