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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.


Related in: MedlinePlus

Summary of the deletions and point mutations examined in this study.(A) An alignment of the N-terminal ends of predicted Vas proteins from several Drosophila species. The N-terminal vas open reading frame is often incorrectly annotated in species that have not undergone extensive cDNA sequencing because of confounding factors such as poor sequence conservation, nested genes and the solo alternative splice form (Yan et al., 2010). Therefore, to produce this alignment, vas open reading frames were manually annotated from three-frame translations of genomic DNA, using the short highly-conserved amino-terminal end to identify the putative translational start site. Asterisks, colons and periods indicate full conservation, strong similarity and weak similarity, respectively. RGG motifs are shown in red. (B) Schematic representation of the N-terminal deletions used in this study. The hash box marks amino acids 141-153, which are encoded by a copy of a 39-nucleotide tandem repeat that is absent from some vas cDNA clones (Lasko and Ashburner, 1988). This segment is absent in eGFP-Vas+ and all the N-terminally deleted proteins as the constructs were built from such a cDNA clone. VasΔ17-110, 3xAGG contains a deletion of amino acids 17-110 and three mutations that convert RGG motifs to AGG. (C) The amino acid substitution mutations in conserved DEAD-box helicase motifs that were produced for this study. Motifs are identified as previously defined (Rocak and Linder, 2004). (D) Sequence alignment of the C-terminal region of Vas from D. melanogaster with orthologues from other species. The red box depicts amino acids 636-646, which are conserved among Drosophila species but not beyond. The purple letters show the conserved highly acidic residues found at the C-terminal ends of Vas orthologues from Drosophila and non-Drosophila species. A tryptophan residue (presented in blue) is also highly conserved.
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f01: Summary of the deletions and point mutations examined in this study.(A) An alignment of the N-terminal ends of predicted Vas proteins from several Drosophila species. The N-terminal vas open reading frame is often incorrectly annotated in species that have not undergone extensive cDNA sequencing because of confounding factors such as poor sequence conservation, nested genes and the solo alternative splice form (Yan et al., 2010). Therefore, to produce this alignment, vas open reading frames were manually annotated from three-frame translations of genomic DNA, using the short highly-conserved amino-terminal end to identify the putative translational start site. Asterisks, colons and periods indicate full conservation, strong similarity and weak similarity, respectively. RGG motifs are shown in red. (B) Schematic representation of the N-terminal deletions used in this study. The hash box marks amino acids 141-153, which are encoded by a copy of a 39-nucleotide tandem repeat that is absent from some vas cDNA clones (Lasko and Ashburner, 1988). This segment is absent in eGFP-Vas+ and all the N-terminally deleted proteins as the constructs were built from such a cDNA clone. VasΔ17-110, 3xAGG contains a deletion of amino acids 17-110 and three mutations that convert RGG motifs to AGG. (C) The amino acid substitution mutations in conserved DEAD-box helicase motifs that were produced for this study. Motifs are identified as previously defined (Rocak and Linder, 2004). (D) Sequence alignment of the C-terminal region of Vas from D. melanogaster with orthologues from other species. The red box depicts amino acids 636-646, which are conserved among Drosophila species but not beyond. The purple letters show the conserved highly acidic residues found at the C-terminal ends of Vas orthologues from Drosophila and non-Drosophila species. A tryptophan residue (presented in blue) is also highly conserved.

Mentions: The N-terminal region of Vas evolves rapidly; there are numerous sequence changes even between D. melanogaster and the closely related species D. simulans (Fig. 1A). Nearly all Vas orthologues contain several RGG repeats within this region, although the number of such motifs and their spacing vary considerably. To examine the role of the variable N-terminal domain in Vas function, we produced egfp-vas transgenic constructs deleted for different segments of the N-terminal region (egfp-vasΔ15-75, egfp-vasΔ17-110, egfp-vasΔ94-127, egfp-vasΔ3-139, egfp-vasΔ3-200; Fig. 1B). In addition, to specifically test the role of RGG motifs, we mutated arginine to alanine in the three RGG motifs that remain present in egfp-vasΔ17-110 (egfp-vasΔ17-110, 3xAGG); the three arginines mutated are R115, R122, and R163.


