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. 2008;3(10):e3460.
doi: 10.1371/journal.pone.0003460. Epub 2008 Oct 21.

A distinct translation initiation mechanism generates cryptic peptides for immune surveillance

Affiliations

A distinct translation initiation mechanism generates cryptic peptides for immune surveillance

Shelley R Starck et al. PLoS One. 2008.

Abstract

MHC class I molecules present a comprehensive mixture of peptides on the cell surface for immune surveillance. The peptides represent the intracellular protein milieu produced by translation of endogenous mRNAs. Unexpectedly, the peptides are encoded not only in conventional AUG initiated translational reading frames but also in alternative cryptic reading frames. Here, we analyzed how ribosomes recognize and use cryptic initiation codons in the mRNA. We find that translation initiation complexes assemble at non-AUG codons but differ from canonical AUG initiation in response to specific inhibitors acting within the peptidyl transferase and decoding centers of the ribosome. Thus, cryptic translation at non-AUG start codons can utilize a distinct initiation mechanism which could be differentially regulated to provide peptides for immune surveillance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Ribosome initiation complexes recognize cryptic CUG start codons.
(A) The mRNA sequences of the AUG[YL8] and CUG[YL8] constructs used for toeprinting differ at a single nucleotide in the initiation codon (boxed). The amino acid sequence encoded by AUG[YL8] (MTFNYRNL, MYL8) or the CUG[YL8] (LYL8) are shown above and below the nucleotide sequences respectively. (B) Toeprinting analysis on AUG[YL8] and CUG[YL8] mRNAs using rabbit reticulocyte lysate as the ribosome source (see methods for experimental details). A band at +15–17 nucleotides downstream of the A of the AUG codon (boxed) is the toeprint and represents the leading edge of ribosomal initiation complexes at either the AUG or the cryptic CUG start codons. The bands above the toeprint bands (∼12 nt from the start codon) are non-specific because they were unaffected by translation initiation inhibitors. Sequencing lanes shown are for the CUG[YL8] mRNA. The data shown are representative from 5 independent toeprinting experiments. (C) Toeprint intensity of the AUG and CUG bands from (B) is shown in arbitrary Phosphoimager units (AU). On average, the CUG toeprint represents 18–25% of the intensity of the AUG toeprint.
Figure 2
Figure 2. Initiation at the CUG codon depends upon the Kozak context in vitro and in vivo.
(A) Toeprinting with AUG[YL8] and CUG[YL8] mRNAs in either an “Excellent Kozak” (UCGACC[CUG]A) or a “Poor Kozak” context (GCGUCCCUGA). The toeprints at +15–17 nucleotides downstream of the AUG or CUG start codons are indicated. The bar graph below shows the intensity of toeprints in arbitrary Phosphoimager units (AU). The data shown are representative of three independent toeprinting experiments. (B) The mRNAs used for toeprinting in (A) and an mRNA with the CCC initiation codon as a negative control, were transfected into Kb-L cells. Three hours later the presentation of peptide-Kb complexes on the cell surface was measured using the LYL8-Kb or MYL8-Kb specific BCZ103 hybridoma. The β-galactosidase activity induced in the activated T cell hybridoma was measured using the substrate chlorophenol red-β-D-galactopyranoside, which yields a colored product with an absorbance at 595 nm.
Figure 3
Figure 3. CUG recognition requires 5′-cap and GTP hydrolysis but is independent of Met-tRNAi Met.
(A) Toeprint analysis of AUG[YL8] and CUG[YL8] mRNAs in the presence of translation initiation inhibitors cycloheximide and sparsomycin and the 5′-cap m7GTP analog (1 mM). (B) Toeprint analysis in the presence of the non-hydrolyzable GTP analog, GMP-PNP (0.4 mM). Cycloheximide and sparsomycin were not included in this experiment because GMP-PNP inhibits large ribosomal subunit assembly on the pre-initiation complexes. (C) Methionine-sulfamide (Met-sulfamide) inhibits toeprints in a dose-dependent manner on AUG[YL8] mRNA, but not CUG[YL8] mRNA. Met-sulfamide (along with cycloheximide and sparsomycin) were added during the 5 min preincubation prior to adding mRNA. (D) Phenylalanine-sulfamide (Phe-sulfamide) does not alter toeprints on either AUG or CUG mRNAs. Phe-sulfamide was added to toeprint reactions as carried out for Met-sulfamide described above. (E) Relative toeprint intensity is % of untreated sample from (C) with data from three independent experiments and from (D) with data from two independent experiments (mean+/−standard error).
Figure 4
