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1.
Plant Cell ; 35(6): 1917-1935, 2023 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-36970782

RESUMO

Besides regulating splicing, the conserved spliceosome component SmD1 (Small nuclear ribonucleoprotein D1)b promotes posttranscriptional silencing of sense transgenes (S-PTGS [post-transcriptional genesilencing]). Here, we show that the conserved spliceosome component PRP39 (Pre-mRNA-processing factor 39)a also plays a role in S-PTGS in Arabidopsis thaliana. However, PRP39a and SmD1b actions appear distinct in both splicing and S-PTGS. Indeed, RNAseq-based analysis of expression level and alternative splicing in prp39a and smd1b mutants identified different sets of deregulated transcripts and noncoding RNAs. Moreover, double mutant analyses involving prp39a or smd1b and RNA quality control (RQC) mutants revealed distinct genetic interactions for SmD1b and PRP39a with nuclear RQC machineries, suggesting nonredundant roles in the RQC/PTGS interplay. Supporting this hypothesis, a prp39a smd1b double mutant exhibited enhanced suppression of S-PTGS compared to the single mutants. Because the prp39a and smd1b mutants (i) showed no major changes in the expression of PTGS or RQC components or in small RNA production and (ii) do not alter PTGS triggered by inverted-repeat transgenes directly producing dsRNA (IR-PTGS), PRP39a, and SmD1b appear to synergistically promote a step specific to S-PTGS. We propose that, independently from their specific roles in splicing, PRP39a and SmD1b limit 3'-to-5' and/or 5'-to-3' degradation of transgene-derived aberrant RNAs in the nucleus, thus favoring the export of aberrant RNAs to the cytoplasm where their conversion into double-stranded RNA (dsRNA) initiates S-PTGS.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Transgenes , RNA Interferente Pequeno/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Interferência de RNA
2.
Plant Cell ; 28(2): 426-38, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26842463

RESUMO

RNA quality control (RQC) eliminates aberrant RNAs based on their atypical structure, whereas posttranscriptional gene silencing (PTGS) eliminates both aberrant and functional RNAs through the sequence-specific action of short interfering RNAs (siRNAs). The Arabidopsis thaliana mutant smd1b was identified in a genetic screen for PTGS deficiency, revealing the involvement of SmD1, a component of the Smith (Sm) complex, in PTGS. The smd1a and smd1b single mutants are viable, but the smd1a smd1b double mutant is embryo-lethal, indicating that SmD1 function is essential. SmD1b resides in nucleoli and nucleoplasmic speckles, colocalizing with the splicing-related factor SR34. Consistent with this, the smd1b mutant exhibits intron retention at certain endogenous mRNAs. SmD1 binds to RNAs transcribed from silenced transgenes but not nonsilenced ones, indicating a direct role in PTGS. Yet, mutations in the RQC factors UPFRAMESHIFT3, EXORIBONUCLEASE2 (XRN2), XRN3, and XRN4 restore PTGS in smd1b, indicating that SmD1 is not essential for but rather facilitates PTGS. Moreover, the smd1b mtr4 double mutant is embryo-lethal, suggesting that SmD1 is essential for mRNA TRANSPORT REGULATOR4-dependent RQC. These results indicate that SmD1 interplays with splicing, RQC, and PTGS. We propose that SmD1 facilitates PTGS by protecting transgene-derived aberrant RNAs from degradation by RQC in the nucleus, allowing sufficient amounts to enter cytoplasmic siRNA bodies to activate PTGS.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , RNA Interferente Pequeno/genética , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Genes Reporter , Mutação , Interferência de RNA , Splicing de RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Ribonucleoproteínas/genética , Plântula/citologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Alinhamento de Sequência , Transgenes
3.
Nucleic Acids Res ; 43(17): 8464-75, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26209135

RESUMO

Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3' maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS.


Assuntos
Interferência de RNA , RNA Antissenso/metabolismo , Transgenes , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Mutação , Estabilidade de RNA , RNA Antissenso/biossíntese , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/biossíntese , RNA Polimerase Dependente de RNA/metabolismo
4.
Plant Cell ; 24(9): 3603-12, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23001035

RESUMO

Posttranscriptional gene silencing (PTGS) mediated by sense transgenes (S-PTGS) results in RNA degradation and DNA methylation of the transcribed region. Through a forward genetic screen, a mutant defective in the Histone3 Lysine4 di/trimethyl (H3K4me2/3) demethylase Jumonji-C (JmjC) domain-containing protein14 (JMJ14) was identified. This mutant reactivates various transgenes silenced by S-PTGS and shows reduced Histone3 Lysine9 Lysine14 acetylation (H3K9K14Ac) levels, reduced polymerase II occupancy, reduced transgene transcription, and increased DNA methylation in the promoter region, consistent with the hypothesis that high levels of transcription are required to trigger S-PTGS. The jmj14 mutation also reduces the expression of transgenes that do not trigger S-PTGS. Moreover, expression of transgenes that undergo S-PTGS in a wild-type background is reduced in jmj14 sgs3 double mutants compared with PTGS-deficient sgs3 mutants, indicating that JMJ14 is required for high levels of transcription in a PTGS-independent manner. Whereas endogenous loci regulated by JMJ14 exhibit increased H3K4me2 and H3K4me3 levels in the jmj14 mutant, transgene loci exhibit unchanged H3K4me2 and decreased H3K4me3 levels. Because jmj14 mutations impair PTGS of transgenes expressed under various plant or viral promoters, we hypothesize that JMJ14 demethylation activity is prevented by antagonistic epigenetic marks specifically imposed at transgene loci. Removing JMJ14 likely allows other H3K4 demethylases encoded by the Arabidopsis thaliana genome to act on transgenes and reduce transcription levels, thus preventing the triggering of S-PTGS.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Histona Desmetilases com o Domínio Jumonji/genética , Interferência de RNA , Acetilação , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Metilação de DNA , Histonas/genética , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Mutação , Fenótipo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de Sequência de DNA , Transgenes
5.
New Phytol ; 198(1): 59-70, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23398515

