Characterization of the Regulation of CD46 RNA Alternative Splicing

Abstract

Here we present a detailed analysis of the alternative splicing regulation of human CD46, which generates different isoforms with distinct functions. CD46 is a ubiquitous membrane protein that protects host cells from complement and plays other roles in immunity, autophagy, and cell adhesion. CD46 deficiency causes an autoimmune disorder, and this protein is also involved in pathogen infection and cancer. Before this study, the mechanisms of CD46 alternative splicing remained unexplored even though dysregulation of this process has been associated with autoimmune diseases. We proved that the 5 splice sites of CD46 cassette exons 7 and 8 encoding extracellular domains are defined by noncanonical mechanisms of base pairing to U1 small nuclear RNA. Next we characterized the regulation of CD46 cassette exon 13, whose inclusion or skipping generates different cytoplasmic tails with distinct functions. Using splicing minigenes, we identified multiple exonic and intronic splicing enhancers and silencers that regulate exon 13 inclusion via trans-acting splicing factors like PTBP1 and TIAL1. Interestingly, a common splicing activator such as SRSF1 appears to repress CD46 exon 13 inclusion. We also report that expression of CD46 mRNA isoforms is further regulated by non-sense-mediated mRNA decay and transcription speed. Finally, we successfully manipulated CD46 exon 13 inclusion using antisense oligonucleotides, opening up opportunities for functional studies of the isoforms as well as for therapeutics for autoimmune diseases. This study provides insight into CD46 alternative splicing regulation with implications for its function in the immune system and for genetic disease.CD46 is a ubiquitously expressed type I membrane-bound protein with a main function of protecting human host cells from complement (1). CD46 exerts such function by acting as a cofactor for Factor I-mediated cleavage of C3b and C4b (1). In addition, CD46 acts as a co-stimulator of T and other immune cells (2-6) and plays important roles in epithelial and sperm cells (7-11). Human CD46 deficiency results in a genetic disorder called atypical hemolytic uremic syndrome (12), its overexpression is used by cancer cells to evade the immune system (13,14), and its expression is often altered in autoimmune disorders like multiple sclerosis, rheumatoid arthritis, and asthma (15)(16)(17)(18)(19). Finally, CD46 is used as an entry receptor for several bacteria and viruses (20 -23). All of these studies underline the multiple connections between CD46 and human disease and the relevance of studying the regulation of CD46 expression.The joining of exons in different combinations, by means of alternative splicing, gives rise to multiple mRNA and protein isoforms (24). The human CD46 gene consists of 14 exons, and four of them are alternatively spliced to generate several CD46 isoforms (25). These four exons fall in the category of cassette exons, which can be either included or skipped from the mature messenger RNA (25-27). Cassette exons 7, 8, and 9 encode the extra...

Non-random pairing of CD46 isoforms with skewing towards BC2 and C2 in activated and memory/effector T cells

“…In addition to alternative splicing, the CD46 transcripts might be regulated by transcription speed and non-sense-mediated mRNA decay 16 . For our studies, we assume that the mRNA level of each of the isoforms roughly correlates with the expression at the protein level.…”

“…Recent data demonstrate that the 5′ splice sites of exons 7 and 8, which determine the BC or C isoforms, are defined by base paring to U1 small nuclear RNA, whereas regulation of exon 13 inclusion, which gives rise to CYT-1 isoforms, is under control of multiple exonic and intronic splicing enhancers and silencers. Of these, SRSF1 and PTBP1 act as repressors of exon 13 inclusion, which would favour CYT-2 isoforms 16 .…”

Brain microvascular endothelial cells exhibit lower activation of the alternative complement pathway than glomerular microvascular endothelial cells

“…A possible explanation for this enigma is that the mAb used to detect surface CD46 does not bind and detect the complete array of BMVEC CD46 isoforms. CD46 has multiple isoforms as a result of alternate gene splicing, and identical CD46 isoforms are not present in cerebral and renal tissue (56). TNF resulted in statistically significant increases of detectable CD46 on the surface of both cell types (1.6-fold on GMVECs and 2.1-fold on BMVECs) but did not result in significant changes in CD55 on either cell type ( Fig.…”

Human PRPF40B regulates hundreds of alternative splicing targets and represses a hypoxia expression signature

“…We seeded nonedited K562, K562-PRPF40B-KO and rescued cells at a concentration of 0.5 × 10 6 cells/mL and incubated with either dimethyl sulfoxide (DMSO), 50 µM DRB or 1 µM CPT for 5 h to inhibit transcription (Tang et al 2016). After RNA extraction and cDNA synthesis, we used primers designed in exons flanking the alternative exon 8 of hnRNPDL to detect the efficiency of the drugs by RT-PCR (FW-hnRNPDL.Ex7 5 ′ -GCCAACAGAGCCAC TTATGG-3 ′ , RW-hnRNPDL.Ex9 5 ′ -ACAGTTGGACACAATGGT GTC-3 ′ ).…”

“…Cotranscriptional AS was evidenced by promoter-dependent AS events, and exons in genomic regions are enriched in nucleosomes and certain chromatin modifications. The speed of Pol II elongation affects AS, as found by Pol II mutants with slow polymerization, and by small molecules that affect its kinetics such as 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) and camptothecin (CPT) (Dujardin et al 2014;Tang et al 2016). The main kinetic model of AS proposes that slow and fast Pol II elongation respectively determine high or low inclusion of cassette exons, by positive AS regulators only assembling onto the substrates with weak splice sites or other signals under slow Pol II elongation (de la Mata et al 2003).…”