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1.
PLoS Genet ; 20(3): e1011200, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38470914

RESUMEN

Long terminal repeat retrotransposons (LTR-RTs) are powerful mutagens regarded as a major source of genetic novelty and important drivers of evolution. Yet, the uncontrolled and potentially selfish proliferation of LTR-RTs can lead to deleterious mutations and genome instability, with large fitness costs for their host. While population genomics data suggest that an ongoing LTR-RT mobility is common in many species, the understanding of their dual role in evolution is limited. Here, we harness the genetic diversity of 320 sequenced natural accessions of the Mediterranean grass Brachypodium distachyon to characterize how genetic and environmental factors influence plant LTR-RT dynamics in the wild. When combining a coverage-based approach to estimate global LTR-RT copy number variations with mobilome-sequencing of nine accessions exposed to eight different stresses, we find little evidence for a major role of environmental factors in LTR-RT accumulations in B. distachyon natural accessions. Instead, we show that loss of RNA polymerase IV (Pol IV), which mediates RNA-directed DNA methylation in plants, results in high transcriptional and transpositional activities of RLC_BdisC024 (HOPPLA) LTR-RT family elements, and that these effects are not stress-specific. This work supports findings indicating an ongoing mobility in B. distachyon and reveals that host RNA-directed DNA methylation rather than environmental factors controls their mobility in this wild grass model.


Asunto(s)
Brachypodium , Retroelementos , Retroelementos/genética , Genoma de Planta/genética , Brachypodium/genética , ARN Interferente Pequeño , Variaciones en el Número de Copia de ADN , Secuencias Repetidas Terminales/genética , Filogenia , Evolución Molecular
2.
Genes Dev ; 31(15): 1601-1614, 2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28882854

RESUMEN

In eukaryotes, transcriptionally inactive loci are enriched within highly condensed heterochromatin. In plants, as in mammals, the DNA of heterochromatin is densely methylated and wrapped by histones displaying a characteristic subset of post-translational modifications. Growing evidence indicates that these chromatin modifications are not sufficient for silencing. Instead, they are prerequisites for further assembly of higher-order chromatin structures that are refractory to transcription but not fully understood. We show that silencing of transposons in the pericentromeric heterochromatin of Arabidopsis thaliana requires SMC4, a core subunit of condensins I and II, acting in conjunction with CG methylation by MET1 (DNA METHYLTRANSFERASE 1), CHG methylation by CMT3 (CHROMOMETHYLASE 3), the chromatin remodeler DDM1 (DECREASE IN DNA METHYLATION 1), and histone modifications, including histone H3 Lys 27 monomethylation (H3K27me1), imparted by ATXR5 and ATXR6. SMC4/condensin also acts within the mostly euchromatic chromosome arms to suppress conditionally expressed genes involved in flowering or DNA repair, including the DNA glycosylase ROS1, which facilitates DNA demethylation. Collectively, our genome-wide analyses implicate condensin in the suppression of hundreds of loci, acting in both DNA methylation-dependent and methylation-independent pathways.


Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Centrosoma/metabolismo , Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica de las Plantas , Complejos Multiproteicos/genética , Cromatina/metabolismo , Metilación de ADN/genética , Reparación del ADN/genética , Silenciador del Gen/fisiología , Estudio de Asociación del Genoma Completo , Técnicas de Genotipaje , Heterocromatina/metabolismo , Histonas/metabolismo , Metiltransferasas/genética , Mutación/genética , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN
3.
Genes Dev ; 30(2): 177-90, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26744421

RESUMEN

In eukaryotes, scores of excess ribosomal RNA (rRNA) genes are silenced by repressive chromatin modifications. Given the near sequence identity of rRNA genes within a species, it is unclear how specific rRNA genes are reproducibly chosen for silencing. Using Arabidopsis thaliana ecotype (strain) Col-0, a systematic search identified sequence polymorphisms that differ between active and developmentally silenced rRNA gene subtypes. Recombinant inbred mapping populations derived from three different ecotype crosses were then used to map the chromosomal locations of silenced and active RNA gene subtypes. Importantly, silenced and active rRNA gene subtypes are not intermingled. All silenced rRNA gene subtypes mapped to the nucleolus organizer region (NOR) on chromosome 2 (NOR2). All active rRNA gene subtypes mapped to NOR4. Using an engineered A. thaliana line in which a portion of Col-0 chromosome 4 was replaced by sequences of another ecotype, we show that a major rRNA gene subtype silenced at NOR2 is active when introgressed into the genome at NOR4. Collectively, these results reveal that selective rRNA gene silencing is not regulated gene by gene based on mechanisms dependent on subtle gene sequence variation. Instead, we propose that a subchromosomal silencing mechanism operates on a multimegabase scale to inactivate NOR2.


