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Drosophila Ge-1 promotes P body formation and oskar mRNA localization.

Fan SJ, Marchand V, Ephrussi A - PLoS ONE (2011)

Bottom Line: Several P body components are involved in osk mRNA localization and translational repression, suggesting a link between P bodies and osk RNPs.In cultured mammalian cells, Ge-1 protein is required for P body formation.Our findings suggest an important role of dGe-1 in optimization of the osk mRNA localization process required for patterning the Drosophila embryo.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
mRNA localization coupled with translational control is a widespread and conserved strategy that allows the localized production of proteins within eukaryotic cells. In Drosophila, oskar (osk) mRNA localization and translation at the posterior pole of the oocyte are essential for proper patterning of the embryo. Several P body components are involved in osk mRNA localization and translational repression, suggesting a link between P bodies and osk RNPs. In cultured mammalian cells, Ge-1 protein is required for P body formation. Combining genetic, biochemical and immunohistochemical approaches, we show that, in vivo, Drosophila Ge-1 (dGe-1) is an essential gene encoding a P body component that promotes formation of these structures in the germline. dGe-1 partially colocalizes with osk mRNA and is required for osk RNP integrity. Our analysis reveals that although under normal conditions dGe-1 function is not essential for osk mRNA localization, it becomes critical when other components of the localization machinery, such as staufen, Drosophila decapping protein 1 and barentsz are limiting. Our findings suggest an important role of dGe-1 in optimization of the osk mRNA localization process required for patterning the Drosophila embryo.

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dGe-1 is required for assembly of osk RNPs.(A) Drosophila wt (upper panel) or dGe-1Δ5 GLC (lower panel) ovarian extracts treated with cycloheximide and analyzed by centrifugation in a 10–45% sucrose density gradient. osk and actin mRNAs in the gradient fractions were detected by RNase protection assay. Absorption at 260 nm was measured to situate the monoribosome peak (80S) and polysomes. (B) Quantification of the RNase protection assays presented in (A). The relative levels of osk and actin mRNAs present in each fraction are indicated as a % of the total levels of both mRNAs present in wt (upper panel) and dGe-1Δ5 GLC (lower panel) ovarian extracts. (C–D) Distribution of Stau protein in starved wt (C) or dGe-1Δ5 GLC (D) ovaries. In nutritionally restricted wt oocytes, Stau (green) is present in large particles, which fail to form in starved dGe-1Δ5 GLC ovaries. Bar, 50 µm.
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pone-0020612-g005: dGe-1 is required for assembly of osk RNPs.(A) Drosophila wt (upper panel) or dGe-1Δ5 GLC (lower panel) ovarian extracts treated with cycloheximide and analyzed by centrifugation in a 10–45% sucrose density gradient. osk and actin mRNAs in the gradient fractions were detected by RNase protection assay. Absorption at 260 nm was measured to situate the monoribosome peak (80S) and polysomes. (B) Quantification of the RNase protection assays presented in (A). The relative levels of osk and actin mRNAs present in each fraction are indicated as a % of the total levels of both mRNAs present in wt (upper panel) and dGe-1Δ5 GLC (lower panel) ovarian extracts. (C–D) Distribution of Stau protein in starved wt (C) or dGe-1Δ5 GLC (D) ovaries. In nutritionally restricted wt oocytes, Stau (green) is present in large particles, which fail to form in starved dGe-1Δ5 GLC ovaries. Bar, 50 µm.

Mentions: dGe-1 protein is required for P body formation (Figure 2E–J) and is associated with osk RNPs (Figure 4). To determine if dGe-1 might be required for osk RNP assembly, we examined the distribution on sucrose gradients of osk mRNA in cytoplasmic extracts of wt and dGe-1Δ5 GLC ovaries. In wt extracts osk mRNA is broadly distributed and enriched in fractions 5 to 7 (Figure 5A–B). In comparison, translationally active actin mRNA (FlyBase: CG4027) displays a typical polysome profile, with much of the mRNA enriched in the polysomal fractions (Figure 5A–B, fractions 10 to 18). While in dGe-1Δ5 GLC extracts osk mRNA is also distributed broadly, it is most enriched in fractions 3 to 6 (Figure 5A–B). This shift of osk mRNA to the lighter sucrose gradient fractions in dGe-1Δ5 mutant extracts is significant, as no obvious change in the sedimentation profile of actin mRNA was observed. Hence, in the dGe-1Δ5 mutant background the size of osk RNPs is selectively affected, revealing a role of dGe-1 in their assembly or stability. Consistent with this, large osk granules (represented by Stau) induced by nutritional restriction fail to form in dGe-1Δ5 GLC (Figure 5C–D). In the future, it could be of interest to assess the size of osk RNPs in wt and dGe-1 mutant oocytes, for instance by observing their ultrastructure using electron microscopy.


