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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.


HeT-A expression in ovaries of vasPH165 females carrying different egfp-vas constructs.Red bars indicate the expression level of HeT-A normalized to that of wild-type ovaries. Data from vasPH165 and vasPH165/Df(2L)A267 controls are presented at the far right. Asterisks indicate a significant increase compared to vas+ (p<0.05). Each bar represents the average of at least three biological replicates, error bars indicate SEM.
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f05: HeT-A expression in ovaries of vasPH165 females carrying different egfp-vas constructs.Red bars indicate the expression level of HeT-A normalized to that of wild-type ovaries. Data from vasPH165 and vasPH165/Df(2L)A267 controls are presented at the far right. Asterisks indicate a significant increase compared to vas+ (p<0.05). Each bar represents the average of at least three biological replicates, error bars indicate SEM.

Mentions: Females lacking vas function overexpress mRNA from a subset of transposon families in their ovaries (Zhang et al., 2012). We examined levels of HeT-A, a retrotransposon that is highly expressed in vas deficient ovaries, in vasPH165 females carrying different egfp-vas constructs. Consistent with a previous report (Zhang et al. 2012), quantitative RT-PCR (RT-qPCR) analyses indicated that the expression level of HeT-A in the ovaries of homozygous vasPH165 or vasPH165/Df(2L)A267 females is more than 100-fold higher than in wild-type controls (Fig. 5). eGFP-Vas+, as well as the N-terminally truncated proteins lacking the interval 17-110 or shorter, could suppress HeT-A expression in the vasPH165 ovaries to within an order of magnitude of the wild-type level. On the other hand, expression of HeT-A in ovaries expressing the largest N-terminal deletion (eGFP-VasΔ3-200) was about 50-fold higher than wild-type (p = 0.0039), indicating some requirement for N-terminal sequences between amino acids 111-200 in repression of transposon-encoded gene expression. HeT-A expression in vas- ovaries expressing constructs that delete part of this region (egfp-vasΔ3-139 or egfp-vasΔ94-127) was highly variable from experiment to experiment, ranging from near wild-type levels to levels similar to vas Δ3-200, and because of this variability the difference from egfp-vas+ was not statistically significant (supplementary material Table S1). We observed that HeT-A expression in vasPH165; egfp-vasΔ17-110, 3xAGG ovaries was also highly variable (Fig. 5).


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

Dehghani M, Lasko P - Biol Open (2015)

HeT-A expression in ovaries of vasPH165 females carrying different egfp-vas constructs.Red bars indicate the expression level of HeT-A normalized to that of wild-type ovaries. Data from vasPH165 and vasPH165/Df(2L)A267 controls are presented at the far right. Asterisks indicate a significant increase compared to vas+ (p<0.05). Each bar represents the average of at least three biological replicates, error bars indicate SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f05: HeT-A expression in ovaries of vasPH165 females carrying different egfp-vas constructs.Red bars indicate the expression level of HeT-A normalized to that of wild-type ovaries. Data from vasPH165 and vasPH165/Df(2L)A267 controls are presented at the far right. Asterisks indicate a significant increase compared to vas+ (p<0.05). Each bar represents the average of at least three biological replicates, error bars indicate SEM.
Mentions: Females lacking vas function overexpress mRNA from a subset of transposon families in their ovaries (Zhang et al., 2012). We examined levels of HeT-A, a retrotransposon that is highly expressed in vas deficient ovaries, in vasPH165 females carrying different egfp-vas constructs. Consistent with a previous report (Zhang et al. 2012), quantitative RT-PCR (RT-qPCR) analyses indicated that the expression level of HeT-A in the ovaries of homozygous vasPH165 or vasPH165/Df(2L)A267 females is more than 100-fold higher than in wild-type controls (Fig. 5). eGFP-Vas+, as well as the N-terminally truncated proteins lacking the interval 17-110 or shorter, could suppress HeT-A expression in the vasPH165 ovaries to within an order of magnitude of the wild-type level. On the other hand, expression of HeT-A in ovaries expressing the largest N-terminal deletion (eGFP-VasΔ3-200) was about 50-fold higher than wild-type (p = 0.0039), indicating some requirement for N-terminal sequences between amino acids 111-200 in repression of transposon-encoded gene expression. HeT-A expression in vas- ovaries expressing constructs that delete part of this region (egfp-vasΔ3-139 or egfp-vasΔ94-127) was highly variable from experiment to experiment, ranging from near wild-type levels to levels similar to vas Δ3-200, and because of this variability the difference from egfp-vas+ was not statistically significant (supplementary material Table S1). We observed that HeT-A expression in vasPH165; egfp-vasΔ17-110, 3xAGG ovaries was also highly variable (Fig. 5).

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.