Publications by authors named "Allison C Mallory"

29 Publications

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Author Correction: In-cell identification and measurement of RNA-protein interactions.

Nat Commun 2020 07 8;11(1):3498. Epub 2020 Jul 8.

Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, 75005, France.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-17282-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343817PMC
July 2020

In-cell identification and measurement of RNA-protein interactions.

Nat Commun 2019 11 22;10(1):5317. Epub 2019 Nov 22.

Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, 75005, France.

Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.
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http://dx.doi.org/10.1038/s41467-019-13235-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876571PMC
November 2019

MicroRNA degradation by a conserved target RNA regulates animal behavior.

Nat Struct Mol Biol 2018 03 26;25(3):244-251. Epub 2018 Feb 26.

Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, France.

microRNAs (miRNAs) repress target transcripts through partial complementarity. By contrast, highly complementary miRNA-binding sites within viral and artificially engineered transcripts induce miRNA degradation in vitro and in cell lines. Here, we show that a genome-encoded transcript harboring a near-perfect and deeply conserved miRNA-binding site for miR-29 controls zebrafish and mouse behavior. This transcript originated in basal vertebrates as a long noncoding RNA (lncRNA) and evolved to the protein-coding gene NREP in mammals, where the miR-29-binding site is located within the 3' UTR. We show that the near-perfect miRNA site selectively triggers miR-29b destabilization through 3' trimming and restricts its spatial expression in the cerebellum. Genetic disruption of the miR-29 site within mouse Nrep results in ectopic expression of cerebellar miR-29b and impaired coordination and motor learning. Thus, we demonstrate an endogenous target-RNA-directed miRNA degradation event and its requirement for animal behavior.
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http://dx.doi.org/10.1038/s41594-018-0032-xDOI Listing
March 2018

Comparative analyses of super-enhancers reveal conserved elements in vertebrate genomes.

Genome Res 2017 02 13;27(2):259-268. Epub 2016 Dec 13.

Institut Curie, PSL Research University, INSERM U934, CNRS UMR 3215, F-75005, Paris, France.

Super-enhancers (SEs) are key transcriptional drivers of cellular, developmental, and disease states in mammals, yet the conservational and regulatory features of these enhancer elements in nonmammalian vertebrates are unknown. To define SEs in zebrafish and enable sequence and functional comparisons to mouse and human SEs, we used genome-wide histone H3 lysine 27 acetylation (H3K27ac) occupancy as a primary SE delineator. Our study determined the set of SEs in pluripotent state cells and adult zebrafish tissues and revealed both similarities and differences between zebrafish and mammalian SEs. Although the total number of SEs was proportional to the genome size, the genomic distribution of zebrafish SEs differed from that of the mammalian SEs. Despite the evolutionary distance separating zebrafish and mammals and the low overall SE sequence conservation, ∼42% of zebrafish SEs were located in close proximity to orthologs that also were associated with SEs in mouse and human. Compared to their nonassociated counterparts, higher sequence conservation was revealed for those SEs that have maintained orthologous gene associations. Functional dissection of two of these SEs identified conserved sequence elements and tissue-specific expression patterns, while chromatin accessibility analyses predicted transcription factors governing the function of pluripotent state zebrafish SEs. Our zebrafish annotations and comparative studies show the extent of SE usage and their conservation across vertebrates, permitting future gene regulatory studies in several tissues.
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http://dx.doi.org/10.1101/gr.203679.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5287231PMC
February 2017

Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses.

PLoS Biol 2015 Dec 22;13(12):e1002326. Epub 2015 Dec 22.

Institut Jean-Pierre Bourgin, UMR 1318, INRA AgroParisTech CNRS, Université Paris-Saclay, Versailles, France.

Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant-virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.
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http://dx.doi.org/10.1371/journal.pbio.1002326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687873PMC
December 2015

LncRNAs in vertebrates: advances and challenges.

Biochimie 2015 Oct 24;117:3-14. Epub 2015 Mar 24.

