Paul M. Macdonald

The gradient morphogen bicoid is a concentration-dependent transcriptional activator.

The bicoid (bcd) protein is expressed in an anteroposterior gradient in early Drosophila embryos and controls the zygotic activation of the segmentation gene hunchback (hb) in a broad but precisely bounded anterior domain. Here we show that the hb gene contains multiple regulatory elements that mediate transcriptional activation in response to bcd protein. Further, we demonstrate that the resulting patterns of expression in vivo depend critically on both the bcd gradient profile and the number and quality of these hb elements. Finally, we show that these same elements mediate bcd-dependent transcriptional activation in yeast and that this interaction requires distinct DNA binding and activating regions in the bcd protein. Our results argue that bcd protein normally binds and activates the hb gene in a concentration-dependent fashion, thereby allowing the gradient of bcd protein to dictate where the hb gene is initially turned on in early embryos. They also suggest that the bcd gradient has the instructive capacity to activate other subordinate control genes by the same mechanism, each in a distinct spatial domain according to its affinity for bcd protein.

oskar mRNA is localized to the posterior pole of the Drosophila oocyte

Mutants of the maternal posterior-group genes of Drosophila lack posterior body pattern elements and germ cells, both of which form through the action of determinants localized to the posterior pole of the oocyte. We report that transcripts of one of these genes, oskar, become localized to the posterior pole of oocytes shortly after the oocyte begins to differentiate visibly. Analysis of various posterior-group mutants reveals that localization of oskar mRNA is an early step in the posterior localization pathway. In addition, we find that nonsense oskar mutations disrupt osk mRNA localization, while missense oskar mutations do not.

Overexpression of oskar directs ectopic activation of nanos and presumptive pole cell formation in Drosophila embryos

In Drosophila, a small group of maternal effect genes, including oskar, defines a shared pathway leading to the provision of two determinants at the posterior pole of the embryo. One determinant is the posterior body patterning morphogen nanos, and the other directs germ cell formation. Overexpression of oskar causes the shared pathway to be hyperactivated, with excess nanos activity present throughout the embryo and a superabundance of posterior pole cells. In addition, presumptive pole cells appear at a novel anterior position. Strikingly, formation of these ectopic pole cells is enhanced in nanos mutants. This observation may reflect competition between nanos and the germ cell determinant for a shared and limiting precursor.

Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro.

Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. Here we describe cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, we demonstrate that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor.

Live imaging of nuage and polar granules : evidence against a precursor-product relationship and a novel role for Oskar in stabilization of polar granule components

Nuage, a germ line specific organelle, is remarkably conserved between species, suggesting that it has an important germline cell function. Very little is known about the specific role of this organelle, but in Drosophila three nuage components have been identified, the Vasa, Tudor and Aubergine proteins. Each of these components is also present in polar granules, structures that are assembled in the oocyte and specify the formation of embryonic germ cells. We used GFP-tagged versions of Vasa and Aubergine to characterize and track nuage particles and polar granules in live preparations of ovaries and embryos. We found that perinuclear nuage is a stable structure that maintains size, seldom detaches from the nuclear envelope and exchanges protein components with the cytoplasm. Cytoplasmic nuage particles move rapidly in nurse cell cytoplasm and passage into the oocyte where their movements parallel that of the bulk cytoplasm. These particles do not appear to be anchored at the posterior or incorporated into polar granules, which argues for a model where nuage particles do not serve as the precursors of polar granules. Instead, Oskar protein nucleates the formation of polar granules from cytoplasmic pools of the components shared with nuage. Surprisingly, Oskar also appears to stabilize at least one shared component, Aubergine, and this property probably contributes to the Oskar-dependent formation of polar granules. We also find that Bruno, a translational control protein, is associated with nuage, which is consistent with a model in which nuage facilitates post transcriptional regulation by promoting the formation or reorganization of RNA-protein complexes.

