Constructs and methods for hairpin RNA-mediated gene silencing in plants

Double-stranded RNA (dsRNA) induces an endogenous sequence-specific RNA degradation mechanism in most eukaryotic cells. The mechanism can be harnessed to silence genes in plants by expressing self-complementary single-stranded (hairpin) RNA in which the duplexed region has the same sequence as part of the target gene's mRNA. We describe a number of plasmid vectors for generating hairpin RNAs, including those designed for high-throughput cloning, and provide protocols for their use.

Gene silencing: Fleshing out the bones

Genetic studies are revealing the pathway for RNA-mediated gene silencing. Short RNA molecules are the key, giving sequence specificity for RNA degradation and mediating communication within and between cells; these short RNAs are common to transcriptional and post-transcriptional silencing pathways. The expression of transgenes in plants varies between independent transformants and there are many examples where the transgenic trait is not expressed, or disappears in subsequent generations, despite the presence of the transgene. This loss of a trait, but not of the transgene, has become known as gene silencing and can take two forms, transcriptional or post-transcriptional. As their names imply, transcriptional gene silencing occurs when a transgene is not transcribed, whereas in post-transcriptional gene silencing, the transgene mRNA is produced but degraded before it is translated (reviewed in [1]). Both forms of silencing seem to be the result of inherent mechanisms for protecting plants against mobile or invading DNA — for example, transposable elements or the T-DNA of Agrobacterium — or RNA viruses. Plants are not alone in their capacity for transgene silencing; both forms of silencing occur in flies and fungi, where it is known as RIP or quelling, while nematodes exhibit post-transcriptional silencing, generally referred to as RNA interference (RNAi). A clearer picture of the mechanisms and relationships of the different types of transgene silencing is beginning to emerge from a number of recent studies [2], [3], [4], [5], [6], [7] and [8]. Some of these studies [2], [3], [4] and [5] have enhanced our understanding of the steps within the post-transcriptional silencing pathway, and others [6], [7] and [8] have demonstrated that the two forms of silencing may be mechanistically linked.

A 22-nt artificial microRNA mediates widespread RNA silencing in Arabidopsis

It is known that 22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger phased small-interfering RNA (phasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA)-mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA-mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis thaliana due to the obvious and non-lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over-expression of CHS in the pap1-D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA-DEPENDENT RNA POLYMERASE6-mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA-directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families.

Small RNA sequencing of potato leafroll virus-infected plants reveals an additional subgenomic RNA encoding a sequence-specific RNA-binding protein

Potato leafroll virus (PLRV) is a positive-strand RNA virus that generates subgenomic RNAs (sgRNA) for expression of 3' proximal genes. Small RNA (sRNA) sequencing and mapping of the PLRV-derived sRNAs revealed coverage of the entire viral genome with the exception of four distinctive gaps. Remarkably, these gaps mapped to areas of PLRV genome with extensive secondary structures, such as the internal ribosome entry site and 5' transcriptional start site of sgRNA1 and sgRNA2. The last gap mapped to ~500. nt from the 3' terminus of PLRV genome and suggested the possible presence of an additional sgRNA for PLRV. Quantitative real-time PCR and northern blot analysis confirmed the expression of sgRNA3 and subsequent analyses placed its 5' transcriptional start site at position 5347 of PLRV genome. A regulatory role is proposed for the PLRV sgRNA3 as it encodes for an RNA-binding protein with specificity to the 5' of PLRV genomic RNA. © 2013.

Vectors and methods for hairpin RNA and artificial microRNA-mediated gene silencing in plants

In plant cells, DICER-LIKE4 processes perfectly double-stranded RNA (dsRNA) into short interfering (si) RNAs, and DICER-LIKE1 generates micro (mi) RNAs from primary miRNA transcripts (pri-miRNA) that form fold-back structures of imperfectly dsRNA. Both si and miRNAs direct the endogenous endonuclease, ARGONAUTE1 to cleave complementary target single-stranded RNAs and either small RNA (sRNA)-directed pathway can be harnessed to silence genes in plants. A routine way of inducing and directing RNA silencing by siRNAs is to express self-complementary single-stranded hairpin RNA (hpRNA), in which the duplexed region has the same sequence as part of the target gene's mRNA. Artificial miRNA (amiRNA)-mediated silencing uses an endogenous pri-miRNA, in which the original miRNA/miRNA* sequence has been replaced with a sequence complementary to the new target gene. In this chapter, we describe the plasmid vector systems routinely used by our research group for the generation of either hpRNA-derived siRNAs or amiRNAs.