Institut Curie, 26 Rue d'Ulm, 75248 Paris Cedex 05, France; CNRS UMR3215, 75248 Paris Cedex 05, France; INSERM U934, 75248 Paris Cedex 05, France. Electronic address:

Beyond the handful of classic and well-characterized long noncoding RNAs (lncRNAs), more recently, hundreds of thousands of lncRNAs have been identified in multiple species including bacteria, plants and vertebrates, and the number of newly annotated lncRNAs continues to increase as more transcriptomes are analyzed. In vertebrates, the expression of many lncRNAs is highly regulated, displaying discrete temporal and spatial expression patterns, suggesting roles in a wide range of developmental processes and setting them apart from classic housekeeping ncRNAs. In addition, the deregulation of a subset of these lncRNAs has been linked to the development of several diseases, including cancers, as well as developmental anomalies. However, the majority of vertebrate lncRNA functions remain enigmatic. As such, a major task at hand is to decipher the biological roles of lncRNAs and uncover the regulatory networks upon which they impinge. This review focuses on our emerging understanding of lncRNAs in vertebrate animals, highlighting some recent advances in their functional analyses across several species and emphasizing the current challenges researchers face to characterize lncRNAs and identify their in vivo functions.
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http://dx.doi.org/10.1016/j.biochi.2015.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125525PMC
October 2015

In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs.

Nucleic Acids Res 2015 Mar 18;43(5):2902-13. Epub 2015 Feb 18.

Institut des Sciences du Végétal, CNRS UPR 2355, SPS Saclay Plant Sciences, Gif-sur-Yvette, France Center for Organismal Studies, University of Heidelberg, Germany

Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5' cap structure and their subsequent 5' to 3' degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs.
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http://dx.doi.org/10.1093/nar/gkv119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357720PMC
March 2015

Chromatin meets the cell cycle.

J Exp Bot 2014 Jun 4;65(10):2677-89. Epub 2014 Feb 4.

Institut de Biologie des Plantes, UMR8618 Université Paris-Sud XI, 91405 Orsay, France

The cell cycle is one of the most comprehensively studied biological processes, due primarily to its significance in growth and development, and its deregulation in many human disorders. Studies using a diverse set of model organisms, including yeast, worms, flies, frogs, mammals, and plants, have greatly expanded our knowledge of the cell cycle and have contributed to the universally accepted view of how the basic cell cycle machinery is regulated. In addition to the oscillating activity of various cyclin-dependent kinase (CDK)-cyclin complexes, a plethora of proteins affecting various aspects of chromatin dynamics has been shown to be essential for cell proliferation during plant development. Furthermore, it was reported recently that core cell cycle regulators control gene expression by modifying histone patterns. This review focuses on the intimate relationship between the cell cycle and chromatin. It describes the dynamics and functions of chromatin structures throughout cell cycle progression and discusses the role of heterochromatin as a barrier against re-replication and endoreduplication. It also proposes that core plant cell cycle regulators control gene expression in a manner similar to that described in mammals. At present, our challenge in plants is to define the complete set of effectors and actors that coordinate cell cycle progression and chromatin structure and to understand better the functional interplay between these two processes.
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http://dx.doi.org/10.1093/jxb/ert433DOI Listing
June 2014

Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants.

Nucleic Acids Res 2013 Apr 12;41(8):4699-708. Epub 2013 Mar 12.

Institut des Sciences du Végétal, CNRS UPR 2355, SPS Saclay Plant Sciences, 91198 Gif-sur-Yvette, France.

Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5'-3' exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat-PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm: siRNA-bodies and processing-bodies. We propose a model of single-stranded RNA tug-of-war between RQC and S-PTGS that ensures the correct partitioning of RNA substrates among these RNA degradation pathways.
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http://dx.doi.org/10.1093/nar/gkt152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3632135PMC
April 2013

The miRNA pathway limits AGO1 availability during siRNA-mediated PTGS defense against exogenous RNA.

Nucleic Acids Res 2011 Nov 3;39(21):9339-44. Epub 2011 Aug 3.

Institut Jean-Pierre Bourgin, UMR1318, INRA, 78026 Versailles Cedex, France.

