Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms

Abstract

Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.

Identification and characterization of a novel geminivirus with a monopartite genome infecting apple trees

“…BLASTN searches identified several contigs homologous to apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic ringspot virus (ACLSV) and ASSVd. Moreover, a novel small circular viroid-like RNA was identified using computational algorithms (Wu et al, 2012;Zhang et al, 2014). The actual presence of these infectious viral and subviral agents in the source plant was verified by reverse transcription (RT)-PCR and/or Northern blotting (Zhang et al, 2014).…”

“…Moreover, a novel small circular viroid-like RNA was identified using computational algorithms (Wu et al, 2012;Zhang et al, 2014). The actual presence of these infectious viral and subviral agents in the source plant was verified by reverse transcription (RT)-PCR and/or Northern blotting (Zhang et al, 2014). In addition, four contigs with similarities to genomic regions of several members of the family Geminiviridae were also identified (Table S1, available in the online Supplementary Material), suggesting the presence of an additional virus with circular DNA genomic component(s) in the apple tree analysed.…”

Placental transcriptome in development and pathology: expression, function, and methods of analysis

“…39 Without a 3' tail, they cannot be polyadenylated, and therefore are missed by typical RNA-seq library preparations that rely on purification using the polyadenylated feature of classic RNA sequences. 40,41 CircRNAs are resistant to cytoplasmic exonucleases and this property is now exploited in their detection.…”

Viroid quasispecies revealed by deep sequencing

“…21,22 In this study, we have gone a step further; that is, we have used small RNA data to compare the mutational landscapes of different infecting viroid variants. These landscapes reflect a combination of replication efficiency, selection, and sequencing errors.…”