Nuclear Export of NF90 Is Required for Interleukin-2 mRNA Stabilization  Jaekyung Shim, Hanjo Lim, John R.Yates, Michael Karin  Molecular Cell  Volume 10, Issue 6, Pages 1331-1344 (December 2002) DOI: 10.1016/S1097-2765(02)00730-X

Figure 1 Identification of NF90 as an IL-2 mRNA Binding Protein (A) Autoradiogram of a Northwestern blot in which cytoplasmic extracts (S-100) of Jurkat T cells that were either unstimulated or stimulated with TPA and A23187 (T/A) for 2 hr were probed with a 32P-labeled total IL-2 mRNA. The arrows indicate proteins whose abundance or activities were increased after cell stimulation. (B) UV-crosslinking assay. Protein fractions eluted at different KCl concentrations from the IL-2 mRNA affinity column were incubated with 32P-labeled total IL-2 mRNA probe. Following UV-crosslinking and SDS-PAGE RNA-protein complexes were identified by autoradiography. H, the load; FT, flow through. The migration positions of NF90 and NF45 are indicated. (C and D) The amino acid sequences of NF90 (C) and NF45 (D). Boldface type represents peptides that were identified by μLC-ESI-MS/MS. (E and F) The MS/MS spectra of NF90 (E) and NF45 (F). (G) Schematic representation of structural motifs present in NF90 and NF45. NES, nuclear export signal; DZF, DSRM or Zn finger C2H2 domain; NLS, nuclear localization signal; RGG, arginine, glycine-rich domain. Molecular Cell 2002 10, 1331-1344DOI: (10.1016/S1097-2765(02)00730-X)

Figure 2 The Cytoplasmic Abundance of NF90 Increases in Response to T Cell Activation (A) UV-crosslinking assay (upper panels) and Northwestern blot analysis (lower panels) of cytoplasmic extracts of Jurkat or HeLa cells prepared at the indicated times poststimulation with TPA+A23187. (B) Cytosolic extracts were prepared at the indicated times after TPA+A23187 stimulation of Jurkat cells, incubated with 32P-labeled IL-2 RNA probe (E1 in Figure 3A), UV crosslinked and immunoprecipitated with anti-NF90 (αMPP4) antibody. (C) The levels of NF90 in the cytoplasmic and nuclear fractions of TPA+A23187-stimulated Jurkat cells were examined by immunoblotting. (D) Total cell extracts were prepared from TPA+A23187-stimulated Jurkat cells at the indicated times, incubated with anti-NF90 (αMPP4) antibody to coimmunoprecipitate the NF90:NF45 heterodimer, and subjected to immunoblot analysis with anti-NF90 and anti-NF45 antibodies. Lower panel, the levels of total NF90, NF110, and NF45 were determined by direct immunoblotting. Molecular Cell 2002 10, 1331-1344DOI: (10.1016/S1097-2765(02)00730-X)

Figure 3 Identification of NF90 Binding Site(s) in the IL-2 3′UTR (A) Schematic representation of the different IL-2 mRNA probes. The locations of the AREs are indicated by the dots, and the results of Northwestern blot and REMSA analysis examining the binding of NF90 to the different RNAs are indicated on the right. (B) Recombinant MBP-NF90 was incubated with the 32P-labeled IL-2 3′UTR probe depicted in (A). The reaction mixtures were digested with RNase T1, and the digests were resolved by REMSA. The migration position of the major T1-resistant species is indicated as “shift band.” (C) RNase T1 digests of 32P-labeled 3′UTR, E1, E2, and E3 RNAs (see [A]) were separated on a denaturing polyacrylamide gel along with a secondary T1 digest of the gel-eluted “shift band” shown in (B) and RNA size markers. (D) Recombinant MBP-NF90 was incubated with a 32P-labeled IL-2 5′UTR, E1, E2, or E3 probes, and the RNA-protein complexes were resolved by REMSA. The shifted E1:NF90 complex is indicated with an arrow. (E) NF90 was incubated with the 32P-labeled E1 IL-2 RNA probe in the presence of the indicated molar excess of the ARE-containing regions of the c-Fos and IL-2 3′UTRs. The RNA:protein complexes were identified by UV crosslinking. Molecular Cell 2002 10, 1331-1344DOI: (10.1016/S1097-2765(02)00730-X)

Figure 4 Identification of Sequences within the E1 Region of the IL-2 3′UTR Required for NF90 Binding (A) The sequences of the E1 region of the IL-2 3′UTR and its mutant derivatives. (B) MBP-NF90 (0.25, 0.5, 1 μg) was incubated with equal amounts of wt and mutant E1 probes (10 ng), and the RNA:protein complexes were identified by UV crosslinking. (C) Cytoplasmic proteins (12, 25, 50 μg) of TPA+A23187-stimulated Jurkat cells were separated by SDS-PAGE, transferred to a PVDF membrane, and analyzed by immunoblotting with anti-NF90 antibody (upper panel) and Northwestern blotting with 10 ng/ml of wt and mutant E1 RNA probes (lower panel). Molecular Cell 2002 10, 1331-1344DOI: (10.1016/S1097-2765(02)00730-X)