Asunto(s)
Arabidopsis/genética , Dosificación de Gen , Silenciador del Gen , Genes de ARNr/genética , Región Organizadora del Nucléolo/genética , Arabidopsis/crecimiento & desarrollo , Cruzamiento , Cromosomas de las Plantas/genética , Genoma de Planta/genética , Polimorfismo Genético/genética
4.
Planta ; 258(4): 81, 2023 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-37715842

RESUMEN

MAIN CONCLUSION: The genus Camellia underwent extensive natural transformation by Agrobacterium. Over a period of 15 million years, at least 12 different inserts accumulated in 72 investigated Camellia species. Like a wide variety of other wild and cultivated plants, Camellia species carry cellular T-DNA sequences (cT-DNAs) in their nuclear genomes, resulting from natural Agrobacterium-mediated transformation. Short and long DNA sequencing reads of 435 accessions belonging to 72 Camellia species (representing 12 out of 14 sections) were investigated for the occurrence of cT-DNA insertions. In all, 12 different cT-DNAs were recovered, either completely or partially, called CaTA to CaTL. Divergence analysis of internal cT-DNA repeats revealed that the insertion events span a period from 0.075 to 15 Mio years ago, and yielded an average transformation frequency of one event per 1.25 Mio years. The two oldest inserts, CaTA and CaTD, have been modified by spontaneous deletions and inversions, and by insertion of various plant sequences. In those cases where enough accessions were available (C. japonica, C. oleifera, C. chekiangoleosa, C. sasanqua and C. pitardii), the younger cT-DNA inserts showed a patchy distribution among different accessions of each species, indicating that they are not genetically fixed. It could be shown that Camellia breeding has led to intersectional transfer of cT-DNAs. Altogether, the cT-DNAs cover 374 kb, and carry 47 open reading frames (ORFs). Two Camellia cT-DNA genes, CaTH-orf358 and CaTK-orf8, represent new types of T-DNA genes. With its large number of cT-DNA sequences, the genus Camellia constitutes an interesting model for the study of natural Agrobacterium transformants.


Asunto(s)
Camellia , Fitomejoramiento , Agrobacterium/genética , Camellia/genética , Sistemas de Lectura Abierta , Análisis de Secuencia de ADN
5.
Mol Cell ; 54(1): 30-42, 2014 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-24657166

RESUMEN

In Arabidopsis, multisubunit RNA polymerases IV and V orchestrate RNA-directed DNA methylation (RdDM) and transcriptional silencing, but what identifies the loci to be silenced is unclear. We show that heritable silent locus identity at a specific subset of RdDM targets requires HISTONE DEACETYLASE 6 (HDA6) acting upstream of Pol IV recruitment and siRNA biogenesis. At these loci, epigenetic memory conferring silent locus identity is erased in hda6 mutants such that restoration of HDA6 activity cannot restore siRNA biogenesis or silencing. Silent locus identity is similarly lost in mutants for the cytosine maintenance methyltransferase, MET1. By contrast, pol IV or pol V mutants disrupt silencing without erasing silent locus identity, allowing restoration of Pol IV or Pol V function to restore silencing. Collectively, these observations indicate that silent locus specification and silencing are separable steps that together account for epigenetic inheritance of the silenced state.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , ARN Polimerasas Dirigidas por ADN/genética , Epigénesis Genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Histona Desacetilasas/genética , Interferencia de ARN , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Citosina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Elementos Transponibles de ADN , ARN Polimerasas Dirigidas por ADN/metabolismo , Sitios Genéticos , Genotipo , Herencia , Histona Desacetilasas/metabolismo , Mutación , Fenotipo , ARN Interferente Pequeño/biosíntesis
6.
Nucleic Acids Res ; 47(17): 9037-9052, 2019 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-31372633

RESUMEN

RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV's largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , ARN Polimerasas Dirigidas por ADN/genética , Regulación de la Expresión Génica de las Plantas , Secuencias de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Metilación de ADN/genética , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Silenciador del Gen , Genoma de Planta , Plantas Modificadas Genéticamente , Dominios Proteicos/genética , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Retroelementos/genética
7.
Genes Dev ; 27(14): 1545-50, 2013 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-23873938

