SRSF is a member of Arginine Serine rich

SRSF4 is a member of Arginine-Serine-rich (SR) protein family, that is essential for constitutive splicing and regulates alternative splicing [14]. SR proteins include one or two RNA recognition motifs (RRM) at their amino terminus and RS domains at carboxyl terminus [[15], [16], [17], [18]]. Whereas RRM domain provides the binding for RNA, RS domain was shown to function as an activator by promoting the nhs chemical mg pairing between U1 snRNA and 5’ splice-site, branch point and polypyrimidine tract (PPT) [19,20]. Individual-nucleotide resolution UV cross-linking and Immnuoprecipitation (iCLIP) of SRSF4 demonstrated that SRSF4 contacts intronless and intron-containing mRNAs as well as non-coding RNAs with its sequence specificities [21]. Pre-mRNA splicing is a fundamental process required for the gene expression of higher eukaryotes [22,23]. The Two-step transesterification reactions that are necessary to accurately remove introns are conserved from yeast to mammals. In the first step, 5′ splice-site is cleaved through the nucleophilic attack of 2′ hydroxyl group of the branch point on the first nucleotide of the intron at the 5′ splice-site forming the lariat intermediate. In the second step, 3′ splice-site is cleaved by another nucleophilic attack at the last nucleotide of the intron at the 3′ splice-site [24]. Spliceosome where splicing takes place recognizes four splicing signals at pre-mRNA: 5′ splice-site at the downstream end of intron, 3′ splice-site at the upstream end of intron, the polypyrimidine tract (PPT) at immediate upstream of 3’ splice-site and branch-point at immediate upstream of PPT sequence [25,26]. Alternative splicing of pre-mRNA allows increased proteome diversity and therefore contributes to biological complexity of mammals [[27], [28], [29], [30]]. Cis-regulatory RNA sequences in exons or introns function as enhancers or silencer of exon recognition through activating or inhibiting splicing of alternative exons [31,32]. At most instances, the enhancers or silencers provide binding sites for RNA binding proteins to regulate alternative exon splicing [33,34]. Previously our research group identified hnRNP A1 as an activator for alternative splicing of exon 6 in Fas pre-mRNA [10]. In this manuscript, we identified a novel enhancer RNA sequence on exon 6 that stimulates exon 6 splicing of Fas pre-mRNA. Remarkably, we demonstrate that this enhancer sequence provides the binding site for SRSF4. Mutations of the enhancer sequence abolish the binding of SRSF4 and its stimulatory function of SRSF4. Our results present a novel mechanism on how exon 6 splicing of Fas pre-mRNA is regulated.
Materials and methods
Results
Discussion
Acknowledgement This work was supported by the NRF-2017R1A2B2005896 grants to Haihong Shen, the NRF-2016R1A2B1007135 grant to Xuexiu Zheng, Cell Logistics Research Center (grant No.2016R1A5A1007318) funded by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea ; and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C0196). We appreciate Juan valcarcel for providing Fas mutant minigene constructs [11].
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory arthritis that is characterized by inflammatory cell infiltration within the synovium [16]. While the exact mechanism of RA pathogenesis remains under investigation, synovial hyperplasia has been considered the major pathogenic finding in RA and could be associated with the persistent inflammation and joint destruction in RA [16], [29]. Numerous studies have suggested the pathogenic mechanism underlying the hyperplasia of the fibroblast-like synoviocytes (FLS), which populate the lining layers of the synovium and are key to the induction and perpetuation of inflammation in RA [4]. Several lines of evidence show that FLS exhibit sensitivity to Fas (CD95)-mediated apoptotic signaling in vitro, in contrast to the observed lack of apoptotic cells in the RA synovium in vivo [14], [23], [26]. There have been substantial studies directed at this conflicting finding in order to address the underlying mechanism responsible for the resistance to apoptosis in RA synovial cells [10], [15], [17], [22]. The cell surface Fas plays a pivotal role in the apoptosis of inflammatory cells, including FLS, and there are intricate mechanisms regulating the Fas-mediated apoptotic pathway to maintain cellular homeostasis [13], [23], [26]. The soluble form of Fas (sFas) inhibits Fas-induced apoptosis by binding to Fas ligand (FasL), thereby blocking interaction with the membrane Fas (mFas) receptor on the cell surface [6], [7]. However, conflicting data have been reported regarding the levels of sFas and its relation with disease activity in RA [2], [9], [27].