Systemic Lupus Erythematosus (SLE) patients’ Peripheral Blood Mononuclear Cells (PBMCs) display an altered transcript isoform repertoire

Abstract

Alternative splicing (AS) is a key mechanism of biological diversity in eukaryotes allowing a single gene to generate multiple mRNA transcript isoforms, yielding unique proteins that may have distinct or opposing functions. Alterations in AS have been associated with numerous diseases. SLE patients display autoantibodies to components of the spliceosome, chiefly anti-Sm/RNP, anti-Ro/La, and anti-dsDNA. We performed Long-Read sequencing using Pacific Biosciences equipment (LR-Seq), as well as short read RNA-Seq, from adolescent SLE patients’ and healthy controls’ PBMCs, to discover and quantitate splice junctions. A computational pipeline was built to differentially quantify splice junctions in the LR-Seq sequenced isoforms, both known and novel using rMATS (Multivariate Analysis of Transcript Splicing). Compared to Gencode, LR-Seq revealed ~9000 novel transcript isoforms. 55% of all transcripts were expressed in 2 or more samples. A majority of these transcripts belonged to genes associated with cell cycle, metabolism, and inflammation. Additionally, we interrogated our SLE LR-Seq generated transcriptome with publicly available RNA-Seq data from adult SLE and healthy donors. We observed 250 differential expression of exon skipping (ES) splice junctions in transcript isoforms of genes, a majority of which did not exhibit any significant change in gene expression. Of the 250 ES transcript isoforms, 160 exon inclusion events were observed in SLE PBMCs. Fifty percent of the exon inclusion events were found to be in novel transcript isoforms. Thus LR-Seq, besides revealing novel transcripts, allows us to study changes in transcript isoform expression.