Intercellular and systemic movement of RNA silencing signals

In most eukaryotes, double-stranded RNA is processed into small RNAs that are potent regulators of gene expression. This gene silencing process is known as RNA silencing or RNA interference (RNAi) and, in plants and nematodes, it is associated with the production of a mobile signal that can travel from cell-to-cell and over long distances. The sequence-specific nature of systemic RNA silencing indicates that a nucleic acid is a component of the signalling complex. Recent work has shed light on the mobile RNA species, the genes involved in the production and transport of the signal. This review discusses the advances in systemic RNAi and presents the current challenges and questions in this rapidly evolving field.     

Plant Mobile Small RNAs

In plants, RNA silencing is a fundamental regulator of gene expression, heterochromatin formation, suppression of transposable elements, and defense against viruses. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. Although the spreading of RNA silencing across the plant has been recognized for nearly two decades, only recently have sRNAs been formally demonstrated as the mobile silencing signals. Here, we discuss the various types of mobile sRNA molecules, their short- and long-range movement, and their function in recipient cells.RNA silencing is a regulatory mechanism that controls the expression of endogenous genes and exogenous molecular parasites such as viruses, transgenes, and transposable elements. One of the most fascinating aspects of RNA silencing found in plants and invertebrates is its mobile nature—in other words, its ability to spread from the cell where it has been initiated to neighboring cells. This phenomenon relies on the movement of small noncoding RNA molecules (sRNA, 21–24 nucleotides [nt] in length) that provide the sequence specificity of the silencing effects. In plants, there are two major classes of sRNAs: short interfering RNAs (siRNAs) and micro RNAs (miRNAs). These sRNAs are generated by diverse and sometimes interacting biochemical pathways, which may influence their mobility. Movement of plant sRNAs falls into two main categories: cell-to-cell (short-range) and systemic (long-range) movement (Melnyk et al. 2011).     

Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses

RNA silencing is an evolutionarily conserved surveillance system that occurs in a broad range of eukaryotic organisms. In plants, RNA silencing acts as an antiviral system; thus, successful virus infection requires suppression of gene silencing. A number of viral suppressors have been identified so far; however, the molecular bases of silencing suppression are still poorly understood. Here we show that p19 of Cymbidium ringspot virus (CymRSV) inhibits RNA silencing via its small RNA-binding activity in vivo. Small RNAs bound by p19 in planta are bona fide double-stranded siRNAs and they are silencing competent in the in vitro RNA-silencing system. p19 also suppresses RNA silencing in the heterologous Drosophila in vitro system by preventing siRNA incorporation into RISC. During CymRSV infection, p19 markedly diminishes the amount of free siRNA in cells by forming p19-siRNA complexes, thus making siRNAs inaccessible for effector complexes of RNA-silencing machinery. Furthermore, the obtained results also suggest that the p19-mediated sequestration of siRNAs in virus-infected cells blocks the spread of the mobile, systemic signal of RNA silencing.     

Small RNAs in development - insights from plants

microRNAs (miRNAs) and small interfering RNAs (siRNAs), which constitute two major classes of endogenous small RNAs in plants, impact a multitude of developmental and physiological processes by imparting sequence specificity to gene and genome regulation. Although lacking the third major class of small RNAs found in animals, Piwi-interacting RNAs (piRNAs), plants have expanded their repertoire of endogenous siRNAs, some of which fulfill similar molecular and developmental functions as piRNAs in animals. Research on plant miRNAs and siRNAs has contributed invaluable insights into small RNA biology, thanks to the highly conserved molecular logic behind the biogenesis and actions of small RNAs. Here, I review progress in the plant small RNA field in the past two years, with an emphasis on recent findings related to plant development. I do not recount the numerous developmental processes regulated by small RNAs; instead, I focus on major principles that have been derived from recent studies and draw parallels, when applicable, between plants and animals.     

RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct

Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (–)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.     

Trans-species small RNAs move long distances in a parasitic plant complex

Parasitic plants exchange various types of RNAs with their host plants, including mRNA, and small non-coding RNA. Among small non-coding RNAs, miRNA production is known to be induced at the haustorial interface. The induced miRNAs transfer to the host plant and activate secondary siRNA production to silence target genes in the host. In addition to interfacial transfer, long-distance movement of the small RNAs has also been known to mediate signaling and regulate biological processes. In this study, we tested the long-distance movement of trans-species small RNAs in a parasitic-plant complex. Small RNA-Seq was performed using a complex of a stem parasitic plant, Cuscuta campestris, and a host, Arabidopsis thaliana. In the host plant’s parasitized stem, genes involved in the production of secondary siRNA, AtSGS3 and AtRDR6, were upregulated, and 22-nt small RNA was enriched concomitantly, suggesting the activation of secondary siRNA production. Stem-loop RT-PCR and subsequent sequencing experimentally confirmed the mobility of the small RNAs. Trans-species mobile small RNAs were detected in the parasitic interface and also in distant organs. To clarify the mode of long-distance translocation, we examined whether C. campestris-derived small RNA moves long distances in A. thaliana sgs3 and rdr6 mutants or not. Mobility of C. campestris-derived small RNA in sgs3 and rdr6 mutants suggested the occurrence of direct long-distance transport without secondary siRNA production in the recipient plant.     

