Role of RNA silencing in plant-viroid interactions and in viroid pathogenesis.

Viroids are small, single-stranded, non-protein coding and circular RNAs able to infect host plants in the absence of any helper virus. They may elicit symptoms in their hosts, but the underlying molecular pathways are only partially known. Here we address the role of post-transcriptional RNA silencing in plant-viroid-interplay, with major emphasis on the involvement of this sequence-specific RNA degradation mechanism in both plant antiviroid defence and viroid pathogenesis.

Viroid pathogenesis: a critical appraisal of the role of RNA silencing in triggering the initial molecular lesion.

The initial molecular lesions through which viroids, satellite RNAs and viruses trigger signal cascades resulting in plant diseases are hotly debated. Since viroids are circular non-protein-coding RNAs of ∼250-430 nucleotides, they appear very convenient to address this issue. Viroids are targeted by their host RNA silencing defense, generating viroid-derived small RNAs (vd-sRNAs) that are presumed to direct Argonaute (AGO) proteins to inactivate messenger RNAs, thus initiating disease.

How sequence variants of a plastid-replicating viroid with one single nucleotide change initiate disease in its natural host.

Understanding how viruses and subviral agents initiate disease is central to plant pathology. Whether RNA silencing mediates the primary lesion triggered by viroids (small non-protein-coding RNAs), or just intermediate-late steps of a signaling cascade, remains unsolved. While most variants of the plastid-replicating peach latent mosaic viroid (PLMVd) are asymptomatic, some incite peach mosaics or albinism (peach calico, PC).

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing.

Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid.

Retraction: Oncogenic activity of Cdc6 through repression of the INK4/ARF locus.

This corrects the article DOI: 10.1038/nature04585.

The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations.

Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (-) RNAs of ELVd resulting from hammerhead-mediated self-cleavage.

Viroid RNA turnover: characterization of the subgenomic RNAs of potato spindle tuber viroid accumulating in infected tissues provides insights into decay pathways operating in vivo.

While biogenesis of viroid RNAs is well-known, how they decay is restricted to data involving host RNA silencing. Here we report an alternative degradation pathway operating on potato spindle tuber viroid (PSTVd), the type species of nuclear-replicating viroids (family Pospiviroidae). Northern-blot hybridizations with full- and partial-length probes revealed a set of PSTVd (+) subgenomic (sg)RNAs in early-infected eggplant, some partially overlapping and reaching levels comparable to those of the genomic circular and linear forms.

Prednisone in lupus nephritis: how much is enough?

Objective: To assess the effectiveness and safety of a protocol using medium doses of prednisone to treat lupus nephritis.

Methods: Patients receiving the 'Cruces-protocol cohort' (CPC) were paired 1:2 with patients from the 'historic cohort' (HC). The CPC received medium doses of prednisone combined with methyl-prednisolone pulses, hydroxychloroquine and immunosuppressive drugs, usually cyclophosphamide.

Cytopathic Effects Incited by Viroid RNAs and Putative Underlying Mechanisms.

Viroids are infectious agents identified only in plants so far. In contrast to viruses, the genome of viroids is composed of a tiny circular RNA (250-400 nt) not coding for proteins, but containing in its compact structure all the information needed for parasitizing the transcriptional and RNA trafficking machineries of their hosts. Viroid infections are frequently accompanied by cellular and developmental disorders that ultimately result in macroscopic symptoms.

Viroids: how to infect a host and cause disease without encoding proteins.

Despite being composed by a single-stranded, circular, non-protein-coding RNA of just 246-401 nucleotides (nt), viroids can incite in their host plants symptoms similar to those caused by DNA and RNA viruses, which have genomes at least 20-fold bigger and encode proteins. On the other hand, certain non-protein-coding plant satellite RNAs display structural similarities with viroids but for replication and transmission they need to parasitize specific helper viruses (modifying concomitantly the symptoms they induce). While phenotypic alterations accompanying infection by viruses may partly result from expressing the proteins they code for, how the non-protein-coding viroids (and satellite RNAs) cause disease remains a conundrum.

Small RNAs containing the pathogenic determinant of a chloroplast-replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing.

