In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo
- Institute of Immunology (Charité - Universitätsmedizin Berlin) and German Cancer Research Center (DKFZ Heidelberg), Germany
- Institute of Biochemistry (Charité - Universitätsmedizin Berlin), Germany
- Max-Delbrück Center for Molecular Medicine, Germany
- Institut für Physiologie (Charité - Universitätsmedizin Berlin), Germany
- Max Delbrück Center for Molecular Medicine in Helmholtz Association, Germany
- Charité - Universitätsmedizin Berlin, Germany
Abstract
Proteasome catalyzed peptide splicing (PCPS) of cancer-driving antigens could generate attractive neoepitopes to be targeted by TCR-based adoptive T cell therapy. Based on a spliced peptide prediction algorithm TCRs were generated against putative KRASG12V and RAC2P29L derived neo-splicetopes with high HLA-A*02:01 binding affinity. TCRs generated in mice with a diverse human TCR repertoire specifically recognized the respective target peptides with high efficacy. However, we failed to detect any neo-splicetope specific T cell response when testing the in vivo neo-splicetope generation and obtained no experimental evidence that the putative KRASG12V- and RAC2P29L-derived neo-splicetopes were naturally processed and presented. Furthermore, only the putative RAC2P29L-derived neo-splicetopes was generated by in vitro PCPS. The experiments pose severe questions on the notion that available algorithms or the in vitro PCPS reaction reliably simulate in vivo splicing and argue against the general applicability of an algorithm-driven 'reverse immunology' pipeline for the identification of cancer-specific neo-splicetopes.
Data availability
Additional source data comprising databases for ProteomDiscoverer, Kras/RAC2 kinetics, cleavage maps and PD2.1 result files have been submitted to Dryad under DOI:10.5061/dryad.jq2bvq88b
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Willimsky_et_al_11-08-2020-RA-eLife-62019_Supplementary data filesDryad Digital Repository, doi:10.5061/dryad.jq2bvq88b.
Article and author information
Author details
Funding
Deutsche Forschungsgemeinschaft (SFB-TR36)
- Gerald Willimsky
- Thomas Blankenstein
Deutsche Krebshilfe (111546)
- Gerald Willimsky
Berlin Institute of Health (CRG-1)
- Thomas Blankenstein
- Peter M Kloetzel
DKTK joint funding (NEO-ATT)
- Gerald Willimsky
Berliner Krebsgesellschaft
- Peter M Kloetzel
Helmholtz-Gemeinschaft, Zukunftsthema 'Immunology and Inflammation'
- Gerald Willimsky
- Thomas Blankenstein
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experiments were performed according to institutional and national guidelines and regulations. The experiments were approved by the governmental authority (Landesamt für Gesundheit und Soziales, Berlin, H0086/16).
Copyright
© 2021, Willimsky et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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In vitro proteasome processing of neo-splicetopes does not predict their presentation in vivo
eLife 10:e62019.
https://doi.org/10.7554/eLife.62019
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Pre-mRNA splicing is catalyzed in two steps: 5ʹ splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (first and second step factors). We recently identified Fyv6 (FAM192A in humans) as a second step factor in Saccharomyces cerevisiae; however, we did not determine how widespread Fyv6’s impact is on the transcriptome. To answer this question, we have used RNA sequencing (RNA-seq) to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3ʹ SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only second step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the first step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3ʹ SS.
