NF45 functions as an IRES trans-acting factor that is required for translation of cIAP1 during the unfolded protein response

Abstract

Expression of the cellular inhibitor of apoptosis protein 1 (cIAP1) is unexpectedly repressed at the level of translation under normal physiological conditions in many cell lines. We have previously shown that the 5' untranslated region of cIAP1 mRNA contains a stress-inducible internal ribosome entry site (IRES) that governs expression of cIAP1 protein. Although inactive in unstressed cells, the IRES supports cap-independent translation of cIAP1 in response to endoplasmic reticulum stress. To gain an insight into the mechanism of cIAP1 IRES function, we empirically derived the minimal free energy secondary structure of the cIAP1 IRES using enzymatic cleavage mapping. We subsequently used RNA affinity chromatography to identify several cellular proteins, including nuclear factor 45 (NF45) as cIAP1 IRES binding proteins. In this report we show that NF45 is a novel RNA binding protein that enhances IRES-dependent translation of endogenous cIAP1. Further, we show that NF45 is required for IRES-mediated induction of cIAP1 protein during the unfolded protein response. The data presented are consistent with a model in which translation of cIAP1 is governed, at least in part, by NF45, a novel cellular IRES trans-acting factor.

Figure 1

cIAP1 IRES structure model and identification of binding proteins. (a) The cIAP1 IRES RNA secondary structure model (nucleotides −150 to −1) with sites sensitive to RNase T1, T2 and V1 shown as shaded circles. (b) SYPRO Ruby-stained RNA affinity chromatography gel of proteins pulled down with a biotin-tagged cIAP1 IRES RNA probe (probe 1) from an HEK293T cytoplasmic lysate. (c) Peptides identified from excised bands by mass spectrometry were mapped to four unique proteins. (d) To confirm protein binding specificity, RNA affinity chromatography was repeated with a cIAP1 IRES (probe 1) or non-IRES (probe 2) RNA, followed by immunoblotting with antibodies specific for RHA, NF90, IGF2BP1 and NF45

Figure 2

NF45 regulates cIAP1 mRNA translation. (a) Western blots illustrating differential expression of endogenous NF45 and NF90 in HEK293T and U373 MG cells. (b) Metabolic labeling and immunoprecipitation of cIAP1 shows impaired translation of cIAP1 in U373 MG relative to HEK293T cells. (c) NF45 increases de novo cIAP1 protein synthesis. Top panel: U373 MG cells transiently expressing NF45 or GFP were pulse-labeled 24 h following transfection and newly synthesized cIAP1 and β-actin proteins were co-immunoprecipitated, resolved on SDS-PAGE and detected by autoradiography. Bottom panel: western blot of U373 MG cells transiently transfected with a FLAG-tagged NF45 overexpression plasmid illustrating efficient expression of the transgene. (d) NF45 enhances cIAP1 protein translation but not transcription. Top panel: densitometric analysis of cIAP1 protein expression from experiments performed in panel c (*n =2, P<0.001; mean±S.D.). Bottom Panel: quantitative RT-PCR of total cIAP1 mRNA levels in cells expressing GFP or NF45 (**n =3, P = 0.33; mean±S.D.). (e) NF45 enhances translation efficiency of cIAP1 mRNA as measured by polysome profiling of U373 MG cells transiently expressing GFP or NF45. The efficiency of the separation was assessed by resolving ethidium-bromide-stained 28S and 18S rRNA from each fraction. Individual fractions were probed for cIAP1 or β-actin (control) mRNA expression by RT-PCR. PCR products were resolved by gel electrophoresis and densitometry was performed to determine the percent distribution of specific mRNAs across the gradient (n = 3; mean±S.D.). The amount of specific mRNA present in higher-order polysomes (P) relative to the translationally quiescent pool (M) produces the metric P/M, and quantifies the change in translational efficiency

Figure 3

NF45 regulates IRES-dependent cIAP1 translation. (a) U373 MG cells were assayed 24 h following transient transfection of a bicistronic DNA reporter construct (schematic) containing the cIAP1 5′ UTR. IRES activity is expressed as the ratio of CAT expression over β-GAL activity. (b) U373 MG cells were transiently transfected with GFP or NF45 together with a monocistronic CAT reporter plasmid with either the cIAP1 5′ UTR (1.) or a truncated UTR that does not bind NF45 (2.) inserted upstream of the CAT reporter. A β-GAL reporter was co-transfected to normalize CAT expression across samples. (*P<0.05, **P<0.005, n = 3 mean±S.D.)

Figure 4

NF45 is required for IRES-mediated translation of cIAP1 during ER stress. (a) Knockdown of NF45 specifically impairs cIAP1 IRES activity. IRES activity in d5 (NF45 shRNA) cells relative to C (nontargeting shRNA) cells was measured using a bicistronic assay for cIAP1, APAF1, BclxL, DAP5 and VCIP as detailed in Material and methods (n = 3, mean±S.D.). (b) NF45 is required for ER stress-mediated induction of cIAP1. c or d5 cells were treated with DMSO (D) as a vehicle or 5 μM thapsigargin (T) to induce ER stress for 24 h. Western blots were performed with antibodies against the indicated proteins. (c) Rescue of d5 cells with transient overexpression of NF45 but not NF90 enhances cIAP1 expression. d5 cells were transiently transfected with an OMNI-tagged NF90 construct (O-NF90c) or a FLAG-tagged NF45 construct harboring a silent mutation that renders it resistant to NF45 shRNA (F.NF45r). Cells were then treated with DMSO (D) or 5 μM thapsigargin (T) for 24 h. Western blots were performed with antibodies against the indicated proteins. Blots shown are representative of at least three experiments

Figure 5

NF45 interacts directly with the cIAP1 IRES. (a) Recombinant GST-NF45 used for in vitro binding studies was expressed in E. coli using IPTG, affinity purified and assessed by SDS-PAGE as shown. (b) Recombinant NF45 (300 ng) was UV cross-linked to a ³²P-labeled cIAP1 IRES RNA probe (nucleotides −150 to −63, probe 3) or a portion of the 5′ UTR with no IRES activity (nucleotides −80 to −1, probe 2). The RNA–protein complex was resolved by SDS-PAGE and detected by autoradiography. (c) Determination of NF45 binding sites on the cIAP1 IRES using masking DNA oligonucleotides. Experiments were performed as in b using RNA probe 3, except that the probe was first hybridized with each of eight competition DNA oligonucleotides (100-fold molar excess). The top panel shows a primary structure of the probe with the location of the masking oligos and the bottom panel illustrates oligo locations on the cIAP1 IRES secondary structure. (d) Confirmation of NF45 binding sites on the cIAP1 IRES by mutational analysis. Experiments were performed as in b with probe 3 (3^{wild type}) or a mutant probe 3 with the bulge of SLI removed (3^ΔCUUA). Alternatively, a probe corresponding to SLI of the cIAP1 IRES (SLI^{wild type}) was used together with a sequence mutant that preserves the stem-loop structure (SLI^s), or a sequence mutant that also disrupts base pairing at the base of SLI (SLI^ss)