Browsing by Subject "Oskar"
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Item Biophysical studies of an expanded RNA recognition motif from the Bruno protein(2009-08) Lyon, Angeline Marie; Hoffman, David W., Ph. D.; Robertus, Jon D.; Browning, Karen S.; Macdonald, Paul M.; Stevens, Scott W.RNA recognition motifs (RRMs) are a ubiquitous class of proteins which bind RNA in a sequence-specific fashion, often with high affinity. The mechanisms through which this single protein domain recognizes diverse RNA sequences is not fully understood. High-resolution three-dimensional structures are particularly important in understanding the structural features required for RNA recognition and binding. This work presents the structure of an expanded RRM domain from the Drosophila melanogaster Bruno protein. The Bruno protein is involved in establishing proper body patterning during development. This is accomplished through the translational repression of several mRNAs, in particular, the oskar mRNA. Previous work has identified an expanded RRM domain within the Bruno protein. This RRM requires an additional forty amino acids prior to the start of the canonical RRM domain for high affinity RNA binding. The protein was found to contain a canonical RRM domain comprised of four anti-parallel [beta] strands and two [alpha] helices. The RRM is preceded by a ten amino acid loop that interacts with [alpha]₁ and [beta]₂, while the remaining amino acids are flexible in solution. Interestingly, the deletion of these residues does not alter the fold or stability of the RRM domain. Thus, these additional residues must be involved in RNA binding, as they are not required for structure. From these studies, the Bruno RRM represents a new example of protein features required for recognition and high affinity binding of RNA.Item Bruno contributes to osk mRNA localization during Drosophila oogenesis(2011-08) Gim, Youme; Macdonald, Paul M.; Fischer, JaniceOskar is a body pattern and germ cell determining protein in Drosophila melanogaster. This protein must be properly expressed to ensure correct formation of the posterior pole of the animal. Bruno is a RNA binding protein known to regulate osk mRNA translation during Drosophila oogenesis. In vitro work has implicated Bru in oligomerizing osk mRNA into silencing particles and thereby preventing osk mRNA from accessing translational machinery and inhibiting translation. In attempts to further investigate Bru mediated translational regulation, reporter transcripts with Bruno binding regions from the osk mRNA were analyzed for translation and localization of the transcript. Localization of these reporter transcripts has shown the first in vivo evidence for the Bru mediated silencing particle assembly model. In this thesis, I report on the distribution of reporter transcripts in the Drosophila egg chamber.Item Bruno regulates mRNA translation by binding to multiple sequence motifs(2010-08) Reveal, Bradley Steven; Macdonald, Paul M.; Fischer, Janice; Russell, Rick; Stein, Dave; Stevens, ScottOskar (Osk) is a posterior body patterning determinant in Drosophila melanogaster oocytes. oskar (osk) mRNA is translationally repressed until it reaches the posterior of the oocyte where Osk protein accumulates. Translational repression of osk prior to posterior localization is mediated by the RNA binding protein, Bruno (Bru). To better define Bru binding sites, I performed in vitro selections using full length Bru and the fragments containing either the first two RRMs (RRM1+2) or the third RRM (RRM3+). The aptamers from the final round from each of the selections produced a multitude of overrepresented primary sequence motifs. Examples of each of these motifs were found in the 3’UTRs of the mRNAs that Bru is known to regulate during oogenesis. GFP reporter transgenes under the control of the UAS-Gal4 expression system were constructed with each class of the binding sites within the reporter transgenes’ 3’UTRs to test the motifs’ ability to repress the reporters in vivo. In a wildtype background, the GFP reporters containing the binding sites were translationally repressed. In the aret mutant background, the GFP levels of the repressed GFP reporters increased with reduced Bru activity, suggesting the transgenes’ repression is mediated by Bru. Three of the motifs isolated in the in vitro selections reside in the AB and C regions of the osk 3’UTR, and the three classes of sites were mutated in the AB and C regions. The mutated AB and C regions were used to assay for a reduction of Bru binding affinity for the mutant RNAs. Additionally, the mutations were incorporated into an osk genomic transgene that was introduced into an osk RNA null as well as an Osk protein null background. The mutations reduced Bru binding to the AB and C regions. The transgenes containing the mutated Bru binding sites could not fully rescue the osk RNA null phenotype but can fully rescue the Osk protein null phenotype, suggesting an osk transcript can regulate other osk mRNAs in trans.Item Characterization of oskar translational activation and the oskar RNA function(2014-12) Kanke, Matthew Robert; Macdonald, Paul M.; Fischer, Janice; Johnson, Arlen; Raab-Graham, Kimberly; Stein, DavidOskar (Osk) protein is required for posterior body patterning and establishment of the germline in Drosophila. Coordination of osk mRNA localization and translational regulation ensures Osk protein expression is confined to the oocyte posterior. Proper expression requires repression of osk RNA during transport and activation upon localization. Once activated, osk mRNA is translated into two protein isoforms, Long and Short Osk. Here I describe an element in the 5’ end of osk mRNA that is highly conserved across multiple Drosophila species and required for osk translational activation. This 5’ element is located in a region that is also protein coding for the longer Osk isoform and assays were designed to disentangle the effects that mutations had on protein and RNA function. The 5’ element is needed for efficient Osk translation, but only in the absence of Long Osk translation from the same transcript, suggesting a redundant