Showing papers on "RNA-induced transcriptional silencing published in 2005"
TL;DR: It is shown that RISC is composed of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2 and it is demonstrated that this complex can cleave target RNA using precursor microRNA (pre-miRNA) hairpin as the source of siRNA.
1,545 citations
TL;DR: These results provide an alternative strategy for eliciting RNAi-mediated target cleavage using low concentrations of synthetic RNA as substrates for cellular Dicer-mediated cleavage.
Abstract: RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing triggered by double-stranded RNAs. In attempts to identify RNAi triggers that effectively function at lower concentrations, we found that synthetic RNA duplexes 25–30 nucleotides in length can be up to 100-fold more potent than corresponding conventional 21-mer small interfering RNAs (siRNAs). Some sites that are refractory to silencing by 21-mer siRNAs can be effectively targeted by 27-mer duplexes, with silencing lasting up to 10 d. Notably, the 27-mers do not induce interferon or activate protein kinase R (PKR). The enhanced potency of the longer duplexes is attributed to the fact that they are substrates of the Dicer endonuclease, directly linking the production of siRNAs to incorporation in the RNA-induced silencing complex. These results provide an alternative strategy for eliciting RNAi-mediated target cleavage using low concentrations of synthetic RNA as substrates for cellular Dicer-mediated cleavage.
1,028 citations
TL;DR: It is shown that AGO1 selectively recruits certain classes of short silencing-related RNA, and it is predicted that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.
Abstract: ARGONAUTE (AGO) RNA-binding proteins are involved in RNA silencing. They bind to short interfering RNAs (siRNAs) and microRNAs (miRNAs) through a conserved PAZ domain, and, in animals, they assemble into a multisubunit RNA-induced silencing complex (RISC). The mammalian AGO2, termed Slicer, directs siRNA- and miRNA-mediated cleavage of a target RNA. In Arabidopsis, there are 10 members of the AGO family, and the AGO1 protein is potentially the Slicer component in different RNA-silencing pathways. Here, we show that AGO1 selectively recruits certain classes of short silencing-related RNA. AGO1 is physically associated with miRNAs, transacting siRNAs, and transgene-derived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs involved in chromatin silencing. We also show that AGO1 has Slicer activity. It mediates the in vitro cleavage of a mir165 target RNA in a manner that depends on the sequence identity of amino acid residues in the PIWI domain that are predicted by homology with animal Slicer-competent AGO proteins to constitute the RNase catalytic center. However, unlike animals, we find no evidence that AGO1 Slicer is in a high molecular weight RNA-induced silencing complex. The Slicer activity fractionates as a complex of approximately 150 kDa that likely constitutes the AGO1 protein and associated RNA without any other proteins. Based on sequence similarity, we predict that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.
1,022 citations
TL;DR: The current understanding of how small RNAs are produced, how they are loaded into protein complexes, and how they repress gene expression is reviewed.
Abstract: RNA silencing pathways convert the sequence information in long RNA, typically double-stranded RNA, into ∼21-nt RNA signaling molecules such as small interfering RNAs (siRNAs) and microRNAs (miRNAs). siRNAs and miRNAs provide specificity to protein effector complexes that repress mRNA transcription or translation, or catalyze mRNA destruction. Here, we review our current understanding of how small RNAs are produced, how they are loaded into protein complexes, and how they repress gene expression.
1,015 citations
TL;DR: By mutation of the two largest subunits (NRPD1a and NRPD2), it is shown that Pol IV silences certain transposons and repetitive DNA in a short interfering RNA pathway involving RNA-dependent RNA polymerase 2 and Dicer-like 3.
Abstract: Plants encode subunits for a fourth RNA polymerase (Pol IV) in addition to the well-known DNA-dependent RNA polymerases I, II, and III. By mutation of the two largest subunits (NRPD1a and NRPD2), we show that Pol IV silences certain transposons and repetitive DNA in a short interfering RNA pathway involving RNA-dependent RNA polymerase 2 and Dicer-like 3. The existence of this distinct silencing polymerase may explain the paradoxical involvement of an RNA silencing pathway in maintenance of transcriptional silencing.
