Major source of antigenic peptides for the MHC class I pathway is produced during the pioneer round of mRNA translation

Abstract

The MHC class I antigen presentation pathway allows the immune system to distinguish between self and nonself. Despite extensive research on the processing of antigenic peptides, little is known about their origin. Here, we show that mRNAs carrying premature stop codons that prevent the production of full-length proteins via the nonsense-mediated decay pathway still produce a majority of peptide substrates for the MHC class I pathway by a noncanonical mRNA translation process. Blocking the interaction of the translation initiation factor eIF4E with the cap structure suppresses the synthesis of full-length proteins but has only a limited effect on the production of antigenic peptides. These results reveal an essential cell biological function for a class of translation products derived during the pioneer round of mRNA translation and will have important implications for understanding how the immune system detects cells harboring pathogens and generates tolerance.

Fig. 1.

Antigenic peptides are produced by noncanonical mRNA translation initiation. (A) Western blot showing the rapid degradation of properly folded full-length IκBα-wt-SL8 and endogenous IκBα in H1299 cells following treatment with TNF-α (Fig. S1). The expression of the IκBα-mut-SL8, which carries mutations (Ser to Ala) in the IKK phosphorylation sites at Ser32 and Ser36, is not affected by TNF-α treatment. Actin is shown as a loading control. W.B., Western blot. (B) Effect on SL8 epitope presentation after IκBα-wt-SL8 degradation in H1299 cells expressing exogenous K^b after TNF-α treatment for indicated time periods, during which cells were washed and incubated with the B3Z reporter CD8⁺ T cells. Control cells were exposed to exogenous SL8 peptide for 16 h (peptide control). The difference in antigen presentation between IκBα-wt-SL8 and IκBα-mut-SL8 after TNF-α treatment is not significant. N.S., not significant. (C) Cartoon illustrating indicated codon positions in the MBP or in a chicken Ova construct. WT codons (Upper, black) and codon changes (Lower, red) are shown. (D, Upper) Autoradiograph of [³⁵S]methionine metabolic pulse labeling, followed by MBP immunoprecipitation in the presence of 25 μM proteasome inhibitor MG132. The two upper bands are derived from AUG codons at position 1 (full length) or codon 22 (MBP 22AUG). Changing codon 39 from CUU to CUG results in the appearance of a translation product (Codon 39) without affecting the synthesis of the two AUG-initiated polypeptides. The corresponding effect of indicated codon changes on the presentation of the MBP(79–87) epitope in H1299 cells coexpressing the K^k molecule is shown below using MBP(79–87) reporter CD8⁺ T cells. F.L., full length. (E) Effect on MBP(79–87) antigen presentation after indicated codon changes in the MBP ORF. (F, Upper) Autoradiograph of metabolic pulse labeling followed by Ova IP in the presence of proteasome inhibitors of cells expressing Ova constructs with indicated silent point mutations. (Lower) Corresponding T-cell assay using B3Z reporter T cells. Antigen presentation was carried out in human H1299 cells expressing the mouse K^b MHC class I molecule. The results are representative of at least three independent experiments plus SD. **P < 0.005; ***P < 0.001.

Fig. 2.

Synthesis of antigenic peptides and full-length proteins is temporally distinct. (A) Turnover rate of the endogenous K^b MHC class I-SL8 complex in EL4 mouse cells, or mouse K^b-SL8 complex in human H1299 cells, was estimated by measuring SL8 presentation on K^b molecules. Synthetic SL8 peptide was added for 1 h, followed by extensive washing (time 0) and cell fixation at the indicated time points before incubation with B3Z reporter cells. (B) Levels of capped Ova mRNA in H1299 cells following RNA transfection were determined using qRT-PCR. The arbitrary value of 100% was given at 1 h. (C) Antigen presentation after transfection of Ova mRNA in H1299 cells expressing K^b was determined on fixed cells at the indicated time points. Maximum antigen presentation (100%) was observed at 2.5 h. (D) Western blot shows the expression of Ova at different time points after mRNA transfection in H1299 cells. Endogenous actin is used as a loading control. W.B., Western blot. The results are representative of at least three independent experiments plus SD (Fig. S3).

