Article Figures & Tables
Figures
- Fig. 1.
Construction of mutant TRBP lacking the interaction with Dicer. (A) Domain structure of the HIV-1 genome. HIV-1 has TAR regions at the 5′- and 3′-LTRs. The HIV-1 genome has genes encoding gag, pol, vif, vpr, rev, tat, vpu, env and nef. (B) Predicted sites of the TRBP interaction with Dicer and the domain structure of TRBP and amino acid sequence of dsRBD3. TRBP is a dsRNA-binding protein consisting of 366 amino acid residues with three dsRBDs (dsRBD1, dsRBD2 and dsRBD3). dsRBD1 and dsRBD2 interact with dsRNA, while dsRBD3 is necessary for the interaction with Dicer. The complex structure of TRBP dsRBD3 and the ATPase/helicase domain of Dicer have been clarified by Wilson et al. (2015). From this structural analysis, the amino acid residues L326, V336, H338 and Y358 (pink circles in sequence) in TRBP were expected to be important for the interaction with Dicer. The interaction of these four amino acids (pink) in TRBP protein (green) with Dicer (blue) are in the crystal structure showing Dicer-TRBP interface (PDB, 4WYQ). The boxes i–iii indicate the region containing these amino acids (pink) and the boxes on the right are enlarged structures. (C) Analysis of the interaction between wild-type or mutant TRBPs with Dicer via immunoprecipitation (IP). TRBP−/− HeLa cells were transfected with each expression plasmid (FLAG-tagged TRBP-WT, TRBP-dsRBDΔ3, TRBP-L326A, TRBP-V336A/H338A, TRBP-Y358A, TRBP-L326A/V336A/H338A/Y358A, TRBP-L326A/V336A/H338A, TRBP-V336A/V336A/H338A/Y358A, TRBPV336A/H338A/Y358A and TRBP-L326A/Y358A) and control Luc, and immunoprecipitated with anti-FLAG antibody. Finally, the TRBP–Dicer interaction was detected using anti-Dicer antibody. Asterisks indicate non-specific band.
- Fig. 2.
Establishment of the Flp-In 293 cell line expressing wild-type and mutant TRBP. (A) T-REx 293 cells contain a TRE promoter capable of inducing downstream gene expression with tetracycline or Dox addition. Using this system, cell lines introduced with FLAG-tag alone (Control), FLAG-tagged TRBP-WT, TRBP-L326A/V336A/H338A, TRBPL326A/Y358A, TRBP-V336A/H338A/Y358A or TRBP-L326A/V336A/H338A/Y358A were established. (B) Confirmation of TRBP expression in Flp-In 293 cells. The established Flp-In 293 cells introducing each plasmid were cultured in medium containing Dox, and western blotting and IP using anti-FLAG antibody were performed. (Left) All incorporated FLAG-tagged TRBP genes were successfully expressed. The interaction with Dicer was attenuated in all cell lines except for cells with TRBP-WT. Asterisk indicates non-specific band. (Right) The intensities in the western blot bands in the left panels were measured using ImageJ, and showed the relative levels of immunoprecipitated Dicer compared to the immunoprecipitated TRBP and its mutants. These results were obtained under nearly identical conditions to Fig. 1C. Asterisks indicate non-specific band. (C) Effect on RNAi activity of TRBP-WT and its mutant TRBPs. Flp-In 293 cells expressing each TRBP were transfected with firefly luciferase and Renilla luciferase expression plasmids with the shRNA expression plasmid against firefly luciferase (pSUPER-FL774). Cells were harvested after 24 h and luciferase luminescence intensity was measured. The luciferase activity of each cell type was normalized using the value obtained by transfection of control shRNA expression plasmid against green fluorescent protein (pSUPER-GY441) in control cells. The data represent the mean±s.d. of two independent experiments; each experiment was carried out using three wells. Student's t-test, *P<0.01.
- Fig. 3.
Translational regulation of HIV-1 TAR RNA by TRBP. (A) Schematic representation of pGL2-TAR-Luciferase expression plasmid. pGL2-TAR-Luciferase contained the 5′-LTR of the HIV-1 genome containing TAR downstream of the CMV promoter and fused with the firefly luciferase gene. (B) Luciferase mRNA levels in Flp-In 293 cells determined with real-time PCR. Flp-In 293 cells with control FLAG-tag alone, FLAG-tagged TRBP-WT, TRBP-dsRBDΔ3 or TRBP-L326A/Y358A were transfected with pGL2-TAR-Luciferase along with pRL-SV40. The cells were recovered after 24 h, and the amount of luciferase mRNA was measured. mRNA levels were normalized using the mRNA level of endogenous GAPDH. The amount of luciferase mRNA in the control cells transfected with pGL2-TAR-Luciferase was set as 1. (C) Luciferase activity in Flp-In 293 cells based on the luciferase reporter assay system. Using cells transfected as described in B, the intensity of luciferase luminescence was measured. The relative luciferase activity in the control cells transfected with pGL2-TAR-Luciferase was set as 1. (D) Luciferase activities shown in C were normalized by their mRNA amounts in B. The luciferase activity normalized by mRNA amount in the control cells transfected with pGL2-TAR-Luciferase was set as 1. The data represent the mean±s.d. of three independent experiments. Student's t-test, **P<0.01, *P<0.05.
