SRSF6 Regulates the Alternative Splicing of the Apoptotic Fas Gene by Targeting a Novel RNA Sequence

SIMPLE SUMMARY: Alternative splicing (AS) produces multiple mRNA isoforms from a gene to make a large number of proteins. Fas (Apo-1/CD95) pre-mRNA, a member of TNF receptor family that mediates apoptosis, can generate pro-apoptotic and anti-apoptotic proteins through AS. Here, we identified SRSF6 as an essential regulator protein in Fas AS. We further located a new functional target sequence of SRSF6 in Fas splicing. In addition, our large-scale RNA-seq analysis using GTEX and TCGA indicated that while SRSF6 expression was correlated with Fas expression in normal tissues, the correlation was disrupted in tumors. Our results suggest a novel regulatory mechanisms of Fas AS. ABSTRACT: Alternative splicing (AS) is a procedure during gene expression that allows the production of multiple mRNAs from a single gene, leading to a larger number of proteins with various functions. The alternative splicing (AS) of Fas (Apo-1/CD95) pre-mRNA can generate membrane-bound or soluble isoforms with pro-apoptotic and anti-apoptotic functions. SRSF6, a member of the Serine/Arginine-rich protein family, plays essential roles in both constitutive and alternative splicing. Here, we identified SRSF6 as an important regulatory protein in Fas AS. The cassette exon inclusion of Fas was decreased by SRSF6-targeting shRNA treatment, but increased by SRSF6 overexpression. The deletion and substitution mutagenesis of the Fas minigene demonstrated that the UGCCAA sequence in the cassette exon of the Fas gene causes the functional disruption of SRSF6, indicating that these sequences are essential for SRSF6 function in Fas splicing. In addition, biotin-labeled RNA-pulldown and immunoblotting analysis showed that SRSF6 interacted with these RNA sequences. Mutagenesis in the splice-site strength alteration demonstrated that the 5′ splice-site, but not the 3′ splice-site, was required for the SRSF6 regulation of Fas pre-mRNA. In addition, a large-scale RNA-seq analysis using GTEX and TCGA indicated that while SRSF6 expression was correlated with Fas expression in normal tissues, the correlation was disrupted in tumors. Furthermore, high SRSF6 expression was linked to the high expression of pro-apoptotic and immune activation genes. Therefore, we identified a novel RNA target with 5′ splice-site dependence of SRSF6 in Fas pre-mRNA splicing, and a correlation between SRSF6 and Fas expression.