Polarization of the microtubule cytoskeleton during early oogenesis is required to specify the posterior of the Drosophila oocyte: this is essential for asymmetric mRNA localization during mid-oogenesis and for embryonic axis specification. The posterior determinant oskar mRNA is translationally silent until mid-oogenesis. Mutations in armitage (armi) and in three components of the RNAi pathway disrupt oskar mRNA translational silencing, polarization of the microtubule cytoskeleton, and posterior localization of oskar mRNA. armitage encodes a homolog of SDE3, a presumptive RNA helicase involved in posttranscriptional gene silencing (RNAi) in Arabidopsis, and is required for RNAi in Drosophila ovaries. Armitage forms an asymmetric network associated with the polarized microtubule cytoskeleton and is concentrated with translationally silent oskar mRNA in the oocyte. It is concluded that RNA silencing is essential for establishment of the cytoskeletal polarity that initiates embryonic axis specification and for translational control of oskar mRNA (Cook, 2004).

As a RNA helicase involved in posttranscriptional gene silencing, armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi. These results suggest that armi is required for RISC maturation (Tomari, 2004).

Asymmetric mRNA localization produces local protein concentrations that are critical to processes ranging from mating-type switching in yeast to synaptic plasticity in mammals. To produce protein at the right time and place within the cell, transcripts must be translationally silent during transport and remain silent until the protein products are needed. Embryonic axis specification in Drosophila is a well-studied developmental process that depends on spatial and temporal coordination of mRNA localization and translation. bicoid (bcd) mRNA encodes the primary anterior morphogen and is localized to the anterior of the developing oocyte during stage 9. However, bcd is not translated until egg activation, when the transcript is polyadenylated in the cytoplasm, recruited to polysomes, and translated to produce an anterior to posterior protein gradient. Asymmetric localization of oskar (osk) mRNA during mid-oogenesis is essential for posterior patterning and for germ cell formation. osk transcript is produced throughout oogenesis, but remains translationally silent until localization to the oocyte posterior pole during stage 9. Following localization and translational activation, Osk protein triggers the assembly of pole plasm, which specifies the germline and is required for abdominal patterning. Translational repression of osk during stages 7 and 8 is mediated by cis-elements in the osk 3' UTR that are bound by, which interacts with Cup, an eIF4E binding protein. Cup is also required for osk translational silencing and may function with Bruno to silence osk mRNA translation by blocking eIF4E interactions with other components of the translation initiation machinery (Cook, 2004).

Unlike bcd and osk mRNAs, grk mRNA is translated throughout oogenesis, producing a TGFα-related growth factor that initiates two spatially and temporally distinct signaling events that specify the anterior-posterior (A/P) and dorsal-ventral (D/V) axes. During early oogenesis, microtubules originate from the posterior of the oocyte and mediate posterior localization of grk mRNA and Grk protein. Grk signals to the overlying follicle cells to induce posterior differentiation. During mid-oogenesis, the posterior follicle cells signal back to the oocyte, inducing reorganization of the oocyte microtubule network, which is essential for the asymmetric localization of bcd, osk, and grk mRNAs. Following reorganization of the microtubule network, grk mRNA localizes to the dorsal-anterior corner of the oocyte, and local Grk signaling induces dorsal differentiation of the adjacent follicle cells. The correct organization of oocyte microtubules early in oogenesis thus initiates a series of signaling events that specify the axes of the oocyte (Cook, 2004).

