RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21-to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.A major focus of current biology is to understand how a pathogen has evolved mechanisms to achieve a balance among several interrelated activities that are crucial to establish a full infection: evading or suppressing host defense, minimizing destructive interference of host metabolism, and maximizing utilization of host factors to support replication and systemic spread. A full understanding of these mechanisms is not only necessary to build a foundation for developing technologies to combat pathogen diseases but also can provide fundamental mechanistic insights into the regulation of basic cellular processes.Recent studies have discovered small RNA-mediated gene silencing as a powerful antiviral mechanism in plants and animals (6,22,25,47,49,50,72,77,84,(87)(88)(89)98). Furthermore, small RNA-mediated gene silencing plays essential roles in regulating a wide variety of growth and development processes (4-6, 11, 13, 17, 23, 28, 42). A key mediator of RNA silencing is several classes of 21-to 24-nucleotide (nt) small RNAs.MicroRNAs (miRNAs) are produced by cleavage of hairpin RNA precursors encoded by the genome of an organism. Short interfering RNAs (siRNAs) are generated by cleavage of double-stranded RNAs (dsRNAs) that may originate from several sources, including cellular genomes, viral replication intermediates, aberrant cellular RNAs, overexpresse...

Cell-to-cell trafficking of RNA is an emerging biological principle that integrates systemic gene regulation, viral infection, antiviral response, and cell-to-cell communication. A key mechanistic question is how an RNA is specifically selected for trafficking from one type of cell into another type. Here, we report the identification of an RNA motif in Potato spindle tuber viroid (PSTVd) required for trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana leaves. This motif, called loop 6, has the sequence 59-CGA-39...59-GAC-39 flanked on both sides by cis Watson-Crick G/C and G/U wobble base pairs. We present a three-dimensional (3D) structural model of loop 6 that specifies all non-Watson-Crick base pair interactions, derived by isostericity-based sequence comparisons with 3D RNA motifs from the RNA x-ray crystal structure database. The model is supported by available chemical modification patterns, natural sequence conservation/variations in PSTVd isolates and related species, and functional characterization of all possible mutants for each of the loop 6 base pairs. Our findings and approaches have broad implications for studying the 3D RNA structural motifs mediating trafficking of diverse RNA species across specific cellular boundaries and for studying the structure-function relationships of RNA motifs in other biological processes.

Viroids are noncoding RNAs that infect plants. In order to establish systemic infection, these RNAs must traffic from an initially infected host cell into neighboring cells and ultimately throughout a whole plant. Recent studies have identified structural motifs in a viroid that are required for trafficking, enabling further studies on the mechanisms of their function. Some cellular proteins interact with viroids in vivo and may play a role in viroid trafficking, which can now be directly tested by using a virus-induced gene silencing system that functions efficiently in plant species from which these factors were identified. This review discusses these recent advances, unanswered questions and the use of viroid infection as an highly productive model to elucidate mechanisms of RNA trafficking that is of broad biological significance.

Intercellular RNA trafficking has been shown as a widely-existing phenomenon that has significant functions in many aspects of biology. Viroids, circular noncoding RNAs that cause plant diseases, have been a model to dissect the role of RNA structural motifs in regulating intercellular RNA trafficking in plants. Recent studies on potato spindle tuber viroid (PSTVd) showed that the RNA motif loop 19 is important for PSTVd to spread from palisade to spongy mesophyll in infected leaves. Here, we performed saturated mutational analysis to uncover all possible functional variants of loop 19 and exploit this data to pinpoint to a three-dimensional structural model of this motif. Interestingly, we found that two distinct structural motifs can replace loop 19 and retain the systemic trafficking capacity. One of the alternative structures rapidly emerged from the inoculation using a loop 19 abolished mutant that is not capable of systemic trafficking. Our observation indicates the flexibility of multiple structural arrangements interchangeably exerting similar function at a particular RNA locus. Taken together, this study deepens the understanding of RNA structural motifs-regulated viroid RNA trafficking, which has broad implications for studying RNA intercellular trafficking as well.

Soga (2021) Size-controlled bimodal invivo nanoprobes as near-infrared phosphors and positive contrast agents for magnetic