Direct evidence of nuclear Argonaute distribution during transcriptional silencing links the actin cytoskeleton to nuclear RNAi machinery in human cells.
ABSTRACT: Mammalian RNAi machinery facilitating transcriptional gene silencing (TGS) is the RNA-induced transcriptional gene silencing-like (RITS-like) complex, comprising of Argonaute (Ago) and small interfering RNA (siRNA) components. We have previously demonstrated promoter-targeted siRNA induce TGS in human immunodeficiency virus type-1 (HIV-1) and simian immunodeficiency virus (SIV), which profoundly suppresses retrovirus replication via heterochromatin formation and histone methylation. Here, we examine subcellular co-localization of Ago proteins with promoter-targeted siRNAs during TGS of SIV and HIV-1 infection. Analysis of retrovirus-infected cells revealed Ago1 co-localized with siRNA in the nucleus, while Ago2 co-localized with siRNA in the inner nuclear envelope. Mismatched and scrambled siRNAs were observed in the cytoplasm, indicating sequence specificity. This is the first report directly visualizing nuclear compartment distribution of Ago-associated siRNA and further reveals a novel nuclear trafficking mechanism for RITS-like components involving the actin cytoskeleton. These results establish a model for elucidating mammalian TGS and suggest a fundamental mechanism underlying nuclear delivery of RITS-like components.
Project description:In fission yeast, factors involved in the RNA interference (RNAi) pathway including Argonaute, Dicer, and RNA-dependent RNA polymerase are required for heterochromatin assembly at centromeric repeats and the silent mating-type region. Previously, we have shown that RNA-induced initiation of transcriptional gene silencing (RITS) complex containing the Argonaute protein and small interfering RNAs (siRNAs) localizes to heterochromatic loci and collaborates with heterochromatin assembly factors via a self-enforcing RNAi loop mechanism to couple siRNA generation with heterochromatin formation. Here, we investigate the role of RNA-dependent RNA polymerase (Rdp1) and its polymerase activity in the assembly of heterochromatin. We find that Rdp1, similar to RITS, localizes to all known heterochromatic loci, and its localization at centromeric repeats depends on components of RITS and Dicer as well as heterochromatin assembly factors including Clr4/Suv39h and Swi6/HP1 proteins. We show that a point mutation within the catalytic domain of Rdp1 abolished its RNA-dependent RNA polymerase activity and resulted in the loss of transcriptional silencing and heterochromatin at centromeres, together with defects in mitotic chromosome segregation and telomere clustering. Moreover, the RITS complex in the rdp1 mutant does not contain siRNAs, and is delocalized from centromeres. These results not only implicate Rdp1 as an essential component of a self-enforcing RNAi loop but also ascribe a critical role for its RNA-dependent RNA polymerase activity in siRNA production necessary for heterochromatin formation.
Project description:RNA interference (RNAi) plays a pivotal role in the formation of heterochromatin at the fission yeast centromeres. The RNA-induced transcriptional silencing (RITS) complex, composed of heterochromatic small interfering RNAs (siRNAs), the siRNA-binding protein Ago1, the chromodomain protein Chp1, and the Ago1/Chp1-interacting protein Tas3, provides a physical tether between the RNAi and heterochromatin assembly pathways. Here, we report the structural and functional characterization of a C-terminal Tas3 alpha-helical motif (TAM), which self-associates into a helical polymer and is required for cis spreading of RITS in centromeric DNA regions. Site-directed mutations of key residues within the hydrophobic monomer-monomer interface disrupt Tas3-TAM polymeric self-association in vitro and result in loss of gene silencing, spreading of RITS, and a dramatic reduction in centromeric siRNAs in vivo. These results demonstrate that, in addition to the chromodomain of Chp1 and siRNA-loaded Ago1, Tas3 self-association is required for RITS spreading and efficient heterochromatic gene silencing at centromeric repeat regions.
Project description:RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of AROGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing (TGS) of homologous promoter sequences. AGO proteins act in silencing effector complexes by anchoring the 3â and 5â ends of the guide siRNAs at their N-terminal PAZ domain and MID domain, respectively. In addition, many AGO proteins cleave complementary target RNAs through an endonuclease (âslicerâ) activity in their C-terminal PIWI domain. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and TGS in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points and the preferential association of Pol V with AGO6. Examination of siRNA abundance in the trasngenic wild type plant (contains trigger and silencer transgenes) and the ago6-4 mutant.
Project description:RNA interference (RNAi) is heritable in Caenorhabditis elegans; the progeny of C. elegans exposed to dsRNA inherit the ability to silence genes that were targeted by RNAi in the previous generation. Here we investigate the mechanism of RNAi inheritance in C. elegans. We show that exposure of animals to dsRNA results in the heritable expression of siRNAs and the heritable deposition of histone 3 lysine 9 methylation (H3K9me) marks in progeny. siRNAs are detectable before the appearance of H3K9me marks, suggesting that chromatin marks are not directly inherited but, rather, reestablished in inheriting progeny. Interestingly, H3K9me marks appear more prominently in inheriting progeny than in animals directly exposed to dsRNA, suggesting that germ-line transmission of silencing signals may enhance the efficiency of siRNA-directed H3K9me. Finally, we show that the nuclear RNAi (Nrde) pathway maintains heritable RNAi silencing in C. elegans. The Argonaute (Ago) NRDE-3 associates with heritable siRNAs and, acting in conjunction with the nuclear RNAi factors NRDE-1, NRDE-2, and NRDE-4, promotes siRNA expression in inheriting progeny. These results demonstrate that siRNA expression is heritable in C. elegans and define an RNAi pathway that promotes the maintenance of RNAi silencing and siRNA expression in the progeny of animals exposed to dsRNA.
