Limits...
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.

Show MeSH

Related in: MedlinePlus

dGe-1 is a P body component in the Drosophila germline.(A) Scheme representing the dGe-1 locus (polytene band 32D3). Numbers indicate genomic positions along chromosome 2L. 5′UTR, coding region, and 3′UTR: grey, red and blue boxes, respectively. The two dGe-1 transcripts, dGe-1-A and dGe-1-B, the insertion site of the P-element transposon (GS5005, red triangle) used for generation of dGe-1 deletions, and the region deleted in dGe-1Δ5 (double-headed arrow) are represented. Positions of primers used for RT-PCR (Figure S1B) are indicated (blue, red, green arrows). (B) Distribution of dGe-1 protein in wt egg-chambers during early and mid-oogenesis. Immunodetection of dGe-1 using rat anti-dGe-1 antibody. (C) HA-dGe1 protein distribution in wt egg-chamber at stage 6 (S6). HA-dGe-1 detected using mouse anti-HA antibody. (D–I) Colocalization of dGe-1 protein and two P body components. Double-staining of two wt S7 egg-chambers using rat anti-dGe1 (green, D and G) and rabbit anti-Me31B (red, E) or rabbit anti-Tral (red, H) antibodies. Overlays (F and I). (J) Association of dGe-1 and dDcp1 in ovarian extract. Western blot of anti-GFP immunoprecipitates of GFP-FWS and YFP-dDcp1 ovaries. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow (see legend to Figure 2A). (K) RNA-independent association of dGe-1 and dDcp1. Western blot of anti-GFP immunoprecipitates from YFP-dDcp1 ovarian extract with or without RNase A treatment prior to immunoprecipitation, probed with rabbit anti-dGe-1 and anti-Exu antibodies. dGe-1 is indicated with an arrow. (L) Exu and Me31B associate with dGe-1 in the ovary. Endogeneous dGe-1 protein was immunoprecipitated from wt ovarian extract and bound and soluble fractions were subjected to western blot analysis. Before immunoprecipitation, rabbit anti-dGe-1 was pre-incubated or not with dGe-1 blocking peptide. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow. Bar, 10 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105097&req=5

pone-0020612-g001: dGe-1 is a P body component in the Drosophila germline.(A) Scheme representing the dGe-1 locus (polytene band 32D3). Numbers indicate genomic positions along chromosome 2L. 5′UTR, coding region, and 3′UTR: grey, red and blue boxes, respectively. The two dGe-1 transcripts, dGe-1-A and dGe-1-B, the insertion site of the P-element transposon (GS5005, red triangle) used for generation of dGe-1 deletions, and the region deleted in dGe-1Δ5 (double-headed arrow) are represented. Positions of primers used for RT-PCR (Figure S1B) are indicated (blue, red, green arrows). (B) Distribution of dGe-1 protein in wt egg-chambers during early and mid-oogenesis. Immunodetection of dGe-1 using rat anti-dGe-1 antibody. (C) HA-dGe1 protein distribution in wt egg-chamber at stage 6 (S6). HA-dGe-1 detected using mouse anti-HA antibody. (D–I) Colocalization of dGe-1 protein and two P body components. Double-staining of two wt S7 egg-chambers using rat anti-dGe1 (green, D and G) and rabbit anti-Me31B (red, E) or rabbit anti-Tral (red, H) antibodies. Overlays (F and I). (J) Association of dGe-1 and dDcp1 in ovarian extract. Western blot of anti-GFP immunoprecipitates of GFP-FWS and YFP-dDcp1 ovaries. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow (see legend to Figure 2A). (K) RNA-independent association of dGe-1 and dDcp1. Western blot of anti-GFP immunoprecipitates from YFP-dDcp1 ovarian extract with or without RNase A treatment prior to immunoprecipitation, probed with rabbit anti-dGe-1 and anti-Exu antibodies. dGe-1 is indicated with an arrow. (L) Exu and Me31B associate with dGe-1 in the ovary. Endogeneous dGe-1 protein was immunoprecipitated from wt ovarian extract and bound and soluble fractions were subjected to western blot analysis. Before immunoprecipitation, rabbit anti-dGe-1 was pre-incubated or not with dGe-1 blocking peptide. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow. Bar, 10 µm.

Mentions: The dGe-1 locus encodes two transcripts, dGe-1-A and dGe-1-B, which differ only in their 5′ UTRs and are transcribed during oogenesis (Figure 1A and S1A). To address the function of dGe-1 during oogenesis, we generated deletion mutants by imprecise excision of a P element in the locus (Figure 1A). One of these, dGe-1Δ5 is a recessive lethal mutation whose lethality was rescued by transgenic expression of a dGe-1-B cDNA, proving is a lethal dGe-1 allele.


