Article

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

ABSTRACT

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.

Download full-text

Full-text

Available from: Alexander Goldshmidt, Feb 16, 2015
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Mar 2015 · The Plant Journal
  • Source
    • "The premiR-PIP1 and synthetic genes were synthesized by DNA 2.0, based on a premiR164 backbone (Alvarez et al., 2006). We used the Web-based mfold program (http://mfold.rna.albany.edu) to produce premiRNA stem-loop representations (Zuker, 2003). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydraulics, in general, and extravascular, radial hydraulic conductance in leaves (Kleaf), in particular, is still fairly limited. We hypothesized that the aquaporins of the vascular bundle sheath (BS) cells regulate Kleaf. To examine this hypothesis, AQP genes were silenced using artificial microRNAs (amiRNAs) that were expressed constitutively or specifically targeted to the BS. MicroRNA sequences were designed to target all five AQP genes from thePIP1 subfamily. Our results show that the constitutively silenced PIP1 (35S promoter) plants had decreased PIP1 transcript and protein levels and decreased mesophyll and BS osmotic water permeability (Pf), mesophyll conductance of CO2 (gm), photosynthesis (AN), Kleaf, transpiration and shoot biomass. Plants in which the PIP1 subfamily was silenced only in the BS (SCR:mir plants) exhibited decreased mesophyll and BS Pf and decreased Kleaf, but no decreases in the rest of the parameters listed above, with the net result of increased shoot biomass. We excluded the possibility of SCR promoter activity in the mesophyll. Hence, the fact that SCR:mir mesophyll exhibited reduced Pf, but not reduced gm suggests that the BS-mesophyll hydraulic continuum acts as a feed-forward control signal. The role of AQPs in the hierarchy of the hydraulic signal pathway controlling leaf water status under normal and limited-water conditions is discussed. Keywords: Plasma membrane intrinsic proteins (PIPs), Aquaporins (AQPs), Bundle sheath, Leaf hydraulic conductivity (Kleaf), Artificial microRNA (amiRNA).
    Full-text · Article · Sep 2014 · Plant physiology
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jul 2014 · Plant Molecular Biology
Show more