Antagonism between RSF1 and SR proteins for both splice-site recognition in vitro and Drosophila development

Abstract

Specific recognition of splice sites within metazoan mRNA precursors (pre-mRNAs) is a potential stage for gene regulation by alternative splicing. Splicing factors of the SR protein family play a major role in this regulation, as they are required for early recognition of splice sites during spliceosome assembly. Here, we describe the characterization of RSF1, a splicing repressor isolated from Drosophila, that functionally antagonizes SR proteins. Like the latter, RSF1 comprises an amino-terminal RRM-type RNA-binding domain, whereas its carboxy-terminal part is enriched in glycine (G), arginine (R), and serine (S) residues (GRS domain). RSF1 induces a dose-sensitive inhibition of splicing for several reporter pre-mRNAs, an inhibition that occurs at the level of early splicing complexes formation. RSF1 interacts, through its GRS domain, with the RS domain of the SR protein SF2/ASF and prevents the latter from cooperating with the U1 small nuclear ribonucleoprotein particle (U1 snRNP) in binding pre-mRNA. Furthermore, overproduction of RSF 1 in the fly rescues several developmental defects caused by overexpression of the splicing activator SR protein B52/ SRp55. Therefore, RSF1 may correspond to the prototypical member of a novel family of general splicing repressors that selectively antagonize the effect of SR proteins on 5 splice-site recognition.[Key Words: pre-mRNA splicing; RNA-binding proteins; SF2/ASF; U1 snRNP; RSF1; B52/SRp55] Received September 25, 1998; revised version accepted January 28, 1999.Pre-mRNA splicing is an essential step in the expression of most metazoan protein-coding genes, often regulated in a cell-type specific or developmental manner. The precision and efficiency of the splicing reaction result from a dynamic series of interactions among the U1, U2, U4/ U6, and U5 small nuclear ribonucleoprotein (snRNP) particles, non-snRNPs, and the pre-mRNA that lead to the formation of the spliceosome, the ribonucleoprotein structure in which the accurate excision of intervening sequences (introns) takes place (Sharp 1994; Krämer 1996;Staley and Guthrie 1998). Although most of the biochemical events involved in constitutive pre-mRNA splicing are being elucidated, the complex regulation underlying the selection of alternative exons is less well understood (Adams et al. 1996;Wang and Manley 1997). Both structural features and sequence content of premRNAs appear to be involved (Green 1991). In the past few years, a class of purine-rich exonic elements known as exonic splicing enhancers (ESEs) has been identified in a number of pre-mRNAs (Katz and Skalka 1990;Lavigueur et al. 1993; Sun et al. 1993a,b;Watakabe et al. 1993;Xu et al. 1993;Caputi et al. 1994;Dirksen et al. 1994Dirksen et al. , 1995Tanaka et al. 1994;Ramchatesingh et al. 1995;Staffa and Cochrane 1995;Staffa et al. 1997). These sequences are likely to play a critical role in the initial recognition and pairing of the 5Ј and 3Ј splice sites (Tian and Maniatis 1993;Staknis and Reed 1994;Berget 1995). They are expected to bind a set of RNA-bin...

Splicing Error in E1α Pyruvate Dehydrogenase mRNA Caused by Novel Intronic Mutation Responsible for Lactic Acidosis and Mental Retardation

“…Further studies will be necessary to confirm our hypothesis by performing in vitro splicing experiments (27).…”

