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. 2005 Aug;25(16):6956-63.
doi: 10.1128/MCB.25.16.6956-6963.2005.

ADAR1 interacts with NF90 through double-stranded RNA and regulates NF90-mediated gene expression independently of RNA editing

Yongzhan Nie  1 , Li Ding, Peter N Kao, Robert Braun, Jing-Hua Yang
Affiliations

ADAR1 interacts with NF90 through double-stranded RNA and regulates NF90-mediated gene expression independently of RNA editing

Yongzhan Nie et al. Mol Cell Biol. 2005 Aug.

Abstract

The RNA-editing enzyme ADAR1 modifies adenosines by deamination and produces A-to-I mutations in mRNA. ADAR1 was recently demonstrated to function in host defense and in embryonic erythropoiesis during fetal liver development. The mechanisms for these phenotypic effects are not yet known. Here we report a novel function of ADAR1 in the regulation of gene expression by interacting with the nuclear factor 90 (NF90) proteins, known regulators that bind the antigen response recognition element (ARRE-2) and have been demonstrated to stimulate transcription and translation. ADAR1 upregulates NF90-mediated gene expression by interacting with the NF90 proteins, including NF110, NF90, and NF45. A knockdown of NF90 with small interfering RNA suppresses this function of ADAR1. Coimmunoprecipitation and double-stranded RNA (dsRNA) digestion demonstrate that ADAR1 is associated with NF110, NF90, and NF45 through the bridge of cellular dsRNA. Studies with ADAR1 deletions demonstrate that the dsRNA binding domain and a region covering the Z-DNA binding domain and the nuclear export signal comprise the complete function of ADAR1 in upregulating NF90-mediated gene expression. These data suggest that ADAR1 has the potential both to change information content through editing of mRNA and to regulate gene expression through interacting with the NF90 family proteins.