植物小RNA荧光原位杂交实验方法

小RNA是对植物生长发育十分重要的一类小分子核苷酸,在多种生命过程以及胁迫响应中发挥重要调控作用。对小RNA的定位研究有助于揭示它们的功能,而小RNA荧光原位杂交(sRNA-FISH)是一种通过荧光检测技术对生物体内小RNA进行定性或半定量分析的技术,目前该技术已经在动物体内被广泛应用,而在植物体内的应用还比较少。该文...     

Characterization and application of small RNAs and RNA silencing mechanisms in fungi

Although extensively cataloged and functionally diverse in plants and animals, the role and targets of small RNAs remain mostly uncharacterized in filamentous fungi. To date, much of the knowledge of small RNAs in filamentous fungi has been derived from studies of a limited group of fungi, most notably in Neurospora crassa. While most of the recently discovered classes of small RNAs appear to be unique to fungi some are commonly found in eukaryotes. It is noteworthy that the RNA silencing protein machinery involved in small RNA biogenesis has also diverged greatly, particularly within filamentous fungi, and may explain the diversity of small RNA classes. In this review, we summarize important classes of eukaryotic small RNAs and provide a current analysis of the RNA silencing machinery based on available fungal genome sequences. Finally, we discuss opportunities for exploiting knowledge of small RNAs and RNA silencing for practical application such as engineering plants resistant to fungal pathogens.     

Characterization and application of small RNAs and RNA silencing mechanisms in fungi

Although extensively cataloged and functionally diverse in plants and animals, the role and targets of small RNAs remain mostly uncharacterized in filamentous fungi. To date, much of the knowledge of small RNAs in filamentous fungi has been derived from studies of a limited group of fungi, most notably in Neurospora crassa. While most of the recently discovered classes of small RNAs appear to be unique to fungi some are commonly found in eukaryotes. It is noteworthy that the RNA silencing protein machinery involved in small RNA biogenesis has also diverged greatly, particularly within filamentous fungi, and may explain the diversity of small RNA classes. In this review, we summarize important classes of eukaryotic small RNAs and provide a current analysis of the RNA silencing machinery based on available fungal genome sequences. Finally, we discuss opportunities for exploiting knowledge of small RNAs and RNA silencing for practical application such as engineering plants resistant to fungal pathogens.     

Argonaute Proteins and Mechanisms of RNA Interference in Eukaryotes and Prokaryotes

Noncoding RNAs play essential roles in genetic regulation in all organisms. In eukaryotic cells, many small non-coding RNAs act in complex with Argonaute proteins and regulate gene expression by recognizing complementary RNA targets. The complexes of Argonaute proteins with small RNAs also play a key role in silencing of mobile genetic elements and, in some cases, viruses. These processes are collectively called RNA interference. RNA interference is a powerful tool for specific gene silencing in both basic research and therapeutic applications. Argonaute proteins are also found in prokaryotic organisms. Recent studies have shown that prokaryotic Argonautes can also cleave their target nucleic acids, in particular DNA. This activity of prokaryotic Argonautes might potentially be used to edit eukaryotic genomes. However, the molecular mechanisms of small nucleic acid biogenesis and the functions of Argonaute proteins, in particular in bacteria and archaea, remain largely unknown. Here we briefly review available data on the RNA interference processes and Argonaute proteins in eukaryotes and prokaryotes.     

Small RNAs in viral infection and host defense

Small RNAs are the key mediators of RNA silencing and related pathways in plants and other eukaryotic organisms. Silencing pathways couple the destruction of double-stranded RNA with the use of the resulting small RNAs to target other nucleic acid molecules that contain the complementary sequence. This discovery has revolutionized our ideas about host defense and genetic regulatory mechanisms in eukaryotes. Small RNAs can direct the degradation of mRNAs and single-stranded viral RNAs, the modification of DNA and histones, and the inhibition of translation. Viruses might even use small RNAs to do some targeting of their own to manipulate host gene expression. This review highlights the current understanding and new insights concerning the roles of small RNAs in virus infection and host defense in plants.     

Gene silencing in Arabidopsis spreads from the root to the shoot, through a gating barrier, by template-dependent, nonvascular, cell-to-cell movement

Upward long-distance mobile silencing has been shown to be phloem mediated in several different solanaceous species. We show that the Arabidopsis (Arabidopsis thaliana) seedling grafting system and a counterpart inducible system generate upwardly spreading long-distance silencing that travels not in the phloem but by template-dependent reiterated short-distance cell-to-cell spread through the cells of the central stele. Examining the movement of the silencing front revealed a largely unrecognized zone of tissue, below the apical meristem, that is resistant to the silencing signal and that may provide a gating or protective barrier against small RNA signals. Using a range of auxin and actin transport inhibitors revealed that, in this zone, alteration of vesicular transport together with cytoskeleton dynamics prevented or retarded the spread of the silencing signal. This suggests that small RNAs are transported from cell to cell via plasmodesmata rather than diffusing from their source in the phloem.