How viroids, tiny non-protein-coding RNAs (~250-400 nt), incite disease is unclear. One hypothesis is that viroid-derived small RNAs (vd-sRNAs; 21-24 nt) resulting from the host defensive response, via RNA silencing, may target for cleavage cell mRNAs and trigger a signal cascade, eventually leading to symptoms. Peach latent mosaic viroid (PLMVd), a chloroplast-replicating viroid, is particularly appropriate to tackle this question because it induces an albinism (peach calico, PC) strictly associated with variants containing a specific 12-14-nt hairpin insertion.

Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: variations on a theme.

Viroids and viroid-like satellite RNAs from plants, and the human hepatitis delta virus (HDV) RNA share some properties that include small size, circularity and replication through a rolling-circle mechanism. Replication occurs in different cell compartments (nucleus, chloroplast and membrane-associated cytoplasmatic vesicles) and has three steps: RNA polymerization, cleavage and ligation. The first step generates oligomeric RNAs that result from the reiterative transcription of the circular templates of one or both polarities, and is catalyzed by either the RNA-dependent RNA polymerase of the helper virus on which viroid-like satellite RNAs are functionally dependent, or by host DNA-dependent RNA polymerases that, remarkably, viroids and HDV redirect to transcribe RNA templates.

Deep sequencing of the small RNAs derived from two symptomatic variants of a chloroplastic viroid: implications for their genesis and for pathogenesis.

Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21-24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations.

A viroid RNA with a specific structural motif inhibits chloroplast development.

Peach latent mosaic viroid (PLMVd) is a chloroplast-replicating RNA that propagates in its natural host, peach (Prunus persica), as a complex mixture of variants, some of which are endowed with specific structural and pathogenic properties. This is the case of variant PC-C40, with an insertion of 12 to 13 nucleotides that folds into a hairpin capped by a U-rich loop, which is responsible for an albino-variegated phenotype known as peach calico (PC). We have applied a combination of ultrastructural, biochemical, and molecular approaches to dissect the pathogenic effects of PC-C40.

Role of prostacyclin (epoprostenol) as anticoagulant in continuous renal replacement therapies: efficacy, security and cost analysis.

Background: Heparin remains the drug most commonly used for anticoagulation in continuous renal replacement therapies (CRRTs). However, in patients with hypercoagulability, heparin is insufficient or, in cases with an increased risk of bleeding or thrombocytopenia, it may be contraindicated. Epoprostenol, a potent vasodilator, antithrombotic and antiplatelet agent, could be an alternative.

Oncogenic activity of Cdc6 through repression of the INK4/ARF locus.

The INK4/ARF locus encodes three tumour suppressors (p15(INK4b), ARF and p16(INK4a)) and is among the most frequently inactivated loci in human cancer. However, little is known about the mechanisms that govern the expression of this locus. Here we have identified a putative DNA replication origin at the INK4/ARF locus that assembles a multiprotein complex containing Cdc6, Orc2 and MCMs, and that coincides with a conserved noncoding DNA element (regulatory domain RD(INK4/ARF)).

Variants of Peach latent mosaic viroid inducing peach calico: uneven distribution in infected plants and requirements of the insertion containing the pathogenicity determinant.

Previous characterization of Peach latent mosaic viroid (PLMVd) variants from a single peach calico (PC) isolate showed that PC symptoms are induced by variants with a 12-13 nt insertion at a specific position and folding into a hairpin with a U-rich loop. Here, this study was extended to two other PC isolates. PLMVd variants with insertions similar to those reported previously (type 1), predominated in one isolate (PC-P2).

A short double-stranded RNA motif of Peach latent mosaic viroid contains the initiation and the self-cleavage sites of both polarity strands.

The transcription initiation sites of viroid RNAs, despite their relevance for replication and in vivo folding, are poorly characterized. Here we have examined this question for Peach latent mosaic viroid (PLMVd), which belongs to the family of chloroplastic viroids with hammerhead ribozymes (Avsunviroidae), by adapting an RNA ligase-mediated rapid amplification of cDNA ends methodology developed for mapping the genuine capped 5' termini of eukaryotic messenger RNAs. To this aim, the characteristic free 5'-triphosphate group of chloroplastic primary transcripts from PLMVd-infected young fruits was previously capped in vitro with GTP and guanylyltransferase.

Viroids: the minimal non-coding RNAs with autonomous replication.

Viroids are small (246-401 nucleotides), non-coding, circular RNAs able to replicate autonomously in certain plants. Viroids are classified into the families Pospiviroidae and Avsunviroidae, whose members replicate in the nucleus and chloroplast, respectively. Replication occurs by an RNA-based rolling-circle mechanism in three steps: (1).