The roles of plant dsRNA-binding proteins in RNAi-like pathways

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway. © 2008 Federation of European Biochemical Societies.

Cloning and characterization of microRNAs from Brassica napus

A library containing approximately 40,000 small RNA sequences was constructed for Brassica napus. Analysis of 3025 sequences obtained from this library resulted in the identification of 11 conserved miRNA families, which were validated by secondary structure prediction using surrounding sequences in the Brassica genome. Two 21 nt small RNA sequences reside within the arm of a pre-miRNA like stem-loop structure, making them likely candidates for novel non-conserved miRNAs in B. napus. Most of the conserved miRNAs were expressed at similar levels in a F1 hybrid B. napus line and its four double haploid progeny that showed marked variations in phenotypes, but many were differentially expressed between B. napus and Arabidopsis. The miR169 family was expressed at high levels in young leaves and stems, but was undetectable in roots and mature leaves, suggesting that miR169 expression is developmentally regulated in B. napus. © 2007 Federation of European Biochemical Societies.

Posttranscriptional gene silencing is not compromised in the Arabidopsis CARPEL FACTORY (DICER-LIKE1) mutant, a homolog of dicer-1 from Drosophila

Posttranscriptional silencing (PTGS) in plants, nematodes, Drosophila, and perhaps all eukaryotes operates by sequence-specific degradation or translational inhibition of the target mRNA. These processes are mediated by duplexed RNA. In Drosophila and nematodes, double-stranded (ds)RNA or self-complementary RNA is processed into fragments of approximately 21 nt by Dicer-1 [1, 2]. These small interfering RNAs (siRNAs) serve as guides to target degradation of homologous single-stranded (ss)RNA [1, 3]. In some cases, the approximately 21 nt guide fragments derived from endogenous, imperfectly self-complementary RNAs cause translational inhibition of their target mRNAs, with which they have substantial, but not perfect sequence complementarity [4-6]. These small temporal RNAs (stRNAs) belong to a class of noncoding microRNAs (miRNAs), 20-24 nt in length, that are found in flies, plants, nematodes, and mammals [4, 6-12]. In nematodes, the Dicer-1 enzyme catalyzes the production of both siRNA and stRNA [2, 13-15]. Mutation of the Arabidopsis Dicer-1 homolog, CARPEL FACTORY (CAF), blocks miRNA production [1, 4, 16-18]. Here, we report that the same caf mutant does not block either PTGS or siRNA production induced by self-complementary hairpin RNA. This suggests either that this mutation only impairs miRNA formation or, more interestingly, that plants have two distinct dicer-like enzymes, one for miRNA and another for siRNAi production.

High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation

Two transgenic callus lines of rice, stably expressing a β-glucuronidase (GUS) gene, were supertransformed with a set of constructs designed to silence the resident GUS gene. An inverted-repeat (i/r) GUS construct, designed to produce mRNA with self-complementarity, was much more effective than simple sense and antisense constructs at inducing silencing. Supertransforming rice calluses with a direct-repeat (d/r) construct, although not as effective as those with the i/r construct, was also substantially more effective in silencing the resident GUS gene than the simple sense and antisense constructs. DNA hybridisation analyses revealed that every callus line supertransformed with either simple sense or antisense constructs, and subsequently showing GUS silencing, had the silence-inducing transgenes integrated into the plant genome in inverted-repeat configurations. The silenced lines containing i/r and d/r constructs did not necessarily have inverted-repeat T-DNA insertions. There was significant methylation of the GUS sequences in most of the silenced lines but not in the unsilenced lines. However, demethylation treatment of silenced lines with 5-azacytidine did not reverse the post-transcriptional gene silencing (PTGS) of GUS. Whereas the levels of RNA specific to the resident GUS gene were uniformly low in the silenced lines, RNA specific to the inducer transgenes accumulated to a substantial level, and the majority of the i/r RNA was unpolyadenylated. Altogether, these results suggest that both sense- and antisense-mediated gene suppression share a similar molecular basis, that unpolyadenylated RNA plays an important role in PTGS, and that methylation is not essential for PTGS.

