Direct MHC Class I Antigen Presentation Regulation by Ribosomal Biogenesis Inhibitor CX-5461

Abstract

Abstract Direct MHC class I antigen presentation relies on on-going protein synthesis by the antigen presenting cell. This is especially true for the presentation of peptides derived from Defective Ribosomal Products (DRiPs) which are inherently defective proteins rapidly degraded following their synthesis. Recently an inhibitor of RNA polymerase I, known as CX-5461, has been described which can inhibit ribosome biogenesis. Here we queried if interruption of ribosome biogenesis could impact direct antigen presentation using a reporter protein whose stability can be controlled post-translationally. We first determined a dose and time of treatment with CX-5461 which was not toxic to cells, but greatly decreased the levels of RNA within EL4 cells. Once an acceptable dose was determined, we examined presentation of a model peptide derived from the Shield-controlled recombinant antigenic protein (SCRAP) which is a fusion protein consisting of a destabilization domain, the SIINFEKL peptide, and a fluorescent reporter protein. SCRAP levels can be controlled through the addition of the small molecule Shield-1, which stabilizes SCRAP and prevents its degradation. After treatment with CX-5461, we found the levels of SCRAP protein partially decreased within the cell, indicating a loss in protein synthesis. Concurrently, presentation of the reporter peptide was diminished from both the DRiP and non-DRiP form of SCRAP. These data indicate that interruption of ribosome biogenesis has a minor impact on direct antigen presentation and protein synthesis.