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.
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http://dx.doi.org/10.1093/nar/gkr590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241636PMC
November 2011

A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5'->3' exoribonuclease (XRN) activities in Arabidopsis thaliana roots.

PLoS One 2011 Feb 3;6(2):e16724. Epub 2011 Feb 3.

CEA, DSV IBEB, Laboratoire de Biologie du Développement des Plantes, UMR 6191 CNRS, CEA, Aix-Marseille II, Saint-Paul-lez-Durance, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016724PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033419PMC
February 2011

Redundant and specific roles of the ARGONAUTE proteins AGO1 and ZLL in development and small RNA-directed gene silencing.

PLoS Genet 2009 Sep 18;5(9):e1000646. Epub 2009 Sep 18.

Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, INRA, Versailles Cedex, France.

The Arabidopsis ARGONAUTE1 (AGO1) and ZWILLE/PINHEAD/AGO10 (ZLL) proteins act in the miRNA and siRNA pathways and are essential for multiple processes in development. Here, we analyze what determines common and specific function of both proteins. Analysis of ago1 mutants with partially compromised AGO1 activity revealed that loss of ZLL function re-establishes both siRNA and miRNA pathways for a subset of AGO1 target genes. Loss of ZLL function in ago1 mutants led to increased AGO1 protein levels, whereas AGO1 mRNA levels were unchanged, implicating ZLL as a negative regulator of AGO1 at the protein level. Since ZLL, unlike AGO1, is not subjected to small RNA-mediated repression itself, this cross regulation has the potential to adjust RNA silencing activity independent of feedback dynamics. Although AGO1 is expressed in a broader pattern than ZLL, expression of AGO1 from the ZLL promoter restored transgene PTGS and most developmental defects of ago1, whereas ZLL rescued only a few AGO1 functions when expressed from the AGO1 promoter, suggesting that the specific functions of AGO1 and ZLL are mainly determined by their protein sequence. Protein domain swapping experiments revealed that the PAZ domain, which in AGO1 is involved in binding small RNAs, is interchangeable between both proteins, suggesting that this common small RNA-binding domain contributes to redundant functions. By contrast, the conserved MID and PIWI domains, which are involved in 5'-end small RNA selectivity and mRNA cleavage, and the non-conserved N-terminal domain, to which no function has been assigned, provide specificity to AGO1 and ZLL protein function.
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http://dx.doi.org/10.1371/journal.pgen.1000646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2730571PMC
September 2009

ARGONAUTE 1 homeostasis invokes the coordinate action of the microRNA and siRNA pathways.

EMBO Rep 2009 May 3;10(5):521-6. Epub 2009 Apr 3.

Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, INRA, Route de Saint-Cyr, 78026 Versailles, France.

ARGONAUTE 1 (AGO1) slices endogenous messenger RNAs (mRNAs) during both microRNA (miRNA)- and short interfering RNA (siRNA)-guided post-transcriptional silencing. We have previously reported that AGO1 homeostasis is maintained through the repressive action of miR168 on AGO1 mRNA and the stabilizing effect of AGO1 protein on miR168, but siRNA-mediated AGO1 regulation has not been reported. Here, we show that AGO1-derived siRNAs trigger RNA DEPENDENT RNA POLYMERASE 6 (RDR6)-, SUPPRESSOR OF GENE SILENCING 3 (SGS3)- and SILENCING DEFECTIVE 5 (SDE5)-dependent AGO1 silencing, which also requires DICER-LIKE 2 (DCL2) and DCL4. By varying the efficacy of miR168-guided AGO1 mRNA cleavage, we show that siRNA-mediated AGO1 silencing depends on correct miRNA targeting, pointing to coordinated regulatory actions of the miRNA and siRNA pathways during the maintenance of AGO1 homeostasis. Finally, our results reveal that dcl2, dcl3 and dcl4 mutations similarly affect post-transcriptional gene silencing (PTGS) mediated by a sense transgene and PTGS mediated by inverted repeats, validating the branched pathway model proposed previously.
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http://dx.doi.org/10.1038/embor.2009.32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680873PMC
May 2009

Criteria for annotation of plant MicroRNAs.