749 citations
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TL;DR: The implications for noncoding RNAs and the formation of specialized chromatin domains in various epigenetic processes as diverse as dosage compensation, RNA interference-mediated heterochromatin assembly and gene silencing, and programmed DNA elimination are discussed.
Abstract: In the universe of science, two worlds have recently collided-those of RNA and chromatin. The intersection of these two fields has been impending, but evidence for such a meaningful collision has only recently become apparent. In this review, we discuss the implications for noncoding RNAs and the formation of specialized chromatin domains in various epigenetic processes as diverse as dosage compensation, RNA interference-mediated heterochromatin assembly and gene silencing, and programmed DNA elimination. While mechanistic details as to how the RNA and chromatin worlds connect remain unclear, intriguing parallels exist in the overall design and machinery used in model organisms from all eukaryotic kingdoms. The role of potential RNA-binding chromatin-associated proteins will be discussed as one possible link between RNA and chromatin.
654 citations
TL;DR: The structure of the Piwi–RNA complex provides direct support for the 5′ region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5′ end of the guided RNA.
Abstract: RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism1,2,3 mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold4,5 critical for the endonuclease cleavage activity of RISC4,5,6. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5′-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5′ end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5′ end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5′ phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi–RNA complex, and that determined elsewhere7, provide direct support for the 5′ region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5′ end of the guide RNA.
653 citations
TL;DR: The authors' biochemical analysis revealed that Ago2 is present in a pre-miRNA processing complex that is able to transfer the miRNA into a target-mRNA cleaving complex, and these proteins are functionally required to mediate miRNA-guided mRNA cleavage.
581 citations
TL;DR: The first structural information on protein components of the RNA interference (RNAi) and miRNA machineries is emerging, and provides some insight into the mechanism of RNA-silencing reactions.
557 citations
TL;DR: This review describes the discovery of two ribonuclease machines and discusses future lines of work on this amazing biochemical pathway, which is triggered by double‐stranded RNA.
548 citations
TL;DR: Insight is provided into mechanisms of target mRNA recognition and cleavage by an Argonaute–siRNA guide complex and a highly conserved metal-binding site that anchors the 5′ nucleotide of the guide RNA.
Abstract: RNA interference and related RNA silencing phenomena use short antisense guide RNA molecules to repress the expression of target genes. Argonaute proteins, containing amino-terminal PAZ (for PIWI/Argonaute/Zwille) domains and carboxy-terminal PIWI domains, are core components of these mechanisms. Here we show the crystal structure of a Piwi protein from Archaeoglobus fulgidus (AfPiwi) in complex with a small interfering RNA (siRNA)-like duplex, which mimics the 5' end of a guide RNA strand bound to an overhanging target messenger RNA. The structure contains a highly conserved metal-binding site that anchors the 5' nucleotide of the guide RNA. The first base pair of the duplex is unwound, separating the 5' nucleotide of the guide from the complementary nucleotide on the target strand, which exits with the 3' overhang through a short channel. The remaining base-paired nucleotides assume an A-form helix, accommodated within a channel in the PIWI domain, which can be extended to place the scissile phosphate of the target strand adjacent to the putative slicer catalytic site. This study provides insights into mechanisms of target mRNA recognition and cleavage by an Argonaute-siRNA guide complex.
TL;DR: It is found that Clr4/Suv39h predominantly silenced repeat elements whose derived transcripts, transcribed mainly by RNA polymerase II, serve as a source for siRNAs and an important role for the RNAi machinery in maintaining genomic integrity is uncovered.
Abstract: The organization of eukaryotic genomes into distinct structural and functional domains is important for the regulation and transduction of genetic information. Here, we investigated heterochromatin and euchromatin profiles of the entire fission yeast genome and explored the role of RNA interference (RNAi) in genome organization. Histone H3 methylated at Lys4, which defines euchromatin, was not only distributed across most of the chromosomal landscape but was also present at the centromere core, the site of kinetochore assembly. In contrast, histone H3 methylated at Lys9 and its interacting protein Swi6/HP1, which define heterochromatin, coated extended domains associated with a variety of repeat elements and small islands corresponding to meiotic genes. Notably, RNAi components were distributed throughout all these heterochromatin domains, and their localization depended on Clr4/Suv39h histone methyltransferase. Sequencing of small interfering RNAs (siRNAs) associated with the RITS RNAi effector complex identified hot spots of siRNAs, which mapped to a diverse array of elements in these RNAi-heterochromatin domains. We found that Clr4/Suv39h predominantly silenced repeat elements whose derived transcripts, transcribed mainly by RNA polymerase II, serve as a source for siRNAs. Our analyses also uncover an important role for the RNAi machinery in maintaining genomic integrity.
