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SR proteins are a family of structurally related RNA binding proteins, highly conserved in metazoan cells that play multiple roles in splicing and in general mRNA metabolism (Graveley 2000; Huang and Steitz 2005). They contain one or two N- terminal RNA-recognition motifs (RRM) and a specific C-terminal domain rich in repeating arginines and serines, the “RS” domain (Bimey, Kumar et al. 1993). The RNA binding and the RS domains are modular structures and they can be exchanged between different SR proteins. Differences among SR proteins structure depend on the length of the RS domain and the second RRM domain; when it is present, the sequence is often divergent from the canonical consensus sequence (Graveley 2000).

In addition to SR proteins other factors involved in pre-mRNA metabolism can contain a RS domain. These proteins are usually referred as SR-related proteins and they include both subunits of U2AF, the U1-70K protein and several ATPases (Boucher, Ouzounis et al. 2001).

The structural organization of SR proteins suggests a model for their function. The RRM mediates sequence-specific binding to the mRNA, whereas the RS domain seems to be involved mainly in protein-protein interactions (Graveley 2000;

Cartegni, Chew et al. 2002). However, a recent work has reported that the RS domains can also be involved in direct RNA contacts during splicing. A specific interaction between the RS domain and the branch point was described as promoting spliceosome formation (Shen and Green 2004).

The sequence-specific binding to pre-mRNAs is crucial for the function of SR proteins in the earliest step of spliceosome assembly (Graveley 2000; Sanford, Ellis et al. 2005). The binding specificity of individual SR protein has been studied using the SELEX technique showing that the consensus sequences recognized are highly degenerated (Liu, Zhang et al. 1998; Liu, Chew et al. 2000). Other SR proteins, indicating a redundancy in their functions, can also bind a target sequence recognized by one SR protein. In agreement with this observation, RNAi depletion of most SR proteins in C. elegans resulted in no detectable phenotype, suggesting a functional overlap among these factors (Longman, Johnstone et al. 2000). However, the lethality caused by loss of a SR protein in Drosophila (Ring and Lis 1994) or in the chicken DT40 cell line (Wang, Takagaki et al. 1996) argue against a large redundancy of SR proteins. Similarly deletion of SRp20, ASF/SF2 and SC35in the germ line of mice led to embryonic lethality indicating an essential role of this factors in the early embryonic development (Jumaa, Wei et al. 1999; Wang, Xu et al.

2001; Xu, Yang et al. 2005).

The SR proteins are required both for constitutive and alternative splicing events (Sanford, Ellis et al. 2005). Two non-exclusive models have been proposed to explain the role of SR protein in pre-mRNA splicing (Fig. 1.6). One model is based on the ability of these splicing factors to bind exonic splicing enhancers (ESEs) and through their RS domain to recruit and stabilize U l snRNP and U2AF binding to the 5’ and 3’ss respectively. SR proteins have also been suggested to allow protein- protein interactions across introns binding the U1-70K factor and the U2AF35 and stimulating the usage of the 5’ and 3’ss (Fig.l.6a) (Cartegni, Chew et al. 2002;

Sanford, Ellis et al. 2005). In addition SR proteins have also been described in the recruitment of the U4/U6 U5 tri-snRNP to the spliceosome during the formation of the B complex (Roscigno and Garcia-Bianco 1995). The second model proposes that

a SR protein, bound to an ESE, can antagonize the negative effect of a juxtaposed silencer element (Fig. 1.6b) (Cartegni, Chew et al. 2002; Sanford, Ellis et al. 2005).

Due to their involvement in splice site selection, specifically in promoting the selection of the proximal 5’ (Mayeda and Krainer 1992; Caceres, Stamm et al. 1994) and 3’ss (Caceres, Stamm et al. 1994; Bai, Lee et al. 1999), SR proteins have been reported to be important players in regulating alternative splicing. They exert a role in promoting U2AF binding to weak 3’ss (Wu and Maniatis 1993; Zuo and Maniatis 1996) or antagonizing the activity of negative splicing factor such as hnRNP A l (Caceres, Stamm et al. 1994) or other SR proteins (Gallego, Gattoni et al. 1997). SR proteins have shown to promote exon inclusion when bound to a target site within exons (ESEs); however, in some cases they can act in a negative fashion. The negative effect on splicing can be mediated by the binding to an intronic sequence (ISS) (Ibrahim el, Schaal et al. 2005; Buratti, Stuani et al. 2007) or by the inhibitory property of the protein itself, as reported for SRp 38 (Barnard, Li et al. 2002).