Figure 5 Ectopic Expression of NF90 Results in IL-2 mRNA Stabilization (A) RNase protection analysis. Reporter plasmids containing a GFP-IL-2 3′UTR fusion gene (WT) or a GFP expression cassette without the IL-2 3′UTR (GFP) controlled by a Tet-regulated promoter was transfected into Jurkat-Tet-off cells. When indicated, these reporters were cotransfected with a pCMV-NF90 expression vector. The transfected cells were either left untreated (control) or stimulated with either TPA+A23187 or anti-CD3+anti-CD28. The amounts of GFP-IL-2 transcripts present at a given time after repression of the Tet-regulated promoter was determined by RPA and quantified by phosphoimaging. The results shown in the graphs are averages of three different experiments, including the one shown above, in which the level of GFP-IL-2 mRNA at any given point was normalized to the level of GAPDH mRNA. Straight lines were fitted using a linear regression program. (B) NF90 binding is required for optimal mRNA stabilization. Reporter plasmids containing mutant versions of the IL-2 3′UTR reporter (M1–M4 and M1/3) were transfected into Jurkat-Tet-off cells without or with a pCMV-NF90 expression vector. The amounts of GFP-IL-2 transcripts were determined at various time points after repression of the Tet-regulated promoter as in (A). The graphs represent averages of three separate determinations, including the one shown in this figure, in which the levels of GFP-IL-2 mRNA were normalized to those of GAPDH mRNA. Molecular Cell 2002 10, 1331-1344DOI: (10.1016/S1097-2765(02)00730-X)

Figure 6 Cell Stimulation Enhances the Nuclear Export of NF90 (A) Amino acid sequences of the leucine-rich NES of NF90 and those of several other proteins and the NES-disruption mutant of NF90. (B) The subcellular distribution of GFP-NF90 and GFP-NF45 in COS-7 cells. The cells were transfected with GFP fusion protein expression vectors including GFP-NF90, GFP-NF90(ΔNLS), GFP-NF90(NES-M), and GFP-NF45 expression vectors, and after 48 hr were either left unstimulated or treated with TPA+A23187 in the absence or presence of leptomycin B (LMB) as indicated. After 4 hr, the cells were fixed, and the subcellular distribution of the GFP fusion proteins was determined by fluorescent microscopy. The nuclei were counterstained with DAPI. (C) Immunoblot analysis of the subcellular distribution of endogenous NF90 and NF45 in Jurkat cells. The cells were stimulated or not with TPA+A23187 in the absence or presence of LMB. After 2 hr the cells were collected and nuclear and cytoplasmic extracts were prepared. The relative amounts of NF90 and NF45 as well as those of the cytoplasmic and nuclear markers, actin and histone H3, respectively, were determined by immunoblotting. Molecular Cell 2002 10, 1331-1344DOI: (10.1016/S1097-2765(02)00730-X)

Figure 7 LMB Treatment and Reduced NF90 Expression Prevent IL-2 mRNA Stabilization (A) Tet-off Jurkat cells were transfected with the GFP- IL2 reporter plasmid without or with the indicated pCMV-NF90 expression vectors (wt, ΔNLS, and NES-M). Cells that did not receive pCMV-NF90 were either left unstimulated (control) or stimulated with either TPA+A23187 or anti-CD3+anti-CD28. Cells were also pretreated or not with LMB for 2 hr. At the indicated time points after shutting off GFP-IL2 transcription by addition of doxycyclin (Dox), total cytoplasmic RNA was extracted. The relative abundance of GFP-IL-2 and GAPDH mRNAs was determined by RPA. The graphs in the right panels show averages of three different experiments, including the one shown here. Lines were fitted using a linear regression program. (B) Jurkat cells were left unstimulated (control) or stimulated with either TPA+A23187 in the absence or presence of LMB. At the 0 time point actinomycin D (ActD) was added, and total cellular RNA was extracted at the indicated time points. The relative levels of IL-2 and GAPDH mRNAs were determined by RPA. The values shown are averages of three different experiments. (C) Jurkat cells were stably transfected with either an empty expression vector (control) or an antisense NF90 RNA expression vector. Individual clones were examined for the levels of NF90 expression, and one clone, N29, expressing much less NF90 was chosen for further analysis. Shown are the levels of NF110, NF90, and NF45 in N29 and parental Jurkat cells. (D) IL-2 mRNA decay in nonstimulated or TPA+A23187-stimulated parental and N29 Jurkat cells. The values shown are averages of three separate determinations, in which the levels of IL-2 mRNA were normalized to those of GAPDH mRNA. Molecular Cell 2002 10, 1331-1344DOI: (10.1016/S1097-2765(02)00730-X)