Viroids, Satellite RNAs and Prions: Folding of Nucleic Acids and Misfolding of Proteins.

Theodor ("Ted") Otto Diener (* 28 February 1921 in Zürich, Switzerland; † 28 March 2023 in Beltsville, MD, USA) pioneered research on viroids while working at the Plant Virology Laboratory, Agricultural Research Service, USDA, in Beltsville. He coined the name viroid and defined viroids' important features like the infectivity of naked single-stranded RNA without protein-coding capacity. During scientific meetings in the 1970s and 1980s, viroids were often discussed at conferences together with other "subviral pathogens". This term includes what are now called satellite RNAs and prions. Satellite RNAs depend on a helper virus and have linear or, in the case of virusoids, circular RNA genomes. Prions, proteinaceous infectious particles, are the agents of scrapie, kuru and some other diseases. Many satellite RNAs, like viroids, are non-coding and exert their function by thermodynamically or kinetically controlled folding, while prions are solely host-encoded proteins that cause disease by misfolding, aggregation and transmission of their conformations into infectious prion isoforms. In this memorial, we will recall the work of Ted Diener on subviral pathogens.

"Pathomorphogenic" Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco.

Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.

The HHIP-AS1 lncRNA promotes tumorigenicity through stabilization of dynein complex 1 in human SHH-driven tumors.

Most lncRNAs display species-specific expression patterns suggesting that animal models of cancer may only incompletely recapitulate the regulatory crosstalk between lncRNAs and oncogenic pathways in humans. Among these pathways, Sonic Hedgehog (SHH) signaling is aberrantly activated in several human cancer entities. We unravel that aberrant expression of the primate-specific lncRNA HedgeHog Interacting Protein-AntiSense 1 (HHIP-AS1) is a hallmark of SHH-driven tumors including medulloblastoma and atypical teratoid/rhabdoid tumors. HHIP-AS1 is actively transcribed from a bidirectional promoter shared with SHH regulator HHIP. Knockdown of HHIP-AS1 induces mitotic spindle deregulation impairing tumorigenicity in vitro and in vivo. Mechanistically, HHIP-AS1 binds directly to the mRNA of cytoplasmic dynein 1 intermediate chain 2 (DYNC1I2) and attenuates its degradation by hsa-miR-425-5p. We uncover that neither HHIP-AS1 nor the corresponding regulatory element in DYNC1I2 are evolutionary conserved in mice. Taken together, we discover an lncRNA-mediated mechanism that enables the pro-mitotic effects of SHH pathway activation in human tumors.

The Splicing Variant TFIIIA-7ZF of Viroid-Modulated Transcription Factor IIIA Causes Physiological Irregularities in Transgenic Tobacco and Transient Somatic Depression of "Degradome" Characteristic for Developing Pollen.

Viroids are small, non-coding, pathogenic RNAs with a significant ability of adaptation to several basic cellular processes in plants. TFIIIA-7ZF, a splicing variant of transcription factor IIIA, is involved in replication of nuclear-replicating viroids by DNA-dependent polymerase II. We overexpressed NbTFIIIA-7ZF from Nicotiana benthamiana in tobacco (Nicotiana tabacum) where it caused morphological and physiological deviations like plant stunting, splitting of leaf petioles, pistils or apexes, irregular branching of shoots, formation of double-blade leaves, deformation of main stems, and modification of glandular trichomes. Plant aging and senescence was dramatically delayed in transgenic lines. Factors potentially involved in viroid degradation and elimination in pollen were transiently depressed in transgenic leaves. This depressed "degradome" in young plants involved NtTudor S-like nuclease, dicers, argonoute 5, and pollen extracellular nuclease I showing expression in tobacco anthers and leaves. Analysis of the "degradome" in tobacco leaves transformed with either of two hop viroids confirmed modifications of the "degradome" and TFIIIA expression. Thus, the regulatory network connected to TFIIIA-7ZF could be involved in plant pathogenesis as well as in viroid adaptation to avoid its degradation. These results support the hypothesis on a significant impact of limited TFIIIA-7ZF on viroid elimination in pollen.

Design of a DNAzyme : Prediction of mRNA Regions Accessible to a DNAzyme.

The efficiency of RNA-cleaving DNAzymes depends on a large extent on complex formation with their RNA targets. We describe available prediction tools that should help in the design of efficient DNAzymes and show some experimental methods to test the predictions. The main example is for a 10-23 DNAzyme, but the procedure works as well for the 8-17 DNAzyme family.

