mRNA Decay of Most Arabidopsis miRNA Targets Requires Slicer Activity of AGO1

Arribas-Hernández, Laura; Kielpinski, Lukasz Jan; Brodersen, Peter

doi:10.1104/pp.16.00231

Cited by 44 publications

(50 citation statements)

References 79 publications

(99 reference statements)

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“…Even when all three functional members of the AGO4 clade (Vaucheret, ) are removed in the ago4‐4 / ago6‐2 / ago9‐1 triple mutant, accumulation of 24 nt siRNAs from most loci is unaffected. This situation seems to contrast with the relationship between the major Arabidopsis miRNA‐binding Argonaute AGO1 and miRNAs: In the null mutant ago1‐3 , accumulation of the majority of miRNAs is decreased (Vaucheret et al ., ; Arribas‐Hernández et al ., ). Why might the majority of 24 nt siRNAs maintain stable accumulation levels in the absence of AGO4, AGO6, and AGO9?…”

Section: Discussionmentioning

confidence: 97%

“…In other systems, two general functions of AGO‐catalyzed slicing have been described: Slicing of passenger strands during AGO‐loading of a small RNA duplex (Matranga et al ., ), and slicing of target RNAs (Qi et al ., ). For Arabidopsis AGO1, both in vitro and in vivo experiments demonstrate that AGO1‐catalyzed slicing is not required for miRNA loading, but is required for many aspects of target regulation (Iki et al ., ; Carbonell et al ., ; Arribas‐Hernández et al ., ,b). In contrast, in vitro and in vivo data have demonstrated that AGO4‐catalyzed slicing is required for passenger‐strand removal during siRNA loading and subsequent nuclear localization of the AGO4‐siRNA complex (Ye et al ., ).…”

Section: Discussionmentioning

confidence: 99%

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AGO4 is specifically required for heterochromatic siRNA accumulation at Pol V‐dependent loci in Arabidopsis thaliana

Wang

Axtell

2017

The Plant Journal

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SUMMARYIn plants, 24 nucleotide long heterochromatic siRNAs (het-siRNAs) transcriptionally regulate gene expression by RNA-directed DNA methylation (RdDM). The biogenesis of most het-siRNAs depends on the plantspecific RNA polymerase IV (Pol IV), and ARGONAUTE4 (AGO4) is a major het-siRNA effector protein.Through genome-wide analysis of sRNA-seq data sets, we found that AGO4 is required for the accumulation of a small subset of het-siRNAs. The accumulation of AGO4-dependent het-siRNAs also requires several factors known to participate in the effector portion of the RdDM pathway, including RNA POLYMERASE V (POL V), DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and SAWADEE HOMEODOMAIN HOMOLOGUE 1 (SHH1). Like many AGO proteins, AGO4 is an endonuclease that can 'slice' RNAs. We found that a slicing-defective AGO4 was unable to fully recover AGO4-dependent het-siRNA accumulation from ago4 mutant plants. Collectively, our data suggest that AGO4-dependent siRNAs are secondary siRNAs dependent on the prior activity of the RdDM pathway at certain loci.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

AGO4 is specifically required for heterochromatic siRNA accumulation at Pol V‐dependent loci in Arabidopsis thaliana

Wang

Axtell

2017

The Plant Journal

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“…This approach enabled us to FLAG affinitypurify AGO1 protein for biochemical analysis from inflorescence tissue of ago1-3 heterozygotes and to analyze phenotypes in ago1-3 homozygotes expressing the different wild-type or point mutant constructs. FLAG-AGO1 WT fully complemented ago1-3, while the expression of each of the four point mutants in ago1-3 caused strong developmental defects similar to, albeit slightly distinct from, the null (Arribas-Hernández et al, 2016). The residues Asp-762 and Asp-848 have been shown directly to be required for slicing (Baumberger and Baulcombe, 2005;Iki et al, 2010), and indirect tests indicate a requirement for His-988 (Carbonell et al, 2012).…”

