The RNA binding protein nuclear factor 90 functions as both a positive and negative regulator of gene expression in mammalian cells

Abstract

Nuclear factor 90 (NF90) was originally isolated in a complex that binds to the antigen recognition response element (ARRE-2) present in the interleukin-2 promoter. To characterize the transcriptional properties of NF90 in mammalian cells, we examined its ability to modulate promoter function in cellular transfection assays. NF90-Gal4 fusion proteins inhibited transcription from the adenovirus major late promoter in a fashion that was dependent on Gal4 targeting. Conversely, NF90 activated the cytomegalovirus immediate-early promoter, to which it was not targeted. These effects required distinct but overlapping domains in the C terminus of NF90, which contains a functional nuclear localization signal and two double-stranded-RNA binding motifs. NF90 is present in cellular complexes together with the NF45 protein. Transfection assays showed that NF45 binds NF90 strongly and stimulates its ability to activate but not to inhibit gene expression. This report characterizes NF90 as both a positive and negative regulator of gene expression, depending on the promoter context, and suggests a role for NF45 as a regulator of NF90.

FIG. 1.

NF90 has an NLS. (A) Comparison of the putative bipartite NLS in NF90 with established NLS sequences in p53 and nucleoplasmin. (B) Schematic diagram of plasmid constructs used for NF90 subcellular localization studies. (C) Localization of EGFP-NF90 and EGFP-NF90 mutants in mouse NIH 3T3 cells. Cells were transfected with the indicated plasmids, fixed after 24 h, and then stained with DAPI and rhodamine-phalloidin. Slides were viewed with an inverted microscope.

FIG. 2.

Dose-dependent effects of NF90 on reporter gene expression. (A) Schematic representation of plasmids used in transfection assays. Human 293-T cells in 12-well plates were transfected with increasing concentrations of NF90-BD plasmid together with 300 ng of pGL3-G5ML1-F.luc, 10 ng of CMV-R.luc, and 600 ng of total pcDNA3.1 plasmid. After 24 h, cell extracts were assayed for (B) firefly luciferase activity (from pGL3-G5ML1-F.luc) and (C) Renilla luciferase activity (from pCMV-R.luc). Luciferase expression is displayed relative to that observed with BD (50 ng) and is normalized to total protein in the extracts. The data represent results of at least three independent transfections performed in duplicate, with the error bars representing standard deviation. Asterisks (*) indicate P < 0.05 for indicated samples versus BD (50 ng) alone. The pound symbols (#) indicate P < 0.05 for NF90-BD (300 ng) versus BD (300 ng). In panel B, the Gal4-BD construct gave a luciferase expression level 1.2-fold higher than that with empty vector.

FIG. 3.

Identification of effector domain in NF90. (A) Schematic depiction of NF90-BD mutants. Shaded boxes represent the dsRBMs, black boxes represent the NLS, and cross-hatched boxes represent the BD. Human 293-T cells in 12-well plates were transfected with 300 ng of pG5-ML1-F.luc, 10 ng of CMV-R.luc, 50 ng of RSV-βgal, 200 ng of expression plasmid [pBD, pNF90-BD, or pNF90(mut)-BD], and 400 ng of pcDNA3.1 plasmid. At 24 h posttransfection, cell extracts were assayed for (B) firefly luciferase, (C) Renilla luciferase, and (D) β-galactosidase activities. Reporter gene expression is presented as in Fig. 2. Asterisks (*) indicate P < 0.05 for indicated samples versus BD alone.

FIG. 3.

Continued.

FIG. 4.

Detailed mapping of NF90 effector domain. (A) Schematic representation of the NF90-BD mutants as in Fig. 3A. Human 293-T cells in 12-well plates were transfected as described for Fig. 3. After 24 h, cell extracts were assayed for (B) firefly luciferase and (C) Renilla luciferase activities as in Fig. 2. Asterisks (*) indicate P < 0.05 for indicated samples versus BD alone.

FIG. 5.

NF90 regulates gene expression at the level of transcription. Human 293-T cells in 60-mm plates were transfected with 1.2 μg of pG5-ML1-F.luc, 40 ng of CMV-R.luc, 40 ng of pSG, 800 ng of pBD or pNF90-BD, and 1.6 μg of empty pcDNA3.1 plasmid. After 24 h, cells were harvested for RNA and then hybridized with RNA probes for the different reporter transcripts. The pSG plasmid encodes a nonfunctional mutant of VA RNA_I and served as a transfection control. (A) Autoradiogram of protected RNA fragments. (B) Quantitation of two experiments like that shown in panel A. The firefly and Renilla RNA levels were normalized to the VA RNA signal; error bars indicate standard deviations.

FIG. 6.

NF45 modulates the activity of NF90. Human 293-T cells in 12-well plates were transfected with 300 ng of pG5-ML1-F.luc, 10 ng of CMV-R.luc, and 200 ng of expression plasmid (pBD or pNF90-BD), pNF45, and empty pcDNA3.1. A total amount of 400 ng of plasmid (pNF45 + pcDNA3.1 empty vector) was used. After 24 h, cell extracts were assayed for (A) firefly luciferase and (B) Renilla luciferase activities as in Fig. 2. Asterisks indicate P < 0.05 for indicated samples versus BD alone. The pound symbol indicates P < 0.05 for NF90-BD + NF45 (100 ng) versus NF90-BD alone.

FIG. 7.

Complex formation between NF90 mutants and NF45. Human 293-T cells in 60-mm plates were transfected with 1.2 μg of pUC19, 800 ng of the indicated NF90-BD plasmid, and 1.6 μg of empty pcDNA3.1 plasmid. Following transfection, cells were lysed, and equivalent amounts of cell extracts were subjected to immunoprecipitation with anti-BD antibody-Sepharose for 3 h. Complexes were resolved in a 10% polyacrylamide-SDS gel and transferred to a nitrocellulose membrane. Western blot analysis was conducted (A) with anti-NF45 antibody (αNF45) and, after stripping, (B) with anti-BD antibody (αBD). The BD protein fragment (≈15 kDa) was too small to be resolved in the 10% polyacrylamide gel used.

FIG. 8.

Models for NF90 structure and function. (A) Functional domains of NF90 and proposed mechanisms of transcriptional activation (B) and inhibition (C) by NF90 and the NF90/NF45 complex. The black box indicates the NLS, and the grey boxes denote the dsRBMs. See Discussion for details.