The long term goals of this project are to understand how the antigen processing pathway generates peptide/MHC class I complexes (pMHC I) by cryptic translation. Numerous studies, from many different laboratories, have shown that naturally processed pMHC I on the surface of tumors, virally infected cells, transfected or even normal cells arise from regions of mRNAs that were not expected to be translated. The mechanism that allows cells to sample this cryptic source of antigenic precursors versus other conventional sources is obscure. In addition, to what extent cryptic peptides arise from normal mRNAs and contribute to the diverse pool of peptides presented by MHC class I molecules is unknown. Here we will test the hypotheses that (a) these unusual, cryptic peptides arise not only from mutated or exogenously introduced genes but also from normal genes and (b) their expression may be regulated under stress. Furthermore, we will test the hypothesis that (c) cryptic translation results from a novel set of ribosomes that initiate translation not at conventional AUG codons, but at cryptic non-AUG codons. We will characterize these ribosomes using primer extension inhibition (toeprinting) and by direct analyses of their t-RNAs, proteins and RNA constituents. We anticipate that an improved understanding of how cryptic pMHC I are generated will not only provide a means to tap into a novel sources of antigenic peptides and new approaches to vaccine design, but will also yield insights into protein translation and immune surveillance mechanisms.