Section: Different Catalytic Roles Of Mg 2+ -Coordinating Active-sitementioning

confidence: 97%

“…Data for additional TAS transcripts can be found in Supplemental Figure 4, and counts of tasiRNAs are detailed in Supplemental Data Set 1. Membranes 1 to 4 that were used to hybridize siRNA probes in Figure 3A are the same as the ones used for miRNA analysis in Figure 2A of Arribas-Hernández et al (2016). Accordingly, the U6 loading controls are the same in the corresponding panels.…”

Section: Mir173 Does Not Guide Cleavage Of Tas1/2 Precursors In Seedlmentioning

confidence: 99%

The Slicer Activity of ARGONAUTE1 is Required Specifically for the Phasing, Not Production, of Trans-Acting Short Interfering RNAs in Arabidopsis

et al. 2016

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ARGONAUTE1 (AGO1) mediates posttranscriptional silencing by microRNAs (miRNAs) and short interfering RNAS (siRNAs). AGO1-catalyzed RNA cleavage (slicing) represses miRNA targets, but current models also highlight the roles of slicing in formation of siRNAs and siRNA-AGO1 complexes. miRNA-guided slicing is required for biogenesis of phased, trans-acting siRNAs (tasiRNAs), whose cleaved precursor fragments are converted to double-stranded RNA by RNA-dependent RNA polymerase 6 (RDR6). In addition, unwinding of duplex siRNA bound to AGO1 requires passenger strand cleavage in vitro. In this study, we analyze how mutation of four metal ion-coordinating residues of Arabidopsis thaliana AGO1 affects slicer activity in vitro and siRNA function in vivo. We show that while all four residues are required for slicer activity, they do not contribute equally to catalysis. Moreover, passenger strand cleavage is required for assembly of active AGO1-siRNA complexes in vivo, and many AGO1-bound siRNAs are trimmed in the absence of slicer activity. Remarkably, seedlings defective in AGO1 slicer activity produce abundant siRNAs from tasiRNA loci in vivo. These siRNAs depend on RDR6 and SUPPRESSOR OF GENE SILENCING3, but unlike wild-type tasiRNAs, they are unphased. These results demonstrate that slicing is solely required for phase definition of tasiRNAs, and they strongly support recruitment of RDR6 by AGO1 rather than by cleavage fragments.

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“…One of several possible pitfalls of this experiment is that miR166 also associates efficiently with AGO10 . Since ago10 mutants are not viable in an ago1 slicer deficient background , we analysed the mutant phb‐1d in which incorrect splicing leads to complete disruption of the miR165/166 target site . Thus, in phb‐1d all miR165/166 guided cleavage of the PHB mRNA is abolished, but miRNA‐guided regulation of other targets, including of other miR165/166 targets, is not directly affected by this mutation.…”

Section: Resultsmentioning

confidence: 99%

A new class of genic nuclear RNA species in Arabidopsis

et al. 2018

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Targeting of ArabidopsisPHABULOSA (PHB) mRNA by miR166 has been implicated in gene body methylation at the PHB locus. We report that the PHB locus produces an array of stable nuclear RNA species that are neither polyadenylated nor capped. Their biogenesis requires neither RNA polymerases IV/V nor miR166-guided cleavage. The PHB RNAs are insensitive to mutation of nuclear RNA decay pathways and are conserved in several Brassicaceae species, suggesting functional relevance. Similar RNA species are also produced by another body-methylated locus encoding the miR414 target eIF2. Our data reveal the existence of a new class of genic nuclear RNA species.

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mRNA Decay of Most Arabidopsis miRNA Targets Requires Slicer Activity of AGO1

Cited by 44 publications

References 79 publications

AGO4 is specifically required for heterochromatic siRNA accumulation at Pol V‐dependent loci in Arabidopsis thaliana

AGO4 is specifically required for heterochromatic siRNA accumulation at Pol V‐dependent loci in Arabidopsis thaliana

The Slicer Activity of ARGONAUTE1 is Required Specifically for the Phasing, Not Production, of Trans-Acting Short Interfering RNAs in Arabidopsis

A new class of genic nuclear RNA species in Arabidopsis

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