The armitage gene has been shown to be required for anterior-posterior polarization of the microtubule cytoskeleton and translational silencing of osk mRNA during early oogenesis and for osk mRNA localization and posterior and dorsal-ventral patterning during mid-oogenesis. The armi gene encodes a putative RNA helicase most closely related to SDE3, which is required for posttranscriptional gene silencing (PTGS) in Arabidopsis (Dalmay, 2001; Willmann, 2001). PTGS is an evolutionarily conserved RNA silencing mechanism closely related to RNA interference (RNAi). Since armi is required for RNAi and efficient assembly of the RNA-induced silencing complex (RISC) in ovary extracts (Tomari, 2004), Armi/SDE3 class RNA helicases appear to have a conserved role in RNAi. Three additional genes shown to be implicated in RNAi are also required for osk mRNA translational silencing and microtubule reorganization during early oogenesis, indicating that the RNAi system is required for axial polarization of the oocyte. Finally, Armi protein is shown to be concentrated in the oocyte with osk mRNA. It is speculated that this asymmetric localization may spatially restrict RNA silencing activity and increase the efficiency of target recognition and thus help to establish the functional asymmetries that initiate embryonic axis specification (Cook, 2004).

Thus armi is required for initial polarization of the microtubule cytoskeleton and for temporal regulation of osk mRNA translation. Armi is required to repress osk translation during early oogenesis, but does not alter osk mRNA levels. armi is also required for Stellate silencing during spermatogenesis (Tomari, 2004), which requires small homologous miRNAs and the RNAi components Spn-E and Aub. Armi is also required for RNAi and efficient RISC assembly in ovary extracts (Tomari, 2004). These findings strongly suggest that the RNAi system is required for an early step in the axis specification pathway (Cook, 2004).

Consistent with this hypothesis, the RNAi components Spn-E, Aub, and Mael are also required for osk mRNA silencing and for polarization of the microtubule cytoskeleton during early oogenesis. Aub enhances osk translation during mid-oogenesis. However, aub disrupts posterior localization of osk mRNA during mid-oogenesis, and posterior localization is required for efficient osk translation. It is therefore speculated that the reduced Osk protein levels during later stages of oogenesis are secondary to defects in posterior patterning during early oogenesis, when Aub and other RNA silencing components are required to establish the microtubule asymmetries required to specify the posterior pole (Cook, 2004).

While the RNA silencing system represses osk during stages 1 to 6, other studies indicate that products of the bruno/arrest, cup, and Bicaudal-C genes are essential for osk silencing during stages 6 to 8. The role of Bicaudal-C in osk silencing is unclear. However, Bruno binds to three sites in the osk 3' UTR, called Bruno response elements (BREs), and deletion of the BREs leads to osk translation during stages 7 and 8 and severe patterning defects. Recent data show that Cup associates with both Bruno and the 5'-cap binding factor eIF4E (Nakamura, 2004). This suggests that Cup and Bruno may function together to repress osk translation by sequestering the 5' end of osk mRNA, thereby blocking translation initiation. Bruno and Cup do not appear to play a role in osk mRNA translational repression during stages 1-6, when the RNAi pathway is required for silencing. The biological reason for this two-step translational control mechanism is unclear, but may be linked to changing functions for the RNAi system during oogenesis (Cook, 2004).

All of the RNAi mutations examined in this study produce nearly identical defects in microtubule polarization and osk silencing during early oogenesis and posterior and D/V patterning during mid-oogenesis. The early defects in osk silencing and microtubule organization appear to reflect independent functions for the RNAi system, since mutations in osk do not suppress the cytoskeletal defects in armi mutants, and forced premature expression of Osk protein from a transgene does not induce changes in microtubule organization. The defects in anterior-posterior polarization of the microtubule cytoskeleton during early oogenesis, by contrast, may directly lead to the posterior and D/V patterning defects observed in mid-oogenesis. Microtubule-dependent posterior localization of grk mRNA in early oogenesis is thought to facilitate Grk signaling from the oocyte to the posterior follicle cells: this initiates a chain of signaling events that trigger microtubule reorganization and mRNA localization during mid-oogenesis. Thus, defects in microtubule polarization associated with RNAi mutations are likely the primary cause of later defects in axial patterning (Cook, 2004).

