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.

RNA affinity chromatography

“…The MALDI-TOF mass spectrometry analysis of cIAP1 IRES captured proteins identified them as RNA Helicase A (RHA), insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), NF90 and NF45 ( Figure 5C). Furthermore, Western blot analysis using antibodies against RHA, IGF2BP1, NF90 and NF45 confirmed that these proteins specifically interact with the cIAP1 IRES but not with the non-IRES portion of the cIAP1 5' UTR ( Figure 5D) (Graber et al, 2010). …”

“…Exactly how ITAFs modulate IRES activity is not clear; ITAFs were suggested to act as adapter proteins which act as a bridge between the ribosome and mRNA (Mitchell et al, 2005), or as RNA chaperones which remodel mRNA into a conformation that permits ribosome recruitment (Yaman et al, 2003). We have been studying IRES-mediated translation of cellular mRNAs during stress using two anti-apoptotic proteins, X chromosome-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis protein 1 (cIAP1), as model systems (Holcik et al, 1999;Graber et al, 2010;Riley et al, 2010;Thakor & Holcik, 2011). To gain full understanding of the regulation and function of XIAP and cIAP1 IRESes, it was necessary to isolate and identify ITAFs which modulate IRES activity.…”

“…To illustrate the utility of the above-described approach we have previously used the minimal functional XIAP IRES (-162 to +1 nt of 5' UTR of XIAP) and cIAP1 IRES (-150 to +1 nt of 5' UTR of cIAP1) (Graber et al, 2010) elements to isolate and identify XIAP and cIAP1 ITAFs, respectively. When XIAP IRES RNA conjugated beads were used in an affinity pulldown, at least six distinct proteins were isolated (Figure 4A Lane # 2) .…”

“…The 5' end biotinylated RNA is preferred over RNA carrying biotin substitutions along its length because internally tagged RNA can bind to the matrix using random sites which limits the protein binding sites. Therefore, we have used biotin-avidin RNA-affinity chromatography approach followed by mass-peptide fingerprinting to isolate and indentify ITAFs which modulate XIAP and cIAP1 IRES activities (Baird et al, 2007;Lewis et al, 2007;Graber et al, 2010;Durie et al, 2011). A general scheme for avidin-biotin RNA affinity chromatography is illustrated in Figure 2.…”

“…(C) GST-NF45 was mixed with radiolabeled cIAP1 IRES RNA probe and UVcrosslinked. It was then resolved on a SDS-PAGE gel and visualised by autoradiography (Adapted from Lewis et al, 2007;Graber et al, 2010;Durie et al, 2011).…”