Fig. 3.

mRNAs targeted for NMD produce antigenic peptides but not full-length proteins. (A) Cartoon illustrating different NMD reporter constructs and the position of the PTC and the SL8 or MBP(79–87) epitope. (B) Ratios of normal and PTC-carrying mRNAs from constructs in which the SL8 is inserted in exon 1 or 2 to the mouse urinary protein (MUP) control mRNA are shown. Numbers show the relative amounts of indicated mRNAs 48 h after transfection in HeLa cells. MUP. (C) Western blot shows the expression of YFP-globin fusion proteins in HeLa cells from indicated constructs using α-YFP mAb. Insertion of SL8 in exon 2 just downstream of the PTC (Glob2-PTC-SL8) partially disrupts the NMD process, allowing the expression of a truncated product that terminates at the PTC. TP, truncated product; W.B., Western blot. (D) Antigen presentation of the SL8 epitope from an increasing amount of indicated constructs in HeLa cells expressing mouse K^b. Similar results were obtained using H1299, a HEK293-TK^b stable cell line, and B6 mouse fibroblast (Fig. S4). Approximately 85% antigen presentation is retained from Glob1-SL8-PTC mRNAs that are targeted for NMD compared with the non-NMD Glob1-SL8 construct. The Glob2-PTC-SL8 construct does not produce antigenic peptide substrates because the PTC stops ribosomal read-through of the SL8 epitope. *P < 0.05. (E) Presentation of the MBP(79–87) epitope on K^k class I molecules from the Glob1-MBP-PTC construct (1 μg cDNA) in L929 mouse fibroblasts reached ∼90% compared with the Glob1-MBP. Similar data were obtained using H1299 and HEK293 cells expressing mouse K^k. The results are representative of at least three independent experiments plus SD.

Fig. 4.

eIF4G but not eIF4E is required for the synthesis of antigenic peptides. (A) Cells were transfected with the indicated YFP-globin reporter construct for 24 h, followed by expression of the α-eIF4E aptamer (24 h), which prevents interaction between the cap structure and eIF4E. Autoradiograph of a [³⁵S]methinonine metabolic pulse labeling experiment (Left) and corresponding Western blot (Right) in HeLa cells expressing indicated amounts of the α-eIF4E RNA aptamer. W.B., Western blot. (B) Western blot of Ova expression under similar conditions. (C) RNA stability of indicated globin NMD reporter constructs in the presence or absence of the α-eIF4E aptamer. The numbers indicate the relative amounts of mRNAs with SD. (D) Presentation of the SL8 epitope expressed from indicated mRNA constructs in the presence of α-eIF4E aptamer. Data are presented as relative to presentation of the exogenous SL8 peptide. (E) Western blot of YFP-globin expression from the Glob1-SL8 construct in the presence of increasing amounts of 4E-BP1. (F) Corresponding antigen presentation from indicated constructs under similar conditions as in E. (G) Western blot shows that increasing amounts of protease 2A, which cleaves the eIF4G translation factor (Fig. S5), lead to a reduction in YFP-globin protein expression levels. (H) Relative presentation of SL8 epitope derived from indicated constructs compared with cells being exposed to exogenous SL8 peptide for 16 h in the presence of increasing amounts of protease 2A. All results are representative of three independent experiments and SD.

Fig. 5.

Model for the synthesis and selection of antigenic peptide substrates during the pioneer round of translation that precedes NMD. mRNAs are checked for PTCs during the initial quality control translation that follows newly synthesized and spliced mRNAs. This translation is initiated by the CBC, including CBP20/80, and produces PTPs that are instantly degraded and selected for antigenic peptide processing for the MHC class I pathway. mRNAs found to contain PTCs are targeted for the NMD pathway (1), and those mRNAs that pass the quality control mature into mRNP complexes that bind the eIF4E cap-binding factor and initiate canonical translation complexes that govern the synthesis of full-length proteins (2). Degradation of full-length proteins plays only a minor role as a source of antigenic peptides for the MHC class I pathway.