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

Alejandro Olmedo-Velarde,Beatriz Navarro,Francesco Di Serio,Michael J Melzer,John S Hu

doi:10.3389/fmicb.2020.01903

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-protein-coding 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 rod-like 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.

Highlights

Viroids are small, infectious, non-protein-coding circular RNAs, so far identified only in plants (Kovalskaya and Hammond, 2014; Flores et al, 2015; Gago-Zachert, 2016)
Starting with high-throughput sequencing (HTS) data, we report the identification of Vd-LRNAs containing hammerhead ribozyme (HRz) in fig
A short region of 40– 50 nt of both contigs shared 84–87% sequence identity with HRzs contained in eggplant latent viroid (Fadda et al, 2003), grapevine hammerhead Vd-LRNA (Wu et al, 2012), and cscRNAs (Di Serio et al, 1997, 2006)

Summary

Introduction

Infectious, non-protein-coding circular RNAs, so far identified only in plants (Kovalskaya and Hammond, 2014; Flores et al, 2015; Gago-Zachert, 2016). While viroids replicate and systemically infect their host plants autonomously (in the absence of any helper virus), the infectivity of Vd-LsatRNAs depends on a coinfecting helper virus (Navarro et al, 2017), Vd-LsatRNAs are encapsidated by the helper virus capsid proteins and have been named virusoids (Symons and Randles, 1999) Both viroids and Vd-LsatRNAs differ from carnation small Vd-LRNA, which is non-infectious and has a DNA counterpart integrated in the genome of a plant pararetrovirus (Daròs and Flores, 1995; Vera et al, 2000) or in the plant genome (Hegedus et al, 2004). Carnation small Vd-LRNA is considered a retroviroid-like element

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