RESUMEN

Eukaryotes can have thousands of 45S ribosomal RNA (rRNA) genes, many of which are silenced during development. Using fluorescence-activated sorting techniques, we show that active rRNA genes in Arabidopsis thaliana are present within sorted nucleoli, whereas silenced rRNA genes are excluded. DNA methyltransferase (met1), histone deacetylase (hda6), or chromatin assembly (caf1) mutants that disrupt silencing abrogate this nucleoplasmic-nucleolar partitioning. Bisulfite sequencing data indicate that active nucleolar rRNA genes are nearly completely demethylated at promoter CGs, whereas silenced genes are nearly fully methylated. Collectively, the data reveal that rRNA genes occupy distinct but changeable nuclear territories according to their epigenetic state.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Epigénesis Genética , ARN Ribosómico/genética , Metilación de ADN , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Modelos Genéticos , Mutación
8.
Proc Natl Acad Sci U S A ; 114(14): 3702-3707, 2017 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-28270603

RESUMEN

Hybrid incompatibility resulting from deleterious gene combinations is thought to be an important step toward reproductive isolation and speciation. Here, we demonstrate involvement of a silent epiallele in hybrid incompatibility. In Arabidopsis thaliana accession Cvi-0, one of the two copies of a duplicated histidine biosynthesis gene, HISN6A, is mutated, making HISN6B essential. In contrast, in accession Col-0, HISN6A is essential because HISN6B is not expressed. Owing to these differences, Cvi-0 × Col-0 hybrid progeny that are homozygous for both Cvi-0 HISN6A and Col-0 HISN6B do not survive. We show that HISN6B of Col-0 is not a defective pseudogene, but a stably silenced epiallele. Mutating HISTONE DEACETYLASE 6 (HDA6), or the cytosine methyltransferase genes MET1 or CMT3, erases HISN6B's silent locus identity, reanimating the gene to circumvent hisn6a lethality and hybrid incompatibility. These results show that HISN6-dependent hybrid lethality is a revertible epigenetic phenomenon and provide additional evidence that epigenetic variation has the potential to limit gene flow between diverging populations of a species.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Epigénesis Genética , Transaminasas/genética , Alelos , Arabidopsis/genética , Quimera , ADN (Citosina-5-)-Metiltransferasas/genética , ADN-Citosina Metilasas/genética , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes Letales , Histona Desacetilasa 6/genética , Mutación
9.
Genes Dev ; 26(9): 945-57, 2012 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-22549957

RESUMEN

Eukaryotes have hundreds of nearly identical 45S ribosomal RNA (rRNA) genes, each encoding the 18S, 5.8S, and 25S catalytic rRNAs. Because cellular demands for ribosomes and protein synthesis vary during development, the number of active rRNA genes is subject to dosage control. In genetic hybrids, one manifestation of dosage control is nucleolar dominance, an epigenetic phenomenon in which the rRNA genes of one progenitor are repressed. For instance, in Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa, the A. thaliana-derived rRNA genes are selectively silenced. An analogous phenomenon occurs in nonhybrid A. thaliana, in which specific classes of rRNA gene variants are inactivated. An RNA-mediated knockdown screen identified SUVR4 {SUPPRESSOR OF VARIEGATION 3-9 [SU(VAR)3-9]-RELATED 4} as a histone H3 Lys 9 (H3K9) methyltransferase required for nucleolar dominance in A. suecica. H3K9 methyltransferases are also required for variant-specific silencing in A. thaliana, but SUVH5 [SU(VAR)3-9 HOMOLOG 5] and SUVH6, rather than SUVR4, are the key activities in this genomic context. Mutations disrupting the H3K27 methyltransferases ATXR5 or ATXR6 affect which rRNA gene variants are expressed or silenced, and in atxr5 atxr6 double mutants, dominance relationships among variants are reversed relative to wild type. Interestingly, these changes in gene expression are accompanied by changes in the relative abundance of the rRNA gene variants at the DNA level, including overreplication of the normally silenced class and decreased abundance of the normally dominant class. Collectively, our results indicate that histone methylation can affect both the doses of different variants and their differential silencing through the choice mechanisms that achieve dosage control.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas , Genes de ARNr , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Arabidopsis/genética , Nucléolo Celular/enzimología , Metilación de ADN , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo
10.
Genes Dev ; 24(11): 1119-32, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-20516197

