Genetic analysis of the SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing

A large number of protein factors play important roles in the splicing of mRNA precursors (for review, see Moore et al. 1993; Kramer 1996). Among them, a family of closely related splicing factors, collectively named SR proteins (Zahler et al. 1992), have been investigated intensively over the past several years (for review, see Fu 1995; Manley and Tacke 1996; Valcarcel and Green 1996). At least nine members of the SR family have been identified and characterized, all of which contain one or two RNP-type RNA-binding domains (RBDs) at their amino terminus and a carboxy-terminal arginine/serine-rich region (RS domain). Comparison of SR proteins from different species reveals that they are highly conserved evolutionarily throughout metazoans. SR proteins are essential splicing factors in vitro, as they are required for an early step(s) in the process of spliceosome assembly (e.g., Krainer et al. 1990a; Fu and Maniatis 1990; Fu 1993). By interacting with the U1 70K protein, a component of the U1 small nuclear ribonucleoprotein particle (snRNP), SR proteins are thought to help recruit the U1 snRNP to the 5′ splice site of the pre-mRNA to form the commitment complex (Kohtz et al. 1994; Jamison et al. 1995). In addition, SR proteins may help bring the 5′ and 3′ splice sites together by interacting simultaneously with U1 70K and U2AF35 (the small subunit of splicing factor U2AF) at the 5′ and 3′ splice sites, respectively (Wu and Maniatis 1993). RS domains have been shown to be necessary but not sufficient for these protein–protein interactions (Kohtz et al. 1994; Xiao and Manley 1997). SR proteins also participate in splicing control. For example, they can modulate alternative splicing, when added to nuclear extracts (Ge and Manley 1990; Krainer et al. 1990b; Fu et al. 1992; Zahler et al. 1993) or transiently overexpressed in cultured cells (Caceres et al. 1994; Screaton et al. 1995; Wang and Manley 1995; Zhang and Wu 1996). In the case of alternative 5′ splice sites, they likely function by recruiting U1 snRNP to the alternative splice sites (Eperon et al. 1993; Zahler and Roth 1995). When bound to RNA elements known as exonic splicing enhancers, SR proteins can stimulate splicing of an upstream intron (e.g., Sun et al. 1993; Tian and Maniatis 1993; Staknis and Reed 1994; Ramachatesingh et al. 1995). Evidence has been presented that this event involves stabilization of U2AF binding to the polypyrimidine tract of the intron (Wang et al. 1995; Zuo and Maniatis 1996). The functions of SR proteins in constitutive splicing appear to be largely redundant, at least in vitro, as essentially any individual SR protein can restore splicing activity to cytoplasmic S100 extracts, which lack all SR proteins (Krainer et al. 1990a, 1991; Ge et al. 1991; Fu et al. 1992; Zahler et al. 1992). However, distinct functions of SR proteins have been described in several assays. First, certain SR proteins can commit specific pre-mRNAs to the splicing pathway, whereas others cannot (Fu 1993). Second, individual SR proteins influence selection of alternative splice sites in different ways (e.g., Kim et al. 1992; Zahler et al. 1993). Third, SR proteins recognize distinct RNA sequences. For example, SR proteins display specificity in the recognition of various splicing enhancer elements (Sun et al. 1993; Tian and Maniatis 1993; Staknis and Reed 1994; Lynch and Maniatis 1995; Ramachatesingh et al. 1995; Tacke and Manley 1995). Additionally, the RNA sequences selected by several SR proteins through in vitro enrichment experiments are distinct (Heinrichs and Baker 1995; Tacke and Manley 1995; Shi et al. 1997; Tacke et al. 1997). Finally, genetic analyses of SR proteins have demonstrated that at least two of them perform nonredundant functions in vivo that are essential for Drosophila development (Ring and Lis 1994; Peng and Mount 1995) or for viability of cultured cells (Wang et al. 1996). RS domains are essential for SR protein function. The ASF/SF2 RS domain is required for activation of constitutive splicing in vitro (Zuo and Manley 1993; Caceres and Krainer 1993), and mutant ASF/SF2 proteins with partially or completely deleted RS domains could not rescue cell death following ASF/SF2 depletion, indicating that the RS domain is essential for ASF/SF2 function in vivo (Wang et al. 1996). Many splicing factors outside the SR family also contain RS-like domains (for review, see Fu 1995). For instance, the protein encoded by Drosophila transformer-2 (tra-2; Amrein et al. 1988; Goralski et al. 1989), a gene involved in the sex determination pathway (for review, see Baker 1989), contains two RS domains, one of which is essential for its function in vivo (Amrein et al. 1994). Several lines of evidence have suggested that RS domains mediate protein–protein interactions (Wu and Maniatis 1993; Kohtz et al. 1994; Amrein et al. 1994; Xiao and Manley 1997) and also direct proteins to specific subnuclear locations (Li and Bingham 1991; Hedley et al. 1995; Caceres et al. 1997). However, U2AF65, the large subunit of U2AF, has an amino-terminal RS-like domain (Zamore et al. 1992) that appears to function by RNA–protein, as opposed to protein–protein, interactions (Valcarcel et al. 1996). Whether the RS domains of different SR proteins perform distinct functions that contribute to the specific roles of SR proteins in vivo is not known. We tested this idea by using a genetic complementation assay in a derivative of the chicken B cell line DT40 (Wang et al. 1996). Our results show that most RS domains, despite their high degree of sequence conservation throughout evolution, are functionally interchangeable. In addition, we analyzed alternative and constitutive splicing of specific endogenous transcripts and several well-studied model pre-mRNAs in cells depleted of ASF/SF2. We provide evidence that ASF/SF2 is not essential for all splicing events, but that the levels of ASF/SF2 can modulate alternative splicing of specific pre-mRNAs in vivo, and that repression of splicing can also be a natural function of ASF/SF2.