- Fig. 4.
Northern blot analysis of the excision of TAR miRNA. Flp-In 293 cells with control FLAG-tag alone, FLAG-tagged TRBP-WT, TRBP-dsRBDΔ3 or TRBP-L326A/Y358A were transfected with pGL2-TAR-Luciferase, and IP was performed with anti-FLAG antibody. Northern blotting was performed using purified total RNA from input samples and IP samples with a probe for detecting the 3′ stem region of TAR RNA (red). A band of approximately 60 bases corresponding to the cleaved stem-loop-structured TAR RNA was observed in the IP sample of TRBP-WT, but not in those of TRBP-dsRBDΔ3 or TRBPL326A/Y358A. The right figure presents a model of TAR RNA cleavage via the TRBP–Dicer interaction. Asterisk indicates non-specific band.
- Fig. 5.
Regulation of apoptosis by the TRBP–Dicer interaction. (A) Morphological changes in TRBP−/− HeLa cells transfected with pGL2-TAR-Luciferase along with each expression plasmid of control FLAG-tag alone, FLAG-tagged TRBP-WT, TRBPdsRBDΔ3 or TRBP-L326A/Y358A at 0, 6 and 10 h after treatment with or without TNFα/CHX. (B,C) Western blots of the apoptosis marker proteins PARP, caspase-3 and IER3 at 0, 6 and 10 h after treatment with TNFα/CHX (B) or without TNFα/CHX (C). β-actin was used as control. Processing of PARP or caspase-3 was confirmed in cells with induced apoptosis following TNFα/CHX treatment (B), but processing was not observed in cells without TNFα/CHX (C). (D) Quantification of cleaved PARP protein at 6 and 10 h after addition of TNFα/CHX. The western blot result shown in B was quantified using ImageJ. The proportions of processed PARP relative to the total amount of PARP protein including full-length and processed PARP were calculated as a percentage. The amount of cleaved PARP in control cells at 6 h after TNFα/CHX treatment was set as 1. (E) GIT2 and IER3 mRNA levels measured by real-time PCR. mRNA levels were normalized to the amount of tublinβ mRNA. The amount of GIT2 or IER3 mRNA at 6 h without TNFα/CHX treatment was set as 1. (F) Quantification of IER3 protein at 0, 6 and 10 h after the addition of TNFα/CHX. The western blot results shown in C were quantified using ImageJ. The amount of IER3 protein at 0 h was set as 1. Two independent experiments were performed, and their results were almost similar. The results shown in B and D were used for quantification in D, E and F.
- Fig. 6.
Regulation of GIT2 or IER3 expression without TAR miRNA expression. (A) Western blots of IER3 at 0, 6 and 10 h after treatment with TNFα/CHX. α-tubulin was used as control. (B) GIT2 and IER3 mRNA levels measured by real-time PCR. mRNA levels were normalized to the amount of tublinβ mRNA. The amount of GIT2 or IER3 mRNA at 6 h without TNFα/CHX treatment was set as 1. (C) Quantification of IER3 protein at 0, 6 and 10 h after the addition of TNFα/CHX. The western blot results shown in A were quantified using ImageJ. The amount of IER3 protein at 0 h was set as 1. Two independent experiments were performed, and their results were similar. The results shown in A were used for quantification in B and C.
- Fig. 7.
Model of TRBP function in HIV-1 infection. TRBP is essential for excision of TAR miRNA in addition to Dicer. Furthermore, Dicer is essential for translational activation of TAR RNA in addition to TRBP. Thus, the TRBP–Dicer interaction is necessary for both processing of TAR miRNA and translational activation of TAR-containing RNA.
Article Navigation
Related articles
Cited by...
More in this TOC section
Similar articles
Other journals from The Company of Biologists
Biology Open and COVID-19
We are aware that the COVID-19 pandemic is having an unprecedented impact on researchers worldwide. The Editors of all The Company of Biologists’ journals have been considering ways in which we can alleviate concerns that members of our community may have around publishing activities during this time. Read about the actions we are taking at this time.
Please don’t hesitate to contact the Editorial Office if you have any questions or concerns.
2020 at The Company of Biologists
Despite 2020’s challenges, we achieved a lot at The Company of Biologists. In the midst of the pandemic, we have seen long-term projects and new ventures come to fruition. Read our full lowdown of 2020.
Interview- Sebastian Markert
Sebastian Markert is first author of a paper in BiO using C. elegans to model amyotrophic lateral sclerosis. In an interview, he talks about the potential implications of his work and his future plans.
Three communities to support biologists to everywhere
Online communities have never been more important. If you’re looking for somewhere to meet fellow scientists, take part in topical discussions and find virtual events in your field, take a look at each of our community sites:
- The Node: the community site for and by developmental biologists
- preLights: the preprint highlights service run by the biological community
- FocalPlane: the community site for microscopists and biologists alike