RNA silencing platforms in plants

Since the discovery of RNAi, its mechanism in plants and animals has been intensively studied, widely exploited as a research tool, and used for a number of potential commercial applications. In this article, we discuss the platforms for delivering RNAi in plants. We provide a brief background to these platforms and concentrate on discussing the more recent advances, comparing the RNAi technologies used in plants with those used in animals, and trying to predict the ways in which RNAi technologies may further develop. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The complete nucleotide sequence of subterranean clover mottle virus

The complete nucleotide sequence of Subterranean clover mottle virus (SCMoV) genomic RNA has been determined. The SCMoV genome is 4,258 nucleotides in length. It shares most nucleotide and amino acid sequence identity with the genome of Lucerne transient streak virus (LTSV). SCMoV RNA encodes four overlapping open reading frames and has a genome organisation similar to that of Cocksfoot mottle virus (CfMV). ORF1 and ORF4 are predicted to encode single proteins. ORF2 is predicted to encode two proteins that are derived from a -1 translational frameshift between two overlapping reading frames (ORF2a and ORF2b). A search of amino acid databases did not find a significant match for ORF1 and the function of this protein remains unclear. ORF2a contains a motif typical of chymotrypsin-like serine proteases and ORF2b has motifs characteristically present in positive-stranded RNA-dependent RNA polymerases. ORF4 is likely to be expressed from a subgenomic RNA and encodes the viral coat protein. The ORF2a/ORF2b overlapping gene expression strategy used by SCMoV and CfMV is similar to that of the poleroviruses and differ from that of other published sobemoviruses. These results suggest that the sobemoviruses could now be divided into two distinct subgroups based on those that express the RNA-dependent RNA polymerase from a single, in-frame polyprotein, and those that express it via a -1 translational frameshifting mechanism.

RNA Silencing in Plants

RNA silencing-related mechanisms have been documented in almost all living organisms and RNA silencing is now used as board term to describe the vast array of related processes involving RNA–RNA, RNA–DNA, RNA–protein or protein–protein interactions that ultimately result in the repression of gene expression. In plants, the parallel RNA silencing pathways have evolved to extraordinary levels of complexity and diversity, playing crucial roles in providing protection against invading nucleic acids derived from viruses or replicating transposons, controlling chromatin modifications as well as regulating endogenous gene expression to ensure normal plant growth and development. The aims of this chapter are (1) to provide an overview of the initial curious observations of RNA silencing-related phenomena in plants, (2) to outline the parallel gene silencing pathways of plants, and (3) to discuss current applications of RNA silencing technologies to not only study but also modify plant development

Construct design for efficient, effective and high-throughput gene silencing in plants

Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.

A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus

Barley yellow dwarf virus-PAV (BYDV-PAV) is the most serious and widespread virus of cereals worldwide. Natural resistance genes against this luteovirus give inadequate control, and previous attempts to introduce synthetic resistance into cereals have produced variable results. In an attempt to generate barley with protection against BYDV-PAV, plants were transformed with a transgene designed to produce hairpin (hp)RNA containing BYDV-PAV sequences. From 25 independent barley lines transformed with the BYDV-PAV hpRNA construct, nine lines showed extreme resistance to the virus and the majority of these contained a single transgene. In the progeny of two independent transgenic lines, inheritance of a single transgene consistently correlated with protection against BYDV-PAV. This protection was rated as immunity because the virus could not be detected in the challenged plants by ELISA nor recovered by aphid feeding experiments. In the field, BYDV-PAV is sometimes associated with the related luteovirus Cereal yellow dwarf virus-RPV (CYDV-RPV). When the transgenic plants were challenged with BYDV-PAV and CYDV-RPV together, the plants were susceptible to CYDV-RPV but immune to BYDV-PAV. This shows that the immunity is virus-specific and not broken down by the presence of CYDV. It suggests that CYDV-RPV does not encode a silencing-suppressor gene or that its product does not protect BYDV-PAV against the plant's RNAi-like defence mechanism. Either way, our results indicate that the BYDV-PAV immunity will be robust in the field and is potentially useful in minimizing losses in cereal production worldwide.