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

Dehghani M, Lasko P - Biol Open (2015)

Summary of the deletions and point mutations examined in this study.(A) An alignment of the N-terminal ends of predicted Vas proteins from several Drosophila species. The N-terminal vas open reading frame is often incorrectly annotated in species that have not undergone extensive cDNA sequencing because of confounding factors such as poor sequence conservation, nested genes and the solo alternative splice form (Yan et al., 2010). Therefore, to produce this alignment, vas open reading frames were manually annotated from three-frame translations of genomic DNA, using the short highly-conserved amino-terminal end to identify the putative translational start site. Asterisks, colons and periods indicate full conservation, strong similarity and weak similarity, respectively. RGG motifs are shown in red. (B) Schematic representation of the N-terminal deletions used in this study. The hash box marks amino acids 141-153, which are encoded by a copy of a 39-nucleotide tandem repeat that is absent from some vas cDNA clones (Lasko and Ashburner, 1988). This segment is absent in eGFP-Vas+ and all the N-terminally deleted proteins as the constructs were built from such a cDNA clone. VasΔ17-110, 3xAGG contains a deletion of amino acids 17-110 and three mutations that convert RGG motifs to AGG. (C) The amino acid substitution mutations in conserved DEAD-box helicase motifs that were produced for this study. Motifs are identified as previously defined (Rocak and Linder, 2004). (D) Sequence alignment of the C-terminal region of Vas from D. melanogaster with orthologues from other species. The red box depicts amino acids 636-646, which are conserved among Drosophila species but not beyond. The purple letters show the conserved highly acidic residues found at the C-terminal ends of Vas orthologues from Drosophila and non-Drosophila species. A tryptophan residue (presented in blue) is also highly conserved.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f01: Summary of the deletions and point mutations examined in this study.(A) An alignment of the N-terminal ends of predicted Vas proteins from several Drosophila species. The N-terminal vas open reading frame is often incorrectly annotated in species that have not undergone extensive cDNA sequencing because of confounding factors such as poor sequence conservation, nested genes and the solo alternative splice form (Yan et al., 2010). Therefore, to produce this alignment, vas open reading frames were manually annotated from three-frame translations of genomic DNA, using the short highly-conserved amino-terminal end to identify the putative translational start site. Asterisks, colons and periods indicate full conservation, strong similarity and weak similarity, respectively. RGG motifs are shown in red. (B) Schematic representation of the N-terminal deletions used in this study. The hash box marks amino acids 141-153, which are encoded by a copy of a 39-nucleotide tandem repeat that is absent from some vas cDNA clones (Lasko and Ashburner, 1988). This segment is absent in eGFP-Vas+ and all the N-terminally deleted proteins as the constructs were built from such a cDNA clone. VasΔ17-110, 3xAGG contains a deletion of amino acids 17-110 and three mutations that convert RGG motifs to AGG. (C) The amino acid substitution mutations in conserved DEAD-box helicase motifs that were produced for this study. Motifs are identified as previously defined (Rocak and Linder, 2004). (D) Sequence alignment of the C-terminal region of Vas from D. melanogaster with orthologues from other species. The red box depicts amino acids 636-646, which are conserved among Drosophila species but not beyond. The purple letters show the conserved highly acidic residues found at the C-terminal ends of Vas orthologues from Drosophila and non-Drosophila species. A tryptophan residue (presented in blue) is also highly conserved.
Mentions: The N-terminal region of Vas evolves rapidly; there are numerous sequence changes even between D. melanogaster and the closely related species D. simulans (Fig. 1A). Nearly all Vas orthologues contain several RGG repeats within this region, although the number of such motifs and their spacing vary considerably. To examine the role of the variable N-terminal domain in Vas function, we produced egfp-vas transgenic constructs deleted for different segments of the N-terminal region (egfp-vasΔ15-75, egfp-vasΔ17-110, egfp-vasΔ94-127, egfp-vasΔ3-139, egfp-vasΔ3-200; Fig. 1B). In addition, to specifically test the role of RGG motifs, we mutated arginine to alanine in the three RGG motifs that remain present in egfp-vasΔ17-110 (egfp-vasΔ17-110, 3xAGG); the three arginines mutated are R115, R122, and R163.

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.


Related in: MedlinePlus