The genome organization and affinities of an Australian isolate of carrot mottle umbravirus

The genomic sequence of an Australian isolate of carrot mottle umbravirus (CMoV-A) was determined from cDNA generated from dsRNA. This provides the first data on the genome organization and phylogeny of an umbravirus. The 4201-nucleotide genome contains four major open reading frames (ORFs). Analysis suggests that ORF2 encodes an RNA-dependent RNA polymerase, that ORF4 encodes a movement protein, and that the virus has no coat protein gene. The functions of ORFs 1 and 3 remain unknown. ORF2 is probably translated following ribosomal frameshifting. ORFs 3 and 4 are probably translated from a subgenomic mRNA. Sequence comparisons showed CMoV-A to be closely related to pea enation mosaic RNA2 NA2), but also to have affinities with the Bromoviridae. These findings shed light on the relationships between the luteoviruses, PEMV, and the umbraviruses and on the relationships between the carmo-like viruses and the Bromoviridae.

Infectious in vitro transcripts from a cloned cDNA of barley yellow dwarf virus

A full-length cDNA clone of barley yellow dwarf virus (BYDV-PAV serotype) has been constructed and fused to the bacteriophage T7 RNA polymerase promoter. RNA transcripts produced in vitro, either capped or uncapped, were infectious in Triticum monococcum protoplasts. Protoplasts inoculated with in vitro-transcribed BYDV RNA accumulated coat protein, synthesized new viral RNAs, and produced virus particles. Aphid feeding on extracts from protoplasts inoculated with in vitro RNA transcripts can be used to transfer the virus progeny to whole plants. Introduction of mutations which interrupt specific BYDV-PAV open reading frames (ORFs) V and VI eliminated infectivity while an ORF I mutant remained infectious. Infectious RNA transcripts derived from BYDV cDNA clones will facilitate analysis of the molecular aspects of BYDV infection and further enhance our understanding of this economically important virus.

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.

Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.

Global analysis of serum microRNAs as potential biomarkers for lung adenocarcinoma

Early diagnosis and the ability to predict the most relevant treatment option for individuals is essential to improve clinical outcomes for non-small cell lung cancer (NSCLC) patients. Adenocarcinoma (ADC), a subtype of NSCLC, is the single biggest cancer killer and therefore an urgent need to identify minimally invasive biomarkers to enable early diagnosis. Recent studies, by ourselves and others, indicate that circulating miRNA s have potential as biomarkers. Here we applied global profiling approaches in serum from patients with ADC of the lung to explore if miRNA s have potential as diagnostic biomarkers. This study involved RNA isolation from 80 sera specimens including those from ADC patients (equal numbers of stages 1, 2, 3, and 4) and age- and gender-matched controls (n = 40 each). Six hundred and sixty-seven miRNA s were co-analyzed in these specimens using TaqMan low density arrays and qPCR validation using individual miRNA s. Overall, approximately 390 and 370 miRNA s were detected in ADC and control sera, respectively. A group of 6 miRNA s, miR-30c-1* (AU C = 0.74; P < 0.002), miR-616(AU C = 0.71; P = 0.001), miR-146b-3p (AU C = 0.82; P < 0.0001), miR-566 (AU C = 0.80; P < 0.0001), miR-550 (AU C = 0.72; P = 0.0006), and miR-939 (AU C = 0.82; P < 0.0001) was found to be present at substantially higher levels in ADC compared with control sera. Conversely, miR-339-5p and miR-656 were detected at substantially lower levels in ADC sera (co-analysis resulting in AU C = 0.6; P = 0.02). Differences in miRNA profile identified support circulating miRNA s having potential as diagnostic biomarkers for ADC. More extensive studies of ADC and control serum specimens are warranted to independently validate the potential clinical relevance of these miRNA s as minimally invasive biomarkers for ADC.

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.