role. Although the 5’ element was previously implicated in a posterior-specific relief of repression, here I provide evidence that the 5’ element acts as a general enhancer of translation, independent of localization and repression. In addition to its protein coding role, osk mRNA has a non-coding function. Egg chambers lacking osk mRNA fail to form a karyosome and arrest mid-oogenesis. RNA function depends on the presence of the osk 3’ UTR in the oocyte. Here I demonstrate that osk mRNA influences distribution of regulators. In the absence of osk mRNA these regulators dissociate from ribonucleoproteins in the germ cells and accumulate in the follicle cells. I find that the osk 3’ UTR performs multiple roles contributing to RNA function. Multiple binding sites act to sequester the translational repressor Bruno in one role. Another involves sequences not bound by Bruno near the 3’ end of osk. In contrary to disruption of Bruno sequestration, which requires mutation of multiple binding sites, mutation of a single site was sufficient to disrupt RNA function. However, disruption of either role recapitulates the failure of karyosome formation and the accumulation of regulators in the follicle cells.Item Determination of how miRNAs mediate repression in Drosophila and the essential role of the oskar mRNA in egg chamber development(2009-12) Reich, John Curtis; Macdonald, Paul M.; Fischer, Janice; Stein, David; Sullivan, Christopher; Gross, JeffreymiRNAs are important regulators of gene expression. These small RNAs function throughout development and regulate translation of a number mRNAs. miRNAs exert their affect on translation as part of the RNP complex RISC. RISC can affect translation of transcripts at both the level of translation initiation, and post-initiation. Although mechanisms of repression mediated by miRNAs have been intensively studied, repression is not well characterized. In order to understand how miRNAs regulate translation in Drosophila, we first characterized miRNA-mediated repression in the ovary. We developed an ovarian assay sensitive to regulation by miRNAs and found that regulated transcripts localize to cytoplasmic puncta distinct from sponge bodies, cytoplasmic RNP structures consisting of proteins implicated in miRNA-mediated regulation. In addition, we devised a genetic screen to identify genes involved in miRNA-mediated regulation. Seven mutants were isolated from the screen, and two mutants were subsequently mapped to separate 1Mb genomic regions. Both these regions are devoid of genes implicated in miRNA-mediated regulation, suggesting our mutants identify novel components involved in repression. The oskar mRNA encodes for the Oskar protein, which is vital in establishing the posterior axis of the Drosophila embryo. In addition to its protein coding function, the osk mRNA has another essential role: it is required for egg chamber progression through oogenesis. This role of oskar is mediated by its 3ʼ UTR, but how it functions in this role is unknown. Here, we investigate the function of the 3ʼ UTR and discover that the well-defined BRE sequences are required for egg chamber progression through oogenesis. The BREs mediate translational repression of the highly regulated oskar mRNA and were previously defined by their ability to bind Bruno, which represses translation of the oskar mRNA. We also provide evidence that the osk BREs sequester Bruno, potentially inhibiting Bruno from binding and misregulating other mRNAs. Our results suggest a novel regulatory loop, where oskar sequesters and inhibits Bruno from misregulating mRNAs, and Bruno, in turn, regulates translation of the oskar mRNA.Item Investigating the role of Bruno interactions with oskar regulatory proteins(2014-08) Kim, Goheun; Macdonald, Paul M.; Browning, Karen; Fischer, Janice; Stein, David; Stevens, ScottOskar (Osk) is a posterior body-patterning determinant in Drosophila melanogaster and is highly concentrated at the posterior pole of the oocyte. osk mRNA is translationally repressed until it reaches the posterior of the oocyte where Osk protein is made. Bruno (Bru) represses translation during osk mRNA localization by direct binding, but how Bru-mediated repression is relieved at the posterior of the oocyte is unknown. Two types of Bru protein interactions are implicated in repression of osk: Bru-Cup interaction and Bru dimerization. By mapping the Bru domains that are important for these interactions, I found that the amino-terminal domain of Bru contributes to both interactions, and deletion of this domain caused a defect in translational repression. However point mutations, within the amino-terminal domain, that disrupt both types of interaction in vitro did not interfere with translational repression in vivo. The difference may be due to other factors stabilizing the Bru-Cup interaction in vivo, as the mutant Bru still associates with Cup in vivo. My work supports the model of repression that relies on Bru interaction with Cup. I also build a new model in which Bru dimerization promotes translational activation of osk, based on my unexpected results: dimerization-defective Bru only weakly accumulated Osk::GFP fusion protein encoded by an osk::GFP reporter RNA bearing a Bru-binding region, while dimerization-competent Bru showed the opposite effect. This suggests that dimerization may contribute to switching Bru from a repressor to an activator, with dimerization controlled via a post-translational modification. Consistent with this, I found that a small fraction of Bru in ovaries is phosphorylated. PKA is a positive regulator of osk expression and phosphorylates Bru in vitro. To test if PKA regulation of osk is mediated through Bru, I examined the effect of altering PKA activity on Bru phosphorylation and Bru-mediated repression. Modulating PKA activity caused small, yet detectable changes in Bru phosphorylation and Bru-dependent translational repression using an osk::GFP reporter. However, while the studies with Bru mutants suggest that phosphorylation promotes repression by Bru, these studies argue for a role in promoting activation. Further work will be required to explain these phenomena.