Project description:In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.
Project description:In Arabidopsis thaliana, functionally diverse small RNA (smRNA) pathways bring about decreased RNA accumulation of target genes via several different mechanisms. Cytological experiments have suggested that the processing of microRNAs (miRNAs) and heterochromatic small interfering RNAs (hc-siRNAs) occurs within a specific nuclear domain that can present Cajal Body (CB) characteristics. It is unclear whether single or multiple smRNA-related domains are found within the same CB and how specialization of the smRNA pathways is determined within this specific sub-compartment. To ascertain whether nuclear smRNA centers are spatially related, we localized key proteins required for siRNA or miRNA biogenesis by immunofluorescence analysis. The intranuclear distribution of the proteins revealed that hc-siRNA, miRNA and trans-acting siRNA (ta-siRNA) pathway proteins accumulate and colocalize within a sub-nuclear structure in the nucleolar periphery. Furthermore, colocalization of miRNA- and siRNA-pathway members with CB markers, and reduced wild-type localization patterns in CB mutants indicates that proper nuclear localization of these proteins requires CB integrity. We hypothesize that these nuclear domains could be important for RNA silencing and may partially explain the functional redundancies and interactions among components of the same protein family. The CB may be the place in the nucleus where Dicer-generated smRNA precursors are processed and assigned to a specific pathway, and where storage, recycling or assembly of RNA interference components takes place.
Project description:In humans, there are four Ago proteins (Ago1M-bM-^@M-^S4) and AGO1- and 2 were previously implicated in TGS induced by exogenous siRNAs and microRNAs (miRs) directed against gene promoter transcripts via promotion of changes in histone covalent modifications and DNA methylation. Not-with-standing, many mechanistic details of this process remain poorly defined in human cells, and very little is known about the identity of possible endogenous signals, which may drive this process in human cells. Given the evolutionary conserved role of siRNAs and AGO proteins in TGS and heterochromatin formation, we set out to analyse their possible involvement in senesence-associated repression of E2F target genes. To obtain a detailed picture of AGO-immunoprecipitating miRs (RIP) in senescent cells, we used next-generation sequencing (NGS)(RIP-Seq). We also included histone H3 dimethylated on lysine 9 (H3K9me2) in this analysis to assign potential AGO2-interacting miRs to a repressive chromatin state and unfractionated, cellular RNA from senescent cells for normalisation. Determination of AGO AGO-immunoprecipitating miRs in WI-38 senescent human fibroblast
Project description:Transcriptional gene silencing (TGS) in plants is a defense mechanism against DNA virus infection. The genomes of viruses in the <i>Geminiviridae</i> family encode several TGS suppressors. In this study, we induced de novo TGS against the transgenic <i>GFP</i> gene encoding green fluorescent protein by expressing a hairpin-shaped self-complementary RNA corresponding to the enhancer region of the 35S promoter (hpE35S). In addition, we examined the TGS suppression activity of proteins encoded in the genome of <i>Tobacco yellow dwarf virus</i> (TYDV, genus <i>Mastrevirus</i>). The results show that the replication-associated protein (Rep) and RepA encoded by TYDV have TGS suppressor activity and lead to decreased accumulation of 24-nt siRNAs. These results suggest that Rep and RepA can block the steps before the loading of siRNAs into Argonaute (AGO) proteins. This is the first report of TGS suppressors in the genus <i>Mastrevirus</i>.
Project description:Heterochromatic gene silencing at the pericentromeric DNA repeats in fission yeast requires the RNA interference (RNAi) machinery. The RNA-induced transcriptional silencing (RITS) complex mediates histone H3 lysine 9 (H3K9) methylation and recruits the RNA-dependent RNA polymerase complex (RDRC) to promote double-stranded RNA (dsRNA) synthesis and siRNA generation. Here we show that ectopic expression of a long hairpin RNA bypasses the requirement for chromatin-dependent steps in siRNA generation. The ability of hairpin-produced siRNAs to silence homologous sequences in trans is subject to local chromatin structure, requires HP1, and correlates with antisense transcription at the target locus. Furthermore, although hairpin siRNAs can be produced in the absence of RDRC, trans-silencing of reporter genes by hairpin-produced siRNAs is completely dependent on the dsRNA synthesis activity of RDRC. These results provide insights into the regulation of siRNA action and reveal roles for cis-dsRNA synthesis and HP1 in siRNA-mediated heterochromatin assembly.
Project description:In plants, it is possible to induce heritable transcriptional gene silencing (TGS) via RNA-directed DNA methylation (RdDM) using artificially synthesized small RNA (siRNA) homologous to the 5'-flanking region of the target gene. As the siRNA signal with a specific RNA determinant moves through plasmodesmata and sieve elements, we attempted to induce TGS of a transgene and an endogenous gene of potato (Solanum tuberosum) rootstock by grafting using siRNA produced in a tobacco (Nicotiana benthamiana) scion. Our results provide evidence that this system can induce TGS of target genes in tubers formed on potato rootstock. The TGS is maintained in the progeny tubers lacking the transported siRNAs. Our findings reveal that epigenome editing using mobile RNA has the potential to allow breeding of artificial sport cultivars in vegetative propagation crops.