Investigating the role of Bruno interactions with oskar regulatory proteins

textOskar (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.Cellular and Molecular Biolog

Twist-dependent ratchet functioning downstream from Dorsal revealed using a light-inducible degron

Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal–ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal. This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets

Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo

Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster.

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo

Region-Specific Activation of <i>oskar</i> mRNA Translation by Inhibition of Bruno-Mediated Repression

<div><p>A complex program of translational repression, mRNA localization, and translational activation ensures that Oskar (Osk) protein accumulates only at the posterior pole of the <i>Drosophila</i> oocyte. Inappropriate expression of Osk disrupts embryonic axial patterning, and is lethal. A key factor in translational repression is Bruno (Bru), which binds to regulatory elements in the <i>osk</i> mRNA 3′ UTR. After posterior localization of <i>osk</i> mRNA, repression by Bru must be alleviated. Here we describe an <i>in vivo</i> assay system to monitor the spatial pattern of Bru-dependent repression, separate from the full complexity of <i>osk</i> regulation. This assay reveals a form of translational activation—region-specific activation—which acts regionally in the oocyte, is not mechanistically coupled to mRNA localization, and functions by inhibiting repression by Bru. We also show that Bru dimerizes and identify mutations that disrupt this interaction to test its role <i>in vivo</i>. Loss of dimerization does not disrupt repression, as might have been expected from an existing model for the mechanism of repression. However, loss of dimerization does impair regional activation of translation, suggesting that dimerization may constrain, not promote, repression. Our work provides new insight into the question of how localized mRNAs become translationally active, showing that repression of <i>osk</i> mRNA is locally inactivated by a mechanism acting independent of mRNA localization.</p></div

PKA phosphorylates Bru in the amino-terminal domain and Bru phosphosilent mutations disrupt phosphorylation by PKA.

<p>A. Left: Western blot of <i>wild-type</i> ovary extract after incubation with phosphatase and/or phosphatase inhibitors as indicated above. Proteins were detected using anti-Bru antibody. Right: Western blot of ovary extract from flies expressing MCP::HA₃::Bru1–146 protein, with treatments noted as above. Proteins were detected using anti-HA antibody. Inhibitors used were sodium vanadate and beta-glycero phosphate, which are competitive inhibitors of the alkaline phosphatase. B. <i>In vitro</i> phosphorylation assay using gamma ₃₂P-ATP, purified mouse PKA catalytic subunit and purified Bru proteins as labeled (as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004992#pgen.1004992.g001" target="_blank">Fig. 1B</a>). BSA was used as a negative control. Top: autoradiogram to detect phosphorylation. Bottom: Coomassie staining of proteins used for the phosphorylation assay to show the relative amounts of input proteins. The upper band in the 1–146 lane is a contaminating bacterial protein. C. <i>In vitro</i> phosphorylation assay using gamma ₃₂P-ATP, purified mouse PKA catalytic subunit and purified phosphosilent (Ala) mutant Bru proteins as labeled. The positions of amino acids predicted to be candidates for phosphorylation by PKA are shown in the schematic (D). The point-mutated Bru proteins have the Δ334–416 deletion, which does not affect phosphorylation (panel B). Top: autoradiogram to detect phosphorylation. A similar assay using the same mutations in the context of the full-length Bru is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004992#pgen.1004992.s006" target="_blank">S4B Fig</a>. Bottom: Western blot of proteins used in the phosphorylation assay to show the relative amounts of input proteins. D. A schematic diagram of Bru showing PKA phosphorylation sites predicted by NetPhosK and KinasePhos. Three amino acids, S4, S7 and T135, depicted as black circles, were tested in different experiments by mutating them to either alanine (phosphosilent) or glutamate (phosphomimetic).</p

Osk expression in <i>aret</i> replacement lines.

<p>A-F, D’-F’. Egg chambers expressing the <i>oskT140</i>::<i>HA</i> genomic transgene (whose expression mimics that of <i>osk</i>, Methods and Materials), in different genetic backgrounds with engineered <i>aret</i> alleles, as labeled. All have a single copy of <i>aret</i> in <i>trans</i> to <i>Df(2L)aret</i> and two copies of <i>oskT140</i>::<i>HA</i>. (A-C) are stage 8, and (D-F) are stage 9 egg chambers. (D’-F’) show Osk::HA in green and nuclei in red. All samples were fixed in parallel and imaged together under the same settings. G-I. Late-stage egg chambers expressing the <i>oskT140</i>::<i>GFP</i> genomic transgene (whose expression mimics that of <i>osk</i>) in different genetic backgrounds with engineered <i>aret</i> alleles, as labeled. All have a single copy of <i>aret</i> in <i>trans</i> to <i>Df(2L)aret</i> and two copies of <i>oskT140</i>::<i>GFP</i>. All samples were fixed in parallel and imaged together under the same settings. J. Cuticular phenotypes of embryos from <i>aret</i> mutant mothers. All have <i>aret</i> in <i>trans</i> to <i>Df(2L)aret</i>. <i>aret⁺</i> is the <i>wild-type</i> replacement.</p

Interaction-defective Bru mutants retain strong repressive activity in the tethering assay.