The spectrum of defects observed in RNAi mutants suggests that the RNA silencing machinery targets multiple processes during early oogenesis. The endogenous miRNAs that mediate RNA silencing are predicted to bind complementary sequences in the 3' UTRs of numerous target transcripts, suggesting that they may coordinate translational control of gene cassettes during complex biological processes (Stark, 2003). A computational screen for miRNA targets has identified osk mRNA, kinesin heavy chain mRNA, and transcripts encoding several other cytoskeletal proteins involved in oogenesis as targets for the miR-280 miRNA (Stark, 2003). It is interesting to note that kinesin, like the RNAi components, is required for posterior and D/V axis specification. This motor also drives ooplasmic streaming during late oogenesis, and mutations in mael lead to premature ooplasmic streaming, which could reflect overexpression of kinesin due to defects in silencing. These observations raise the possibility that the RNAi system, through miR-280 and other miRNAs, coordinates axis specification by silencing osk mRNA and simultaneously regulating genes involved in microtubule function (Cook, 2004).

Armi is asymmetrically localized during early oogenesis, when it is concentrated in the oocyte with osk mRNA. This finding raises the possibility that asymmetric Armi localization plays a role establishing developmental asymmetries during early oogeneis. Tomari (2004) has shown that Armi is required for mRNA target cleavage and RISC maturation in vitro. Armi could therefore promote local RISC assembly and thus increase the efficiency of osk mRNA silencing. This could play a role in regulating other transcripts that accumulate in the oocyte. Local increases in RISC activation could also lead to oocyte-specific silencing of transcripts that are uniformly distributed within the oocyte-nurse cell complex. The molecular, genetic, and cytological tools available in Drosophila should allow direct tests of these possibilities (Cook, 2004).

GENE STRUCTURE

cDNA clone length - 4165

Bases in 5' UTR - 478

Exons - 10

Bases in 3' UTR - 125

PROTEIN STRUCTURE

Amino Acids - 1188

Structural Domains

Conceptual translation of the open reading frame in the 4.2 kb armi cDNA produces an 1188 amino acid protein of approximately 136 kDa. The first 500 amino acids show no significant homology to known proteins and do not contain conserved structural motifs. The C-terminal domain, however, shows significant homology to a group of putative RNA helicases from plants, mice, and humans. This domain contains eight motifs characteristic of the Upf1p family of ATP-dependent RNA helicases (Koonin, 1992; Linder, 2000; Tanner, 2001; Weng, 1996). Armi is most closely related to Mov10 (gb110) proteins in mice and humans (33% identity) (Mooslehner, 1991) and to Arabidopsis SDE3 (34% identity) (Dalmay, 2001). A C. elegans homolog has not been identified. The function of mammalian Mov10 is not known. SDE3, however, is required for PTGS (Dalmay, 2001; Himber, 2003), an RNA silencing mechanism related to RNAi in animals. Plant PTGS targets viral mRNAs for degradation and thus provides protection against viral infection. This system can also induce degradation of RNAs encoded by transgenes and by homologous endogenous genes. SDE3 also participates in amplification of the silencing trigger, which is required for long-range cell-to-cell spreading of silencing activity (Himber, 2003). Propagation of RNA silencing occurs in plants and nematodes (Cook, 2004 and references therein).

EVOLUTIONARY HOMOLOGS

Post-transcriptional gene silencing (PTGS) provides protection in plants against virus infection and can suppress expression of transgenes. Arabidopsis plants carrying mutations at the SDE3 locus are defective in PTGS mediated by a green fluorescent protein transgene. However, PTGS mediated by tobacco rattle virus (TRV) was not affected by sde3. From these results it is concluded that SDE3, like the previously described RNA polymerase encoded by SDE1, acts at a stage in the mechanism that is circumvented when PTGS is mediated by TRV. The product of SDE3 is similar to RNA helicase-like proteins including GB110 in mouse and other proteins in Drosophila and humans. These proteins are similar to, but clearly distinct from Upf1p and SMG-2, which are required for nonsense-mediated mRNA decay in yeast and Caenorhabditis elegans and, in the case of SMG-2, for PTGS (Delmay, 2001).

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