Sigova, A. , Rhind, N. & Zamore, P.D. A single argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe. Genes Dev. 18, 2359-2367

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Genes & Development (Impact Factor: 10.8). 11/2004; 18(19):2359-67. DOI: 10.1101/gad.1218004
Source: PubMed


The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. Here, we show that the introduction of a double-stranded RNA (dsRNA) hairpin corresponding to a green fluorescent protein (GFP) transgene triggers classical RNA interference (RNAi) in S. pombe. That is, GFP silencing triggered by dsRNA reflects a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe requires dcr1, rdp1, and ago1, but does not require chp1, tas3, or swi6, genes required for transcriptional silencing. Thus, the RNAi machinery in S. pombe can direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. Our findings suggest that these three proteins fulfill a common biochemical function in distinct siRNA-directed silencing pathways.

    • "Small RNAs are composed of 20–30 nucleotides, containing coding sequences that direct RISC to recognize target genes and information of preference to AGO proteins, which are core elements of RISC (Song et al., 2004; Kim, 2008; Takeda et al., 2008; Thieme et al., 2012). miRNAs are a class of small RNA endogenously generated from stemloop structures, having the ability to regulate both chromatin state and encoding progress of target genes (Hall et al., 2002; Volpe et al., 2002; Sigova et al., 2004; Chellappan et al., 2010). In Arabidopsis, miR168 regulates AGO1, and loss of miR168 function causes developmental defects (Vaucheret et al., 2004). "
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    ABSTRACT: In Arabidopsis thaliana, ARGONAUTE1 (AGO1) interacts with miR168 to modulate the small RNA regulatory pathway. However, the underlying mechanism of regulation and relationship between AGO1 and miR168 is poorly understood in the cash crop Solanum lycopersicum (tomato). We previously found that SlAGO1A and SlAGO1B were cleaved by miR168 in tomato. In this study, we show that SlAGO1A and SlAGO1B accumulate in miR168-sponge transgenic plants, and that expression of miR168-resistant SlAGO1A (4m-SlAGO1A) and SlAGO1B (4m-SlAGO1B) in tomato results in a series of defects affecting growth rate, floral timing, leaves, and fruit. Accumulation of miR156 was found when 4m-SlAGO1A was at an early developmental stage compared to the wild type and original SlAGO1A transgenic plants, and miR172 was highly expressed in adult 4m-SlAGO1A compared to the controls. In addition, the expression of multiple small RNAs was altered in 4m-SlAGO1A. Taken together, our data provide novel insights into the interaction between SlAGO1s and miR168 in determining growth rate, phase change, leaf epinasty, fruit initiation and expansion, and other developmental processes in tomato.
    No preview · Article · Nov 2014 · Journal of Experimental Botany
    • "Different hpRNA constructs targeting endogenous genes or transgenes have been tested in several fungal species with varying success (reviewed by [22,24]). In addition, hpRNA expression in some fungi such as fission yeast resulted in not only post-transcriptional silencing, but also heterochromatin formation [14,25,26]. "
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    ABSTRACT: Hairpin RNA (hpRNA) transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms. However, in fungi, expression of hairpin RNA transcripts can induce post-transcriptional gene silencing, but in some species can also lead to transcriptional gene silencing, suggesting a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a powerful technique to understand gene function, particularly when gene knockouts are difficult to obtain. We investigated whether the plant pathogenic fungus Fusarium oxysporum possesses a functional gene silencing machinery and whether hairpin RNA transcripts can be employed to effectively induce gene silencing. Here we show that, in the phytopathogenic fungus F. oxysporum, hpRNA transgenes targeting either a beta-glucuronidase (Gus) reporter transgene (hpGus) or the endogenous gene Frp1 (hpFrp) did not induce significant silencing of the target genes. Expression analysis suggested that the hpRNA transgenes are prone to transcriptional inactivation, resulting in low levels of hpRNA and siRNA production. However, the hpGus RNA can be efficiently transcribed by promoters acquired either by recombination with a pre-existing, actively transcribed Gus transgene or by fortuitous integration near an endogenous gene promoter allowing siRNA production. These siRNAs effectively induced silencing of a target Gus transgene, which in turn appeared to also induce secondary siRNA production. Furthermore, our results suggested that hpRNA transcripts without poly(A) tails are efficiently processed into siRNAs to induce gene silencing. A convergent promoter transgene, designed to express poly(A)-minus sense and antisense Gus RNAs, without an inverted-repeat DNA structure, induced consistent Gus silencing in F. oxysporum. These results indicate that F. oxysporum possesses functional RNA silencing machineries for siRNA production and target mRNA cleavage, but hpRNA transgenes may induce transcriptional self-silencing due to its inverted-repeat structure. Our results suggest that F. oxysporum possesses a similar gene silencing pathway to other fungi like fission yeast, and indicate a need for developing more effective RNA silencing technology for gene function studies in this fungal pathogen.
    No preview · Article · Jul 2013 · Silence
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    • "There are also cases of over simplification and loss of Argonaute proteins in different lineages. S. pombe has only one AGO protein and plays a role in both post-transcriptional gene silencing and heterochromatin silencing [50]. Whereas, S. cerevisiae, another species of the same genus, and a few parasitic organisms such as T. cruzi and L. major, do not encode Argonaute proteins. "
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    ABSTRACT: Intergenic DNA, often described as "playground of evolution", harbors a plethora of cis and trans regulatory elements in the form of non-coding RNAs (ncRNAs). The evolution of the silencing mechanism mediated by microRNAs (miRNAs), an important class of ncRNA, involves the proliferation of miRNA biogenesis and effector proteins, continuing innovation of novel families by the diversification of established families and spawning additional paralogous family members. Such evolving miRNA pathways for spatiotemporal regulation of the transcriptome have shaped the evolution of eukaryotic genomes and contributed to the complexity of multicellular organisms. Here, we focus on the emergence of new target specificity of the miRNAs along with the proliferation of core biogenesis and effector modules and show how this has contributed to generate diverse miRNA regulatory pathways.
    Full-text · Article · Apr 2012 · Current Genomics
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