TL;DR: A model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves is suggested.
Abstract: One of the functions of RNA silencing in plants is antiviral defense. A hallmark of RNA silencing is spreading of the silenced state through the plant. Little is known about the nature of the systemic silencing signal and the proteins required for its production, transport, and reception in plant tissues. Here, we show that the RNA-dependent RNA polymerase RDR6 in Nicotiana benthamiana is involved in defense against potato virus X at the level of systemic spreading and in exclusion of the virus from the apical growing point. It has no effect on primary replication and cell-to-cell movement of the virus and does not contribute significantly to the formation of virus-derived small interfering (si) RNA in a fully established potato virus X infection. In grafting experiments, the RDR6 homolog was required for the ability of a cell to respond to, but not to produce or translocate, the systemic silencing signal. Taking these findings together, we suggest a model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA. According to this idea, the secondary siRNAs mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves.
TL;DR: Advances have taken mechanistic understanding of RNA interference to a new level and promise to improve the ability to exploit this biological process for use in experimental biology and medicine.
Abstract: In the RNA-interference pathway, double-stranded RNA induces sequence-specific mRNA degradation through the action of the RNA-induced silencing complex (RISC). Recent work has provided our first glimpses of the RISC-assembly pathway and uncovered the biochemical roles of critical RISC components. These advances have taken our mechanistic understanding of RNA interference to a new level and promise to improve our ability to exploit this biological process for use in experimental biology and medicine.
TL;DR: It is shown that the biogenesis and function of trans-acting siRNA can be genetically uncoupled from a bona fide DCL4-dependent pathway that accounts for RNA interference and for production of the 21-nt siRNA component of the plant cell-to-cell silencing signal.
Abstract: In RNA interference1,2, the RNase-III enzyme Dicer3 processes exogenous double-stranded RNA into small interfering RNAs (siRNAs). siRNAs guide RNA-induced silencing complexes to cleave homologous transcripts, enabling gene-specific knock-down4. In plants, double-stranded RNA is processed into siRNA species of 21 nucleotides (nt) and 24 nt (ref. 5), but, unlike in nematodes6, the Dicer enzymes involved in this processing have not been identified. Additionally, in both plants and nematodes, systemic signals7,8,9,10 with RNA components convey the sequence-specific effects of RNA interference between cells. Here, we describe Arabidopsis thaliana mutants with altered silencing cell-to-cell movement beyond the vasculature. At least three SILENCING MOVEMENT DEFICIENT genes (SMD1, SMD2 and SMD3) are required for trafficking, the extent of which correlates with siRNA levels in the veins. Five alleles defective in synthesis of 21-nt, but not 24-nt, siRNAs carry mutations in Dicer-like 4 (DCL4) that are involved in biogenesis of trans-acting siRNAs11,12. We show that the biogenesis and function of trans-acting siRNA can be genetically uncoupled from a bona fide DCL4-dependent pathway that accounts for RNA interference and for production of the 21-nt siRNA component of the plant cell-to-cell silencing signal.
TL;DR: The generation of Arabidopsis extracts that reproduce many aspects of RNA silencing reactions in vitro are described and it is found that specific members of the Dicer and Argonaute families have distinct biochemical activities, which provides insight into their roles within RNAsilencing pathways in Arabidoptera.
TL;DR: The findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation.
Abstract: RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21–22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5 mRNA fragments generated by RISC cleavage are rapidly degraded from their 3 ends by the exosome, whereas the 3 fragments are degraded from their 5 ends by XRN1. Exosome-mediated decay of the 5 fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation.
TL;DR: The data presented show that various attributes of the 3' end structure play a primary role in determining the position of Dicer cleavage in both dsRNA and unimolecular, short hairpin RNA (shRNA).
