Processing of Potato Spindle Tuber Viroid RNAs in Yeast, a Nonconventional Host

Abstract

Potato spindle tuber viroid (PSTVd) is a circular, single-stranded, noncoding RNA plant pathogen that is a useful model for studying the processing of noncoding RNA in eukaryotes. Infective PSTVd circles are replicated via an asymmetric rolling circle mechanism to form linear multimeric RNAs. An endonuclease cleaves these into monomers, and a ligase seals these into mature circles. All eukaryotes may have such enzymes for processing noncoding RNA. As a test, we investigated the processing of three PSTVd RNA constructs in the yeast Of these, only one form, a construct that adopts a previously described tetraloop-containing conformation (TL), produces circles. TL has 16 nucleotides of the 3' end duplicated at the 5' end and a 3' end produced by self-cleavage of a delta ribozyme. The other two constructs, an exact monomer flanked by ribozymes and a trihelix-forming RNA with requisite 5' and 3' duplications, do not produce circles. The TL circles contain nonnative nucleotides resulting from the 3' end created by the ribozyme and the 5' end created from an endolytic cleavage by yeast at a site distinct from where potato enzymes cut these RNAs. RNAs from all three transcripts are cleaved in places not on path for circle formation, likely representing RNA decay. We propose that these constructs fold into distinct RNA structures that interact differently with host cell RNA metabolism enzymes, resulting in various susceptibilities to degradation versus processing. We conclude that PSTVd RNA is opportunistic and may use different processing pathways in different hosts. In higher eukaryotes, the majority of transcribed RNAs do not encode proteins. These noncoding RNAs are responsible for mRNA regulation, control of the expression of regulatory microRNAs, sensing of changes in the environment by use of riboswitches (RNAs that change shape in response to environmental signals), catalysis, and more roles that are still being uncovered. Some of these functions may be remnants from the RNA world and, as such, would be part of the evolutionary past of all forms of modern life. Viroids are noncoding RNAs that can cause disease in plants. Since they encode no proteins, they depend on their own RNA and on host proteins for replication and pathogenicity. It is likely that viroids hijack critical host RNA pathways for processing the host's own noncoding RNA. These pathways are still unknown. Elucidating these pathways should reveal new biological functions of noncoding RNA.

Symptomatic plant viroid infections in phytopathogenic fungi

“…As viroids do not encode any proteins and do not require a helper agent for their multiplication and survival, the biological activities of viroids are entirely dependent on direct interactions of their RNA genome or its derivatives with cellular host components (5)(6)(7)(8)(9). Viroid replication or processing of its RNAs in the yeast, Saccharomyces cerevisiae, and cyanobacterium, Nostoc (Nostocales), have been demonstrated (10)(11)(12).…”

Sequence Similarities between Viroids and Human MicroRNAs

“…Viroids were not detected previously in humans but they could have been missed. Interestingly, although viroids were deemed to only replicate in higher plants, one of them (Avocado sunblotch viroid) was shown to be able to replicate in a cyanobacterium and in the yeast Saccha- romyces cerevisiae [33][34][35]. In addition, exogenous small RNAs such as miRNAs were shown to be capable of spreading between cells [36].…”

“…As a matter of fact, viroid infection was recently associated with the appearance of specific single-stranded RNA, the size of which is similar to that of miRNAs and endogenous small interfering RNAs [7, 29]. Thus, viroid-specific small RNAs comprised by 21–24 nt were detected by Northern blots in infected plants for Potato spindle tuber viroid [28, 30], Citrus exocortis viroid [31], and Peach latent mosaic viroid [32], while Potato spindle tuber viroid RNA was deemed to be opportunistic and suspected as capable of using different processing pathways in different hosts [33].…”

Potato spindle tuber viroid

“…Interestingly, the PSTVd genome contains 17 G/U complementary interactions and some of them are conserved and functionally important for replication and systemic spreading throughout plant tissues (Wu J. et al, 2020). Artificial RNA constructs derived from viroid genomes can form circular molecules even in Saccharomyces cerevisiae, suggesting that their processing mechanisms are highly conservative (Friday et al, 2017).…”