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In vivo mapping of the functional regions of the DEAD-box helicase Vasa.

Dehghani M, Lasko P - Biol Open (2015)

Bottom Line: We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification.One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases.Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 3649 Promenade Sir William Osler, Montréal, QC H3G 0B1, Canada.

No MeSH data available.


Germ cell formation in embryos from vas1 females expressing different eGFP-Vas proteins.Red bars indicate the percentage of the embryos with germ cells. Data from the vas1 control is presented at the far right. Asterisks show a significant difference from vas+ (p<0.05). Error bars represent SEM from at least three biological replicates each with more than 50 embryos.
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f06: Germ cell formation in embryos from vas1 females expressing different eGFP-Vas proteins.Red bars indicate the percentage of the embryos with germ cells. Data from the vas1 control is presented at the far right. Asterisks show a significant difference from vas+ (p<0.05). Error bars represent SEM from at least three biological replicates each with more than 50 embryos.

Mentions: eGFP-Vas deleted for amino acids 15-75, 17-110, or 94-127 retained the ability to induce germ cell formation in vas1 embryos to a similar or even greater extent to eGFP-Vas+ (Fig. 6). However, deletion of amino acids 3-139 clearly impacted the ability of eGFP-Vas to restore pole cell formation to vas1 embryos (p = 0.018 when compared with eGFP-Vas+), with only 7±1.1% of embryos from females expressing this construct forming pole cells. This was not due to low expression level from this construct (supplementary material Fig. S1), but we did find that this protein is unstable in embryos (supplementary material Fig. S3). Next, we examined embryos produced from vas1 females expressing eGFP-VasΔ17-110, 3xAGG (Fig. 6; supplementary material Fig. S3). In this line the frequency of embryos forming germ cells was only 14±4.9% (p = 0.022 when compared with eGFP-Vas+), suggesting that the RGG motifs in the N-terminal region of Vas play a role in germ cell formation. Importantly, all of these forms of eGFP-Vas localize normally to the posterior pole plasm (Fig. 4A). vas1 embryos expressing eGFP-VasΔ3-200 never produced germ cells, further implicating N-terminal motifs in pole cell specification.


In vivo mapping of the functional regions of the DEAD-box helicase Vasa.

Dehghani M, Lasko P - Biol Open (2015)

Germ cell formation in embryos from vas1 females expressing different eGFP-Vas proteins.Red bars indicate the percentage of the embryos with germ cells. Data from the vas1 control is presented at the far right. Asterisks show a significant difference from vas+ (p<0.05). Error bars represent SEM from at least three biological replicates each with more than 50 embryos.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400588&req=5

f06: Germ cell formation in embryos from vas1 females expressing different eGFP-Vas proteins.Red bars indicate the percentage of the embryos with germ cells. Data from the vas1 control is presented at the far right. Asterisks show a significant difference from vas+ (p<0.05). Error bars represent SEM from at least three biological replicates each with more than 50 embryos.
Mentions: eGFP-Vas deleted for amino acids 15-75, 17-110, or 94-127 retained the ability to induce germ cell formation in vas1 embryos to a similar or even greater extent to eGFP-Vas+ (Fig. 6). However, deletion of amino acids 3-139 clearly impacted the ability of eGFP-Vas to restore pole cell formation to vas1 embryos (p = 0.018 when compared with eGFP-Vas+), with only 7±1.1% of embryos from females expressing this construct forming pole cells. This was not due to low expression level from this construct (supplementary material Fig. S1), but we did find that this protein is unstable in embryos (supplementary material Fig. S3). Next, we examined embryos produced from vas1 females expressing eGFP-VasΔ17-110, 3xAGG (Fig. 6; supplementary material Fig. S3). In this line the frequency of embryos forming germ cells was only 14±4.9% (p = 0.022 when compared with eGFP-Vas+), suggesting that the RGG motifs in the N-terminal region of Vas play a role in germ cell formation. Importantly, all of these forms of eGFP-Vas localize normally to the posterior pole plasm (Fig. 4A). vas1 embryos expressing eGFP-VasΔ3-200 never produced germ cells, further implicating N-terminal motifs in pole cell specification.

Bottom Line: We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification.One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases.Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 3649 Promenade Sir William Osler, Montréal, QC H3G 0B1, Canada.

No MeSH data available.