RESUMEN

The Arabidopsis histone deacetylase HDA6 is required to silence transgenes, transposons, and ribosomal RNA (rRNA) genes subjected to nucleolar dominance in genetic hybrids. In nonhybrid Arabidopsis thaliana, we show that a class of 45S rRNA gene variants that is normally inactivated during development fails to be silenced in hda6 mutants. In these mutants, symmetric cytosine methylation at CG and CHG motifs is reduced, and spurious RNA polymerase II (Pol II) transcription occurs throughout the intergenic spacers. The resulting sense and antisense spacer transcripts facilitate a massive overproduction of siRNAs that, in turn, direct de novo cytosine methylation of corresponding gene sequences. However, the resulting de novo DNA methylation fails to suppress Pol I or Pol II transcription in the absence of HDA6 activity; instead, euchromatic histone modifications typical of active genes accumulate. Collectively, the data reveal a futile cycle of unregulated transcription, siRNA production, and siRNA-directed DNA methylation in the absence of HDA6-mediated histone deacetylation. We propose that spurious Pol II transcription throughout the intergenic spacers in hda6 mutants, combined with losses of histone deacetylase activity and/or maintenance DNA methylation, eliminates repressive chromatin modifications needed for developmental rRNA gene dosage control.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Citosina/metabolismo , ADN Polimerasa II/metabolismo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de ARNr/genética , Histona Desacetilasas/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Arabidopsis/genética , ADN Intergénico/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Histona Desacetilasas/genética , Histonas/metabolismo , Metilación , Mutación
11.
PLoS Pathog ; 8(9): e1002941, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23028332

RESUMEN

In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3'-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5'-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.


Asunto(s)
Arabidopsis/virología , Geminiviridae/genética , Interferencia de ARN , ARN Bicatenario/genética , ARN Interferente Pequeño/genética , ARN Viral/genética , Regiones no Traducidas 3' , Regiones no Traducidas 5'/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , ARN Polimerasa II/metabolismo , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo
12.
Nucleic Acids Res ; 39(12): 5003-14, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21378120

RESUMEN

To successfully infect plants, viruses must counteract small RNA-based host defense responses. During infection of Arabidopsis, Cauliflower mosaic pararetrovirus (CaMV) is transcribed into pregenomic 35S and subgenomic 19S RNAs. The 35S RNA is both reverse transcribed and also used as an mRNA with highly structured 600 nt leader. We found that this leader region is transcribed into long sense- and antisense-RNAs and spawns a massive quantity of 21, 22 and 24 nt viral small RNAs (vsRNAs), comparable to the entire complement of host-encoded small-interfering RNAs and microRNAs. Leader-derived vsRNAs were detected bound to the Argonaute 1 (AGO1) effector protein, unlike vsRNAs from other viral regions. Only negligible amounts of leader-derived vsRNAs were bound to AGO4. Genetic evidence showed that all four Dicer-like (DCL) proteins mediate vsRNA biogenesis, whereas the RNA polymerases Pol IV, Pol V, RDR1, RDR2 and RDR6 are not required for this process. Surprisingly, CaMV titers were not increased in dcl1/2/3/4 quadruple mutants that accumulate only residual amounts of vsRNAs. Ectopic expression of CaMV leader vsRNAs from an attenuated geminivirus led to increased accumulation of this chimeric virus. Thus, massive production of leader-derived vsRNAs does not restrict viral replication but may serve as a decoy diverting the silencing machinery from viral promoter and coding regions.


Asunto(s)
Arabidopsis/virología , Caulimovirus/genética , ARN Pequeño no Traducido/biosíntesis , ARN Viral/biosíntesis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas , Caulimovirus/fisiología , ADN Viral/biosíntesis , Mutación , Enfermedades de las Plantas/virología , Ribonucleasa III/genética , Replicación Viral
13.
PLoS Genet ; 6(11): e1001225, 2010 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-21124873

RESUMEN

In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilación de ADN/genética , Regulación de la Expresión Génica de las Plantas , Genes de ARNr/genética , Mutación/genética , Fosfoproteínas/metabolismo , ARN de Planta/genética , Proteínas de Unión al ARN/metabolismo , Arabidopsis/enzimología , ADN Espaciador Ribosómico/genética , Perfilación de la Expresión Génica , Histonas/metabolismo , Región Organizadora del Nucléolo/genética , Nucleosomas/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , ARN Polimerasa I/metabolismo , ARN Ribosómico/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Transcripción Genética , Nucleolina
14.
bioRxiv ; 2023 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-38234754

RESUMEN

Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a "one CLSY per Pol IV" model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.