Abstract: Dicer processes long double-stranded RNA (dsRNA) and pre-microRNAs to generate the functional intermediates (short interfering RNAs and microRNAs) of the RNA interference pathway. Here we identify features of RNA structure that affect Dicer specificity and efficiency. The data presented show that various attributes of the 3′ end structure, including overhang length and sequence composition, play a primary role in determining the position of Dicer cleavage in both dsRNA and unimolecular, short hairpin RNA (shRNA). We also demonstrate that siRNA end structure affects overall silencing functionality. Awareness of these new features of Dicer cleavage specificity as it is related to siRNA functionality provides a more detailed understanding of the RNAi mechanism and can shape the development of hairpins with enhanced functionality.
TL;DR: It is shown 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.
Abstract: 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.
TL;DR: Results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHv small interfering RNAs into the RNA-induced silencing complex.
Abstract: As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.
TL;DR: It is shown that Tudor-SN specifically interacts with and promotes cleavage of model hyper-edited dsRNA substrates containing multiple I·U and U·I pairs, suggesting a novel unsuspected interplay between the two pathways that is more complex than mutual antagonism.
Abstract: Long perfect double-stranded RNA (dsRNA) molecules play a role in various cellular pathways dsRNA may undergo extensive covalent modification (hyper-editing) by adenosine deaminases that act on RNA (ADARs), resulting in conversion of up to 50% of adenosine residues to inosine (I) Alternatively, dsRNA may trigger RNA interference (RNAi), resulting in silencing of the cognate mRNA These two pathways have previously been shown to be antagonistic We show a novel interaction between components of the ADAR and RNAi pathways Tudor staphylococcal nuclease (Tudor-SN) is a subunit of the RNA-induced silencing complex, which is central to the mechanism of RNAi Here we show that Tudor-SN specifically interacts with and promotes cleavage of model hyper-edited dsRNA substrates containing multiple IU and UI pairs This interaction suggests a novel unsuspected interplay between the two pathways that is more complex than mutual antagonism
TL;DR: The recent developments in the field of RNA silencing in relation to other epigenetic phenomena are reviewed and the significance of this process and its targets in the regulation of modern eukaryotic genomes are discussed.
TL;DR: It is demonstrated here that codelivery of a construct expressing an inverted repeat ADK RNA (dsADK), or addition of an ADK inhibitor (the adenosine analogue A-134974), suppresses GFP-directed silencing in a manner similar to the geminivirus proteins.
Abstract: In eukaryotic cells, homology-dependent silencing that operates at the RNA level is involved in a number of fundamental processes, including cellular defense against viruses, control of transposon mobility, developmental gene regulation via micro-RNAs (miRNAs), de novo histone and DNA methylation, and the establishment of heterochromatin (8, 9, 60, 63). RNA silencing is triggered by double-stranded RNA (dsRNA), and a defining feature is the appearance of short interfering RNA (siRNA), 21- to 26-nucleotide (nt) dsRNA species homologous to the silenced gene (17, 68). These siRNAs are produced from inducing dsRNA by the action of RNase III-like enzymes called Dicer or Dicer-Like (12). In turn, the antisense strands of unwound siRNAs guide another RNase-containing complex, the RNA-induced silencing complex (RISC), to homologous single-stranded RNA (ssRNA) targets (usually mRNA) for degradation (19). Methylation of homologous nuclear DNA corresponding to transcribed regions also occurs, although the role of methylation in RNA silencing is unclear (3, 4, 26).