Plant Cell 2008 Dec 12;20(12):3186-90. Epub 2008 Dec 12.

Department of Plant and Soil Sciences, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711, USA.

MicroRNAs (miRNAs) are approximately 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs. This fact, coupled with the rapidly increasing rate of plant small RNA discovery, demands an increased rigor in miRNA annotations. Herein, we update the specific criteria required for the annotation of plant miRNAs, including experimental and computational data, as well as refinements to standard nomenclature.
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http://dx.doi.org/10.1105/tpc.108.064311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2630443PMC
December 2008

MicroRNA maturation and action--the expanding roles of ARGONAUTEs.

Curr Opin Plant Biol 2008 Oct 6;11(5):560-6. Epub 2008 Aug 6.

Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, INRA, 78026 Versailles Cedex, France.

MicroRNAs are endogenously produced 21-nt riboregulators that associate with ARGONAUTE (AGO) proteins to direct mRNA cleavage or repress translation of complementary RNAs. In addition to protein-coding gene repression, miRNA-directed regulation of non-protein-coding transcripts can incite production of trans-acting siRNA (tasiRNA) populations that themselves direct mRNA repression. Arabidopsis encodes 10 AGO proteins among which, AGO1, AGO7, and AGO10 have been implicated in miRNA-guided gene repression in vivo. Recent work has shown that AGO proteins discriminate their associated small RNA populations on the basis of size and 5'-terminal nucleotide identity, extending the roles of AGO proteins beyond small RNA action. Our expanding appreciation of miRNA-directed regulation during plant development and stress adaptations has placed miRNAs at the forefront of plant biology.
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http://dx.doi.org/10.1016/j.pbi.2008.06.008DOI Listing
October 2008

MicroRNA-directed regulation: to cleave or not to cleave.

Trends Plant Sci 2008 Jul 22;13(7):359-67. Epub 2008 May 22.

Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique (INRA), 78026 Versailles Cedex, France.

Gene expression is regulated by transcriptional and post-transcriptional pathways, which are crucial for optimizing gene output and for coordinating cellular programs. MicroRNAs (miRNAs) regulate gene expression networks necessary for proper development, cell viability and stress responses. In plants and animals, 20-24-nt miRNAs direct cleavage and translational repression of partially complementary mRNA target transcripts, through conserved ARGONAUTE proteins. In plants, certain miRNAs indirectly regulate developmental programs by instigating the production of small interfering RNAs (siRNAs). In addition, non-cleavable plant miRNA targets sequester miRNAs, thus regulating miRNA availability. This review summarizes the complexities and diversity of plant miRNA-directed gene regulatory mechanisms and highlights the use of miRNAs for the specific knockdown of gene expression in plants.
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http://dx.doi.org/10.1016/j.tplants.2008.03.007DOI Listing
July 2008

Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors.

Plant Cell 2007 Nov 9;19(11):3451-61. Epub 2007 Nov 9.

Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique (INRA), 78026 Versailles Cedex, France.

The eukaryotic defense response posttranscriptional gene silencing (PTGS) is directed by short-interfering RNAs and thwarts invading nucleic acids via the RNA slicing activity of conserved ARGONAUTE (AGO) proteins. PTGS can be counteracted by exogenous or endogenous suppressors, including the cytoplasmic exoribonuclease XRN4, which also degrades microRNA (miRNA)-guided mRNA cleavage products but does not play an obvious role in development. Here, we show that the nuclear exoribonucleases XRN2 and XRN3 are endogenous PTGS suppressors. We also identify excised MIRNA loops as templates for XRN2 and XRN3 and show that XRN3 is critical for proper development. Independently, we identified the nucleotidase/phosphatase FIERY1 (FRY1) as an endogenous PTGS suppressor through a suppressor screen in a hypomorphic ago1 genetic background. FRY1 is one of six Arabidopsis thaliana orthologs of yeast Hal2. Yeast hal2 mutants overaccumulate 3'-phosphoadenosine 5'-phosphate, which suppresses the 5'-->3' exoribonucleases Xrn1 and Rat1. fry1 mutant plants recapitulate developmental and molecular characteristics of xrn mutants and likely restore PTGS in ago1 hypomorphic mutants by corepressing XRN2, XRN3, and XRN4, thus increasing RNA silencing triggers. We anticipate that screens incorporating partially compromised silencing components will uncover additional PTGS suppressors that may not be revealed using robust silencing systems.
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http://dx.doi.org/10.1105/tpc.107.055319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2174888PMC
November 2007

Functions of microRNAs and related small RNAs in plants.

