Plant RNAi: How aViral Silencing Suppressor Inactivates siRNA

AbstractThe three-dimensional structure of an siRNA bound to the tombusvirus p19 protein – a suppressor of gene silencing – provides a first glimpse into how plant viruses can defeat their host's anti-viral RNAi defenses

RNA: methods and protocols - a new series

This month, Silence launches a new series on methods and protocols to study silencing pathways and analyze nucleic acids and proteins

Welcome to silence.

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Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour

RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals

Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage

The RNA-Induced Silencing Complex Is a Mg2+-Dependent Endonuclease

AbstractIn the Drosophila and mammalian RNA interference (RNAi) pathways, target RNA destruction is catalyzed by the siRNA-guided, RNA-induced silencing complex (RISC). RISC has been proposed to be an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. Although 5′ cleavage products are readily detected for RNAi in vitro, only 3′ cleavage products have been observed in vivo. Proof that RISC acts as an endonuclease requires detection of both 5′ and 3′ cleavage products in a single experimental system. Here, we show that siRNA-programmed RISC generates both 5′ and 3′ cleavage products in vitro; cleavage requires Mg2+, but not Ca2+, and the cleavage product termini suggest a role for Mg2+ in catalysis. Moreover, a single phosphorothioate in place of the scissile phosphate blocks cleavage; the phosphorothioate effect can be rescued by the thiophilic cation Mn2+, but not by Ca2+ or Mg2+. We propose that during catalysis, a Mg2+ ion is bound to the RNA substrate through a nonbridging oxygen of the scissile phosphate. The mechanism of endonucleolytic cleavage is not consistent with the mechanisms of the previously identified RISC nuclease, Tudor-SN. Thus, the RISC-component that mediates endonucleolytic cleavage of the target RNA remains to be identified

A 5\u27-uridine Amplifies miRNA/miRNA* Asymmetry in Drosophila by Promoting RNA-induced Silencing Complex Formation

BACKGROUND: MicroRNA (miRNA) are diverse in sequence and have a single known sequence bias: they tend to start with uridine (U). RESULTS: Our analyses of fly, worm and mouse miRNA sequence data reveal that the 5\u27-U is recognized after miRNA production. Only one of the two strands can be assembled into Argonaute protein from a single miRNA/miRNA* molecule: in fly embryo lysate, a 5\u27-U promotes miRNA loading while decreasing the loading of the miRNA*. CONCLUSION: We suggest that recognition of the 5\u27-U enhances Argonaute loading by a mechanism distinct from its contribution to weakening base pairing at the 5\u27-end of the prospective miRNA and, as recently proposed in Arabidopsis and in humans, that it improves miRNA precision by excluding incorrectly processed molecules bearing other 5\u27-nt

A Single Mechanism of Biogenesis, Initiated and Directed by PIWI Proteins, Explains piRNA Production in Most Animals [preprint]

In animals, piRNAs guide PIWI-proteins to silence transposons and regulate gene expression. The mechanisms for making piRNAs have been proposed to differ among cell types, tissues, and animals. Our data instead suggest a single model that explains piRNA production in most animals. piRNAs initiate piRNA production by guiding PIWI proteins to slice precursor transcripts. Next, PIWI proteins direct the stepwise fragmentation of the sliced precursor transcripts, yielding tail-to-head strings of phased pre-piRNAs. Our analyses detect evidence for this piRNA biogenesis strategy across an evolutionarily broad range of animals including humans. Thus, PIWI proteins initiate and sustain piRNA biogenesis by the same mechanism in species whose last common ancestor predates the branching of most animal lineages. The unified model places PIWI-clade Argonautes at the center of piRNA biology and suggests that the ancestral animal--the Urmetazoan--used PIWI proteins both to generate piRNA guides and to execute piRNA function

Rethinking the Microprocessor

MicroRNAs (miRNAs) are tiny regulators of gene expression that are processed from longer primary transcripts. In this issue, Han et al. (2006) report some of the structural features of the primary transcript that ensure that the Drosha-DGCR8 enzyme complex liberates precisely the correct precursor sequence, enabling production of a fully functional miRNA