Drosophila Ge-1 promotes P body formation and oskar mRNA localization.

Fan SJ, Marchand V, Ephrussi A - PLoS ONE (2011)

dGe-1 is required for assembly of osk RNPs.(A) Drosophila wt (upper panel) or dGe-1Δ5 GLC (lower panel) ovarian extracts treated with cycloheximide and analyzed by centrifugation in a 10–45% sucrose density gradient. osk and actin mRNAs in the gradient fractions were detected by RNase protection assay. Absorption at 260 nm was measured to situate the monoribosome peak (80S) and polysomes. (B) Quantification of the RNase protection assays presented in (A). The relative levels of osk and actin mRNAs present in each fraction are indicated as a % of the total levels of both mRNAs present in wt (upper panel) and dGe-1Δ5 GLC (lower panel) ovarian extracts. (C–D) Distribution of Stau protein in starved wt (C) or dGe-1Δ5 GLC (D) ovaries. In nutritionally restricted wt oocytes, Stau (green) is present in large particles, which fail to form in starved dGe-1Δ5 GLC ovaries. Bar, 50 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105097&req=5

pone-0020612-g005: dGe-1 is required for assembly of osk RNPs.(A) Drosophila wt (upper panel) or dGe-1Δ5 GLC (lower panel) ovarian extracts treated with cycloheximide and analyzed by centrifugation in a 10–45% sucrose density gradient. osk and actin mRNAs in the gradient fractions were detected by RNase protection assay. Absorption at 260 nm was measured to situate the monoribosome peak (80S) and polysomes. (B) Quantification of the RNase protection assays presented in (A). The relative levels of osk and actin mRNAs present in each fraction are indicated as a % of the total levels of both mRNAs present in wt (upper panel) and dGe-1Δ5 GLC (lower panel) ovarian extracts. (C–D) Distribution of Stau protein in starved wt (C) or dGe-1Δ5 GLC (D) ovaries. In nutritionally restricted wt oocytes, Stau (green) is present in large particles, which fail to form in starved dGe-1Δ5 GLC ovaries. Bar, 50 µm.
Mentions: dGe-1 protein is required for P body formation (Figure 2E–J) and is associated with osk RNPs (Figure 4). To determine if dGe-1 might be required for osk RNP assembly, we examined the distribution on sucrose gradients of osk mRNA in cytoplasmic extracts of wt and dGe-1Δ5 GLC ovaries. In wt extracts osk mRNA is broadly distributed and enriched in fractions 5 to 7 (Figure 5A–B). In comparison, translationally active actin mRNA (FlyBase: CG4027) displays a typical polysome profile, with much of the mRNA enriched in the polysomal fractions (Figure 5A–B, fractions 10 to 18). While in dGe-1Δ5 GLC extracts osk mRNA is also distributed broadly, it is most enriched in fractions 3 to 6 (Figure 5A–B). This shift of osk mRNA to the lighter sucrose gradient fractions in dGe-1Δ5 mutant extracts is significant, as no obvious change in the sedimentation profile of actin mRNA was observed. Hence, in the dGe-1Δ5 mutant background the size of osk RNPs is selectively affected, revealing a role of dGe-1 in their assembly or stability. Consistent with this, large osk granules (represented by Stau) induced by nutritional restriction fail to form in dGe-1Δ5 GLC (Figure 5C–D). In the future, it could be of interest to assess the size of osk RNPs in wt and dGe-1 mutant oocytes, for instance by observing their ultrastructure using electron microscopy.

Bottom Line: Several P body components are involved in osk mRNA localization and translational repression, suggesting a link between P bodies and osk RNPs.In cultured mammalian cells, Ge-1 protein is required for P body formation.Our findings suggest an important role of dGe-1 in optimization of the osk mRNA localization process required for patterning the Drosophila embryo.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

ABSTRACT
mRNA localization coupled with translational control is a widespread and conserved strategy that allows the localized production of proteins within eukaryotic cells. In Drosophila, oskar (osk) mRNA localization and translation at the posterior pole of the oocyte are essential for proper patterning of the embryo. Several P body components are involved in osk mRNA localization and translational repression, suggesting a link between P bodies and osk RNPs. In cultured mammalian cells, Ge-1 protein is required for P body formation. Combining genetic, biochemical and immunohistochemical approaches, we show that, in vivo, Drosophila Ge-1 (dGe-1) is an essential gene encoding a P body component that promotes formation of these structures in the germline. dGe-1 partially colocalizes with osk mRNA and is required for osk RNP integrity. Our analysis reveals that although under normal conditions dGe-1 function is not essential for osk mRNA localization, it becomes critical when other components of the localization machinery, such as staufen, Drosophila decapping protein 1 and barentsz are limiting. Our findings suggest an important role of dGe-1 in optimization of the osk mRNA localization process required for patterning the Drosophila embryo.

Show MeSH
Related in: MedlinePlus