Molecular biology. Amplified silencing

Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK.
Science (Impact Factor: 33.61). 02/2007; 315(5809):199-200. DOI: 10.1126/science.1138030
Source: PubMed
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    • "In RNA interference (RNAi), short interfering RNAs (siRNAs) are produced by endonucleases of the Dicer family from long double-stranded RNA (dsRNA) (4,5). The initial dsRNA triggers may be products of RNA-dependent RNA polymerases (RdRPs), enzymes that also act at downstream steps in some organisms to amplify the silencing response through the synthesis of secondary siRNAs (6,7). "
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    ABSTRACT: In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia.
    Nucleic Acids Research 05/2014; 42(11). DOI:10.1093/nar/gku223 · 9.11 Impact Factor
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    • "In some organisms including nematodes, plants, and fungi, the effect of RNA silencing is amplified by a host RNA-dependent RNA polymerase (RdRp), which converts single-stranded (ss) RNA into dsRNA and thereby promotes accumulation of secondary siRNA (Baulcombe, 2007; Dang et al., 2011). One of the important biological roles of RNA silencing is as a fundamental defense system against viral infections (Wang and Metzlaff, 2005; Ding and Voinnet, 2007). "
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    ABSTRACT: RNA silencing is a fundamental antiviral response in eukaryotic organisms. We investigated the counterdefense strategy of a fungal virus (mycovirus) against RNA silencing in the white root rot fungus, Rosellinia necatrix. We generated an R. necatrix strain that constitutively induced RNA silencing of the exogenous green fluorescent protein (GFP) gene, and infected it with each of four unrelated mycoviruses, including a partitivirus, a mycoreovirus, a megabirnavirus, and a quadrivirus. Infection with a mycoreovirus (R. necatrix mycoreovirus 3; RnMyRV3) suppressed RNA silencing of GFP, while the other mycoviruses did not. RnMyRV3 reduced accumulation of GFP-small interfering (si) RNAs and increased accumulation of GFP-double-stranded (ds) RNA; suggesting that the virus interferes with the dicing of dsRNA. Moreover, an agroinfiltration assay in planta revealed that the S10 gene of RnMyRV3 has RNA silencing suppressor activity. These data corroborate the counterdefense strategy of RnMyRV3 against host RNA silencing.
    Virology 07/2013; 444(1-2). DOI:10.1016/j.virol.2013.07.010 · 3.32 Impact Factor
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    • "Pollen and sperm cells are known to contain 21nt miRNA sequences that are believed to be directed to the male germline (Slotkin et al., 2009); small RNAs associated with AGO9 are specific for the female germline (Olmedo-Monfil et al., 2010). Chromatin condensation driving the formation of heterochromatin in animals silences much of the male genome before fertilization and is augmented by polyamine binding which also inhibits transcription (Baulcombe, 2007). Although polyamines have not been found in nonmotile plant sperm cells, chromatin-silencing gene pathways are highly represented in rice sperm transcripts and could have a similar role in plants. "
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    ABSTRACT: Genomic assay of sperm cell RNA provides insight into functional control, modes of regulation, and contributions of male gametes to double fertilization. Sperm cells of rice (Oryza sativa) were isolated from field-grown, disease-free plants and RNA was processed for use with the full-genome Affymetrix microarray. Comparison with Gene Expression Omnibus (GEO) reference arrays confirmed expressionally distinct gene profiles. A total of 10,732 distinct gene sequences were detected in sperm cells, of which 1668 were not expressed in pollen or seedlings. Pathways enriched in male germ cells included ubiquitin-mediated pathways, pathways involved in chromatin modeling including histones, histone modification and nonhistone epigenetic modification, and pathways related to RNAi and gene silencing. Genome-wide expression patterns in angiosperm sperm cells indicate common and divergent themes in the male germline that appear to be largely self-regulating through highly up-regulated chromatin modification pathways. A core of highly conserved genes appear common to all sperm cells, but evidence is still emerging that another class of genes have diverged in expression between monocots and dicots since their divergence. Sperm cell transcripts present at fusion may be transmitted through plasmogamy during double fertilization to effect immediate post-fertilization expression of early embryo and (or) endosperm development.
    New Phytologist 06/2012; 195(3):560-73. DOI:10.1111/j.1469-8137.2012.04199.x · 7.67 Impact Factor
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