15.
Annu Rev Plant Biol ; 72: 245-271, 2021 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-33752440

RESUMEN

Plants have an extraordinary diversity of transcription machineries, including five nuclear DNA-dependent RNA polymerases. Four of these enzymes are dedicated to the production of long noncoding RNAs (lncRNAs), which are ribonucleic acids with functions independent of their protein-coding potential. lncRNAs display a broad range of lengths and structures, but they are distinct from the small RNA guides of RNA interference (RNAi) pathways. lncRNAs frequently serve as structural, catalytic, or regulatory molecules for gene expression. They can affect all elements of genes, including promoters, untranslated regions, exons, introns, and terminators, controlling gene expression at various levels, including modifying chromatin accessibility, transcription, splicing, and translation. Certain lncRNAs protect genome integrity, while others respond to environmental cues like temperature, drought, nutrients, and pathogens. In this review, we explain the challenge of defining lncRNAs, introduce the machineries responsible for their production, and organize this knowledge by viewing the functions of lncRNAs throughout the structure of a typical plant gene.


Asunto(s)
ARN Largo no Codificante , Núcleo Celular , Cromatina , Plantas
16.
Plant Cell Rep ; 29(12): 1401-10, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20953786

RESUMEN

Plant response to stress has been linked to different RNA-silencing processes and epigenetic mechanisms. Our recent results showed that Arabidopsis thaliana Dicer-like (DCL) mutants were impaired in transgenerational changes, recombination frequency and stress tolerance. We also found that transgenerational changes were dependent on changes in DNA methylation. Here, we hypothesized that plants deficient in the production of small RNAs would show an impaired abiotic stress response. To test this, we exposed A. thaliana dcl2, dcl3, dcl4, dcl2 dcl3 (d2d3), dcl2 dcl4 (d2d4), dcl2 dcl3 dcl4 (d2d3d4), nrpd1a, rdr2 and rdr6 mutants to methyl methane sulfonate (MMS). We found dcl4 and rdr6 to be more sensitive and dcl2, dcl3, d2d3 and rdr2 plants more resistant to MMS, as shown by fresh weight, root length and survival rate. The in vitro repair assay showed the lower ability of dcl2 and dcl3 to repair UV-damaged DNA. To summarize, we found that whereas mutants impaired in transactivating siRNA biogenesis were more sensitive to MMS, mutants impaired in natural antisense siRNA and heterochromatic siRNA biogeneses were more tolerant. Our data suggest that plant response to MMS is in part regulated through biogenesis of various siRNAs.


Asunto(s)
Arabidopsis/genética , Metilmetanosulfonato/toxicidad , Mutágenos/toxicidad , Mutación , ARN Interferente Pequeño , Estrés Fisiológico , Arabidopsis/fisiología , Reparación del ADN , Germinación , Plásmidos , Recombinación Genética
17.
Nucleic Acids Res ; 36(20): 6429-38, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18842626

RESUMEN

In Arabidopsis, canonical 21-nt miRNAs are generated by Dicer-like (DCL) 1 from hairpin precursors. We have identified a novel class of functional 23- to 25-nt long-miRNAs that is generated independently from the same miRNA precursors by DCL3. Long-miRNAs are developmentally regulated and in some cases have been conserved during evolution implying that they have biological functions. Plant microRNA genes (MIR) have been proposed to evolve by inverted duplication of the target gene. We found that recently evolved MIR genes consistently give rise to long-miRNAs, while ancient MIR genes give rise predominantly to canonical miRNAs. Transcripts from inverted repeats representing evolving proto-MIR genes were processed by DCL3 into long-miRNAs and also by DCL1, DCL2 or DCL4 depending on hairpin stem length to produce different sizes of miRNAs. Our results suggest that evolution of MIR genes is associated with gradual, overlapping changes in DCL usage resulting in specific size classes of miRNAs.


Asunto(s)
Arabidopsis/genética , Evolución Molecular , MicroARNs/clasificación , MicroARNs/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , MicroARNs/metabolismo , Precursores del ARN/química , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Ribonucleasa III/metabolismo
18.
Nucleic Acids Res ; 36(18): 5896-909, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18801846