Fluorescence-Based Kinetic Measurements for RNA-Cleaving DNAzymes.

Studying the catalytic behavior of biocatalysts under different conditions including temperature, buffer conditions, and cofactor concentrations is an important tool to understand their reaction mechanism. We describe two protocols that allow for the investigation of the catalysis of RNA-cleaving DNAzymes. The techniques include the use of FRET-labeled RNA substrates for studying the RNA-cleavage reaction in real-time under high throughput as well as RNA substrates labeled with a fluorescein molecule at the 5' end for gel-based assays. Both methods allow for an accurate determination of rate constants given a reaction model.

Conserved Motifs and Domains in Members of Pospiviroidae.

In 1985, Keese and Symons proposed a hypothesis on the sequence and secondary structure of viroids from the family Pospiviroidae: their secondary structure can be subdivided into five structural and functional domains and "viroids have evolved by rearrangement of domains between different viroids infecting the same cell and subsequent mutations within each domain"; this article is one of the most cited in the field of viroids. Employing the pairwise alignment method used by Keese and Symons and in addition to more recent methods, we tried to reproduce the original results and extent them to further members of Pospiviroidae which were unknown in 1985. Indeed, individual members of Pospiviroidae consist of a patchwork of sequence fragments from the family but the lengths of fragments do not point to consistent points of rearrangement, which is in conflict with the original hypothesis of fixed domain borders.

Time-resolved structural analysis of an RNA-cleaving DNA catalyst.

The 10-23 DNAzyme is one of the most prominent catalytically active DNA sequences1,2. Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential2. However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action3. Here we provide high-resolution NMR characterization of all apparent states of the prototypic 10-23 DNAzyme and present a comprehensive survey of the kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites of the precatalytic DNAzyme-RNA complex reveal that the basis of the DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic process via real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance of the DNAzyme, demonstrating that the acquired knowledge of the molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes.

Predicting the Structure of a Viroid : Structure, Structure Distribution, Consensus Structure, and Structure Drawing.

Viroids are small non-coding RNAs that require a special sequence and structure to be replicated and transported by the host machinery. Many of these features can be predicted and later experimentally verified. Here, we will present workflows to predict viroid structures and draw the predicted structures in a pleasing and descriptive way using recently developed software.

Transformation of Seed Non-Transmissible Hop Viroids in Nicotiana benthamiana Causes Distortions in Male Gametophyte Development.

Viroids are small, non-coding, parasitic RNAs that promote developmental distortions in sensitive plants. We analyzed pollen of Nicotiana benthamiana after infection and/or ectopic transformation with cDNAs of citrus bark cracking viroid (CBCVd), apple fruit crinkle viroid (AFCVd) and potato spindle tuber viroid (PSTVd) variant AS1. These viroids were seed non-transmissible in N. benthamiana. All viroids propagated to high levels in immature anthers similar to leaves, while their levels were drastically reduced by approximately 3.6 × 103, 800 and 59 times in mature pollen of CBCVd, AFCVd and PSTVd infected N. benthamiana, respectively, in comparison to leaves. These results suggest similar elimination processes during male gametophyte development as in the Nicotiana tabacum we presented in our previous study. Mature pollen of N. benthamiana showed no apparent defects in infected plants although all three viroids induced strong pathological symptoms on leaves. While Nicotiana species have naturally bicellular mature pollen, we noted a rare occurrence of mature pollen with three nuclei in CBCVd-infected N. benthamiana. Changes in the expression of ribosomal marker proteins in AFCVd-infected pollen were detected, suggesting some changes in pollen metabolism. N. benthamiana transformed with 35S-driven viroid cDNAs showed strong symptoms including defects in pollen development. A large number of aborted pollen (34% and 62%) and a slight increase of young pollen grains (8% and 15%) were found in mature pollen of AFCVd and CBCVd transformants, respectively, in comparison to control plants (3.9% aborted pollen and 0.3% young pollen). Moreover, pollen grains with malformed nuclei or trinuclear pollen were found in CBCVd-transformed plants. Our results suggest that "forcing" overexpression of seed non-transmissible viroid led to strong pollen pathogenesis. Viroid adaptation to pollen metabolism can be assumed as an important factor for viroid transmissibility through pollen and seeds.

Integrated Proteo-Transcriptomic Analyses Reveal Insights into Regulation of Pollen Development Stages and Dynamics of Cellular Response to Apple Fruit Crinkle Viroid (AFCVd)-Infection in Nicotiana tabacum.

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

Molecular Features and Metal Ions That Influence 10-23 DNAzyme Activity.