Comparison of the coat protein, movement protein and RNA polymerase gene sequences of Australian, Chinese, and American isolates of barley yellow dwarf virus transmitted by Rhopalosiphum padi

Barley yellow dwarf luteovirus-GPV (BYDV-GPV) is a common problem in Chinese wheat crops but is unrecorded elsewhere. A defining characteristic of GPV is its capacity to be transmitted efficiently by both Schizaphis graminum and Rhopaloshiphum padi. This dual aphid species transmission contrasts with those of BYDV-RPV and BYDV-SGV, globally distributed viruses, which are efficiently transmitted only by Rhopaloshiphum padi and Schizaphis graminum respectively. The viral RNA sequences encoding the coat protein (22K) gene, the movement protein (17K) gene, the region surrounding the conserved GDD motif of the polymerase gene and the intergenic sequences between these genes were determined for GPV and an Australian isolate of BYDV-RPV (RPVa). In all three genes, the sequences of GPV and RPVa were more similar to those of an American isolate of BYDV-RPV (RPVu) than to any other luteovirus for which there is data available. RPVa and RPVu were very similar, especially their coat proteins which had 97% identity at the amino acid level. The coat protein of GPV had 76% and 78% amino acid identity with RPVa and RPVu respectively. The data suggest that RPVu and RPVa are correctly named as strains of the same serotype and that GPV is sufficiently different from either RPV strain to be considered a distinct BYDV type. The coat protein and movement protein genes of GPV are very dissimilar to SGV. The polymerase sequences of RPVu, RPVa and GPV show close affinities with those of the sobemo-like luteoviruses and little similarity with those of the carmo-like luteoviruses. The sequences of the coat proteins, movement proteins and the polymerase segments of BYDV serotypes, other than RPV and GPV, form a cluster that is separate from their counterpart sequences from dicot-infecting luteoviruses. The RPV and GPV isolates consistently fall within a dicot-infecting cluster. This suggests that RPV and GPV evolved from within this group of viruses. Since these other viruses all infect dicots it seems likely that their common ancestor infected a dicot and that RPV and GPV evolved from a virus that switched hosts from a dicot to a monocot.

Rice ragged stunt oryzavirus genome segment S4 could encode an RNA dependent RNA polymerase and a second protein of unknown function

The complete nucleotide sequence of genome segment S4 of rice ragged stunt oryzavirus (RRSV, Thai-isolate) was determined. The 3823 bp sequence contains two large open reading frames (ORFs). ORF1, spanning nucleotides 12 to 3776, is capable of encoding a protein of M(r) 141,380 (P4a). The P4a amino acid sequence predicted from the nucleotide sequence contains sequence motifs conserved in RNA-dependent RNA polymerases (RDRPs). When compared for evolutionary relationships with RDRPs of other reoviruses using the amino acid sequences around the conserved GDD motif, P4a was shown to be more related to Nilaparvata lugens reovirus and reovirus serotype 3 than to rice dwarf phytoreovirus, bovine rotavirus or bluetongue virus. The ORF2, spanning nucleotides 491 to 1468, is out of frame with ORF1 and is capable of encoding a protein of 36, 920 (P4b). Coupled in vitro transcription-translation from cloned ORF2 in wheat germ extract confirmed the existence of ORF2 but in vivo production and possible function of P4b is yet to be determined.

Nucleotide sequence of coat protein gene for GPV isolate of barley yellow dwarf virus and construction of expression plasmid for plant

GPV is a Chinese serotype isolate of barley yellow dwarf virus (BYDV) that has no reaction with antiserum of MAV, PAV, SGV, RPV and RMV The sequence of the coat protein (CP) of GPV isolate of BYDV was identified and its amino acid sequence was deduced. The coding region for the putative GPV CP is 603 bases nucleotides and encodes a Mr 22 218 (22 ku) protein. The same as MAV, PAV and RPV, GPV contained a second ORF within the coat protein coding region. This protein of 17 024 Mr (17 ku) is thought to correspond to the Virion protein genome linked (Vpg). Sequence comparisons of the CP coding region between the GPV isolate of BYDV and other isolates of BYDV have been done. The nucleotide and amino acid sequence homology of GPV has a greater identity to the sequence of RPV than those of PAV and MAV. The GPV CP sequence stored 83.7% of nucleotide similarity and 77.5% of deduced amino acid similarity, whereas that of the PAV and MAV shared 56.9%, 53.2% and 44.1%, 43.8% respectively. According to BYDV-GPV CP sequence, two primers were designed. The cDNA of CP was produced by RT-PCR. Full-length cDNA of CP was inserted into plasmid to construct expression plasmids named pPPI1, pPPI2 and pPPI5 based on different promoters. The recombinant plasmids were identified by using α-32P-dATP labelled CP probe, α-32P-ATP labelled GPV RNA probe and sequencing to confirm real GPV CP gene cDNA in plasmids.