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Figures

FIG. 1.
FIG. 1.
Interactions between ADAR1 and NF90 proteins. (A) Electrophoretic mobility shift assay. Human 293T cells were transfected with Myc-His-tagged long-form ADAR1 (p150) or the Myc-His tag (tag). Cell lysates were coimmunoprecipitated with an anti-His antibody. Associated proteins were resuspended in the loading buffer, with or without DTT, and resolved by SDS-PAGE (0.1% SDS). Protein complexes containing ADAR1 were identified by immunoblotting with antibodies against the Myc epitope. NS, nonspecific. (B) Coimmunoprecipitation of NF110 and NF90 with ADAR1. Cell lysates (Lys) were coimmunoprecipitated using an immobilized anti-His antibody; the associated proteins (Co-IP) were denatured in loading buffer containing 100 mM DTT and analyzed by immunoblotting using antibodies against NF90 (lower panels) or Myc (upper panels). (C) Coimmunoprecipitation of NF45 with ADAR1. The same membrane was detected with an antibody against NF45.
FIG. 2.
FIG. 2.
Interaction between endogenous ADAR1 and NF90. (A) Cell lysates (Lys) of SGC7901 cells (human gastric cancer cells) were separated with immobilized rabbit IgG or antigen-purified anti-p68 antibody (ap68). Associated proteins were eluted and analyzed by immunoblotting using antibodies against ADAR1 (upper panel), NF90 (middle panel), or NF45 (the lower panel). (B) For negative controls, the cell lysates were immunoprecipitated with an antibody against an unrelated protein, RNA helicase p68 (ap68), followed by immunoblotting to detect ADAR1 (upper panel), NF110/NF90 (middle panel), or another unrelated protein, eIF2a (lower panel).
FIG. 3.
FIG. 3.
dsRNA components in ADAR1-NF90 complex. ADAR1-NF90 complexes immunoprecipitated with an anti-His-tag antibody were digested with 0, 0.006, 0.025, 0.1, or 0.4 units/μl of RNase V1 (lanes 1 to 5, respectively). Proteins resistant (R) or sensitive (S) to RNase V1 digestion were analyzed by immunoblotting using antibodies against NF90 (A), NF45 (B), or ADAR1 (C).
FIG. 4.
FIG. 4.
ADAR1 upregulates NF90-mediated gene expression. (A) Effects of ADAR1 on different promoters. A plasmid containing the IFN-β-, c-fos-, SV40-, RSV-, or CMV-luciferase reporter was cotransfected into 293T cells along with pcDNA3-ADAR1 p150 (LF) or pcDNA3 vector DNA (Vr) for 48 h. Luciferase activities were determined and normalized to the amount of protein in cell lysates. Relative upregulation was calculated in comparison with the vector. Data are expressed as means ± standard deviations (n = 3). (B) ADAR1 upregulates IFN-β-luciferase. 293T cells were cotransfected with the IFN-β-luciferase reporter along with a mixture of pcDNA3-ADAR1 and pcDNA3 at a ratio of 0:0.6, 0.2:0.4, 0.4:0.2, or 0.6:0 (μg:μg). Luciferase activities were determined and normalized as described above. *, P < 0.05 for lane 1 versus lane 2, 3, or 4. (C) ADAR1 upregulates SV40-luciferase. 293T cells were cotransfected with the SV40-luciferase reporter along with a mixture of pcDNA3-ADAR1 and pcDNA3 at a ratio of 0:0.6, 0.2:0.4, 0.4:0.2, or 0.6:0 (μg:μg). Luciferase activities were determined and normalized as described above. *, P < 0.05 for lane 1 versus lane 2, 3, or 4.
FIG. 5.
FIG. 5.
NF90 is required for ADAR1-mediated gene expression. (A) A siRNA specific for a bacterial RNA (siBac) or the NF110/NF90 mRNA (siNF90) was cotransfected into 293T cells along with pcDNA3-ADAR1 p150 (LF) or Myc-His-tagged vector (tag). The protein levels of ADAR1, NF110, NF90, and β-actin were determined by immunoblotting. (B) c-fos- and SV40-luciferase expression was determined and normalized to the amounts of protein in cell lysates. All data are expressed as means ± standard deviations (n = 3). *, P < 0.05 for LF versus tag. (C) A siRNA specific for the 5′ end of ADAR1 p150 (siADAR1) or the control siRNA (siBac) was cotransfected into 293T cells along with the SV40-luciferase reporter plasmid. The protein levels of ADAR1 and β-actin were determined by immunoblotting. (D) SV40-luciferase expression in transfected cells was determined and normalized to the amounts of protein in cell lysates. The relative expression affected by siADAR1 was normalized to that of the siBac control.
FIG.6.
FIG.6.
Selectivity of ADAR1 interaction and function. (A) Double-tagged ADAR1 constructs used for interaction and function assays. p150, p80, Dcat, dsRBD, and Z-DBD/NES are ADAR1 fragments amplified from positions 1 to 3466, 1561 to 3466, 1 to 2263, 748 to 2263, and 1 to 748 of the ADAR1 DNA (GenBank accession no. AF291050), respectively. (B) Expression and interaction of ADAR1 fragments. The double-tagged ADAR1 fragments were transfected into 293T cells, and their expression was detected by immunoblotting using an anti-Myc antibody (upper panels). Interactions of ADAR1 fragments with NF110/NF90 were determined by coimmunoprecipitation using an immobilized anti-His antibody followed by immunoblotting using an anti-NF90 antibody (lower panels). The expression and interaction of the transient ADAR1 p110 were determined and are shown in the right panels. (C) Upregulation of SV40-luciferase by ADAR1 fragments. Luciferase activities in the cell lysates were determined and normalized to the amounts of protein. Relative upregulation was calculated compared to the tag control. Data are expressed as means ± standard deviations (n = 3). *, P < 0.05 for Vr versus Dcat or p150.
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