118 research outputs found
The Nuclear dsRNA Binding Protein HYL1 Is Required for MicroRNA Accumulation and Plant Development, but Not Posttranscriptional Transgene Silencing
AbstractMicroRNAs (miRNAs) are 21–24 nucleotides long molecules processed from imperfect double-stranded RNAs (dsRNAs). They regulate gene expression by targeting complementary mRNA for cleavage or interfering with their translation [1–6]. In Arabidopsis, point mutations in or short truncations of the nuclear DICER-LIKE1 (DCL1) or HEN1 protein reduce miRNA accumulation and increase uncleaved target mRNAs accumulation, resulting in developmental abnormalities [7–12]. Here, we show that miRNA accumulation also depends on the activity of HYL1, a nuclear dsRNA binding protein [13]. hyl1 mutants exhibit developmental defects overlapping with that of dcl1 and hen1 mutants, suggesting that DCL1, HEN1, and HYL1 act together in the nucleus. We validate additional target mRNAs and show that reduced miRNA accumulation in hyl1 correlates with an increased accumulation of uncleaved target mRNAs, including meristem- and auxin-related genes, providing clues for the developmental abnormalities of hyl1 and for the previous identification of hyl1 as a mutant with altered responses to phytohormones [13]. Lastly, we show that posttranscriptional transgene silencing occurs in hyl1, suggesting that HYL1 has specialized function in the plant miRNA pathway, whereas the HYL1-related RDE-4 and R2D2 proteins associate with DICER in the cytoplasm and act in the RNAi pathway in C. elegans and Drosophila, respectively [14–15]
A single transgene locus triggers both transcriptional and post-transcriptional silencing through double-stranded RNA production
Silencing of a target locus by an unlinked silencing locus can result from transcription inhibition (transcriptional gene
silencing; TGS) or mRNA degradation (post-transcriptional gene silencing; PTGS), owing to the production of double-stranded
RNA (dsRNA) corresponding to promoter or transcribed sequences, respectively. The involvement of distinct cellular components
in each process suggests that dsRNA-induced TGS and PTGS likely result from the diversification of an ancient common mechanism.
However, a strict comparison of TGS and PTGS has been difficult to achieve because it generally relies on the analysis of
distinct silencing loci. We describe a single transgene locus that triggers both TGS and PTGS, owing to the production of
dsRNA corresponding to promoter and transcribed sequences of different target genes. We describe mutants and epigenetic variants
derived from this locus and propose a model for the production of dsRNA. Also, we show that PTGS, but not TGS, is graft-transmissible,
which together with the sensitivity of PTGS, but not TGS, to RNA viruses that replicate in the cytoplasm, suggest that the
nuclear compartmentalization of TGS is responsible for cell-autonomy. In contrast, we contribute local and systemic trafficking
of silencing signals and sensitivity to viruses to the cytoplasmic steps of PTGS and to amplification steps that require high
levels of target mRNAs
The origin and effect of small RNA signaling in plants
Given their sessile condition, land plants need to integrate environmental cues rapidly and send signal throughout the organism to modify their metabolism accordingly. Small RNA (sRNA) molecules are among the messengers that plant cells use to carry such signals. These molecules originate from fold-back stem-loops transcribed from endogenous loci or from perfect double-stranded RNA produced through the action of RNA-dependent RNA polymerases. Once produced, sRNAs associate with Argonaute (AGO) and other proteins to form the RNA-induced silencing complex (RISC) that executes silencing of complementary RNA molecules. Depending on the nature of the RNA target and the AGO protein involved, RISC triggers either DNA methylation or chromatin modification (leading to transcriptional gene silencing, TGS) or RNA cleavage or translational inhibition (leading to post-transcriptional gene silencing, PTGS). In some cases, sRNAs move to neighboring cells and/or to the vascular tissues for long-distance trafficking. Many genes are involved in the biogenesis of sRNAs and recent studies have shown that both their origin and their protein partners have great influence on their activity and range. Here we summarize the work done to uncover the mode of action of the different classes of sRNA with special emphasis on their movement and how plants can take advantage of their mobility. We also review the various genetic requirements needed for production, movement and perception of the silencing signal