A satellite RNA of barley yellow dwarf virus contains a novel hammerhead structure in the self-cleavage domain

An RNA molecule with properties of a satellite RNA was found in an isolate of barley yellow dwarf virus (BYDV), RPV serotype. It is 322 nucleotides long, single-stranded, and does not hybridize to the viral genome. Dimers of the RNA, which presumably represent replicative intermediates, were able to self-cleave into monomers. In vitro transcripts from cDNA clones were capable of self-cleavage in both the plus (encapsidated) and minus orientations. The sequence flanking the minus strand cleavage site contained a consensus " hammerhead" structure, similar to those found in other self-cleaving satellite RNAs. Although related to the hammerhead structure, sequences flanking the plus strand termini showed differences from the consensus and may be folded into a different structure containing a pseudoknot. © 1991.

IL-23R is epigenetically regulated and modulated by chemotherapy in non-small cell lung cancer

The Interleukin-23 (IL-23)/IL-23R signaling axis is an important inflammatory pathway, involved in the stimulation and regulation of the T helper (Th) 17 lymphocytes, resulting in the production of IL-17. Aside from auto-immunity, this cytokine has also been linked to carcinogenesis and polymorphisms in the IL-23R gene are associated with an increased risk for the development of a number of different cancers. Activation of the IL-23 pathway results in the up-regulation of STAT3 and it is thought that the pathological consequences associated with this are in part due to the production of IL-17. We have previously identified IL-23A as pro-proliferative and epigenetically regulated in non-small cell lung cancer (NSCLC). The current study aims to evaluate IL-23R in greater detail in NSCLC. We demonstrate that IL-23R is expressed and epigenetically regulated in NSCLC through histone post-translation modifications and CpG island methylation. In addition, Gemcitabine treatment, a chemotherapy drug used in the treatment of NSCLC, resulted in the up-regulation of the IL-23R. Furthermore, Apilimod (STA 5326), a small molecule which blocks the expression of IL-23 and IL-12, reduced the proliferative capacity of NSCLC cells, particularly in the adenocarcinoma (A549) sub-type. Apilimod is currently undergoing investigation in a number of clinical trials for the treatment of auto-immune conditions such as Crohn's disease and Rheumatoid Arthritis. Our results may have implications for treating NSCLC patients with Gemcitabine or epigenetic targeted therapies. However, Apilimod may possibly provide a new treatment avenue for NSCLC patients. Work is currently ongoing to further delineate the IL-23/IL-23R axis in this disease.

Epigenetic regulation of chemokine/chemokine receptor expression

Epigenetic regulation of gene expression is an important event for normal cellular homeostasis. Gene expression may be "switched" on or "turned" off via epigenetic means through adjustments in DNA architecture. These structural alterations result from changes to the DNA methylation status in addition to histone posttranslational modifications such as acetylation and methylation. Drugs which can alter the status of these epigenetic markers are currently undergoing clinical trials in a wide variety of diseases, including cancer.We illustrate the treatment of cell lines with histone deacetylase (HDi) and DNA methyltransferase inhibitors and the subsequent RNA isolation and reverse transcriptase polymerase chain reaction for several members of the CXC (ELR(+)) chemokine family. In addition we describe a chromatin immunoprecipitation assay to determine the association between chromatin transcription markers and DNA following pretreatment of cell cultures with an HDi, Trichostatin A (TSA). This assay allows us to determine whether treatment with TSA dynamically remodels the promoter region of our selected genes, as judged by the differences in the PCR product between our treated and untreated samples.

Identification of a long non-coding RNA gene, GHSROS (growth hormone secretagogue receptor opposite strand), which stimulates cell migration in non-small cell lung cancer cell lines

The molecular mechanisms involved in non‑small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.

Nedd4-2functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl– channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 μg/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 μg/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.