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posted 27. November 2004 23:45
PLoS Biology November 11 2004
Unanticipated Antigens: Translation Initiation at CUG with Leucine
Susan R. Schwab, Jessica A. Shugart, Tiffany Horng, Subramaniam Malarkannan, Nilabh Shastri
Abstract
Major histocompatibility class I molecules display tens of thousands of peptides on the cell surface for immune surveillance by T cells. The peptide repertoire represents virtually all cellular translation products, and can thus reveal a foreign presence inside the cell. These peptides are derived from not only conventional but also cryptic translational reading frames, including some without conventional AUG codons. To define the mechanism that generates these cryptic peptides, we used T cells as probes to analyze the peptides generated in transfected cells. We found that when CUG acts as an alternate initiation codon, it can be decoded as leucine rather than the expected methionine residue. The leucine start does not depend on an internal ribosome entry site–like mRNA structure, and its efficiency is enhanced by the Kozak nucleotide context. Furthermore, ribosomes scan 5′ to 3′ specifically for the CUG initiation codon in a eukaryotic translation initiation factor 2–independent manner. Because eukaryotic translation initiation factor 2 is frequently targeted to inhibit protein synthesis, this novel translation mechanism allows stressed cells to display antigenic peptides. This initiation mechanism could also be used at non-AUG initiation codons often found in viral transcripts as well as in a growing list of cellular genes.
Received May 24, 2004; Accepted August 24, 2004; Published October 26, 2004
DOI: 10.1371/journal.pbio.0020366
Copyright: © 2004 Schwab et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abbreviations: APC, antigen-presenting cell; CPV-IRES, cricket paralysis virus-like internal ribosome entry site; CTL, cytotoxic T cell; eIF2, eukaryotic translation initiation factor 2; eIF2α, eIF2 α subunit; GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; HPLC, high performance liquid chromatography; IRES, internal ribosome entry site; MHC, major histocompatibility complex; NaAs, sodium arsenite; RNAiMet, methionyl initiator RNA; RP-HPLC, reverse-phase HPLC; UTR, untranslated region
Academic Editor: Marc Jenkins, University of Minnesota
*To whom correspondence should be addressed. E-mail: nshastri@socrates.berkeley.edu
Citation: Schwab SR, Shugart JA, Horng T, Malarkannan S, Shastri N (2004) Unanticipated Antigens: Translation Initiation at CUG with Leucine. PLoS Biol 2(11): e366.
Introduction Paragraphs 2 and 3
The antigen-presenting cells (APCs), which include almost all nucleated cells, are also very efficient in generating peptides for display by MHC class I (Pamer and Cresswell 1998; Princiotta et al. 2003). In fact, they are so efficient that the complex mixture of peptides on the cell surface includes some peptides that should in theory never have been translated in the first place (Shastri et al. 2002). These peptides, referred to as “cryptic,” are derived from the 5′ and 3′ “untranslated” regions of the RNA or from alternate translational reading frames. Cryptic peptides have been identified as targets for CTLs specific for tumors as well as virus-infected cells (Mayrand and Green 1998; Cardinaud et al. 2004). Several studies have shown that these peptides can arise in tumor cells and cultured cell lines despite the absence of conventional AUG codons (Malarkannan et al. 1995a; Dolstra et al. 1999; Malarkannan et al. 1999; Ronsin et al. 1999). Recently, using a transgenic approach we demonstrated that such peptides can also be expressed in a variety of normal cells and can elicit CTL responses (Schwab et al. 2003). Remarkably, a distinct translation mechanism appeared to be responsible for their generation, because it was capable of decoding the CUG initiation codon as leucine rather than the expected methionine residue.
How APCs generate peptides using non-AUG codons remains obscure. It is believed that cells express only one class of initiator tRNA, RNAiMet, which is specific for AUG and is always charged with the methionine residue (Peabody 1989; Rajbhandary and Chow 1995). 40S ribosomes are preloaded with RNAiMet and other initiation factors even before they approach the mRNA to be translated. Initiation at non-AUG codons is therefore thought to be caused by “wobble” in the pairing of the non-AUG codon with the anticodon of the RNAiMet (Peabody 1989). This mispairing results in incorporation of the methionine residue at the non-AUG initiation codon. How cells initiate translation with a nonmethionine residue is thus not explained by current translational theory. It is nevertheless important to understand this mechanism not only because antigenic peptides can arise from non-AUG initiated translation, but also because expression of a growing number of genes appears to be mediated by translation initiated at non-AUG codons.
Results/Discussion - Paragraphs 24 and 25
The CPV-IRESs are to date the only known sequences that allow eIF2-independent initiation in eukaryotic cells. Viruses employ a host of creative strategies to prevent phosphorylation of eIF2α (Kaufman 2000). Initiation at non-AUG codons, which is relatively common in viral transcripts, may provide a way to continue translation despite the lack of eIF2. Similarly, despite general translational inhibition during viral infection, cells need to continue generating antigenic peptides to flag down T cells. In addition to viral proteins and antigenic peptides, a number of regulatory cellular proteins have non-AUG initiation codons. For example, c-Myc has two distinct isoforms, the longer of which is initiated with CUG and may inhibit proliferation, as it is absent in a number of tumor-derived cell lines. Intriguingly, synthesis of the CUG-initiated form increases when cells reach high density, specifically when methionine is limiting (Hann et al. 1988; Hann et al. 1992). It should be interesting to test whether this and other CUG-initiated proteins have a leucine start. In addition, whether other non-AUG initiation codons such as GUG or ACG are decoded in a manner similar to the CUG codon described here remains to be determined.
In summary, we found that when CUG acts as an alternate initiation codon, it can be decoded as leucine as well as methionine. The leucine start does not depend on mRNA structure or sequence, but its efficiency can be enhanced by the Kozak context. A set of ribosomes is scanning 5′ to 3′ specifically for the CUG initiation codon. While the methionine start is inhibited when cells are treated with NaAs, the leucine start is enhanced, suggesting that leucine initiation is independent of eIF2. This novel translation initiation mechanism provides cells not only antigenic peptides but also a potential tool for translational control.
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Some key terms in this summary are defined in the ISCID Encyclopedia of Science and Philosophy:
Methionine
Leucine
Peptide
Major_Histocompatibility_Complex
Codon
[ 03. December 2004, 20:23: Message edited by: ISCID News Editor ]
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