Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression of oskar translation.

Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3' UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells we show that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. We observe increased association of the translational repressor Bru1 with osk mRNA upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3' UTR.

Analysis of RNA Interference Lines Identifies New Functions of Maternally-Expressed Genes Involved in Embryonic Patterning in Drosophila melanogaster.

Embryonic patterning in Drosophila melanogaster is initially established through the activity of a number of maternally expressed genes that are expressed during oogenesis. mRNAs from some of these genes accumulate in the posterior pole plasm of the oocyte and early embryo and localize further into RNA islands, which are transient ring-like structures that form around the nuclei of future primordial germ cells (pole cells) at stage 3 of embryogenesis. As mRNAs from several genes with known functions in anterior-posterior patterning and/or germ cell specification accumulate in RNA islands, we hypothesized that some other mRNAs that localize in this manner might also function in these developmental processes. To test this, we investigated the developmental functions of 51 genes whose mRNAs accumulate in RNA islands by abrogating their activity in the female germline using RNA interference. This analysis revealed requirements for ttk, pbl, Hip14, eIF5, eIF4G, and CG9977 for progression through early oogenesis. We observed dorsal appendage defects in a proportion of eggs produced by females expressing double-stranded RNA targeting Mkrn1 or jvl, implicating these two genes in dorsal-ventral patterning. In addition, posterior patterning defects and a reduction in pole cell number were seen in the progeny of Mkrn1 females. Because the mammalian ortholog of Mkrn1 acts as an E3 ubiquitin ligase, these results suggest an additional link between protein ubiquitination and pole plasm activity.

Mextli is a novel eukaryotic translation initiation factor 4E-binding protein that promotes translation in Drosophila melanogaster.

Translation is a fundamental step in gene expression, and translational control is exerted in many developmental processes. Most eukaryotic mRNAs are translated by a cap-dependent mechanism, which requires recognition of the 5'-cap structure of the mRNA by eukaryotic translation initiation factor 4E (eIF4E). eIF4E activity is controlled by eIF4E-binding proteins (4E-BPs), which by competing with eIF4G for eIF4E binding act as translational repressors. Here, we report the discovery of Mextli (Mxt), a novel Drosophila melanogaster 4E-BP that in sharp contrast to other 4E-BPs, has a modular structure, binds RNA, eIF3, and several eIF4Es, and promotes translation. Mxt is expressed at high levels in ovarian germ line stem cells (GSCs) and early-stage cystocytes, as is eIF4E-1, and we demonstrate the two proteins interact in these cells. Phenotypic analysis of mxt mutants indicates a role for Mxt in germ line stem cell (GSC) maintenance and in early embryogenesis. Our results support the idea that Mxt, like eIF4G, coordinates the assembly of translation initiation complexes, rendering Mxt the first example of evolutionary convergence of eIF4G function.

Vasa promotes Drosophila germline stem cell differentiation by activating mei-P26 translation by directly interacting with a (U)-rich motif in its 3' UTR.

Vasa (Vas) is a DEAD-box RNA-binding protein required in Drosophila at several steps of oogenesis and for primordial germ cell (PGC) specification. Vas associates with eukaryotic initiation factor 5B (eIF5B), and this interaction has been implicated in translational activation of gurken mRNA in the oocyte. Vas is expressed in all ovarian germline cells, and aspects of the vas-null phenotype suggest a function in regulating the balance between germline stem cells (GSCs) and their fate-restricted descendants. We used a biochemical approach to recover Vas-associated mRNAs and obtained mei-P26, whose product represses microRNA activity and promotes GSC differentiation. We found that vas and mei-P26 mutants interact, and that mei-P26 translation is substantially reduced in vas mutant cells. In vitro, Vas protein bound specifically to a (U)-rich motif in the mei-P26 3' untranslated region (UTR), and Vas-dependent regulation of GFP-mei-P26 transgenes in vivo was dependent on the same (U)-rich 3' UTR domain. The ability of Vas to activate mei-P26 expression in vivo was abrogated by a mutation that greatly reduces its interaction with eIF5B. Taken together, our data support the conclusion that Vas promotes germ cell differentiation by directly activating mei-P26 translation in early-stage committed cells.

Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins.

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components we isolated proteins that associate in early embryos with Vasa (VAS) and Tudor (TUD), two known polar granule associated molecules. We identified Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) as components of both VAS and TUD complexes and confirmed their localization to polar granules by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. Our recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly.

Fat facets interacts with vasa in the Drosophila pole plasm and protects it from degradation.

Anterior-posterior patterning and germ cell specification in Drosophila requires the establishment, during oogenesis, of a specialized cytoplasmic region termed the pole plasm. Numerous RNAs and proteins accumulate to the pole plasm and assemble in polar granules. Translation of some of these RNAs is generally repressed and active only in pole plasm. Vasa (VAS) protein, an RNA helicase and a component of polar granules, is essential maternally for posterior patterning and germ cell specification, and VAS is a candidate translational activator in the pole plasm. VAS is stabilized within the pole plasm in that it is initially present throughout the entire embryo but strictly limited to the pole cells by the cellular blastoderm stage. hsp83 mRNA, which accumulates in the pole plasm through a stabilization-degradation mechanism, is another example. Here, we used a biochemical approach to identify proteins that copurify with VAS in crosslinked extracts. Prominent among these proteins was the ubiquitin-specific protease Fat facets (FAF), a pole plasm component [7], but one whose roles in posterior patterning and germ line specification have remained unclear. We present evidence that FAF interacts with VAS physically and reverses VAS ubiquitination, thereby stabilizing VAS in the pole plasm.