The role of armitage in RNAi and Drosophila development.
Authors: Tingting Du
Journal: Cancer Research (2004)

In eukaryotes, long double-stranded RNA (dsRNA) silences genes homologous in sequence, a process termed RNA interference (RNAi). RNAi and other examples of RNA silencing have been observed in animals, plants, protozoa, and fungi. In plants, green algae, and invertebrates, RNAi defends the genome against mobile genetic elements, such as transposons and viruses, whose expression and activity increase in RNAi-defective mutants. The RNAi pathway also regulates endogenous gene expression for at least one Drosophilagene, Stellate(Ste), which is targeted for destruction by dsRNA transcribed from the Suppressor-of-Stellate (Su(Ste)) locus.  Long dsRNA is converted into small interfering RNAs (siRNAs) by Dicer, a multidomain ribonuclease III enzyme. siRNAs serve as the specificity determinants of the RNAi pathway. siRNAs direct mRNA cleavage as part of a protein-siRNA complex called the RNA-induced silencing complex (RISC).  Members of the Argonaute family of proteins are core components of RISC or RISC-like complexes in flies, worms, and humans and are required genetically for RNA silencing in every organism where their function has been studied. Genetic studies also reveal the importance of helicase-domain proteins in the RNAi pathway. Putative DEA(H/D) box helicases are required for post-transcriptional gene silencing (PTGS) in the green alga Chlamydomonas reinhardtii and RNAi in C. elegans. In Drosophila, mutations in spindle-E(spn-E), a gene encoding a putative DEAD-box helicase, abrogate endogenous RNAi regulation of the Stelocus and trigger expression of retrotransposon mRNA in the germline. In cultured DrosophilaS2 cells, the putative helicase Dmp68 is a component of affinity purified RISC. Similarly, a putative DEAD-box RNA helicase, Gemin3, is a component of human RISC. Dicer, too, contains a putative ATP-dependent RNA helicase domain. Except for Dicer, no specific biochemical function in RNAi has been ascribed to any of these helicase proteins.  armitage(armi) mutants were identified in a screen for maternal effect genes in Drosophila. armimutants are defective in dorsoventral and anteroposterior axis specification during oogenesis. Armitage protein (Armi) is a member of a family of putative ATP-dependent helicases distinct from the DEA(H/D) box proteins. Armi is homologous to SDE3, which is required for PTGS in Arabidopsis. Because PTGS in plants is mechanistically related to RNAi in animals, Armi may play a role in RNAi in flies. Our data show that Armi is required for Ste Silencing, a form of natural RNAi. Ste is silenced by siRNAs derived from the highly homologous Y-linked Su(Ste) locus, which is transcribed on both strands to produce dsRNA. armi mutants, like known RNAi pathway mutants, fail to silence Ste, as evidenced by the accumulation of Ste protein crystals in the testes. The desilencing of Stein armimutants suggests a role for Armi in Drosophila RNAi.  We have shown that armi mutant ovaries are also defective in RNAi. We developed methods to prepare lysates from hand-dissected ovaries. These lysates support RNAi in vitro; that is, when siRNA is added to the lysate, it is assembled into the RNAi effector complex, RISC, and can direct endonucleolytic cleavage of a complementary RNA target, the hallmark of RNAi in Drosophila. We tested RNAi activity in lysates prepared from armi ovaries. The RNAi defect in different armi alleles correlated with their allele strength, demonstrating that the defect in the mutants is caused by mutation of armi, not an unlinked gene. The RNAi defect in armi ovary lysates occurs because armi mutants are impaired in RISC assembly. To demonstrate this, we developed new, small-scale methods to measure RISC formation. Double-stranded siRNA was incubated with ovary lysate in a standard RNAi reaction. Then a 5′-32P-radiolabled, 2′-O-methyl oligonucleotide complementary to the anti-sense strand of the siRNA was added. 2′-O-methyl oligonucleotides bind irreversibly to RISC containing a complementary siRNA, but unlike RNA targets, cannot be cleaved by RISC. Finally, RISC/2′-O-methyl oligonucleotide complexes were resolved by electrophoresis through an agarose gel. We detected at least two distinct RISC-like complexes in ovary lysates. Formation of both complexes is reduced significantly in armi mutants, and the extent of the deficit correlated with allele strength.  We further identified the intermediates in RISC assembly and determined the specific step in RISC assembly at which armi is blocked. We designed a native gel assay to detect intermediates in the assembly of RISC. In this assay, one of the two siRNA strands is radiolabeled, allowing detection of complexes containing either single-stranded or double-stranded siRNA. We used functionally asymmetric siRNAs to distinguish between complexes containing single- and double-stranded siRNA. Using this assay, we identified two intermediates in the RISC assembly pathway. Complex B forms rapidly upon incubation of siRNA in lysate, in the absence of ATP. The siRNA is then transferred to complex A, which contains the previously identified R2D2/Dcr-2 heterodimer. The siRNA is double-stranded in both B and A. RISC is formed from complex A by a process that requires both siRNA unwinding and ATP.  We determined that RISC does not form in ovary lysates from armimutants. However, both complex B and A are readily detected in armimutants. Thus, armimutants are impaired in a step in RISC assembly after binding of the siRNA to the Dcr-2/R2D2 heterodimer (Complex A). Complex A contains both strands of siRNA, while RISC contains only a single siRNA strand. To test the possibility that Armi might act after the formation of complex A to unwind siRNA duplexes prior to their assembly into RISC, we examined if single-stranded siRNA circumvents the requirement for Armi. armi ovary lysates failed to support RNAi when the reactions were programmed with single-stranded siRNA. The requirement for a putative ATPase — Armi — in RNAi triggered by single-stranded siRNA led to our discovery of an additional ATP-dependent step in the RISC assembly, after siRNA unwinding.  In summary, we have shows that armi is required for RNAi. armimutant male germ cells fail to silence Ste, a gene regulated endogenously by RNAi, and lysates from armimutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armimutant ovary lysates suggests that armimutants support early steps in the RNAi pathway, but are defective in the production of the active RISC, which mediates target RNA destruction in RNAi. Our results suggest that armiis required for RISC maturation.