4E-BP1 inhibits the production of luc activity without abrogating NMD. COS cells were transiently transfected with pmCMV-Gl (Norm or Ter), phCMV-MUP, pGL2, and either pACTAG2 empty vector (-) or expressing 4E-BP1 (+). (A) 4E-BP1 is detected in cells transfected with a 4E-BP1 expression vector (+) but not in cells transfected with empty vector (-) as determined by Western blotting, where the level of endogenous calnexin controlled for variations in the amount of cellular protein analyzed. (B) Luc activity (act), which was normalized to the level of LUC mRNA, is inhibited in cells expressing 4E-BP1. (C) Production of the secreted protein MUP, which was measured relative to the level of calnexin, is inhibited in cells expressing 4E-BP1. (D) The level of nonsense-containing Gl mRNA (Ter) is the same percentage of the level of nonsense-free Gl (Norm) in cells regardless of 4E-BP1 expression. For each lane, the level of Gl mRNA was normalized to the level of MUP mRNA, and the normalized level of Norm was defined as 100. Serial dilutions of RNA in the left four lanes demonstrate that the RT-PCR analysis is semiquantitative. (E) α-eIF4E antibody but not α-CBP80 antibody, or, as a control for nonspecific IP, normal rabbit serum (NRS), immunopurifies 4E-BP1. Serial dilutions of protein in the left three lanes demonstrate that the Western blot analyses of immunopurified proteins using antibodies against CBP80, eIF4E, or 4E-BP1 are semiquantitative. All results are representative of at least two independently performed experiments.

eIF2α MUT abrogates NMD, which indicates that eIF2α functions in NMD. COS cells were transiently transfected with pmCMV-Gl (Norm or Ter), phCMV-MUP, pGL2, and pMT2-HA-eIF2α, either WT (wild type) or S51D MUT. (A) Western blotting of total-cell protein using antibodies against HA and calnexin demonstrates that HA-eIF2α WT and HA-eIF2α MUT are expressed. (B) Assays of luc activity, where activity in total-cell protein was normalized to the level of LUC mRNA, demonstrate that eIF2α MUT inhibits activity relative to eIF2α WT. (C) Production of the secreted protein MUP, which was measured relative to the level of calnexin, is inhibited in cells expressing eIF2α MUT. (D) RT-PCR analysis of total-cell RNA using primers specific for Gl and MUP mRNAs demonstrates that eIF2α MUT inhibits NMD relative to eIF2α WT. Analyses were as in Figure 1D.

Evidence that CBP80-bound mRNAs are translated less efficiently than the corresponding eIF4E-bound mRNAs. 293T cells were transiently transfected with pmCMV-Gl and pmCMV-GPx1. Cells were lysed, and polysomes were separated according to size in a 10%-50% sucrose gradient. Each gradient fraction was analyzed using rabbit α-CBP80 or mouse α-eIF4E antibody. Proteins were analyzed using Western blotting and antibodies against CBP80, eIF4E, PABP1, PABP2, Upf2, or Upf3/3X. After IP using antibody against eIF4E or CBP80, CBP80 and eIF4E were analyzed using Western blotting, and Gl, GPx1, and SMG7 mRNAs were analyzed using RT-PCR. Results are representative of at least three independently performed experiments.

Steady-state translation initiation factors coimmunopurify not only with eIF4E but also with CBP80. (A) α-eIF4E antibody as well as α-CBP80 antibody immunopurify eIF4GI and PABP1. COS cells were lysed, and proteins were immunopurified using rabbit α-CBP80 or mouse α-eIF4E antibody, where rabbit α-VSV or mouse α-Flag antibody was used to control for the IP specificity, respectively. Immunopurified proteins were analyzed by Western blotting using antibodies against CBP80, eIF4E, eIF4GI, and PABP1. (B) α-eIF4E antibody as well as α-CBP80 antibody immunopurify subunits of eIF3. As in A except that rabbit α-VSV antibody was used to control for IP specificity of rabbit α-eIF4E, and immunopurified proteins were analyzed by Western blotting using antibody against eIF3. (C) eIF4AI and eIF2α coimmunopurify with CBP80 and eIF4E, whereas eIF4AIII coimmunopurifies with CBP80 and not eIF4E. COS cells were transfected with a vector expressing HA-eIF4AI, HA-eIF4AIII, or HA-eIF2α, and cell lysates were immunopurified using α-HA antibody or, as a control for IP specificity, rIgG. Immunopurified proteins were analyzed by Western blotting using antibody against HA, CBP80, or eIF4E. (D) PABP1 and PABP2 coimmunopurify in an RNase-insensitive manner. COS cells were transfected with a vector expressing PABP2-HA, and cell lysates were immunopurified using α-HA antibody or, as a control for the IP specificity, rIgG. Lysates were (+) or were not (-) exposed to RNase A prior to IP and analyzed by Western blotting using antibody against HA or PABP1. (E) Purified CBP80-His interacts directly with cellular eIF4GI and exogenously produced myc-eIF4GI using Far-Western analysis. Using untransfected cells (-) or cells transfected with an expression vector encoding myc-eIF4GI (+) at a level that was 2.5-fold above the level of endogenous eIF4GI, total-cell protein was subjected to Far-Western blotting (FW) using bacculovirus-produced CBP80-His. CBP80-His was subsequently detected by Western blotting (WB) using α-His antibody (left). Reactivity was removed, and blots were subjected to Western blotting using α-eIF4GI antibody (right). For all panels except E, serial dilutions of protein in the leftmost three lanes, which start with different amounts of protein based on antibody reactivity, demonstrate that the Western blot analyses are semiquantitative.

Paip2 abrogates NMD, which indicates that PABP1 functions in NMD. COS cells were transiently transfected with pmCMV-Gl (Norm or Ter), phCMV-MUP, pGL2, and either pcDNA3 (-) or pcDNA3-HA-Paip2 (+). (A) Western blotting of total-cell protein using antibodies against Paip2 and calnexin demonstrates that plasmid-encoded Paip2 is sevenfold more abundant than endogenous Paip2. (B) Assays of luc activity, where activity in total-cell protein was normalized to the level of LUC mRNA, demonstrate that Paip2 inhibits activity. (C) Production of the secreted protein MUP, which was measured relative to the level of calnexin, is inhibited in cells expressing Paip2. (D) RT-PCR analysis of total-cell RNA using primers specific for Gl and MUP mRNAs demonstrates that Paip2 inhibits NMD. Analyses were as in Figure 1D.