Sizolwenkosi Mlotshwa

RNA Silencing and the Mobile Silencing Signal

RNA silencing is a sequence-specific RNA degradation mechanism that occurs in a broad range of eukaryotic organisms including fungi (quelling), animals (RNA interference [RNAi]), and plants (post-transcriptional gene silencing). In all these organisms, the process is triggered by double-stranded RNA

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.

DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis.

Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2–but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.

Oncogenic Wip1 Phosphatase Is Inhibited by miR-16 in the DNA Damage Signaling Pathway

Wild-type p53-induced phosphatase 1 (Wip1) was identified as an oncogene amplified and overexpressed in several human cancers. Recent evidence suggested that Wip1 is a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 dephosphorylates several key DNA damage-responsive proteins and reverses DNA damage-induced cell cycle checkpoints. Previous reports showed that Wip1 was transcriptionally induced by p53 at the early stage of the DNA damage response. To investigate the temporal and functional regulation of Wip1, we identified a microRNA, miR-16, that specifically targets the mRNA of Wip1 and thus negatively regulates the expression level of Wip1. miR-16 itself is induced immediately after DNA damage. Therefore, the increase in Wip1 protein level is significantly postponed compared with that of its mRNA level, preventing a premature inactivation of ATM/ATR signaling and allowing a functional completion of the early DNA damage response. To better understand miR-16 biological functions in the context of cancer cells, we examined its expression in mammary tumor stem cells and found it to be markedly downregulated in mammary tumor stem cells. Overexpression of miR-16 or inhibition of Wip1 suppresses the self-renewal and growth of mouse mammary tumor stem cells and sensitizes MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin. Together, our results suggest an important role of miR-16 in the regulation of Wip1 phosphatase in the DNA damage response and mammary tumorigenesis.

Two plant viral suppressors of silencing require the ethylene-inducible host transcription factor RAV2 to block RNA silencing.

RNA silencing is a highly conserved pathway in the network of interconnected defense responses that are activated during viral infection. As a counter-defense, many plant viruses encode proteins that block silencing, often also interfering with endogenous small RNA pathways. However, the mechanism of action of viral suppressors is not well understood and the role of host factors in the process is just beginning to emerge. Here we report that the ethylene-inducible transcription factor RAV2 is required for suppression of RNA silencing by two unrelated plant viral proteins, potyvirus HC-Pro and carmovirus P38. Using a hairpin transgene silencing system, we find that both viral suppressors require RAV2 to block the activity of primary siRNAs, whereas suppression of transitive silencing is RAV2-independent. RAV2 is also required for many HC-Pro-mediated morphological anomalies in transgenic plants, but not for the associated defects in the microRNA pathway. Whole genome tiling microarray experiments demonstrate that expression of genes known to be required for silencing is unchanged in HC-Pro plants, whereas a striking number of genes involved in other biotic and abiotic stress responses are induced, many in a RAV2-dependent manner. Among the genes that require RAV2 for induction by HC-Pro are FRY1 and CML38, genes implicated as endogenous suppressors of silencing. These findings raise the intriguing possibility that HC-Pro-suppression of silencing is not caused by decreased expression of genes that are required for silencing, but instead, by induction of stress and defense responses, some components of which interfere with antiviral silencing. Furthermore, the observation that two unrelated viral suppressors require the activity of the same factor to block silencing suggests that RAV2 represents a control point that can be readily subverted by viruses to block antiviral silencing.

Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana.

