Accurate recognition of splice sites is essential for pre-messenger RNA splicing. Mammalian 5′ splice sites are mainly recognized by canonical base-pairing to the 5′ end of U1 small nuclear RNA, yet we described multiple noncanonical base-pairing registers by shifting base-pair positions or allowing one-nucleotide bulges. By systematic mutational and suppressor U1 analyses, we prove three registers involving asymmetric loops and show that two-nucleotide bulges but not longer can form in this context. Importantly, we established that a noncanonical uridine-pseudouridine interaction in the 5′ splice site/U1 helix contributes to the recognition of certain 5′ splice sites. Thermal melting experiments support the formation of noncanonical registers and uridine-pseudouridine interactions. Overall, we experimentally validated or discarded the majority of predicted noncanonical registers, to derive a list of 5′ splice sites using such alternative mechanisms that is much different from the original. This study allows not only the mechanistic understanding of the recognition of a wide diversity of mammalian 5′ splice sites, but also the future development of better splice-site scoring methods that reliably predict the effects of disease-causing mutations at these sequences.

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...