Altered protein structures and neoepitopes in lupus neutrophils from dysregulated splicing of messenger RNA - Project Summary/Abstract The goal of our research is to better define the molecular mechanisms cause and perpetuate systemic lupus erythematosus (SLE). Based on our discovery that transcripts from a large number of genes are spliced differently in neutrophils from SLE patients compared to neutrophils from healthy donors or COVID-19 patients, we propose a novel hypothesis of SLE pathogenesis, namely that dysregulated mRNA splicing in SLE granulocytes can affect their function and generate (neo)autoantigens not seen by the immune system before. We propose to expand and substantiate the analysis of altered mRNA splicing and begin to search for its cause(s). We will define how specific it is to SLE, understand the relation of altered splicing to sex, age, disease activity, type I interferons, and patient heterogeneity. Our work will also embark on the quest to elucidate why mRNA slicing is dysregulated in SLE. Next, we propose to explore the consequences of altered mRNA splicing in SLE. Although we have high confidence in the alignment of RNAseq reads to support all the splicing events we detect, and the transcripts are present at good read counts, it needs to be demonstrated that at least some are translated into altered proteins. We will continue to use targeted LC-MS/MS to find evidence for those with novel amino acid sequences. The proteolytic processing into antigenic peptides presented on MHC seen by T cells will then be explored. We know the MHC haplotypes of all our donors from the RNAseq data, so can match putative neoepitopes with the right MHC. Our work will clarify whether the production of novel, and perhaps individual, neo-autoantigens are a feature of SLE. If so, the production of such antigenic peptides would drive an immune response against the specific cell type that produces them (rather than all cells expressing the gene), adding a new element of tissue or cell linage-targeting to the autoimmune response. This model also introduces a new avenue for the development of therapeutics: modulating mRNA splicing to eliminate the production of neo-autoantigens. This will only be possible once the mechanism(s) underpinning abnormal splicing have been elucidated.