Nat Genet 2006 Jun;38 Suppl:S31-6

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.

MicroRNAs (miRNAs) and short interfering RNAs (siRNAs), 20- to 27-nt in length, are essential regulatory molecules that act as sequence-specific guides in several processes in most eukaryotes (with the notable exception of the yeast Saccharomyces cerevisiae). These processes include DNA elimination, heterochromatin assembly, mRNA cleavage and translational repression. This review focuses on the regulatory roles of plant miRNAs during development, in the adaptive response to stresses and in the miRNA pathway itself. This review also covers the regulatory roles of two classes of endogenous plant siRNAs, ta-siRNAs and nat-siRNAs, which participate in post-transcriptional control of gene expression.
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http://dx.doi.org/10.1038/ng1791DOI Listing
June 2006

AGO1 homeostasis entails coexpression of MIR168 and AGO1 and preferential stabilization of miR168 by AGO1.

Mol Cell 2006 Apr;22(1):129-36

Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, INRA, 78026 Versailles Cedex, France.

Arabidopsis ARGONAUTE1 (AGO1) encodes the RNA slicer enzyme of the microRNA (miRNA) pathway and is regulated by miR168-programmed, AGO1-catalyzed mRNA cleavage. Here, we describe two additional regulatory processes required for AGO1 homeostasis: transcriptional coregulation of MIR168 and AGO1 genes, and posttranscriptional stabilization of miR168 by AGO1. Disrupting any of these regulatory processes by using mutations or transgenes disturbs a proper functioning of the miRNA pathway. In contrast, minor perturbation leads to fine-tuned posttranscriptional adjustment of miR168 and AGO1 levels, thereby maintaining a proper balance of other miRNAs, which, together with AGO1, control the mRNA levels of miRNA targets. We suggest that miR168 stabilization occurs at the level of silencing-complex assembly and that modulating the efficiency of assembling miRNA-programmed silencing complexes will also be important in other contexts.
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http://dx.doi.org/10.1016/j.molcel.2006.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323247PMC
April 2006

Ectopic DICER-LIKE1 expression in P1/HC-Pro Arabidopsis rescues phenotypic anomalies but not defects in microRNA and silencing pathways.

Plant Cell 2005 Nov 7;17(11):2873-85. Epub 2005 Oct 7.

Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208, USA.

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.
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http://dx.doi.org/10.1105/tpc.105.036608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1276016PMC
November 2005

Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs.

Curr Biol 2005 Aug;15(16):1494-500

Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique, 78026 Versailles Cedex, France.

Arabidopsis encodes four DICER-like (DCL) proteins. DCL1 produces miRNAs, DCL2 produces some virus-derived siRNAs, and DCL3 produces endogenous RDR2-dependent siRNAs, but the role of DCL4 is unknown. We show that DCL4 is the primary processor of endogenous RDR6-dependent trans-acting siRNAs (tasiRNAs). Molecular and phenotypic analyses of all dcl double mutants also revealed partially compensatory functions among DCL proteins. In the absence of DCL4, some RDR6-dependent siRNAs were produced by DCL2 and DCL3, and in the absence of DCL3, some RDR2-dependent siRNAs were produced by DCL2 and DCL4. Consistent with partial redundancies, dcl2 and dcl3 mutants developed normally, whereas dcl4 and dcl3 dcl4 mutants had weak and severe rdr6 phenotypes, respectively, and increased tasiRNA target mRNA accumulation. After three generations, dcl3 dcl4 and dcl2 dcl3 mutants exhibited stochastic developmental phenotypes, some of which were lethal, likely owing to the accumulated loss of heterochromatic siRNA-directed marks. dcl1 dcl3 and dcl1 dcl4, but not dcl1 dcl2 mutants, had phenotypes more severe than dcl1 mutants, consistent with DCL1, DCL3, and DCL4 acting as the primary processors of the three respective classes of endogenous silencing RNAs and DCL2 acting to produce viral-derived siRNAs and as an alternative DCL for endogenous siRNA production.
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http://dx.doi.org/10.1016/j.cub.2005.07.024DOI Listing
August 2005

MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes.

Plant Cell 2005 May 13;17(5):1360-75. Epub 2005 Apr 13.

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.

The phytohormone auxin plays critical roles during plant growth, many of which are mediated by the auxin response transcription factor (ARF) family. MicroRNAs (miRNAs), endogenous 21-nucleotide riboregulators, target several mRNAs implicated in auxin responses. miR160 targets ARF10, ARF16, and ARF17, three of the 23 Arabidopsis thaliana ARF genes. Here, we describe roles of miR160-directed ARF17 posttranscriptional regulation. Plants expressing a miRNA-resistant version of ARF17 have increased ARF17 mRNA levels and altered accumulation of auxin-inducible GH3-like mRNAs, YDK1/GH3.2, GH3.3, GH3.5, and DFL1/GH3.6, which encode auxin-conjugating proteins. These expression changes correlate with dramatic developmental defects, including embryo and emerging leaf symmetry anomalies, leaf shape defects, premature inflorescence development, altered phyllotaxy along the stem, reduced petal size, abnormal stamens, sterility, and root growth defects. These defects demonstrate the importance of miR160-directed ARF17 regulation and implicate ARF17 as a regulator of GH3-like early auxin response genes. Many of these defects resemble phenotypes previously observed in plants expressing viral suppressors of RNA silencing and plants with mutations in genes important for miRNA biogenesis or function, providing a molecular rationale for phenotypes previously associated with more general disruptions of miRNA function.
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http://dx.doi.org/10.1105/tpc.105.031716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1091760PMC
May 2005

Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs.

Mol Cell 2004 Oct;16(1):69-79

Laboratoire de physiologie de la différenciation végétale, UPRES-EA3569, IFR118, EPT1016, Université des Sciences et Technologies de Lille, Bât. SN2, cité scientifique, 59650 Villeneuve d'Ascq Cedex, France.

Here we describe a set of endogenous short interfering RNAs (siRNAs) in Arabidopsis, some of which direct the cleavage of endogenous mRNAs. These siRNAs correspond to both sense and antisense strands of a noncoding RNA (At2g27400) that apparently is converted to double-stranded RNA and then processed in 21 nt increments. These siRNAs differ from previously described regulatory small RNAs in two respects. First, they require components of the cosuppression pathway (RDR6 and SGS3) and also components of the microRNA (miRNA) pathway (AGO1, DCL1, HEN1, and HYL1) but not components needed for heterochromatic siRNAs (DCL3 and RDR2), another class of endogenous plant siRNAs. Second, these siRNAs repress the expression of genes that have little overall resemblance to the genes from which they originate, a characteristic previously reported only for miRNAs. The identification of this silencing pathway provides yet another dimension to posttranscriptional mRNA regulation in plants.
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http://dx.doi.org/10.1016/j.molcel.2004.09.028DOI Listing
October 2004

MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5' region.

EMBO J 2004 Aug 29;23(16):3356-64. Epub 2004 Jul 29.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142-1479, USA.

MicroRNAs (miRNAs) are approximately 22-nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA-guided cleavage of these mRNAs in vitro. Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb-d mutants. In planta, disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA-directed cleavage efficiency in vitro, where mismatch scanning revealed more tolerance for mismatches at the center and 3' end of the miRNA compared to mismatches to the miRNA 5' region. In this respect, miR165/166 resembles animal miRNAs in its pairing requirements. Pairing to the 5' portion of the small silencing RNA appears crucial regardless of the mode of post-transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target.
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http://dx.doi.org/10.1038/sj.emboj.7600340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC514513PMC
August 2004

MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs.