Figure 4. Edeine inhibits AUG but not CUG toeprints.
(A) Toeprints of mRNAs with the indicated initiation codons in the absence or presence of edeine (2 µM). The toeprints at +15–17 nucleotides downstream of the AUG or CUG start codons are boxed. Data are representative of five independent experiments. Sequencing lanes shown are for the CUG[YL8] mRNA. (B) Edeine enhances the toeprints on the cricket paralysis virus (CrPV) IRES mRNA in a dose-dependent manner. (C) Relative toeprint intensity (% of untreated sample) of the AUG[YL8], CUG[YL8], and CrPV IRES mRNAs in the presence of indicated doses of edeine (mean+/−standard error). The intensity of the toeprint in the absence of edeine is set at 100%. Data are from two independent experiments.
Figure 5
Figure 5. Edeine inhibits AUG- but not CUG-specific ribosomal initiation complexes.
Ribosomes bound to the AUG[YL8] or CUG[YL8] mRNAs were fractionated on 10–40% sucrose gradients in the absence or presence of edeine (2 µM). (A) The ultraviolet light absorbance is shown for each fraction for reactions with the AUG[YL8] mRNA. (B) Total RNA from fractions 21–29 of AUG[YL8] and 26–33 of CUG[YL8] samples with or without edeine was fractionated on 1% formaldehyde gels. For each fraction, the ribosomal RNA from the large and small ribosomal subunits was visualized with ethidium bromide (EtBr) and the mRNA was detected by Northern blot. (C) mRNA amounts measured by pixel intensity of peak fractions of AUG versus CUG reactions in (B) is shown in arbitrary units (AU). (D) Sucrose gradient fractionation of AUG[YL8] and CUG[YL8] initiation complexes was carried out as in (A) except the mRNA was directly labeled with [α35S]-CTP and radioactivity (CPM, counts per minute) in each fraction was determined by liquid scintillation. Results are representative of three independent experiments.
Figure 6
Figure 6. The toeprint and translational activity of CUG initiation codon is resistant to bruceantin.
(A) The translation inhibitor bruceantin inhibits AUG toeprints, but not CUG toeprints in a dose-dependent manner. Bruceantin was preincubated with translational extract prior to adding the mRNA with the indicated AUG or CUG initiation codons. Relative toeprint intensity (% of untreated sample) represent three independent experiments (mean+/−standard error). (B) The AUG as well as CUG toeprints are insensitive to emetine, an elongation inhibitor. The data are representative of three independent experiments. (C) [35S]-Methionine labeled translated products of firefly luciferase (Luc) mRNAs with AUG, CUG, and CCC initiation codons in vitro. The translation products were resolved on 4–10% SDS-PAGE gel. Position of the 61 kD luc product is indicated by an arrow. Data are representative of three independent experiments. (D) Translation of AUG-Luc, but not CUG-Luc is inhibited by bruceantin in a dose-dependent manner. Indicated concentrations of bruceantin were added to the translation mix prior to the addition of mRNA. The luciferase activity was determined using a luminometer. Luciferase activity (% of untreated sample) is from two independent experiments (mean+/−standard error).
Figure 7
Figure 7. Generation of pMHC I complex derived from the AUG but not CUG mRNA is inhibited by bruceantin.
(A) Primary cells from a transgenic mouse are sensitive to bruceantin inhibition from AUG but not CUG start codons. The transgene encodes two peptides: WI9 initiated with an AUG and LYL9 initiated with CUG located in the 3′ untranslated region directly downstream from WI9. Spleen cells were washed with mild-acid and allowed to recover for 3 hours in medium+DMSO or in the presence of 25 nM bruceantin. Peptide translation is measured with the 11p9Z and BCZ103 hybridomas for AUG and CUG initiation, respectively. (B) The Kb-L cells were transfected with mRNAs encoding AUG[YL8] or CUG[YL8], and CCC[YL8] as a negative control. After three hours to allow mRNA entry and expression, the cells were incubated with 100 nM bruceantin for another three hours. The indicated numbers of cells were then used as antigen presenting cells for the BCZ103 hybridoma specific for Kb-bound LYL8 or the MYL8 peptides. (C) Antigenic peptides in extracts of mRNA transfected cells in the absence or presence of bruceantin. After transfection, Kb-L cells were acid-washed and allowed to recover for 3 hours in medium+DMSO or in the presence of 100 nM bruceantin. Peptides were extracted from the cells by homogenizing in 10% acetic acid, dried and antigenic activity measured with the BCZ103 hybridoma and Kb-L cells as APC. The data are representative from four independent experiments.

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