BREs mediate both repression and activation of oskar mRNA translation and act in trans

Abstract

Asymmetric positioning of proteins within cells is crucial for cell polarization and function. Deployment of Oskar protein at the posterior pole of the Drosophila oocyte relies on localization of the oskar mRNA, repression of its translation prior to localization, and finally activation of translation. Translational repression is mediated by BREs, regulatory elements positioned in two clusters near both ends of the oskar mRNA 3' UTR. Here we show that some BREs are bifunctional: both clusters of BREs contribute to translational repression, and the 3' cluster has an additional role in release from BRE-dependent repression. Remarkably, both BRE functions can be provided in trans by an oskar mRNA with wild-type BREs that is itself unable to encode Oskar protein. Regulation in trans is likely enabled by assembly of oskar transcripts in cytoplasmic RNPs. Concentration of transcripts in such RNPs is common, and trans regulation of mRNAs may therefore be widespread.

Figure 1. Body patterning activity of osk transgenes

A. Transgenes were tested in the osk^A87/Df(3R)osk background, and progeny embryos examined for cuticular phenotypes. Examples of the classes of phenotypes are shown in Fig. S1. See also Fig. S2 for data on additional transgenes. B. Transcript levels for osk transgenes. All transgenes were tested in the osk^A87/Df(3R)osk (RNA null) background. Top: RNase protection assays of osk and rp49 mRNAs from ovaries of the indicated genotypes. Bottom: Levels of transgene mRNAs relative to the level of endogenous osk mRNA in wild type (w¹¹¹⁸) flies. RNA levels were quantified by phosphorimaging and osk levels normalized using the rp49 signal. Three or more assays were used to generate the average levels (and standard deviations). Flies had one copy of the transgene-bearing chromosome.

Figure 2. Redundant contributions of BREs to translational repression

Panels A-D show stage 8 egg chambers with Osk in green and nuclei in red. A is wild type and B and C are osk^A87/Df(3R)osk with the osk AB^- (B) or osk ABC^- (C) transgenes. Results similar to that shown in B were obtained for the wild type and osk C^- transgenes. D is osk⁵⁴/Df(3R)osk with transgene osk ABC^-. All egg chambers show a low level of green background signal in both germline and somatic cells. The only consistent difference among the different genotypes was the higher level of Osk in the oocytes of osk^A87/Df(3R)osk ovaries with the osk ABC^- transgene present, detected at roughly the level shown in 69% (n=18) of the egg chambers expressing osk ABC^-, with many of the remaining egg chambers showing a lower level. None of the other genotypes ever showed Osk above background. Panels E-G show stage 10A egg chambers expressing a GFP transgene (green, nuclei in red. All samples were fixed in parallel and imaged together with the same laser power and confocal settings. A, UAS-GFP; B, UAS-GFP-AB; C, UAS-GFP-C (AB and C are the eponymous regions of the osk 3′ UTR). Panel D, RNase protection assays of mRNA levels of the transgenes.

Figure 3. Mutation of C region BREs interferes with translational activation, but not cytoplasmic polyadenylation

A. Osk protein in oocytes. Shown at top are posterior portions of stage 10A egg chambers, with examples of the different levels of Osk detected by immunofluorescence. Osk is green and nuclei are red. Transgenes were in the osk^A87/Df(3R)osk background, except for those at the bottom in which osk⁵⁴ was present (as indicated). The osk IBE^- transgene has mutations of the first three IBEs (Munro et al., 2006). The osk ABC^- transgene only poorly rescues the oogenesis progression defects of the osk^A87/Df(3R)osk mutant, and even those egg chambers that develop to later stages often display morphological abnormalities (data not shown). These defects are largely suppressed by an additional copy of the transgene, suggesting that any reduction in posterior Osk is a secondary consequence of the poor rescue of progression through oogenesis (the osk RNA null phenotype). See also Table S1 for data on additional transgenes. B. osk mRNA in oocytes. Shown at top are posterior portions of stage 10A egg chambers, with examples of the different degrees of posterior osk mRNA localization detected by fluorescent in situ hybridization. osk mRNA is red. All transgenes were in the osk^A87/Df(3R)osk background. The absence of localization for a minor fraction of the osk ABC^- egg chambers is likely due to the incomplete rescue of the oogenesis progression defects by this transgene, as explained above. C. Thermal elution assay. Ovarian RNAs were purified, bound to poly U agarose, and eluted at the temperatures indicated. Each fraction was tested by RNase protection assay for the RNA indicated. As indicated, the osk transgenes were tested in the osk^A87/Df(3R)osk background. The eluted fractions of RNA from orb mutant ovaries (orb^MEL/orb^DEC) were tested for both osk and rp49 to confirm that polyadenylated mRNAs were indeed bound to the poly U agarose. D. Ovarian RNAs from osk^A87/Df(3R)osk females expressing the indicated transgenes were subjected to the PAT assay (Salles et al., 1994). The distribution of the signal in each lane reflects the range of poly(A) tail length.

Figure 4. Suppression of regulatory defects by BRE⁺ mRNA

A. Body patterning activities of osk transgenes in the absence of endogenous osk mRNA (upper part) or in the presence of the BRE⁺ osk⁵⁴ mRNA (lower part). The transgenes are indicated at top. For each transgene the percentage of progeny embryos with different levels of Osk activity is indicated in the graph below (shading key at bottom). Levels of Osk activity: low/none, missing or absent abdominal denticle belts; wild type, wild type cuticles; excess, loss of anterior structures or bicaudal phenotypes. 2× osk ABC^- is a transgenic line that expresses twice the level of the line used in all other experiments. Embryos in the ‘excess’ category for the 2× line typically have more extreme phenotypes than for the 1× line (e.g. 56% bicaudal for 2× vs 13% bicaudal for 1×). The osk IBE^- transgene has the 5′ subset of the IBEs mutated. A similar transgene with all IBEs mutated (Munro et al., 2006) yielded identical results (data not shown). See also Fig. S3 for data on additional transgenes. B. Osk protein in embryos from mothers expressing osk transgenes, with or without the BRE⁺ osk⁵⁴ mRNA. Shown at top are posterior portions of early stage embryos, with examples of the different levels of Osk (green). C. Pole cell numbers for embryos from mothers expressing the transgene indicated at top, with or without osk⁵⁴ mRNA as indicated. See also Table. S3 for data on additional transgenes.