Floral patterning and morphogenesis are controlled by many transcription factors including floral homeotic proteins, by which floral organ identity is determined. Recent studies have uncovered widespread regulation of transcription factors by microRNAs (miRNAs), ~21-nucleotide non-coding RNAs that regulate protein-coding RNAs through transcript cleavage and/or translational inhibition. The regulation of the floral homeotic gene APETALA2 (AP2) by miR172 is crucial for normal Arabidopsis flower development and is likely to be conserved across plant species. Here we probe the activity of the AP2/miR172 regulatory circuit in a heterologous Solanaceae species, Nicotiana benthamiana. We generated transgenic N. benthamiana lines expressing Arabidopsis wild type AP2 (35S::AP2), miR172-resistant AP2 mutant (35S::AP2m3) and MIR172a-1 (35S::MIR172) under the control of the cauliflower mosaic virus 35S promoter. 35S::AP2m3 plants accumulated high levels of AP2 mRNA and protein and exhibited floral patterning defects that included proliferation of numerous petals, stamens and carpels indicating loss of floral determinacy. On the other hand, nearly all 35S::AP2 plants accumulated barely detectable levels of AP2 mRNA or protein and were essentially non-phenotypic. Overall, the data indicated that expression of the wild type ArabidopsisAP2 transgene was repressed at the mRNA level by an endogenous N. benthamiana miR172 homologue that could be detected using Arabidopsis miR172 probe. Interestingly, 35S::MIR172 plants had sepal-to-petal transformations and/or more sepals and petals, suggesting interference with N. benthamiana normal floral homeotic gene function in perianth organs. Our studies uncover the potential utility of the ArabidopsisAP2/miR172 system as a tool for manipulation of floral architecture and flowering time in non-model plants.

Ectopic DICER-LIKE1 Expression in P1/HC-Pro Arabidopsis Rescues Phenotypic Anomalies but Not Defects in MicroRNA and Silencing Pathways

Expression of the viral silencing suppressor P1/HC-Pro in plants causes severe developmental anomalies accompanied by defects in both short interfering RNA (siRNA) and microRNA (miRNA) pathways. P1/HC-Pro transgenic lines fail to accumulate the siRNAs that mediate RNA silencing and are impaired in both miRNA processing and function, accumulating abnormally high levels of miRNA/miRNA* processing intermediates as well as miRNA target messages. Both miRNA and RNA silencing pathways require participation of DICER-LIKE (DCL) ribonuclease III-like enzymes. Here, we investigate the effects of overexpressing DCL1, one of four Dicers in Arabidopsis thaliana, on P1/HC-Pro–induced defects in development and small RNA metabolism. Expression of a DCL1 cDNA transgene (35S:DCL1) produced a mild gain-of-function phenotype and largely rescued dcl1 mutant phenotypes. The 35S:DCL1 plants were competent for virus-induced RNA silencing but were impaired in transgene-induced RNA silencing and in the accumulation of some miRNAs. Ectopic DCL1 largely alleviated developmental anomalies in P1/HC-Pro plants but did not correct the P1/HC-Pro–associated defects in small RNA pathways. The ability of P1/HC-Pro plants to suppress RNA silencing and the levels of miRNAs, miRNA*s, and miRNA target messages in these plants were essentially unaffected by ectopic DCL1. These data suggest that P1/HC-Pro defects in development do not result from general impairments in small RNA pathways and raise the possibility that DCL1 participates in processes in addition to miRNA biogenesis.

A novel chemopreventive strategy based on therapeutic microRNAs produced in plants

Dear Editor, MicroRNAs (miRNAs) are small non-coding RNAs that play a critical role in regulation of gene expression in nearly all eukaryotic organisms, including mammals. In humans, an estimated 60% of all protein-coding genes are targeted by miRNAs, affecting virtually every physiological process in the body [1]. In addition, a diverse array of human diseases is associated with dysregulation of miRNAs [2]. In many forms of cancer, for example, certain miRNAs, termed tumor suppressor miRNAs, are downregulated in diseased cells. Restoration of the downregulated tumor suppressor miRNA has been shown to block one or more steps in oncogenesis in animal models and cell culture systems. Thus, the therapeutic potential of tumor suppressor miRNAs has been experimentally confirmed and is now widely recognized. However , systemic delivery of such therapeutic small RNAs in humans is challenging and numerous delivery options are currently under investigation. We have investigated the possibility of an effective oral delivery system for therapeutic miRNAs. It has long been known that ingested RNA from food sources is tak-en up by the digestive system in nematodes and insects and can control the expression of genes in those organisms [3]. More recent evidence suggests that a similar phenomenon might occur in humans and other mammals [4]. These data indicate that plant miRNAs from foods are absorbed by cells of the mammalian digestive tract and packaged into microvesicles, which protect them from degradation. The miRNAs are then trafficked via the bloodstream to a variety of tissues, where they are capable of regulating the expression of mammalian genes. Such work has generated considerable excitement because it raises the possibility of bioengineering edible plants to produce therapeutic miRNAs that could then be delivered to affected tissues by ingestion. However, the work has also generated controversy as several groups have subsequently reported being unable to detect ingest-ed plant miRNAs in mammalian tissues at levels significantly above background [5]. We addressed this controversy in experiments designed to both detect a therapeutic effect of ingested miRNAs and to demonstrate their uptake. Here we report that oral administration of a cocktail of tumor suppressor miRNAs reduced tumor burden in the well-established Apc Min/+ mouse model of colon cancer. The cocktail contains three validated tumor suppressor miRNAs (miR-34a, miR-143, and miR-145), synthesized with the exact nucleotide sequence of the mouse miRNAs, but with a methyl group on the 2′ position of the ribose of the 3′ terminal nucleotide, …

