Alternative splicing and intron retention: Their profiles and roles in cutaneous fibrosis of systemic sclerosis.

Alternative splicing (AS) and intron retention (IR) implicated in multiple pathophysiological processes, have rarely been reported in systemic sclerosis (SSc). We integrated bulk RNA-seq and 4D label-free mass spectrometry to perform a multi-omics analysis of AS and IR in SSc skin tissue and fibroblasts. RMATS and iREAD were used to identify AS and IR, which were validated by real-time PCR. Spearman correlation and the LASSO method were employed to assess correlations among clinical features, introns, splicing factors (regulators of AS) and proteins. AS profiles showed distinct alterations in SSc skin tissue, with the most pronounced changes occurring in IR. AS and IR were associated with total modified Rodnan skin score (mRSS) and local skin score. Upon TGF-β stimulation, fibroblasts exhibited significant alterations in IR profiles, affecting genes related to fibroblast proliferation and collagen fibril organization. A comprehensive integrated analysis of introns, exons, and proteome profiles revealed that IR exerted a negative impact on protein expression, with certain changes being under intronic control. RT-PCR confirmed the presence of intron and exon-derived sequences of CTTN, OGA, MED16 and PHYKPL. Additionally, notable changes were observed in the regulatory network of splicing factors in SSc skin tissues. These factors are also involved in fibrosis pathways and correlated with clinical features. Totally, abnormal AS, IR profiles and splicing factors were identified in SSc, altered IRs and splicing factors participated in fibrosis-related pathways. IR exerted a negative impact on protein expression in TGF-β-stimulated fibroblasts. Clarification of the IR mechanisms will provide new insights into the pathophysiology of SSc. A genome-wide analysis of alternative splicing (AS) and intron retention (IR) was conducted across systemic sclerosis skin (SSc = 48, HC = 33) and TGF-β stimulated fibroblasts based on bulk RNA-seq and 4D label-free mass spectrometry. AS and IR profiles were identified and functional analysis was conducted through Gene Ontology enrichment of gene with differential expressed AS and IR. The correlations between AS/IR and clinical features were assessed by spearman correlation and t -test. LASSO method was employed to assess the correlation between splicing factors and IR. IR profiles were integrated with proteome and transcriptome analysis. Specific IR and protein were validated by RT-PCR and Western blot. [Display omitted] • Alterations of AS/IR were identified across SSc skin and TGF-β-stimulated fibroblasts, related to skin score and ECM score. • Splicing factors were dysregulated and related to IR regulation in the process of skin fibrosis. • IR had a negative impact on protein and were implicated in the processes associated with fibrosis. [ABSTRACT FROM AUTHOR]