RNA silencing acts as an antiviral defense in both plant and animal (insect) cells, and viruses are both inducers and targets of the system and thus determine its specificity (31, 32, 55, 59, 65). To counter this adaptive defense, viruses from different families have elaborated a variety of apparently unrelated suppressor proteins that affect different, and possibly multiple, steps in the silencing pathway (33, 61). For example, P1/HC-Pro (HC-Pro) of Tobacco etch virus (TEV) and related potyviruses can reverse established silencing in plants and suppress local silencing of reporter genes in transient assays (2, 6, 27, 35). HC-Pro interacts with a cellular protein (rgs-CaM) that is itself a silencing suppressor, suggesting that the viral protein stimulates an endogenous regulatory pathway (1). It also at least partially inhibits dsRNA processing by Dicer (14, 37). In addition, HC-Pro appears to block unwinding of siRNA/siRNA* duplexes, thereby preventing the incorporation of targeting information into RISC (10). In contrast, the p19 protein of Cymbidium ringspot virus and related tombusviruses cannot reverse established silencing, although it blocks the production of a systemic silencing signal in plants and can suppress local silencing in transient assays. The activity of p19 is due to its ability to bind and sequester siRNAs, which could also prevent incorporation of siRNA into RISC (29, 46, 58, 67). Interestingly, HC-Pro and p19 impact both siRNA and miRNA metabolism, underscoring the similar and overlapping natures of these pathways (10, 14, 28). Further study of HC-Pro, p19, and other viral suppressor proteins will no doubt provide additional insight into the molecular mechanisms of RNA silencing and related processes.
The geminiviruses package ssDNA that replicates in the host cell nucleus through dsDNA intermediates that assemble into minichromosomes (15, 20, 41). Geminiviruses do not encode polymerases but specify multifunctional proteins that provide a cellular environment favorable to replication, initiate specific steps in replication and/or transcription, potentiate virus spread within and between hosts, and suppress host defenses. For example, the AL2 protein of Tomato golden mosaic virus (TGMV; genus Begomovirus) is a transcription factor required for the expression of late viral genes (49-51). The 15-kDa AL2 protein (also known as AC2, C2, or transcriptional activator protein) has a C-terminal activation domain that is functional in plant, yeast, and mammalian cells (22). AL2 is also a pathogenicity factor, and homologues from several begomoviruses have been shown to reverse RNA silencing in plants and to suppress local silencing in transient assays (56, 57, 61). In addition, TGMV AL2 and the related L2 protein of Beet curly top virus (BCTV; genus Curtovirus) condition a virus-nonspecific enhanced-susceptibility phenotype in transgenic plants which is attributable to their ability to inactivate SNF1-related kinase (21, 52). AL2 and L2 also interact with and inactivate adenosine kinase (ADK), which phosphorylates adenosine to produce 5′-AMP (64). Because AMP can stimulate SNF1 activity, the inactivation of SNF1 and ADK by AL2/L2 may represent a dual mechanism to counter SNF1-mediated antiviral responses.
ADK is generally considered to be a housekeeping enzyme involved in adenosine salvage. More recently, it has also been shown to play a key role in sustaining the methyl cycle and S-adenosylmethionine-dependent methyltransferase activity (30, 39, 45, 66). In yeast, methylation deficiency is the primary defect of ADK-null mutants. ADK deficiency also reduces methyltransferase activity in plants, and observational evidence suggests that this can compromise the maintenance of RNA silencing (39, 64).
In this report, a transient system is used to demonstrate that TGMV AL2 protein and the related L2 protein from the curtovirus BCTV can suppress RNA silencing directed against a green fluorescent protein (GFP) reporter gene. We also demonstrate that inhibiting cellular ADK activity causes similar silencing suppression, providing evidence that the TGMV AL2 and BCTV L2 proteins counter RNA silencing by reducing ADK activity. We further show that the AL2 and L2 proteins operate by mechanisms which differ from those of HC-Pro and p19.
TL;DR: This work defines a centromeric pre-siRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation, and shows that another Pol II subunit, Rpb7 has a specific role in pre-SIRNA transcription.
Abstract: Fission yeast centromeric repeats are transcribed into small interfering RNA (siRNA) precursors (pre-siRNAs), which are processed by Dicer to direct heterochromatin formation. Recently, Rpb1 and Rpb2 subunits of RNA polymerase II (RNA Pol II) were shown to mediate RNA interference (RNAi)-directed chromatin modification but did not affect pre-siRNA levels. Here we show that another Pol II subunit, Rpb7 has a specific role in pre-siRNA transcription. We define a centromeric pre-siRNA promoter from which initiation is exquisitely sensitive to the rpb7-G150D mutation. In contrast to other Pol II subunits, Rpb7 promotes pre-siRNA transcription required for RNAi-directed chromatin silencing.
TL;DR: Research in plants and Caenorhabditis elegans has revealed that non‐cell autonomous silencing is operated through specialized, remarkably sophisticated pathways and serves important biological functions, including antiviral immunity and, perhaps, developmental patterning.