<p>A-G, A’-G’. Egg chambers expressing the <i>GFP-MS2</i> reporter mRNA. (B-G, B’-G’) also express MCP::HA₃::Bru proteins, of the type shown at left. All Bru proteins include point mutations in RRM2 and RRM3 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004992#pgen.1004992.g002" target="_blank">Fig. 2</a> legend). All samples were fixed in parallel and imaged together under the same settings. Expression of the UAS transgenes was driven by the <i>nosGAL4VP16</i> driver. The MCP::HA₃::Bru proteins were expressed at similar levels, except for S4E/S7E/T135E which was slightly elevated (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004992#pgen.1004992.s003" target="_blank">S1B Fig</a>.). H. RNase protection assays: <i>GFP-MS2</i> RNA levels were quantified by ImageJ and normalized using the <i>rp49</i> signal. The value for none, which lacks any MCP::HA₃::Bru proteins, was set to one. The mean and standard deviation were calculated from three independent experiments.</p

Translational repression and activation in <i>aret</i> mutants.

<p>A. Schematic diagram of the <i>aret</i> locus. Numbering is according to the <i>Drosophila</i> genome sequence, R5.48. Orange bars depict exons (the widths of the bars are not to scale), and the red rectangle is the 2.1kb targeted region, which when translated, includes the amino-terminal domain of female Bru.B-D, B’-D’. Stage 6 egg chambers expressing the <i>osk1–534</i>::<i>GFP-AB</i> reporter in different <i>aret</i> mutant backgrounds, as labeled. <i>Df</i> is <i>Df(2L)BSC407</i>. For B-D GFP is in green and nuclei in red. Panels B′-D′ show just the GFP channel. E-G, E’-G’. Examples of phenotypic categories for panel H. For E-G GFP is in green and nuclei in red. Panels E′-G′ show just the GFP channel. All samples were fixed in parallel and imaged together under the same settings. Expression of the UAS transgene was driven by the <i>matα4-GAL-VP16</i> driver. H. Intensity of the posterior zone of GFP from <i>UAS-osk1–534</i>::<i>GFP-AB</i> in <i>aret</i> mutants. Examples of strong, weak and undetectable are shown in E/E′, F/F′ and G/G′, respectively.</p

The Bru amino-terminal domain is essential for translational repression in a tethering assay.

<p>A-E, A’-E’. Egg chambers expressing the <i>GFP-MS2</i> reporter mRNA. (B-E, B’-E’) also express MCP::HA₃::Bru proteins, of the type shown at left. All Bru proteins used include point mutations in RRM2 and RRM3 to inhibit RNA-binding activity, and thus prevent the early arrest of oogenesis caused by ectopic expression of Bru [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004992#pgen.1004992.ref027" target="_blank">27</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004992#pgen.1004992.ref060" target="_blank">60</a>]. The RRM2 and RRM3 mutations have no effect on tethering, which relies on RNA binding by MCP. All samples were fixed in parallel and imaged together under the same settings. Expression of the UAS transgenes was driven by the <i>nosGAL4VP16</i> driver. Expression levels for different MCP::Bru proteins are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004992#pgen.1004992.s003" target="_blank">S1A Fig</a>. F. GFP fluorescence was quantitated using Macnification and the value for none, which lacks any MCP::HA₃::Bru proteins, was set to one. The mean and standard deviation were calculated from at least 40 samples per genotype. The asterisks indicate the Bru proteins with the GFP protein level differing significantly from the Bru+, using the student’s T test (**p≤0.01, ***p≤0.001). G. RNase protection assays: <i>GFP-MS2</i> RNA levels were quantified by ImageJ and normalized using the <i>rp49</i> signal. The value for none, which lacks any MCP::HA₃::Bru proteins, was set to one. The mean and standard deviation were calculated from three independent experiments. The asterisks indicate the Bru proteins with the tethered <i>GFP</i> RNA level differing significantly from the Bru+, using the student’s T test (*p≤0.05, **p≤0.01). H. GFP fluorescence was normalized for the <i>GFP-MS2</i> RNA levels, which were normalized using the <i>rp49</i> RNA levels as in panel (G). The value for none, which lacks any MCP::HA₃::Bru proteins, was set to one. The mean and standard deviation were calculated from at least 40 samples per genotype. The asterisks indicate the Bru proteins with the GFP protein/RNA level differing significantly from the Bru+, using the student’s T test (*p≤0.05, **p≤0.01, ***p≤0.001).</p