The Mammalian Homolog of Suppressor-of-white-apricot Regulates Alternative mRNA Splicing of CD45 Exon 4 and Fibronectin IIICS

Abstract

We have previously described human (HsSWAP) and mouse (MmSWAP) homologs to the Drosophila alternative splicing regulator suppressor-of-white-apricot (su(w a ) or DmSWAP). DmSWAP was formally defined as an alternative splicing regulator by studies showing that it autoregulates splicing of its own pre-mRNA. We report here that mammalian SWAP regulates its own splicing, and also the splicing of fibronectin and CD45. Using an in vivo system of cell transfection, mammalian SWAP regulated 5 splice site selection in splicing of its own second intron. SWAP enhanced splicing to the distal 5 splice site, whereas the SR protein ASF/SF2 enhanced splicing to the proximal site. SWAP also regulated alternative splicing of the fibronectin IIICS region by promoting exclusion of the entire IIICS region. In contrast, ASF/SF2 stimulated inclusion of the entire II-ICS region. Finally, SWAP regulated splicing of CD45 exon 4, promoting exclusion of this exon, an effect also seen with ASF/SF2. Experiments using SWAP deletion mutants showed that splicing regulation of the fibronectin IIICS region and CD45 exon 4 requires a region including a carboxyl-terminal arginine/serine (R/S)-rich motif. Since R/S motifs of various splicing proteins have been shown to interact with each other, these results suggest that the R/S motif in SWAP may regulate splicing, at least in part, through interactions with other R/S containing splicing factors.Recent studies have identified several genes important in regulating alternative mRNA splicing. Members of the SR protein family, characterized by an arginine/serine (R/S)-rich domain in the carboxyl terminus and an RNA-recognition motif in the amino terminus were originally defined by their activity complementing HeLa cell S100 extracts for constitutive splicing, but SR proteins also have distinctive activities in regulating alternative splicing (1-3). ASF/SF2 (4 -6) and SC35 (7), the two SR proteins most completely characterized as splicing regulators, can regulate alternative 5Ј and 3Ј splice site selection (7) and exon inclusion/exclusion (8). SR proteins likely differentially activate splice sites due to different affinity interactions with either 5Ј splice sites or exon enhancers (9) and may similarly affect 3Ј splice site selection (10). In addition to interacting with RNA, SR proteins interact with other constitutive splicing factors containing R/S-rich domains, including U2AF and the U1-70K protein through the R/S-rich region of each protein (11,12). Several heterogeneous nuclear ribonuclear proteins (hnRNPs) 1 can also influence alternative mRNA splicing. hnRNP A1 directly antagonizes the effect of ASF/SF2 on 5Ј splice site selection both in vitro and in vivo: ASF/SF2 (as well as other SR proteins) increases splicing to proximal 5Ј splice sites, while hnRNP A1 increases splicing to distal 5Ј splice sites (8,(13)(14)(15). In some cases it also antagonizes the effect of ASF/SF2 on exon inclusion/exclusion (8). PTB also has activity in regulating alternative mRNA splicing (16). It binds to certain pol...

Regulation of Alternative Splicing of CD45 by Antagonistic Effects of SR Protein Splicing Factors

“…It has recently been shown using a CD45 minigene containing only alternative exon 4 that overexpression of hsSWAP, SF2/ASF, SC35, SRp40, and SRp75 all promoted exclusion of exon 4, which is in agreement with our findings (57,58). Lemaire et al (58) also demonstrated that regulation of CD45 exon 4 splicing is dependent on exon 4 itself and not affected by the presence of the 3Ј constitutive exon, exon 7.…”

Regulation of Alternative Splicing of Human Tau Exon 10 by Phosphorylation of Splicing Factors

“…2C). It should be noted that overexpression of CLKs does not change the splicing patterns of other minigenes (data not shown), e.g., GABA A ␥2 alternative 24-nt exon (Ashiya and Grabowski, 1997), clathrin light-chain B exon EN (Stamm et al, 1992, insulin receptor exon 11 (Kosaki and Webster, 1993), and SWAP exon 2A (Sarkissian et al, 1996). Only with the SRp20 exon 4 was a similar effect on exon skipping found (Stoss et al, 1999).…”

A Model System for Activation-Induced Alternative Splicing of CD45 Pre-mRNA in T Cells Implicates Protein Kinase C and Ras

“…However, previous studies have demonstrated the increased expression of several members of the SR family of general splicing factors upon T-cell activation (28,46). SR proteins have been implicated in the regulation of many alternative splicing events (16,32) and have been shown to influence CD45 alternative splicing in a heterologous system (28,43). Therefore, it is possible that increased synthesis of SR proteins is a required step in the pathway between T-cell activation and CD45 alternative splicing.…”