Novel Fig-Associated Viroid-Like RNAs Containing Hammerhead Ribozymes in Both Polarity Strands Identified by High-Throughput Sequencing

Abstract

Based on high-throughput sequencing (HTS) data, the existence of viroid-like RNAs (Vd-LRNAs) associated with fig trees grown in the Hawaiian Islands has been predicted. One of these RNAs has been characterized as a circular RNA ranging in size from 357 to 360 nucleotides. Structural and biochemical features of this RNA, tentatively named fig hammerhead viroid-like RNA (FHVd-LR), markedly resemble those previously reported for several viroids and viroid-like satellite RNAs (Vd-LsatRNAs), which are non-proteincoding RNAs infecting their hosts autonomously and in combination with a helper virus, respectively. The full-length sequence of FHVd-LR variants was determined by RT-PCR, cloning, and sequencing. Despite a low global sequence identity with known viroids and Vd-LsatRNAs, FHVd-LR contains a hammerhead ribozyme (HRz) in each polarity strand. Northern blot hybridization assays identified the circular and linear forms of both polarity strands of FHVd-LR and showed that one strand, assigned the (+) polarity, accumulates at higher levels than the (−) polarity strand in vivo. The (+) polarity RNA assumes a rodlike secondary structure of minimal free energy with the conserved domains of the HRzs located in opposition to each other, a feature typical of several viroids and Vd-LRNAs. The HRzs of both FHVd-LR polarity strands were shown to be active in vitro during transcription, self-cleaving the RNAs at the predicted sites. These data, together with the sequence variability observed in the cloned and sequenced full-length variants, indicate that FHVd-LR is a novel viroid or Vd-LsatRNA. According to HTS data, the coexistence of FHVd-LR of different sizes in the same host cannot be excluded. The relationships of FHVd-LR with previously reported viroids and Vd-LsatRNAs, and the need to perform bioassays to conclusively clarify the biological nature of this circular RNA, are discussed.

Quality Assessment and Validation of High-Throughput Sequencing for Grapevine Virus Diagnostics

“…For plant certification, sensitivity is the most important performance criteria for a diagnostic assay. The current study demonstrates the comprehensiveness of HTS for the detection of numerous viruses/viroids reflecting the results of several other studies that have used different input templates including total RNA and sRNA [19], dsRNA [8,22], sRNA [17,30], dsRNA and total RNA [44], dsRNA, total RNA, and sRNA [39], and TNA [14,40]. Overall sensitivity for our TNA HTS assay in the broad evaluation of 19 vines was 98%.…”

“…For HTS-based detection, several nucleic acid templates have been used, including small RNA (sRNA), total RNA, TNA, and dsRNA [13]. Since viruses infecting grapevine have either RNA or DNA genomes and viroids have RNA genomes, the use of TNA allows the detection of both nucleic acid types [14,40,41]. Since plants do not contain high molecular weight dsRNA, and dsRNA is formed during replication of RNA and DNA viruses [42,43], it can be utilized as an enriched source for virus detection [8,28,44].…”

CircRNA FUT10 regulates the regenerative potential of aged skeletal muscle stem cells by targeting HOXA9

“…Our results suggest that miR-365a-3p can be negatively regulated by circRNA FUT10. Similarly, this argument was validated by some researches [ 35 – 37 ].…”

A scenario for the emergence of protoviroids in the RNA world and for their further evolution into viroids and viroid-like RNAs by modular recombinations and mutations

“…Recently, a novel viroid-like RNA, tentatively named fig hammerhead viroid-like RNA (FHVd-LR) and containing HHRs in both polarity strands, has been detected in fig trees growing in Hawaii ( Olmedo-Velarde et al. 2020 ).…”