Endogenous and Synthetic MicroRNAs Stimulate Simultaneous, Efficient, and Localized Regulation of Multiple Targets in Diverse Species

Department of Plant Sciences, Weizman Institute of Science, Rehovot, 76100, Israel.
The Plant Cell (Impact Factor: 9.34). 06/2006; 18(5):1134-51. DOI: 10.1105/tpc.105.040725
Source: PubMed


Recent studies demonstrated that pattern formation in plants involves regulation of transcription factor families by microRNAs (miRNAs). To explore the potency, autonomy, target range, and functional conservation of miRNA genes, a systematic comparison between plants ectopically expressing pre-miRNAs and plants with corresponding multiple mutant combinations of target genes was performed. We show that regulated expression of several Arabidopsis thaliana pre-miRNA genes induced a range of phenotypic alterations, the most extreme ones being a phenocopy of combined loss of their predicted target genes. This result indicates quantitative regulation by miRNA as a potential source for diversity in developmental outcomes. Remarkably, custom-made, synthetic miRNAs vectored by endogenous pre-miRNA backbones also produced phenocopies of multiple mutant combinations of genes that are not naturally regulated by miRNA. Arabidopsis-based endogenous and synthetic pre-miRNAs were also processed effectively in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). Synthetic miR-ARF targeting Auxin Response Factors 2, 3, and 4 induced dramatic transformations of abaxial tissues into adaxial ones in all three species, which could not cross graft joints. Likewise, organ-specific expression of miR165b that coregulates the PHABULOSA-like adaxial identity genes induced localized abaxial transformations. Thus, miRNAs provide a flexible, quantitative, and autonomous platform that can be employed for regulated expression of multiple related genes in diverse species.

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Available from: Alexander Goldshmidt, Feb 16, 2015
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    • "Plant miRNAs target transcripts with highly complementary sequence through direct AGO-mediated endonucleolytic cleavage, or through other cleavage-independent mechanisms (Axtell, 2013). Artificial miRNAs (amiRNAs) can be produced accurately by modifying the miRNA/miRNA* sequence within a functional MIRNA precursor (Alvarez et al., 2006; Schwab et al., 2006). AmiRNAs have been used in plants to selectively and effectively knockdown reporter and endogenous genes, non-coding RNAs and viruses (Ossowski et al., 2008; Tiwari et al., 2014). "
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    ABSTRACT: Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distal stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5'-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    The Plant Journal 03/2015; DOI:10.1111/tpj.12835 · 5.97 Impact Factor
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    • "The observations related to effectivity and consistency of different miRNA precursors for amiRNA generation suggest that a particular pri-miRNA is not universally optimal for all the amiR- NAs. Further, some reports also indicated that processing of amiRNAs was shifted one nucleotide ahead during heterologous expression of pre-miR164b (Alvarez et al. 2006). Therefore, choosing a specific pre-miRNA is also a set of rules for silencing of legitimate targets, and despite the frequent use of phylogenetically conserved pre-miRNA, the backbone from same species should be preferred. "
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    ABSTRACT: Homology based gene silencing has emerged as a convenient approach for repressing expression of genes in order to study their functions. For this purpose, several antisense or small interfering RNA based gene silencing techniques have been frequently employed in plant research. Artificial microRNAs (amiRNAs) mediated gene silencing represents one of such techniques which can utilize as a potential tool in functional genomics. Similar to microRNAs, amiRNAs are single-stranded, approximately 21 nt long, and designed by replacing the mature miRNA sequences of duplex within pre-miRNAs. These amiRNAs are processed via small RNA biogenesis and silencing machinery and deregulate target expression. Holding to various refinements, amiRNA technology offers several advantages over other gene silencing methods. This is a powerful and robust tool, and could be applied to unravel new insight of metabolic pathways and gene functions across the various disciplines as well as in translating observations for improving favourable traits in plants. This review highlights general background of small RNAs, improvements made in RNAi based gene silencing, implications of amiRNA in gene silencing, and describes future themes for improving value of this technology in plant science.
    Plant Molecular Biology 07/2014; 86(1-2). DOI:10.1007/s11103-014-0224-7 · 4.26 Impact Factor
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    • "A few classes of plant miRNA precursor have been successfully engineered to silence genes of interest, by replacing natural miRNAs with specifically modified or “designed” miRNA molecules, termed artificial miRNAs (amiRNAs) [24]–[28]. AmiRNAs engineered by modifications of Arabidopsis MIR319a precursor, the precursor backbone most commonly used, have been successfully used to induce gene silencing in Arabidopsis [29], tobacco [25], eggplant [30], soybean [31], Medicago [32], [33], and Physcomitrella patens [34]. Recently, this technology has also been used to silence genes in the vegetative cells of pollen in Petunia inflata [35]. "
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    ABSTRACT: Although artificial microRNA (amiRNA) technology has been used frequently in gene silencing in plants, little research has been devoted to investigating the accuracy of amiRNA precursor processing. In this work, amiRNAchs1 (amiRchs1), based on the Arabidopsis miR319a precursor, was expressed in order to suppress the expression of CHS genes in petunia. The transgenic plants showed the CHS gene-silencing phenotype. A modified 5' RACE technique was used to map small-RNA-directed cleavage sites and to detect processing intermediates of the amiRchs1 precursor. The results showed that the target CHS mRNAs were cut at the expected sites and that the amiRchs1 precursor was processed from loop to base. The accumulation of small RNAs in amiRchs1 transgenic petunia petals was analyzed using the deep-sequencing technique. The results showed that, alongside the accumulation of the desired artificial microRNAs, additional small RNAs that originated from other regions of the amiRNA precursor were also accumulated at high frequency. Some of these had previously been found to be accumulated at low frequency in the products of ath-miR319a precursor processing and some of them were accompanied by 3'-tailing variant. Potential targets of the undesired small RNAs were discovered in petunia and other Solanaceae plants. The findings draw attention to the potential occurrence of undesired target silencing induced by such additional small RNAs when amiRNA technology is used. No appreciable production of secondary small RNAs occurred, despite the fact that amiRchs1 was designed to have perfect complementarity to its CHS-J target. This confirmed that perfect pairing between an amiRNA and its targets is not the trigger for secondary small RNA production. In conjunction with the observation that amiRNAs with perfect complementarity to their target genes show high efficiency and specificity in gene silencing, this finding has an important bearing on future applications of amiRNAs in gene silencing in plants.
    PLoS ONE 06/2014; 9(6):e98783. DOI:10.1371/journal.pone.0098783 · 3.23 Impact Factor
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