Deoxyribozymes (DNAzymes) with RNA hydrolysis activity have a tremendous potential as gene suppression agents for therapeutic applications. The most extensively studied representative is the 10-23 DNAzyme consisting of a catalytic loop and two substrate binding arms that can be designed to bind and cleave the RNA sequence of interest. The RNA substrate is cleaved between central purine and pyrimidine nucleotides. The activity of this DNAzyme in vitro is considerably higher than in vivo, which was suggested to be related to its divalent cation dependency. Understanding the mechanism of DNAzyme catalysis is hindered by the absence of structural information. Numerous biological studies, however, provide comprehensive insights into the role of particular deoxynucleotides and functional groups in DNAzymes. Here we provide an overview of the thermodynamic properties, the impact of nucleobase modifications within the catalytic loop, and the role of different metal ions in catalysis. We point out features that will be helpful in developing novel strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. Consideration of these features will enable to develop improved strategies for structure determination and to understand the mechanism of the 10-23 DNAzyme. These insights provide the basis for improving activity in cells and pave the way for developing DNAzyme applications.

Elimination of Viroids from Tobacco Pollen Involves a Decrease in Propagation Rate and an Increase of the Degradation Processes.

Some viroids-single-stranded, non-coding, circular RNA parasites of plants-are not transmissible through pollen to seeds and to next generation. We analyzed the cause for the elimination of apple fruit crinkle viroid (AFCVd) and citrus bark cracking viroid (CBCVd) from male gametophyte cells of Nicotiana tabacum by RNA deep sequencing and molecular methods using infected and transformed tobacco pollen tissues at different developmental stages. AFCVd was not transferable from pollen to seeds in reciprocal pollinations, due to a complete viroid eradication during the last steps of pollen development and fertilization. In pollen, the viroid replication pathway proceeds with detectable replication intermediates, but is dramatically depressed in comparison to leaves. Specific and unspecific viroid degradation with some preference for (-) chains occurred in pollen, as detected by analysis of viroid-derived small RNAs, by quantification of viroid levels and by detection of viroid degradation products forming "comets" on Northern blots. The decrease of viroid levels during pollen development correlated with mRNA accumulation of several RNA-degrading factors, such as AGO5 nuclease, DICER-like and TUDOR S-like nuclease. In addition, the functional status of pollen, as a tissue with high ribosome content, could play a role during suppression of AFCVd replication involving transcription factors IIIA and ribosomal protein L5.

Expanding crystallization tools for nucleic acid complexes using U1A protein variants.

The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes.

Influence of monovalent metal ions on metal binding and catalytic activity of the 10-23 DNAzyme.

Deoxyribozymes (DNAzymes) are single-stranded DNA molecules that catalyze a broad range of chemical reactions. The 10-23 DNAzyme catalyzes the cleavage of RNA strands and can be designed to cleave essentially any target RNA, which makes it particularly interesting for therapeutic and biosensing applications. The activity of this DNAzyme in vitro is considerably higher than in cells, which was suggested to be a result of the low intracellular concentration of bioavailable divalent cations. While the interaction of the 10-23 DNAzyme with divalent metal ions was studied extensively, the influence of monovalent metal ions on its activity remains poorly understood. Here, we characterize the influence of monovalent and divalent cations on the 10-23 DNAzyme utilizing functional and biophysical techniques. Our results show that Na+ and K+ affect the binding of divalent metal ions to the DNAzyme:RNA complex and considerably modulate the reaction rates of RNA cleavage. We observe an opposite effect of high levels of Na+ and K+ concentrations on Mg2+- and Mn2+-induced reactions, revealing a different interplay of these metals in catalysis. Based on these findings, we propose a model for the interaction of metal ions with the DNAzyme:RNA complex.

Viroid research and its significance for RNA technology and basic biochemistry.

Viroids were described 47 years ago as the smallest RNA molecules capable of infecting plants and autonomously self-replicating without an encoded protein. Work on viroids initiated the development of a number of innovative methods. Novel chromatographic and gelelectrophoretic methods were developed for the purification and characterization of viroids; these methods were later used in molecular biology, gene technology and in prion research. Theoretical and experimental studies of RNA folding demonstrated the general biological importance of metastable structures, and nuclear magnetic resonance spectroscopy of viroid RNA showed the partially covalent nature of hydrogen bonds in biological macromolecules. RNA biochemistry and molecular biology profited from viroid research, such as in the detection of RNA as template of DNA-dependent polymerases and in mechanisms of gene silencing. Viroids, the first circular RNA detected in nature, are important for studies on the much wider spectrum of circular RNAs and other non-coding RNAs.