Curr Biol 2004 Jun;14(12):1035-46

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142 USA.

Background: MicroRNAs (miRNAs) are approximately 21 nucleotide (nt) RNAs that regulate gene expression in plants and animals. Most known plant miRNAs target transcription factors that influence cell fate determination, and biological functions of miRNA-directed regulation have been reported for four of 15 known microRNA gene families: miR172, miR159, miR165, and miR168. Here, we identify a developmental role for miR164-directed regulation of NAC-domain genes, which encode a family of transcription factors that includes CUP-SHAPED COTYLEDON1 (CUC1) and CUC2.

Results: Expression of a miR164-resistant version of CUC1 mRNA from the CUC1 promoter causes alterations in Arabidopsis embryonic, vegetative, and floral development, including cotyledon orientation defects, reduction of rosette leaf petioles, dramatically misshapen rosette leaves, one to four extra petals, and one or two missing sepals. Reciprocally, constitutive overexpression of miR164 recapitulates cuc1 cuc2 double mutant phenotypes, including cotyledon and floral organ fusions. miR164 overexpression also leads to phenotypes not previously observed in cuc1 cuc2 mutants, including leaf and stem fusions. These likely reflect the misregulation of other NAC-domain mRNAs, including NAC1, At5g07680, and At5g61430, for which miR164-directed cleavage products were detected.

Conclusions: These results demonstrate that miR164-directed regulation of CUC1 is necessary for normal embryonic, vegetative, and floral development. They also show that proper miR164 dosage or localization is required for separation of adjacent embryonic, vegetative, and floral organs, thus implicating miR164 as a common regulatory component of the molecular circuitry that controls the separation of different developing organs and thereby exposes a posttranscriptional layer of NAC-domain gene regulation during plant development.
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http://dx.doi.org/10.1016/j.cub.2004.06.022DOI Listing
June 2004

MicroRNAs: something important between the genes.

Curr Opin Plant Biol 2004 Apr;7(2):120-5

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.

Non-coding small endogenous RNAs, of 21-24 nucleotides in length, have recently emerged as important regulators of gene expression in both plants and animals. At least three categories of small RNAs exist in plants: short interfering RNAs (siRNAs) deriving from viruses or transgenes and mediating virus resistance or transgene silencing via RNA degradation; siRNAs deriving from transposons or transgene promoters and controlling transposon and transgene silencing probably via chromatin changes; and microRNAs (miRNAs) deriving from intergenic regions of the genome and regulating the expression of endogenous genes either by mRNA cleavage or translational repression. The disruption of miRNA-mediated regulation causes developmental abnormalities in plants, demonstrating that miRNAs play an important role in the regulation of developmental decisions.
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http://dx.doi.org/10.1016/j.pbi.2004.01.006DOI Listing
April 2004

The capacity of transgenic tobacco to send a systemic RNA silencing signal depends on the nature of the inducing transgene locus.

Plant J 2003 Jul;35(1):82-92

Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, USA.

RNA silencing is a conserved eukaryotic pathway in which double-stranded RNA (dsRNA) triggers destruction of homologous target RNA via production of short-interfering RNA (siRNA). In plants, at least some cases of RNA silencing can spread systemically. The signal responsible for systemic spread is expected to include an RNA component to account for the sequence specificity of the process, and transient silencing assays have shown that the capacity for systemic silencing correlates with the accumulation of a particular class of small RNA. Here, we report the results of grafting experiments to study transmission of silencing from stably transformed tobacco lines in the presence or absence of helper component-proteinase (HC-Pro), a viral suppressor of silencing. The studied lines carry either a tail-to-tail inverted repeat, the T4-IR transgene locus, or one of two different amplicon transgene loci encoding replication-competent viral RNA. We find that the T4-IR locus, like many sense-transgene-silenced loci, can send a systemic silencing signal, and this ability is not detectably altered by HC-Pro. Paradoxically, neither amplicon locus effectively triggers systemic silencing except when suppressed for silencing by HC-Pro. In contrast to results from transient assays, these grafting experiments reveal no consistent correlation between capacity for systemic silencing and accumulation of any particular class of small RNA. In addition, although all transgenic lines used to transmit systemic silencing signals were methylated at specific sites within the transgene locus, silencing in grafted scions occurred without detectable methylation at those sites in the target locus of the scion.
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http://dx.doi.org/10.1046/j.1365-313x.2003.01785.xDOI Listing
July 2003

A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco.