The Conserved RNA Trafficking Proteins HPR1 and TEX1 Are Involved in the Production of Endogenous and Exogenous Small Interfering RNA in Arabidopsis
The miRNA pathway limits AGO1 availability during siRNA-mediated PTGS defense against exogenous RNA
In plants, most microRNAs (miRNAs) and several endogenous small interfering RNAs (siRNAs) bind to ARGONAUTE1 (AGO1) to regulate the expression of endogenous genes through post-transcriptional gene silencing (PTGS). AGO1 also participates in a siRNA-mediated PTGS defense response that thwarts exogenous RNA deriving from viruses and transgenes. Here, we reveal that plants supporting transgene PTGS exhibit increased levels of AGO1 protein. Moreover, increasing AGO1 levels either by mutating miRNA pathway components or, more specifically, by impairing miR168-directed regulation of AGO1 mRNA leads to increased PTGS efficiency, indicating that the miRNA pathway dampens the efficiency of PTGS, likely by limiting the availability of AGO1. We propose that during the transgene PTGS initiation phase, transgene siRNAs and endogenous siRNAs and miRNA compete to bind to AGO1, leading to a transient reduction in AGO1–miR168 complexes and a decline in AGO1 mRNA cleavage. The concomitant increase in AGO1 protein levels would facilitate the formation of AGO1–transgene siRNA complexes and the entry into the PTGS amplification phase. We suggest that the miRNA pathway imposes an important limitation on PTGS efficiency, which could help protect endogenous mRNAs from being routinely targeted by PTGS
A Novel fry1 Allele Reveals the Existence of a Mutant Phenotype Unrelated to 5′->3′ Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots
BACKGROUND Mutations in the FRY1/SAL1 Arabidopsis locus are highly pleiotropic, affecting drought tolerance, leaf shape and root growth. FRY1 encodes a nucleotide phosphatase that in vitro has inositol polyphosphate 1-phosphatase and 3',(2'),5'-bisphosphate nucleotide phosphatase activities. It is not clear which activity mediates each of the diverse biological functions of FRY1 in planta. PRINCIPAL FINDINGS A fry1 mutant was identified in a genetic screen for Arabidopsis mutants deregulated in the expression of Pi High affinity Transporter 1;4 (PHT1;4). Histological analysis revealed that, in roots, FRY1 expression was restricted to the stele and meristems. The fry1 mutant displayed an altered root architecture phenotype and an increased drought tolerance. All of the phenotypes analyzed were complemented with the AHL gene encoding a protein that converts 3'-polyadenosine 5'-phosphate (PAP) into AMP and Pi. PAP is known to inhibit exoribonucleases (XRN) in vitro. Accordingly, an xrn triple mutant with mutations in all three XRNs shared the fry1 drought tolerance and root architecture phenotypes. Interestingly these two traits were also complemented by grafting, revealing that drought tolerance was primarily conferred by the rosette and that the root architecture can be complemented by long-distance regulation derived from leaves. By contrast, PHT1 expression was not altered in xrn mutants or in grafting experiments. Thus, PHT1 up-regulation probably resulted from a local depletion of Pi in the fry1 stele. This hypothesis is supported by the identification of other genes modulated by Pi deficiency in the stele, which are found induced in a fry1 background. CONCLUSIONS/SIGNIFICANCE Our results indicate that the 3',(2'),5'-bisphosphate nucleotide phosphatase activity of FRY1 is involved in long-distance as well as local regulatory activities in roots. The local up-regulation of PHT1 genes transcription in roots likely results from local depletion of Pi and is independent of the XRNs.This work was supported by an ANR-GENOPLANT grant (RIBOROOT-ANR06 GPLA 011) and the CEA agency. Array hybridizations have been partly supported by RNG (Réseau National des Génopoles, Evry, France). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study
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