Deciphering the regulatory programs of RNA binding proteins in rheumatoid arthritis through single-cell transcriptome analysis

Abstract

Single-cell RNA sequencing (scRNA-seq) data from published datasets were obtained to investigate the expression and dysregulation of RNA-binding proteins (RBPs), which are critical for alternative mRNA splicing and translational control in rheumatoid arthritis (RA). How RBP regulation differs between RA and osteoarthritis (OA) was examined using RBP for single-cell sub-clustering. Quantitative polymerase chain reactions (PCRs) were performed to confirm differentially expressed RBPs in RA fibroblast-like synoviocytes (FLSs) and OA-FLSs, as well as in mice with collagen-induced arthritis (CIA) and control mice. Additionally, bulk RNA-seq data were collected and RBP-alternative splicing event (ASE) co-expression analyses were performed to reveal the potential regulatory role of RA-related RBPs on ASEs. Significant variations in relative proportions of cell subtypes were demonstrations between RA and OA with downregulated RBPs outnumbering upregulated RBPs in each cell type and showing high specificity for particular subsets. One hundred five upregulated and 133 downregulated RBPs were identified in fibroblasts. Y-Box binding protein 3 (YBX3) and splicing factor 3b subunit 6 (SF3B6) were confirmed to be upregulated in RA-FLS and CIA mice, while eukaryotic translation initiation factor 4A1 (EIF4A1) and U2 small nuclear RNA auxiliary factor 1 (U2AF1) were downregulated in RA-FLS. The RA group displayed stronger cell type interactions compared to the OA group with enhanced signaling pathways, such as fibronectin 1-cluster of differentiation 44 (FN1-CD44) and C-X-C motif chemokine ligand 12-C-X-C motif chemokine receptor 4 (CXCL12-CXCR4). Furthermore, three upregulated genes (spectrin repeat containing nuclear envelope protein 2 [SYNE2], S100 calcium binding protein A9 [S100A9], and interferon induced protein with tetratricopeptide repeats 3 [IFIT3]) and four downregulated genes (ribonuclease 1 [RNASE1], granulin [GRN], FN1, and sorbin and SH3 domain containing 2 [SORBS2]) were co-expressed in RA-associated RBPs and ASEs. These findings suggest that dysregulation of RBPs may contribute to the development of RA and provide potential targets for therapeutic interventions.