Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. Here, we show that staufen- and FMRP-containing RNPs in Drosophila neurons contain proteins also present in somatic "P bodies," including the RNA-degradative enzymes Dcp1p and Xrn1p/Pacman and crucial components of miRNA (argonaute), NMD (Upf1p), and general translational repression (Dhh1p/Me31B) pathways. Drosophila Me31B is shown to participate (1) with an FMRP-associated, P body protein (Scd6p/trailer hitch) in FMRP-driven, argonaute-dependent translational repression in developing eye imaginal discs; (2) in dendritic elaboration of larval sensory neurons; and (3) in bantam miRNA-mediated translational repression in wing imaginal discs. These results argue for a conserved mechanism of translational control critical to neuronal function and open up new experimental avenues for understanding the regulation of mRNA function within neurons.

Localization and translational control of Drosophila melanogaster gurken and oskar mRNAs rely on the hnRNP proteins Squid and Hrp48, which are complexed with one another in the ovary. Imp, the Drosophila homolog of proteins acting in localization of mRNAs in other species, is also associated with Squid and Hrp48. Notably, Imp is concentrated at sites of gurken and oskar mRNA localization in the oocyte, and alteration of gurken localization also alters Imp distribution. Imp binds gurken mRNA with high affinity in vitro; thus, the colocalization with gurken mRNA in vivo is likely to be the result of direct binding. Imp mutants support apparently normal regulation of gurken and oskar mRNAs. However, loss of Imp activity partially suppresses a gurken misexpression phenotype, indicating that Imp does act in control of gurken expression but has a largely redundant role that is only revealed when normal gurken expression is perturbed. Overexpression of Imp disrupts localization of gurken mRNA as well as localization and translational regulation of oskar mRNA. The opposing effects of reduced and elevated Imp activity on gurken mRNA expression indicate a role in gurken mRNA regulation.The restriction of proteins to discrete subcellular regions can be accomplished by a coordinated program of mRNA localization and translational control. These mechanisms are used prominently during oogenesis in Drosophila melanogaster, where several localized proteins direct body patterning. The dorsoventral axis of the oocyte and later the embryo are established by a process that involves the specific expression of Gurken (Grk) protein at a dorsal position near the anterior of the oocyte. Similarly, patterning along the anteroposterior axis relies on restricted expression of Bicoid (Bcd) and Oskar (Osk) proteins at the anterior and posterior poles, respectively, of the oocyte and embryo. In each case, the deployment of the protein is a consequence of localization of the mRNA to the appropriate region within the oocyte, coupled with translational controls to enhance accumulation of the protein at this destination (reviewed in reference 12).These programs of posttranscriptional control of gene expression require RNA binding proteins that recognize regulatory elements within the mRNAs and mediate association with the localization or translational control machinery. Although it has proven difficult in most cases to demonstrate that a particular protein/RNA interaction contributes to regulation, multiple RNA binding proteins are required for correct expression of grk mRNA; these include Squid (Hrp40), Hrp48 (also known as Hrb27C), Bruno (Bru), Vasa, and Otu (8,9,13,31,41,45,49). Each of these proteins is also required for correct expression of osk (11,14,20,30,32,40,44,47,51), revealing substantial similarities in the control of grk and osk mRNAs.

The central question in stem cell regulation is how the balance between self-renewal and differentiation is controlled at the molecular level. This study uses germline stem cells (GSCs) in the Drosophila ovary to demonstrate that the Drosophila CCR4 homolog Twin is required intrinsically to promote both GSC self-renewal and progeny differentiation. Twin/CCR4 is one of the two catalytic subunits in the highly conserved CCR4-NOT mRNA deadenylase complex. Twin works within the CCR4-NOT complex to intrinsically maintain GSC self-renewal, at least partly by sustaining E-cadherin-mediated GSC-niche interaction and preventing transposable element-induced DNA damage. It promotes GSC progeny differentiation by forming protein complexes with differentiation factors Bam and Bgcn independently of other CCR4-NOT components. Interestingly, Bam can competitively inhibit the association of Twin with Pop2 in the CCR4-NOT complex. Therefore, this study demonstrates that Twin has important intrinsic roles in promoting GSC self-renewal and progeny differentiation by functioning in different protein complexes.

bThe Drosophila melanogaster Myb-MuvB/dREAM complex (MMB/dREAM) participates in both the activation and repression of developmentally regulated genes and origins of DNA replication. Mutants in MMB subunits exhibit diverse phenotypes, including lethality, eye defects, reduced fecundity, and sterility. Here, we used P-element excision to generate mutations in lin-52, which encodes the smallest subunit of the MMB/dREAM complex. lin-52 is required for viability, as null mutants die prior to pupariation. The generation of somatic and germ line mutant clones indicates that lin-52 is required for adult eye development and for early embryogenesis via maternal effects. Interestingly, the maternal-effect embryonic lethality, larval lethality, and adult eye defects could be suppressed by mutations in other subunits of the MMB/dREAM complex. These results suggest that a partial MMB/dREAM complex is responsible for the lethality and eye defects of lin-52 mutants. Furthermore, these findings support a model in which the Lin-52 and Myb proteins counteract the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner.

We thank Will Kruesi and Monica Sullivian for assistance with the screen. We thank the Bloomington, Szeged and Harvard Medical School stock centers for flies, and Trudi Schupbach, David Stein and Daniel St. Johnson for fly stocks, cDNAs and antibodies. The 1D12, 40-1a antibody was from the Developmental Studies Hybridoma Bank, which was developed under the auspices of the NICHD and maintained by The Universities of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was supported by grants GM42612 and GM54409 from the NIH. AbstrACtAxial patterning in Drosophila relies on the deployment of patterning proteins at specific regions within the developing oocyte. This process involves transport of mRNAs from the nurse cells to the oocyte, localization of mRNAs within the oocyte, and translational regulation of these mRNAs to restrict the final distribution of the proteins. Despite extensive analysis, the events of deployment are not fully understood and it seems certain that many contributing factors remain to be identified. We describe the development and application of a sensitized genetic screen to reveal such additional factors. Overexpression of Imp, a factor implicated in regulation of gurken mRNA, causes a weak dorsalization that can be enhanced by reducing the level of other factors acting in the same pathway. A collection of deficiency mutants was screened using this assay, leading to the identification of 5 genes that are candidates to contribute to axial patterning. Three of the genes were characterized in greater detail. The mushroom body expressed gene was implicated in axial patterning, with overexpression leading to a range of patterning abnormalities that can be explained by a more primary defect in organization of the cytoskeleton. Two mitotic cell cycle control factors, cyclin E and E2f1, were also implicated, raising the possibility that a mitotic cell cycle checkpoint may impinge on grk expression, much as meiotic checkpoints can alter expression of this gene.