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The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes.

Haac ME, Anderson MA, Eggleston H, Myles KM, Adelman ZN - Nucleic Acids Res. (2015)

Bottom Line: Conversely, Loqs-PB alone interacted with mosquito dicer-1 and was essential for full miRNA production.Mosquito Loqs interacted with both argonaute 1 and 2 in a manner independent of its interactions with dicer.We conclude that the functional specialization of Loqs-PD in Drosophila is a recently derived trait, and that in other dipterans, including the medically important mosquitoes, Loqs-PA participates in both the miRNA and endo-siRNA based pathways.

View Article: PubMed Central - PubMed

Affiliation: Fralin Life Science Institute and Department of Entomology, Virginia Tech, Blacksburg, VA 24061, USA.

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Interactions between mosquito dsRBPs and RNAi/miRNA components. (A) Schematic representation of the Loqs-PA exon structure showing the location of the two Loqs-PA truncations used (Δ258 and Δ226). Start (1) and Stop (329) positions in the ORF are indicated; gray bars above indicate the locations of the three DRMs. The 32 amino acid spacer between DRMs 2 and 3 is highlighted, along with the location of exon 5 when spliced into Loqs-PB. (B) Co-immunoprecipitation of Dcr and Ago proteins with HA-tagged dsRBPs in Aag2 cells. HA-dsRBPs were expressed in Aag2 cells by transfection of plasmid DNA. (C) Co-Immunoprecipitation of FLAG-Loqs-PA or FLAG-Loqs-PB with HA-tagged proteins. Column headings indicate the overexpressed HA-tagged protein, row headings indicate the antibody used in the corresponding western blot. Input (In), flow-through (FT) and bound (B) fractions are indicated. To increase the amount of detectable R2D2, HA-R2D2 proteins were expressed via infection with dsSINV; all others were expressed by plasmid transfection. Anti-HA and anti-FLAG co-IP assays were run 24 h post-infection (48 h post-transfection). (D) Dimerization of Loqs-PA is unaffected by both the Δ258 and Δ226 deletions as shown by co-IP.
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Figure 3: Interactions between mosquito dsRBPs and RNAi/miRNA components. (A) Schematic representation of the Loqs-PA exon structure showing the location of the two Loqs-PA truncations used (Δ258 and Δ226). Start (1) and Stop (329) positions in the ORF are indicated; gray bars above indicate the locations of the three DRMs. The 32 amino acid spacer between DRMs 2 and 3 is highlighted, along with the location of exon 5 when spliced into Loqs-PB. (B) Co-immunoprecipitation of Dcr and Ago proteins with HA-tagged dsRBPs in Aag2 cells. HA-dsRBPs were expressed in Aag2 cells by transfection of plasmid DNA. (C) Co-Immunoprecipitation of FLAG-Loqs-PA or FLAG-Loqs-PB with HA-tagged proteins. Column headings indicate the overexpressed HA-tagged protein, row headings indicate the antibody used in the corresponding western blot. Input (In), flow-through (FT) and bound (B) fractions are indicated. To increase the amount of detectable R2D2, HA-R2D2 proteins were expressed via infection with dsSINV; all others were expressed by plasmid transfection. Anti-HA and anti-FLAG co-IP assays were run 24 h post-infection (48 h post-transfection). (D) Dimerization of Loqs-PA is unaffected by both the Δ258 and Δ226 deletions as shown by co-IP.

Mentions: To further explore the relationships of Ae. aegypti R2D2, Loqs-PA and Loqs-PB to siRNA and miRNA factors, we employed co-immunoprecipitation (co-IP) assays to test for protein–protein interactions between each dsRBP and Dcr1, Dcr2, Ago1 and Ago2. As both the Drosophila (Loqs) and human (TRBP) orthologs of Ae. aegypti Loqs have been shown to bind Dcr1 through an interaction mediated by the 3rd DRM at the C-terminus of the protein (19,32), two deletion constructs were included with a truncation immediately preceding the third DRM (Δ258) or immediately following the second DRM (Δ226) (Figure 3A). Co-IP experiments confirmed a strong association between R2D2 and Dcr2; this interaction was not affected by pre-treatment with RNaseA and is consistent with the role of this protein in the siRNA pathway (Figure 3B). Likewise, only Loqs-PB interacted with DCR1, consistent with its role in miRNA biogenesis. Interestingly, both Loqs-PA and Loqs-PB interacted with Dcr2, Ago1 and Ago2. Deleting the third DRM had no effect on the interaction between Loqs and Ago proteins. However, this interaction was lost when the 32 a.a. spacer separating the 2nd and 3rd DRMs was deleted (Figure 3B). Neither deletion had any effect on the ability of Loqs to interact with Dcr2.


The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes.