Mutational Analysis of an RNA Recognition Element That Mediates Localization of bicoid mRNA

ABSTRACT Localization signals are RNA regulatory elements that direct the localization of mRNAs to subcellular sites. Localization signals presumably function by mediating RNA recognition events through which the mRNA becomes associated with the localization machinery. At present little is known about individual RNA recognition events, which in turn has limited progress in identifying the trans-acting binding factors involved in these events. Here we describe a detailed characterization of the RNA elements required for the RNA recognition event, event A, that initiates localization of bicoid mRNA in the Drosophila ovary. One element is a helix in which nucleotide identities are not important, suggesting that it plays a primarily structural role. Immediately adjacent to the helix is a recognition domain in which the identities of some, but not all, nucleotides are important for function. Comparison of two related but different RNAs that both support recognition event A further defines the important features of the recognition domain.

Prediction and verification of microRNA targets by MovingTargets, a highly adaptable prediction method

BackgroundMicroRNAs (miRNAs) mediate a form of translational regulation in animals. Hundreds of animal miRNAs have been identified, but only a few of their targets are known. Prediction of miRNA targets for translational regulation is challenging, since the interaction with the target mRNA usually occurs via incomplete and interrupted base pairing. Moreover, the rules that govern such interactions are incompletely defined.ResultsMovingTargets is a software program that allows a researcher to predict a set of miRNA targets that satisfy an adjustable set of biological constraints. We used MovingTargets to identify a high-likelihood set of 83 miRNA targets in Drosophila, all of which adhere to strict biological constraints. We tested and verified 3 of these predictions in cultured cells, including a target for the Drosophila let-7 homolog. In addition, we utilized the flexibility of MovingTargets by relaxing the biological constraints to identify and validate miRNAs targeting tramtrack, a gene also known to be subject to translational control dependent on the RNA binding protein Musashi.ConclusionMovingTargets is a flexible tool for the accurate prediction of miRNA targets in Drosophila. MovingTargets can be used to conduct a genome-wide search of miRNA targets using all Drosophila miRNAs and potential targets, or it can be used to conduct a focused search for miRNAs targeting a specific gene. In addition, the values for a set of biological constraints used to define a miRNA target are adjustable, allowing the software to incorporate the rules used to characterize a miRNA target as these rules are experimentally determined and interpreted.

Recognition of a bicoid mRNA Localization Signal by a Protein Complex Containing Swallow, Nod, and RNA Binding Proteins

Localization of mRNAs, a process essential for embryonic body patterning in Drosophila, requires recognition of cis-acting signals by cellular components responsible for movement and anchoring. We have purified a large multiprotein complex that binds a minimal form of the bicoid mRNA localization signal in a manner both specific and sensitive to inactivating mutations. Identified complex components include the RNA binding proteins Modulo, PABP, and Smooth, the known localization factor Swallow, and the kinesin family member Nod. We demonstrate that localization of bcd mRNA is defective in modulo mutants. The presence of three required localization components (Swallow, Modulo, and specific RNA binding activity) within the recognition complex strongly implicates it in mRNA localization.

BSF binds specifically to the bicoid mRNA 3' untranslated region and contributes to stabilization of bicoid mRNA.

ABSTRACT The early stages of Drosophila melanogaster development rely extensively on posttranscriptional forms of gene regulation. Deployment of the anterior body patterning morphogen, the Bicoid protein, requires both localization and translational regulation of the maternal bicoid mRNA. Here we provide evidence that thebicoid mRNA is also selectively stabilized during oogenesis. We identify and isolate a protein, BSF, that binds specifically to IV/V RNA, a minimal form of the bicoid mRNA 3′ untranslated region that supports a normal program of mRNA localization during oogenesis. Mutations that disrupt the BSF binding site in IV/V RNA or substantially reduce the level of BSF protein lead to reduction in IV/V RNA levels, indicating a role for BSF in RNA stabilization. The BSF protein is novel and lacks all of the characterized RNA binding motifs. However, BSF does include multiple copies of the PPR motif, whose function is unknown but appears in other proteins with roles in RNA metabolism.

Translational regulation of maternal mRNAs

Translational regulation of maternal mRNAs serves to constrain their activities in both time and space. Both types of constraint might be expected to be critical for normal development and the rather short list of maternal mRNAs for which this has been shown to be true has expanded considerably over the past year. Substantial progress has also been reported in the identification and characterization of the cis-acting elements and trans-acting factors that mediate translational regulation and their interactions with one another.