RESUMO

TT8/bHLH042 is a key regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis thaliana. TT8 transcriptional activity has been studied extensively, and relies on its ability to form, with several R2R3-MYB and TTG1 (WD-Repeat protein), different MYB-bHLH-WDR (MBW) protein complexes. By contrast, little is known on how TT8 expression is itself regulated. Transcriptional regulation of TT8 expression was studied using molecular, genetic and biochemical approaches. Functional dissection of the TT8 promoter revealed its modular structure. Two modules were found to specifically drive TT8 promoter activity in PA- and anthocyanin-accumulating cells, by differentially integrating the signals issued from different regulators, in a spatio-temporal manner. Interestingly, this regulation involves at least six different MBW complexes, and an unpredicted positive feedback regulatory loop between TT8 and TTG2. Moreover, the results suggest that some putative new regulators remain to be discovered. Finally, specific cis-regulatory elements through which TT8 expression is regulated were identified and characterized. Together, these results provide a molecular model consistent with the specific and highly regulated expression of TT8. They shed new light into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation in Arabidopsis and other plant species.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Flavonoides/biossíntese , Regulação da Expressão Gênica de Plantas , Transcrição Gênica , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Complexos Multiproteicos/metabolismo , Mutação/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
6.
Plant Cell ; 22(8): 2697-709, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20798330

RESUMO

We previously identified Arabidopsis thaliana mutants defective in sense transgene posttranscriptional gene silencing (S-PTGS) that defined six loci; here, we describe mutants that define nine additional loci, including HYPER RECOMBINATION1 (HPR1), SILENCING DEFECTIVE3 (SDE3), and SDE5. Our analyses extend previous findings by showing that the requirement for the putative RNA helicase SDE3 is inversely proportional to the strength of the PTGS inducer and that the putative RNA trafficking protein SDE5 is an essential component of the trans-acting small interfering RNA (tasiRNA) pathway and is required for S-PTGS but not inverted repeat transgene-mediated PTGS (IR-PTGS). Our screen also identified HPR1 as a PTGS actor. We show that hpr1 mutations negatively impact S-PTGS, IR-PTGS, and tasiRNA pathways, resulting in increased accumulation of siRNA precursors and decreased accumulation of mature siRNA. In animals, HPR1/THO1 is a member of the conserved RNA trafficking THO/TREX complex, which also includes TEX1/THO3. We show that tex1 mutants, like hpr1 mutants, impact TAS precursor and mature tasiRNA levels, suggesting that a THO/TREX complex exists in plants and that this complex is important for the integrity of the tasiRNA pathway. We propose that both HPR1 and TEX1 participate in the trafficking of siRNA precursors to the ARGONAUTE catalytic center.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , RNA Interferente Pequeno/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , DNA de Plantas/genética , Mutagênese Insercional , Mutação , Transporte Proteico , RNA Helicases/metabolismo , Interferência de RNA
7.
Nucleic Acids Res ; 39(21): 9339-44, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21813456

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Mutação , Transgenes
8.
PLoS Genet ; 5(9): e1000646, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19763164

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Inativação Gênica , RNA Interferente Pequeno/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/embriologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Argonautas , Quimera/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Reporter , Meristema/citologia , Meristema/metabolismo , MicroRNAs/metabolismo , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Estrutura Terciária de Proteína , Plântula/metabolismo , Sementes/citologia , Sementes/metabolismo , Supressão Genética , Transgenes
10.
PLoS One ; 7(1): e29785, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22242179

RESUMO

BACKGROUND: RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and SUPPRESSOR of GENE SILENCING 3 (SGS3) are required for DNA methylation and post-transcriptional gene silencing (PTGS) mediated by 21-nt siRNAs produced by sense transgenes (S-PTGS). In contrast, RDR2, but not RDR6, is required for DNA methylation and TGS mediated by 24-nt siRNAs, and for cell-to-cell spreading of IR-PTGS mediated by 21-nt siRNAs produced by inverted repeat transgenes under the control of a phloem-specific promoter. PRINCIPAL FINDINGS: In this study, we examined the role of RDR2 and RDR6 in S-PTGS. Unlike RDR6, RDR2 is not required for DNA methylation of transgenes subjected to S-PTGS. RDR6 is essential for the production of siRNAs by transgenes subjected to S-PTGS, but RDR2 also contributes to the production of transgene siRNAs when RDR6 is present because rdr2 mutations reduce transgene siRNA accumulation. However, the siRNAs produced via RDR2 likely are counteractive in wildtype plants because impairement of RDR2 increases S-PTGS efficiency at a transgenic locus that triggers limited silencing, and accelerates S-PTGS at a transgenic locus that triggers efficient silencing. CONCLUSIONS/SIGNIFICANCE: These results suggest that RDR2 and RDR6 compete for RNA substrates produced by transgenes subjected to S-PTGS. RDR2 partially antagonizes RDR6 because RDR2 action likely results in the production of counteractive siRNA. As a result, S-PTGS efficiency is increased in rdr2 mutants.


Assuntos
Proteínas de Plantas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/biossíntese , Transgenes/genética , Metilação de DNA/genética , DNA de Plantas/metabolismo , RNA de Plantas/metabolismo
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