RESUMEN

Several RNA silencing pathways in plants restrict viral infections and are suppressed by distinct viral proteins. Here we show that the endogenous trans-acting (ta)siRNA pathway, which depends on Dicer-like (DCL) 4 and RNA-dependent RNA polymerase (RDR) 6, is suppressed by infection of Arabidopsis with Cauliflower mosaic virus (CaMV). This effect was associated with overaccumulation of unprocessed, RDR6-dependent precursors of tasiRNAs and is due solely to expression of the CaMV transactivator/viroplasmin (TAV) protein. TAV expression also impaired secondary, but not primary, siRNA production from a silenced transgene and increased accumulation of mRNAs normally silenced by the four known tasiRNA families and RDR6-dependent secondary siRNAs. Moreover, TAV expression upregulated DCL4, DRB4 and AGO7 that mediate tasiRNA biogenesis. Our findings suggest that TAV is a general inhibitor of silencing amplification that impairs DCL4-mediated processing of RDR6-dependent double-stranded RNA to siRNAs. The resulting deficiency in tasiRNAs and other RDR6-/DCL4-dependent siRNAs appears to trigger a feedback mechanism that compensates for the inhibitory effects.


Asunto(s)
Proteínas de Arabidopsis/antagonistas & inhibidores , Arabidopsis/genética , Arabidopsis/virología , Interferencia de ARN , ARN Interferente Pequeño/biosíntesis , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Arabidopsis/metabolismo , Caulimovirus/metabolismo , Precursores del ARN/biosíntesis , ARN Bicatenario/biosíntesis , ARN Interferente Pequeño/química , Ribonucleasa III , Ribonucleasas/metabolismo , Transgenes
19.
Transcription ; 11(3-4): 172-191, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33180661

RESUMEN

Multisubunit RNA polymerase (Pol) complexes are the core machinery for gene expression in eukaryotes. The enzymes Pol I, Pol II and Pol III transcribe distinct subsets of nuclear genes. This family of nuclear RNA polymerases expanded in terrestrial plants by the duplication of Pol II subunit genes. Two Pol II-related enzymes, Pol IV and Pol V, are highly specialized in the production of regulatory, non-coding RNAs. Pol IV and Pol V are the central players of RNA-directed DNA methylation (RdDM), an RNA interference pathway that represses transposable elements (TEs) and selected genes. Genetic and biochemical analyses of Pol IV/V subunits are now revealing how these enzymes evolved from ancestral Pol II to sustain non-coding RNA biogenesis in silent chromatin. Intriguingly, Pol IV-RdDM regulates genes that influence flowering time, reproductive development, stress responses and plant-pathogen interactions. Pol IV target genes vary among closely related taxa, indicating that these regulatory circuits are often species-specific. Data from crops like maize, rice, tomato and Brassicarapa suggest that dynamic repositioning of TEs, accompanied by Pol IV targeting to TE-proximal genes, leads to the reprogramming of plant gene expression over short evolutionary timescales.


Asunto(s)
Elementos Transponibles de ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Regulación de la Expresión Génica de las Plantas/genética , Plantas/genética , ARN no Traducido/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Plantas/metabolismo , ARN no Traducido/metabolismo
20.
Methods Mol Biol ; 2166: 387-411, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32710422

RESUMEN

Cells have sophisticated RNA-directed mechanisms to regulate genes, destroy viruses, or silence transposable elements (TEs). In terrestrial plants, a specialized non-coding RNA machinery involving RNA polymerase IV (Pol IV) and small interfering RNAs (siRNAs) targets DNA methylation and silencing to TEs. Here, we present a bioinformatics protocol for annotating and quantifying siRNAs that derive from long terminal repeat (LTR) retrotransposons. The approach was validated using small RNA northern blot analyses, comparing the species Arabidopsis thaliana and Brachypodium distachyon. To assist hybridization probe design, we configured a genome browser to show small RNA-seq mappings in distinct colors and shades according to their nucleotide lengths and abundances, respectively. Samples from wild-type and pol IV mutant plants, cross-species negative controls, and a conserved microRNA control validated the detected siRNA signals, confirming their origin from specific TEs and their Pol IV-dependent biogenesis. Moreover, an optimized labeling method yielded probes that could detect low-abundance siRNAs from B. distachyon TEs. The integration of de novo TE annotation, small RNA-seq profiling, and northern blotting, as outlined here, will facilitate the comparative genomic analysis of RNA silencing in crop plants and non-model species.


Asunto(s)
Arabidopsis/genética , Northern Blotting/métodos , Brachypodium/genética , Genoma de Planta , ARN de Planta/genética , ARN de Planta/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Retroelementos/genética , Proteínas de Arabidopsis/genética , ARN Polimerasas Dirigidas por ADN/genética , Plantas Modificadas Genéticamente , Interferencia de ARN , ARN Bicatenario/genética , RNA-Seq , Secuencias Repetidas Terminales/genética
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