Proc Natl Acad Sci U S A 2002 Nov 25;99(23):15228-33. Epub 2002 Oct 25.

Department of Biological Sciences, University of South Carolina, Columbia 29208, USA.

Two major classes of small noncoding RNAs have emerged as important regulators of gene expression in eukaryotes, the short interfering RNAs (siRNAs) associated with RNA silencing and endogenous micro-RNAs (miRNAs) implicated in regulation of gene expression. Helper component-proteinase (HC-Pro) is a viral protein that blocks RNA silencing in plants. Here we examine the effect of HC-Pro on the accumulation of siRNAs and endogenous miRNAs. siRNAs were analyzed in transgenic tobacco plants silenced in response to three different classes of transgenes: sense-transgenes, inverted-repeat transgenes, and amplicon-transgenes. HC-Pro suppressed silencing in each line, blocking accumulation of the associated siRNAs and allowing accumulation of transcripts from the previously silenced loci. HC-Pro-suppression of silencing in the inverted-repeat- and amplicon-transgenic lines was accompanied by the apparent accumulation of long double-stranded RNAs and proportional amounts of small RNAs that are larger than the siRNAs that accumulate during silencing. Analysis of these results suggests that HC-Pro interferes with silencing either by inhibiting siRNA processing from double-stranded RNA precursors or by destabilizing siRNAs. In contrast to siRNAs, the accumulation of endogenous miRNAs was greatly enhanced in all of the HC-Pro-expressing lines. Thus, our results demonstrate that accumulation of siRNAs and miRNAs in plants can be differentially regulated by a viral protein. The fact that HC-Pro affects the miRNA pathway raises the possibility that this pathway is targeted by plant viruses as a means to control gene expression in the host.
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http://dx.doi.org/10.1073/pnas.232434999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137572PMC
November 2002

The amplicon-plus system for high-level expression of transgenes in plants.

Nat Biotechnol 2002 Jun;20(6):622-5

University of South Carolina, Columbia, SC 29208, USA.

Many biotechnological applications require high-level expression of transgenes in plants. One strategy to achieve this goal was the production of potato virus X (PVX) "amplicon" lines: transgenic lines that encode a replicating RNA virus vector carrying a gene of interest. The idea was that transcription of the amplicon transgene would initiate viral RNA replication and gene expression, resulting in very high levels of the gene product of interest. This approach failed, however, because every amplicon transgene, in both tobacco and Arabidopsis thaliana, was subject to post-transcriptional gene silencing (PTGS). In PTGS, the transgene is transcribed but the transcripts fail to accumulate as a result of sequence-specific targeting and destruction. Even though the amplicon locus is silenced, the level of beta-glucuronidase (GUS) activity in a PVX/GUS line is similar to that in some transgenic lines expressing GUS from a conventional (not silenced) GUS locus. This result suggested that the very high levels of expression originally envisioned for amplicons could be achieved if PTGS could be overcome and if the resulting plants did not suffer from severe viral disease. Here we report that high-level transgene expression can be achieved by pairing the amplicon approach with the use of a viral suppressor of PTGS, tobacco etch virus (TEV) helper component proteinase (HC-Pro). Leaves of mature tobacco plants co-expressing HC-Pro and a PVX/GUS amplicon accumulate GUS to approximately 3% of total protein. Moreover, high-level expression occurs without viral symptoms and, when HC-Pro is expressed from a mutant transgene, without detrimental developmental phenotypes.
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http://dx.doi.org/10.1038/nbt0602-622DOI Listing
June 2002