Sorting out the complexity of SR protein functions

“…65 (Zamore et al+, 1992) (Zhou & Reed, 1998), HRH1 (Ono et al+, 1994) Liu et al+, 1998Liu et al+, , 2000Cavaloc et al+, 1999;Schaal & Maniatis, 1999b)+ A surprising conclusion from these experiments is that SR proteins recognize a vast array of RNA sequences (see Table 1 (Wu & Maniatis, 1993;Amrein et al+, 1994;Kohtz et al+, 1994)+ Importantly, these protein interactions all require the RS domains of each protein (Wu & Maniatis, 1993;Amrein et al+, 1994;Kohtz et al+, 1994)+ Only recently, however, have the RS domains been shown to be sufficient to mediate protein interactions+ For instance, the RS domain of SF2/ASF has been shown to be sufficient to interact with RSF1, a Drosophila splicing repressor (Labourier et al+, 1999)+ In addition, when artificially tethered to the pre-mRNA, the RS domains of several human SR proteins are sufficient to activate enhancer-dependent splicing (Graveley & Maniatis, 1998), an activity that presumably requires protein interactions+ In contrast, the RS domain of SF2/ASF is unable to interact with U1-70K (Xiao & Manley, 1997)+ These studies indicate that the RS domains of SR pro- a N: any nucleotide; R: purine; Y: pyrimidine; S: G or C; K: U or G; W: A or U; D: A, G, or U; M: A or C+ b "SELEX" indicates that the RNA sequence was determined to be a high affinity binding site for a purified SR protein; "functional" indicates that the RNA sequence was determined to function as an SR protein-specific splicing enhancer+ teins are indeed protein interaction domains, but suggest that different protein interactions may have distinct RS domain sequence requirements+…”

Regulation of alternative splicing by SRrp86 through coactivation and repression of specific SR proteins

“…Given the prevalence of alternative splicing in higher eukaryotes (Croft et al+, 2000;International Human Genome Sequencing Consortium, 2001), it is important to understand the mechanisms that determine tissuespecific alternative splicing+ Members of the SR protein family have been shown to play an important role in regulating splice site selection, but how these proteins are themselves regulated remains unclear+ Regulated phosphorylation of SR proteins is clearly important and characterization of several kinases and phosphatases involved in SR protein modification is underway (reviewed in Gravely, 2000)+ However, the identification of proteins such as p32 (Petersen-Mahrt et al+, 1999), RSF1 (Labourier et al+, 1999), SRrp35, SRrp40 (Cowper et al+, 2001, and now SRrp86 indicates that SR proteins are subject to multiple modes of regulation, consistent with an important role in controlling splicing patterns+…”

“…Changes in the cellular concentration of SRrp86 would be expected to alter the global activity of SR proteins, down-regulating some while up-regulating others+ Although the inhibitory activity of SRrp86 is similar to p32, RSF1, SRrp35, and SRrp40, it appears to be a broader regulator due to its ability to both activate and repress SR protein activity+ It is thought that splicing repression by RSF1 is caused by sequence-specific binding and subsequent interference with the normal protein-protein interactions of SR proteins (Labourier et al+, 1999)+ Inhibition by p32 appears to be due to repression of ASF/SF2 phosphorylation, consistent with p32 binding to only hypophosphorylated ASF/SF2 (Petersen-Mahrt et al+, 1999)+ For SRrp35 and SRrp40, it is not yet known how these proteins repress splicing but their effects are consistent with competition for RNAbinding sites (Cowper et al+, 2001)+ In contrast, SRrp86 directly interacts with target SR proteins (Barnard & Patton, 2000;J+ Li, D+C+ Barnard, & J+G+ Patton, submitted)+ It should be stressed that each of the multiple assays that have been used to examine interaction between SR proteins and SRrp86 suffer from potential pitfalls, making it difficult to quantitate the strength of interaction+ Nevertheless, regulation of SR proteins by SRrp86 appears to be distinct from simple disruption of the normal phosphorylation/dephosphorylation cycle of SR proteins, as the phosphorylation state of the target SR protein does not apparently alter interaction nor does SRrp86 appear to affect the ability of SR proteins to be phosphorylated (Barnard & Patton, 2000;D+C+ Barnard & J+G+ Patton, unpubl+)+ It is still not clear how SRrp86 is able to both activate and repress target SR proteins, but such regulation appears to be distinct from the mechanisms invoked for p32, RSF1, SRrp35, and SRrp40+ The overall picture that emerges is that a variety of mechanisms are utilized to regulate the activity of SR proteins, consistent with an important role for these proteins in controlling splice site selection+…”