Haac ME, Anderson MA, Eggleston H, Myles KM, Adelman ZN - Nucleic Acids Res. (2015)

Interactions between mosquito dsRBPs and RNAi/miRNA components. (A) Schematic representation of the Loqs-PA exon structure showing the location of the two Loqs-PA truncations used (Δ258 and Δ226). Start (1) and Stop (329) positions in the ORF are indicated; gray bars above indicate the locations of the three DRMs. The 32 amino acid spacer between DRMs 2 and 3 is highlighted, along with the location of exon 5 when spliced into Loqs-PB. (B) Co-immunoprecipitation of Dcr and Ago proteins with HA-tagged dsRBPs in Aag2 cells. HA-dsRBPs were expressed in Aag2 cells by transfection of plasmid DNA. (C) Co-Immunoprecipitation of FLAG-Loqs-PA or FLAG-Loqs-PB with HA-tagged proteins. Column headings indicate the overexpressed HA-tagged protein, row headings indicate the antibody used in the corresponding western blot. Input (In), flow-through (FT) and bound (B) fractions are indicated. To increase the amount of detectable R2D2, HA-R2D2 proteins were expressed via infection with dsSINV; all others were expressed by plasmid transfection. Anti-HA and anti-FLAG co-IP assays were run 24 h post-infection (48 h post-transfection). (D) Dimerization of Loqs-PA is unaffected by both the Δ258 and Δ226 deletions as shown by co-IP.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Interactions between mosquito dsRBPs and RNAi/miRNA components. (A) Schematic representation of the Loqs-PA exon structure showing the location of the two Loqs-PA truncations used (Δ258 and Δ226). Start (1) and Stop (329) positions in the ORF are indicated; gray bars above indicate the locations of the three DRMs. The 32 amino acid spacer between DRMs 2 and 3 is highlighted, along with the location of exon 5 when spliced into Loqs-PB. (B) Co-immunoprecipitation of Dcr and Ago proteins with HA-tagged dsRBPs in Aag2 cells. HA-dsRBPs were expressed in Aag2 cells by transfection of plasmid DNA. (C) Co-Immunoprecipitation of FLAG-Loqs-PA or FLAG-Loqs-PB with HA-tagged proteins. Column headings indicate the overexpressed HA-tagged protein, row headings indicate the antibody used in the corresponding western blot. Input (In), flow-through (FT) and bound (B) fractions are indicated. To increase the amount of detectable R2D2, HA-R2D2 proteins were expressed via infection with dsSINV; all others were expressed by plasmid transfection. Anti-HA and anti-FLAG co-IP assays were run 24 h post-infection (48 h post-transfection). (D) Dimerization of Loqs-PA is unaffected by both the Δ258 and Δ226 deletions as shown by co-IP.
Mentions: To further explore the relationships of Ae. aegypti R2D2, Loqs-PA and Loqs-PB to siRNA and miRNA factors, we employed co-immunoprecipitation (co-IP) assays to test for protein–protein interactions between each dsRBP and Dcr1, Dcr2, Ago1 and Ago2. As both the Drosophila (Loqs) and human (TRBP) orthologs of Ae. aegypti Loqs have been shown to bind Dcr1 through an interaction mediated by the 3rd DRM at the C-terminus of the protein (19,32), two deletion constructs were included with a truncation immediately preceding the third DRM (Δ258) or immediately following the second DRM (Δ226) (Figure 3A). Co-IP experiments confirmed a strong association between R2D2 and Dcr2; this interaction was not affected by pre-treatment with RNaseA and is consistent with the role of this protein in the siRNA pathway (Figure 3B). Likewise, only Loqs-PB interacted with DCR1, consistent with its role in miRNA biogenesis. Interestingly, both Loqs-PA and Loqs-PB interacted with Dcr2, Ago1 and Ago2. Deleting the third DRM had no effect on the interaction between Loqs and Ago proteins. However, this interaction was lost when the 32 a.a. spacer separating the 2nd and 3rd DRMs was deleted (Figure 3B). Neither deletion had any effect on the ability of Loqs to interact with Dcr2.

Bottom Line: Conversely, Loqs-PB alone interacted with mosquito dicer-1 and was essential for full miRNA production.Mosquito Loqs interacted with both argonaute 1 and 2 in a manner independent of its interactions with dicer.We conclude that the functional specialization of Loqs-PD in Drosophila is a recently derived trait, and that in other dipterans, including the medically important mosquitoes, Loqs-PA participates in both the miRNA and endo-siRNA based pathways.

View Article: PubMed Central - PubMed

Affiliation: Fralin Life Science Institute and Department of Entomology, Virginia Tech, Blacksburg, VA 24061, USA.

Show MeSH
Related in: MedlinePlus