Schwab et al. generated mice that ubiquitously express a bi-cistronic transgene, encoding a peptide derived from the Uty gene and a peptide derived from the H60 histocompatibility gene (LYL8). The first peptide was initiated by a conventional AUG codon, whereas the second peptide, which was downstream of a stop codon in the 3′ UTR, had CUG as its initiation codon. Cells derived from transgenic mice could present both peptides to peptide-specific T-cell hybridomas, and were lysed by LYL8-specific cytotoxic T lymphocytes (CTLs). Further evidence for the presence of LYL8–MHC class I complexes was provided by the observation that transgenic splenocytes elicited LYL8-specific CTL responses after immunization of non-transgenic animals, but that transgenic mice expressing the cryptic LYL8 peptide were tolerant to the peptide.
Given that LYL8 is encoded downstream of a stop codon in the 3′ UTR, and has a non-AUG start codon, the authors investigated the mechanisms that regulate translation of this cryptic peptide. Analysis of peptide extracts from transgenic splenocytes by chromatography confirmed that the LYL8 initiation codon (CUG) was decoded as leucine, and was not misread as methionine. Interestingly, translation initiation was specific to the leucine-encoding codon CUG, indicating that ribosome read-through of the upstream stop codon was not responsible for LYL8 translation. This was confirmed by the observation that increasing the number of stop codons between the two peptides and shifting the peptides out of frame with the initial AUG codon did not affect MHC class I presentation of LYL8.
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