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

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

dGe-1 is a P body component in the Drosophila germline.(A) Scheme representing the dGe-1 locus (polytene band 32D3). Numbers indicate genomic positions along chromosome 2L. 5′UTR, coding region, and 3′UTR: grey, red and blue boxes, respectively. The two dGe-1 transcripts, dGe-1-A and dGe-1-B, the insertion site of the P-element transposon (GS5005, red triangle) used for generation of dGe-1 deletions, and the region deleted in dGe-1Δ5 (double-headed arrow) are represented. Positions of primers used for RT-PCR (Figure S1B) are indicated (blue, red, green arrows). (B) Distribution of dGe-1 protein in wt egg-chambers during early and mid-oogenesis. Immunodetection of dGe-1 using rat anti-dGe-1 antibody. (C) HA-dGe1 protein distribution in wt egg-chamber at stage 6 (S6). HA-dGe-1 detected using mouse anti-HA antibody. (D–I) Colocalization of dGe-1 protein and two P body components. Double-staining of two wt S7 egg-chambers using rat anti-dGe1 (green, D and G) and rabbit anti-Me31B (red, E) or rabbit anti-Tral (red, H) antibodies. Overlays (F and I). (J) Association of dGe-1 and dDcp1 in ovarian extract. Western blot of anti-GFP immunoprecipitates of GFP-FWS and YFP-dDcp1 ovaries. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow (see legend to Figure 2A). (K) RNA-independent association of dGe-1 and dDcp1. Western blot of anti-GFP immunoprecipitates from YFP-dDcp1 ovarian extract with or without RNase A treatment prior to immunoprecipitation, probed with rabbit anti-dGe-1 and anti-Exu antibodies. dGe-1 is indicated with an arrow. (L) Exu and Me31B associate with dGe-1 in the ovary. Endogeneous dGe-1 protein was immunoprecipitated from wt ovarian extract and bound and soluble fractions were subjected to western blot analysis. Before immunoprecipitation, rabbit anti-dGe-1 was pre-incubated or not with dGe-1 blocking peptide. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow. Bar, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020612-g001: dGe-1 is a P body component in the Drosophila germline.(A) Scheme representing the dGe-1 locus (polytene band 32D3). Numbers indicate genomic positions along chromosome 2L. 5′UTR, coding region, and 3′UTR: grey, red and blue boxes, respectively. The two dGe-1 transcripts, dGe-1-A and dGe-1-B, the insertion site of the P-element transposon (GS5005, red triangle) used for generation of dGe-1 deletions, and the region deleted in dGe-1Δ5 (double-headed arrow) are represented. Positions of primers used for RT-PCR (Figure S1B) are indicated (blue, red, green arrows). (B) Distribution of dGe-1 protein in wt egg-chambers during early and mid-oogenesis. Immunodetection of dGe-1 using rat anti-dGe-1 antibody. (C) HA-dGe1 protein distribution in wt egg-chamber at stage 6 (S6). HA-dGe-1 detected using mouse anti-HA antibody. (D–I) Colocalization of dGe-1 protein and two P body components. Double-staining of two wt S7 egg-chambers using rat anti-dGe1 (green, D and G) and rabbit anti-Me31B (red, E) or rabbit anti-Tral (red, H) antibodies. Overlays (F and I). (J) Association of dGe-1 and dDcp1 in ovarian extract. Western blot of anti-GFP immunoprecipitates of GFP-FWS and YFP-dDcp1 ovaries. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow (see legend to Figure 2A). (K) RNA-independent association of dGe-1 and dDcp1. Western blot of anti-GFP immunoprecipitates from YFP-dDcp1 ovarian extract with or without RNase A treatment prior to immunoprecipitation, probed with rabbit anti-dGe-1 and anti-Exu antibodies. dGe-1 is indicated with an arrow. (L) Exu and Me31B associate with dGe-1 in the ovary. Endogeneous dGe-1 protein was immunoprecipitated from wt ovarian extract and bound and soluble fractions were subjected to western blot analysis. Before immunoprecipitation, rabbit anti-dGe-1 was pre-incubated or not with dGe-1 blocking peptide. Western blot probed with rabbit anti-dGe-1, anti-Exu and anti-Khc, and mouse anti-Me31B and anti-Tub antibodies. dGe-1 is indicated with an arrow. Bar, 10 µm.
Mentions: The dGe-1 locus encodes two transcripts, dGe-1-A and dGe-1-B, which differ only in their 5′ UTRs and are transcribed during oogenesis (Figure 1A and S1A). To address the function of dGe-1 during oogenesis, we generated deletion mutants by imprecise excision of a P element in the locus (Figure 1A). One of these, dGe-1Δ5 is a recessive lethal mutation whose lethality was rescued by transgenic expression of a dGe-1-B cDNA, proving is a lethal dGe-1 allele.

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