SR protein activity is regulated through phosphorylation/dephosphorylation at multiple positions within the RS domain (Stamm 2008). This post-translational modification is a crucial step for the splicing organization inside the cell nucleus by affecting the RNA-binding activity and sub nuclear localization of the SR proteins (Misteli and Spector 1997). While localized predominantly in the nucleus, some (not all) SR proteins shuttle continuously between the nucleus and the cytoplasm (Caceres, Screaton et al. 1998). The RS domain phosphorylation is required for the translocation of the SR proteins from the cytoplasm to the nucleus and also for the recruitment of these factors from nuclear speckles (“splicing factor compartments”) to the sites of pre-mRNA synthesis (Bourgeois, Lejeune et al. 2004).

a Recruiting function: RS-domain dependent

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Figure 1.6: Models of SR protein action in ESE dependent splicing.

a) RS-domain-dependent mechanism. A SR protein bound to an exonic splicing enhancer (ESE) through its RNA-recognition motifs (RRM) contacts the splicing and through its RS domain recruits and stabilizes U l snRNP and U2AF binding to the 5’

and 3’ss, respectively. SR proteins can also allow protein-protein interactions across introns binding the U1-70K factor and the U2AF35 stimulating the usage of the 5’

and 3’ss. For some ESE-dependent pre-mRNAs, indirect interactions (black arrows) are bridged by the splicing co-activator SRml60, which stimulates splicing of some ESE-dependent pre-mRNAs and also interacts with the U2 snRNP.

b) RS-domain-independent mechanism. In this case, the main function of the SR protein bound to an ESE is to antagonize the negative effect on splicing o f an inhibitory protein that is bound to a juxtaposed exonic splicing silencer (ESS). The SR protein is shown without its RS domain, although this domain is normally present and might still promote U2AF binding. Inhibitory interactions are shown in red and the putative stimulatory interactions are double-headed arrow. These models are not mutually exclusive, and some splicing might involve a combination o f these mechanisms. Figure adapted from Cartegni, Chew et al. (Cartegni, Chew et al. 2002).

Phosphorylation is also important for specific RNA recognition, since the high positive charge of unphosphorylated RS domains masks the specificity of the RNP domains and enhances non-specific binding (Tacke, Chen et al. 1997; Stamm 2008).

While the functions of SR proteins in pre-mRNA splicing have been extensively studied, recent work has demonstrated their roles in numerous other steps of mRNA metabolism including mRNA nuclear export, mRNA stability and translation (Huang and Steitz 2005; Sanford, Ellis et al. 2005).

ASF/SF2: One of the best known SR proteins is the alternative splicing factor/splicing factor 2 (ASF/SF2). Different groups have highlighted the two basic properties of this SR protein. ASF/SF2 was described as an essential splicing factor necessary for the early step of splicing (Krainer, Conway et al. 1990) and was also characterized as an alternative splicing factor able to drive splice site selection (Ge and Manley 1990). ASF/SF2 can promote the recruitment of U l snRNP to 5’ss (Kohtz, Jamison et al. 1994) to help 5’ss and 3’ss bridging (Wu and Maniatis 1993), and plays a role in splicing regulation, through binding to exonic splicing enhancers (Sun, Mayeda et al. 1993).

ASF/SF2, together with other SR proteins, is involved in additional roles in gene expression. For example, ASF/SF2 remains associated with the spliced mRNA and is able to shuttle between the nucleus and the cytoplasm (Caceres, Screaton et al.

1998), suggesting a role in mRNA export (Huang and Steitz 2005). In addition ASF/SF2 seems to regulate the mRNA stability by binding to the 3’UTR and enhancing RNA degradation in the cytoplasm (Lemaire, Prasad et al. 2002).

ASF/SF2 can also stimulate translation of reporter mRNAs by associating with translating ribosomes (Sanford, Gray et al. 2004).

Despite these advances in understanding the functions of ASF/SF2 less is known about the physiological roles of this protein. Depletion of ASF/SF2 by RNAi resulted

in lethality in C. elegans (Longmari, Johnstone et al. 2000) and tissue-specific deletion in mice resulted in defects in the developing heart (Xu, Yang et al. 2005).

ASF/SF2 showed also an unexpected role in maintaining genomic stability by protecting cells from the deleterious effects of R-loop formation (Li and Manley 2005). In addition a recent work found that ASF/SF2 is an oncoprotein with roles in both the establishment and the maintenance of cell transformation (Kami, de Stanchina et al. 2007). In particular, ASF/SF2 has been found to control alternative splicing of the oncogene Ron which modulates cell motility (Ghigna, Giordano et al.

2005).