Transcriptome and iRIP-seq reveal RBP-mediated alternative splicing and LGALS3-RMRP interaction in adult degenerative scoliosis

Adult degenerative scoliosis (ADS) significantly impairs the quality of life of patients. Alternative splicing (AS) is a post-transcriptional regulatory mechanism that can produce functionally diverse proteins. The process of AS is regulated by RNA-binding proteins (RBPs). However, our understanding of the role of AS in ADS is limited. This study constructed a gene regulatory network of AS in ADS and explores the potential roles of AS-related genes and RBPs in the disease. We evaluated the public database (GSE209825) in NCBI GEO, which included three ADS patients and three nondegenerative lumbar trauma (NDLT) patients. We identified differentially expressed RNA‑binding proteins (RBPs) between ADS and NDLT groups, and a subset of these RBPs was further validated by quantitative real‑time PCR (qRT‑PCR) in an independent cohort of clinical samples. To assess RBP regulation of AS, we employed the splicing site usage variation analysis (SUVA) software to analyze the transcriptome data and identify AS events in ADS. We then established a co-expression network of RBPs and AS events, predicting the potential functions of RBPs in regulating AS in ADS. Additionally, improved RNA immunoprecipitation (iRIP)-seq was employed to investigate potential binding interactions of LGALS3 in ADS. We analyzed the RNA-seq data in GSE209825 and identified 627 and 178 differentially expressed genes (DEGs) by DESeq2 and edgeR, respectively, enriched in defense response to gram-negative bacterium and adaptive immune response. Subsequently, we collected 2141 RBPs from existing reports and intersected them with DEGs, revealing DE-RBPs enriched mainly in RNA phosphodiester bond hydrolysis and adherens junction pathway. Among them, RBM38, MKRN1, EPB41, TNS1, and PRDX1 were validated by qRT‑PCR. Furthermore, we utilized SUVA and rMATS to identify regulated AS (RAS) events between ADS and NDLT, with splicing patterns primarily including Alternative 5′ Site, Exon Skipping, and Alternative 3′ Site, predominantly enriched in DNA repair and regulation of transcription by RNA polymerase II. Through co-expression network analysis, significant associations between differentially expressed RNA-binding proteins (DE-RBPs) and alternative splicing events (RASs) were identified. A significant association was identified between the RNA-binding protein LGALS3 and ADS. IRIP-seq results demonstrated that LGALS3 interacts with plentiful of mRNAs and genomic location toward the intron and antisense region. As well, motif analysis indicated that LGALS3 bound to CG-rich motifs. Integrated iRIP-seq and RNA-seq analyses identified 7 DEGs that were selectively bound by LGALS3. Notably, RMRP, which was positively associated with ADS. AS events are closely linked to ADS, with RBPs playing crucial regulatory roles. Further exploration of the mechanisms of action of these target genes can offer new directions for the treatment of ADS.