TL;DR: Two RNA silencing‐related phenomena, quelling and meiotic silencing by unpaired DNA (MSUD) have been identified in the fungus Neurospora crassa, indicating that the majority of fungi possess the silencing machinery.
TL;DR: HIV-1 TAR RNA is the binding site of the viral protein Tat, the trans-activator of the HIV-1 LTR, which has a highly folded stem-bulge-loop structure, which also binds cellular proteins to form ribonucleoprotein complexes.
Abstract: HIV-1 TAR RNA is the binding site of the viral protein Tat, the trans-activator of the HIV-1 LTR. It is present at the 5' end of all HIV-1 spliced and unspliced mRNAs in the nucleus as well as in the cytoplasm. It has a highly folded stem-bulge-loop structure, which also binds cellular proteins to form ribonucleoprotein complexes. The Tat-Cyclin T1-CDK9 complex is the main component in the trans-activation of HIV-1 and its affinity for TAR is regulated through Tat acetylation by histone acetyl transferases. Recent studies show that this complex is able to recruit other cellular partners to mediate efficient transcriptional elongation. TRBP, PKR and La bind directly to the TAR RNA structure and influence translation of HIV-1 in either positive or negative manners. Some mutations in TAR RNA severely impair HIV-1 trans-activation, translation and viral production, showing its functional importance. The overexpression or suppression of several TAR RNA- binding proteins has a strong impact on viral replication pointing out their major role in the viral life cycle. TAR RNA has been the target of drug development to inhibit viral replication. Recent data using small molecules or RNA-based technologies show that acting on the TAR RNA or on its viral and cellular binding factors effectively decreases virion production.
TL;DR: The biological process of A-to-I RNA editing mediated by ADAR is discussed with new directions on potentially novel targets, including the widely expressed Alu retrotransposable elements found in noncoding regions of mRNA.
Abstract: Publisher Summary The biological process of A-to-I RNA editing mediated by ADAR is discussed with new directions on potentially novel targets, including the widely expressed Alu retrotransposable elements found in noncoding regions of mRNA. Many events take place after the de novo synthesis of an RNA transcript, leading to alterations from its gene-encoded origin. In addition to posttranslational modification, which occurs after the production of the polypeptide chain, RNA can be modified in several ways as to vary the amino acid sequence before it is even translated. Once transcription has commenced, the newly formed pre-mRNA must be processed by several mechanisms that operate posttranscriptionally. The RNA itself plays a role in this regulatory process by forming an assortment of secondary structures. These complex elements in part are formed by the RNA sequence itself producing double-stranded (ds) RNA, creating a configuration of bulges, stem loops, and hairpins.
TL;DR: It is shown that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV, and this work provides evidence that a class 1 RNase III is involved in suppression of RNA silencing.
Abstract: Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.
TL;DR: An epigenetic model for ensuring that CENP-A is targeted and replenished at the kinetochore domain is discussed, which could have additional roles in centromere architecture and the prevention of merotely.
Abstract: Chromatin at centromeres is distinct from the chromatin in which the remainder of the genome is assembled. Two features consistently distinguish centromeres: the presence of the histone H3 variant CENP-A and, in most organisms, the presence of heterochromatin. In fission yeast, domains of silent ‘heterochromatin’ flank the CENP-A chromatin domain that forms a platform upon which the kinetochore is assembled. Thus, fission yeast centromeres resemble their metazoan counterparts where the kinetochore is embedded in centromeric heterochromatin. The centromeric outer repeat chromatin is underacetylated on histones H3 and H4, and methylated on lysine 9 of histone H3, which provides a binding site for the chromodomain protein Swi6 (orthologue of Heterochromatin Protein 1, HP1). The remarkable demonstration that the assembly of repressive heterochromatin is dependent on the RNA interference machinery provokes many questions about the mechanisms of this process that may be tractable in fission yeast. Heterochromatin ensures that a high density of cohesin is recruited to centromeric regions, but it could have additional roles in centromere architecture and the prevention of merotely, and it might also act as a trigger for kinetochore assembly. In addition, we discuss an epigenetic model for ensuring that CENP-A is targeted and replenished at the kinetochore domain.