Transcriptional silencing induced by Arabidopsis T-DNA mutants is associated with 35S promoter siRNAs and requires genes involved in siRNA-mediated chromatin silencing

The utility of many T-DNA insertion mutant lines of Arabidopsis is compromised by their propensity to trigger transcriptional silencing of transgenes expressed from the CaMV 35S promoter. To try to circumvent this problem, we characterized the genetic requirements for maintenance of 35S promoter homology-dependent transcriptional gene silencing induced by the dcl3-1 (SALK_005512) T-DNA insertion mutant line. Surprisingly, even though DCL3 and RDR2 are known components of the siRNA-dependent transcriptional gene silencing pathway, transcriptional gene silencing of a 35S promoter-driven GUS hairpin transgene did occur in plants homozygous for the dcl3-1 T-DNA insertion and was unaffected by loss of function of RDR2. However, the transcriptional gene silencing was alleviated in dcl2 dcl3 dcl4 triple mutant plants and also by mutations in AGO4, NRPD2, HEN1 and MOM1. Thus, some T-DNA insertion mutant lines induce 35S promoter homology-dependent transcriptional silencing that requires neither DCL3 nor RDR2, but involves other genes known to function in siRNA-dependent transcriptional silencing. Consistent with these results, we detected 35S promoter siRNAs in dcl3-1 SALK line plants, suggesting that the 35S promoter homology-dependent silencing induced by some T-DNA insertion mutant lines is siRNA-mediated.

Developmental Defects Mediated by the P1/HC-Pro Potyviral Silencing Suppressor Are Not Due to Misregulation of AUXIN RESPONSE FACTOR 8

Misregulation of ARF8 does not underlie developmental defects in Arabidopsis expressing a P1/HC-Pro transgene. Plant viral suppressors of RNA silencing induce developmental defects similar to those caused by mutations in genes involved in the microRNA pathway. A recent report has attributed viral suppressor-mediated developmental defects to up-regulation of AUXIN RESPONSE FACTOR 8 (ARF8), a target of miR167. The key piece of evidence was that the developmental defects in transgenic Arabidopsis (Arabidopsis thaliana) expressing viral suppressors were greatly alleviated in the F1 progeny of a cross with plants carrying the arf8-6 mutation. Arf8-6 is a SALK line T-DNA insertion mutant, a class of mutations prone to inducing transcriptional silencing of transgenes expressed from the 35S promoter. We have reinvestigated the role of ARF8 in viral suppressor-mediated developmental defects, using two independent arf8 mutations and the P1/HC-Pro potyviral suppressor of silencing. Progeny of a cross between P1/HC-Pro transgenic Arabidopsis and the arf8-6 T-DNA insertion mutant showed little effect on the P1/HC-Pro phenotype in the F1 generation, but almost all arf8-6/P1/HC-Pro progeny had lost the phenotype in the F2 generation. However, the loss of phenotype in the F2 generation was not correlated with the number of functional copies of the ARF8 gene. Instead, it reflected transcriptional silencing of the P1/HC-Pro transgene, as evidenced by a pronounced decrease in P1/HC-Pro mRNA and the appearance of 35S promoter small interfering RNAs. Furthermore, an independent loss-of-function point mutation, Arf8-8, had no detectable effects on P1/HC-Pro phenotype in either the F1 or F2 generations. Together, these data argue against the previously reported role of increased ARF8 expression in developmental defects caused by P1/HC-Pro.