RESUMO
We recently reported that p28, one of the two turnip crinkle virus (TCV) replication proteins, trans-complemented a defective TCV lacking p28, yet repressed the replication of another TCV replicon encoding wild-type p28 (Zhang et al., 2017). Here we show that p88, the TCV-encoded RNA-dependent RNA polymerase, likewise trans-complemented a p88-defective TCV replicon, but repressed one encoding wild-type p88. Surprisingly, lowering p88 protein levels enhanced trans-complementation, but weakened repression. Repression by p88 was not simply due to protein over-expression, as deletion mutants missing 127 or 224 N-terminal amino acids accumulated to higher levels but were poor repressors. Finally, both trans-complementation and repression by p88 were accompanied by preferential accumulation of subgenomic RNA2, and a novel class of small TCV RNAs. Our results suggest that repression of TCV replication by p88 may manifest a viral mechanism that regulates the ratio of genomic and subgenomic RNAs based on p88 abundance.
Assuntos
Carmovirus/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/genética , Expressão Gênica , Doenças das Plantas/virologia , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/deficiência , RNA Polimerase Dependente de RNA/genética , Replicon/genética , Deleção de Sequência , Proteínas Virais/genéticaRESUMO
Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA-dependent RNA polymerase 2 (RDR2) is a component of the RNA-directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA-seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially expressed DNA TEs (78%) were up-regulated in the mop1 mutant. In contrast, most differentially expressed retrotransposons (68%) were down-regulated. This striking difference suggests that distinct silencing mechanisms are applied to different silencing templates. In addition, >6,000 genes (24% of analyzed genes), including nearly 80% (286/361) of genes in chromatin modification pathways, were differentially expressed. Overall, two-thirds of differentially regulated genes were down-regulated in the mop1 mutant. This finding suggests that RDR2 plays a significant role in regulating the expression of not only transposons, but also of genes. A re-analysis of existing small RNA data identified both RDR2-sensitive and RDR2-resistant species of 24 nt siRNAs that we hypothesize may at least partially explain the complex changes in the expression of genes and transposons observed in the mop1 mutant.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , RNA de Plantas/metabolismo , RNA Polimerase Dependente de RNA/deficiência , Proteínas de Arabidopsis/genética , Pareamento de Bases/genética , Montagem e Desmontagem da Cromatina/genética , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Lasers , Meristema/genética , Microdissecção , Mutação/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNARESUMO
P granules are non-membrane-bound organelles found in the germ-line cytoplasm throughout Caenorhabditis elegans development. Like their "germ granule" counterparts in other animals, P granules are thought to act as determinants of the identity and special properties of germ cells, properties that include the unique ability to give rise to all tissues of future generations of an organism. Therefore, understanding how P granules work is critical to understanding how cellular immortality and totipotency are retained, gained, and lost. Here we report on a genomewide RNAi screen in C. elegans, which identified 173 genes that affect the stability, localization, and function of P granules. Many of these genes fall into specific classes with shared P-granule phenotypes, allowing us to better understand how cellular processes such as protein degradation, translation, splicing, nuclear transport, and mRNA homeostasis converge on P-granule assembly and function. One of the more striking phenotypes is caused by the depletion of CSR-1, an Argonaute associated with an endogenous siRNA pathway that functions in the germ line. We show that CSR-1 and two other endo-siRNA pathway members, the RNA-dependent RNA polymerase EGO-1 and the helicase DRH-3, act to antagonize RNA and P-granule accumulation in the germ line. Our findings strengthen the emerging view that germ granules are involved in numerous aspects of RNA metabolism, including an endo-siRNA pathway in germ cells.
Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Genômica , Interferência de RNA , Transporte Ativo do Núcleo Celular/genética , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Divisão Celular/genética , Polaridade Celular/genética , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Regulação para Baixo , Genoma/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , Homeostase/genética , Proteínas de Domínio MADS/deficiência , Proteínas de Domínio MADS/genética , Fenótipo , Porosidade , Complexo de Endopeptidases do Proteassoma/deficiência , Complexo de Endopeptidases do Proteassoma/genética , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/deficiência , RNA Polimerase Dependente de RNA/genética , Ribossomos/genética , Ubiquitina/deficiência , Ubiquitina/genéticaRESUMO
SUMMARY: In plants, both transcriptional (TGS) and post-transcriptional gene silencing (PTGS) can be self-reinforcing, and this allows maintenance of silencing once the initiator has been removed or suppressed. For TGS, this can be accomplished by the generation of small interfering RNAs (siRNAs) from methylated DNA templates by RNA polymerase IV (PolIV), RNA-dependent RNA polymerase 2 (RDR2), DICER-LIKE 3 (DCL3), and the RNA-directed DNA methylation (RdDM) machinery. Maintenance of PTGS requires RNA-dependent RNA polymerase 6 (RDR6), and may be associated with DNA methylation and transitive production of secondary siRNAs. In this work, mutants defective for the NRPD1a and NRPD1b alternative largest subunits of PolIV were tested for their ability to undergo RdDM, transitive RNA silencing and maintenance of PTGS. PTGS could be initiated in both nrpd1a and nrpd1b mutants, and this was associated with production of secondary siRNAs; silencing was not maintained however. nrpd1a mutants could support RdDM although this was lost upon reversal of silencing, as was methylation in rdr6 mutants. We conclude that components of the machinery that maintain TGS are required for maintenance of PTGS, and that RDR6 uses distinct templates in the initiation and maintenance phases of RNA silencing.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Metilação de DNA , DNA de Plantas/genética , RNA Polimerases Dirigidas por DNA/genética , Inativação Gênica , Processamento Pós-Transcricional do RNA/genética , RNA de Plantas/genética , RNA Polimerase Dependente de RNA/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citosina/metabolismo , DNA de Plantas/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Genes Reporter , Metilação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , RNA Polimerase Dependente de RNA/deficiência , RNA Polimerase Dependente de RNA/genéticaRESUMO
RNA interference (RNAi) is a broadly used reverse genetics method in C. elegans. Unfortunately, RNAi does not inhibit all genes. We show that loss of function of a putative RNA-directed RNA polymerase (RdRP) of C. elegans, RRF-3, results in a substantial enhancement of sensitivity to RNAi in diverse tissues. This is particularly striking in the nervous system; neurons that are generally refractory to RNAi in a wild-type genetic background can respond effectively to interference in an rrf-3 mutant background. These data provide the first indication of physiological negative modulation of the RNAi response and implicate an RdRP-related factor in this effect. The rrf-3 strain can be useful to study genes that, in wild-type, do not show a phenotype after RNAi, and it is probably the strain of choice for genome-wide RNAi screens.
Assuntos
Caenorhabditis elegans/genética , Interferência de RNA , RNA Polimerase Dependente de RNA/metabolismo , Animais , Caenorhabditis elegans/enzimologia , Fenótipo , RNA de Cadeia Dupla/genética , RNA Polimerase Dependente de RNA/deficiência , RNA Polimerase Dependente de RNA/genéticaRESUMO
The insect nodavirus Flock house virus (FHV) has a small genome divided between two segments of positive-sense RNA, RNA1 and RNA2. RNA1 encodes the RNA-dependent RNA polymerase (RdRp) catalytic subunit and templates the synthesis of a subgenomic RNA (RNA3) that encodes two small nonstructural proteins. Replication of RNA2, which encodes a precursor to the viral capsid proteins, suppresses RNA3 synthesis. Here we report that RNA1 mutants deficient in RNA3 synthesis failed to support RNA2 replication. This effect was not caused by alterations in the RdRp catalytic subunit nor by a lack of the proteins encoded by RNA3. Furthermore, RNA3 supplied in trans from an exogenous source restored RNA2 replication. These data indicate that RNA3 transactivates the replication of RNA2, a novel property for a viral RNA. We propose that the RNA3 dependence of RNA2 replication serves to coordinate replication of the FHV genome segments.
Assuntos
Genoma Viral , Nodaviridae/genética , RNA Viral/fisiologia , Ativação Transcricional , Animais , Capsídeo/biossíntese , Capsídeo/genética , Linhagem Celular , Cricetinae , Mutação , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/deficiência , RNA Polimerase Dependente de RNA/genética , Replicon , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genéticaRESUMO
Tomato bushy stunt virus (TBSV) and other tombusviruses are notorious for their propensity to accumulate defective interfering RNAs (DIs) upon serial passage through experimental Nicotiana species. Hallmarks of this occurrence include reduced levels of helper RNA and protein accumulation and amelioration of the lethal necrosis induced upon infection of the host with the helper viruses alone. The objective of this study was to determine whether the prolific trans-accumulation of defective RNAs typically occurs for all replicase-deficient TBSV mutants, or if this process is influenced by internal cis-acting elements that have been excised from DIs. For this purpose, various replicase-deficient TBSV cDNA constructs were generated and their transcripts were tested for trans-accumulation competence in the presence of helper virus. The results revealed that a region of ca. 150 nucleotides near the center of the replicase gene, with a predicted high degree of secondary structure, was a potent inhibitor of trans-rescue (ITR) by TBSV. Relocation of the ITR into efficiently trans-replicating DIs inhibited their accumulation drastically, but only when inserted in the reverse orientation and with an intact 5' ITR-specific predicted hairpin structure. Insertion of the ITR element in the positive orientation yielded DI transcripts that were able to replicate, but failed to interfere noticeably with either accumulation of the helper RNA or the onset of the lethal necrosis phenotype in N. benthamiana. In conclusion, the ITR has an intrinsic capacity to inhibit trans-accumulation of defective RNAs, but its stringency and biological effects are strongly influenced by the overall sequence context.