Inability of DNAzymes to cleave RNA in vivo is due to limited Mg[Formula: see text] concentration in cells.

Sequence specific cleavage of RNA can be achieved by hammerhead ribozymes as well as DNAzymes. They comprise a catalytic core sequence flanked by regions that form double strands with complementary RNA. While different types of ribozymes have been discovered in natural organisms, DNAzymes derive from in vitro selection. Both have been used for therapeutic down-regulation of harmful proteins by reducing drastically the corresponding mRNA concentration. A priori DNAzymes appear advantageous because of the higher haemolytic stability and better cost effectiveness when compared to RNA. In the present work the 10-23 DNAzyme was applied to knockdown expression of the prion protein (PrP), the sole causative agent of transmissible spongiform encephalopathies. We selected accessible target sequences on the PrP mRNA based on a sequential folding algorithm. Very high effectivity of DNAzymes was found for cleavage of RNA in vitro, but activity in neuroblastoma cells was very low. However, siRNA directed to the identical target sequences reduced expression of PrP in the same cell type. According to our analysis, three Mg[Formula: see text] bind cooperatively to the DNAzyme to exert full activity. However, free ATP binds the Mg[Formula: see text] ions more strongly and already stoichiometric amounts of Mg[Formula: see text] and ATP inhibited the activity of DNAzymes drastically. In contrast, natural ribozymes form three-dimensional structures close to the cleavage site that stabilize the active conformation at much lower Mg[Formula: see text] concentrations. For DNAzymes, however, a similar stabilization is not known and therefore DNAzymes need higher free Mg[Formula: see text] concentrations than that available inside the cell.

Modelling the three-dimensional structure of the right-terminal domain of pospiviroids.

Viroids, the smallest know plant pathogens, consist solely of a circular, single-stranded, non-coding RNA. Thus for all of their biological functions, like replication, processing, and transport, they have to present sequence or structural features to exploit host proteins. Viroid binding protein 1 (Virp1) is indispensable for replication of pospiviroids, the largest genus of viroids, in a host plant as well as in protoplasts. Virp1 is known to bind at two sites in the terminal right (TR) domain of pospiviroids; each site consists of a purine- (R-) and a pyrimidine- (Y-)rich motif that are partially base-paired to each other. Here we model the important structural features of the domain and show that it contains an internal loop of two Y · Y cis Watson-Crick/Watson-Crick (cWW) pairs, an asymmetric internal loop including a cWW and a trans Watson/Hoogsteen pair, and a thermodynamically quite stable hairpin loop with several stacking interactions. These features are discussed in connection to the known biological functions of the TR domain.

Viroid quasispecies revealed by deep sequencing.

Viroids are non-coding single-stranded circular RNA molecules that replicate autonomously in infected host plants causing mild to lethal symptoms. Their genomes contain about 250-400 nucleotides, depending on viroid species. Members of the family Pospiviroidae, like the Potato spindle tuber viroid (PSTVd), replicate via an asymmetric rolling-circle mechanism using the host DNA-dependent RNA-Polymerase II in the nucleus, while members of Avsunviroidae are replicated in a symmetric rolling-circle mechanism probably by the nuclear-encoded polymerase in chloroplasts. Viroids induce the production of viroid-specific small RNAs (vsRNA) that can direct (post-)transcriptional gene silencing against host transcripts or genomic sequences. Here, we used deep-sequencing to analyze vsRNAs from plants infected with different PSTVd variants to elucidate the PSTVd quasipecies evolved during infection. We recovered several novel as well as previously known PSTVd variants that were obviously competent in replication and identified common strand-specific mutations. The calculated mean error rate per nucleotide position was less than [Formula: see text], quite comparable to the value of [Formula: see text] reported for a member of Avsunviroidae. The resulting error threshold allows the synthesis of longer-than-unit-length replication intermediates as required by the asymmetric rolling-circle mechanism of members of Pospiviroidae.

Structure and Associated Biological Functions of Viroids.

Mature viroids consist of a noncoding, covalently closed circular RNA that is able to autonomously infect respective host plants. Thus, they must utilize proteins of the host for most biological functions such as replication, processing, transport, and pathogenesis. Therefore, viroids can be regarded as minimal parasites of the host machinery. They have to present to the host machinery the appropriate signals based on either their sequence or their structure. Here, we summarize such sequence and structural features critical for the biological functions of viroids.