The two well-known trans-acting factor protein classes (SR and hnRNP proteins) are conserved between plants and humans; they have a regulatory role in AS, often in an antagonistic manner (M. Chen & Manley, 2009; Reddy, 2007; Simpson et al., 2010). SR proteins are known to facilitate splice site recognition by binding to ESEs and can act as a general splicing factor or specific splicing modulators in a range of organisms from plants to humans (M. Chen & Manley, 2009; Lopato et al., 1999; Reddy, 2007).
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HnRNP-like proteins repress splice site recognition by binding to splicing silencers in a variety of organisms, they are thought to play a similar role in plants (Blanchette & Chabot, 1999; M. Chen & Manley, 2009; B.-B. Wang & Brendel, 2004b).
1.2.2.2.1 SR proteins
SR proteins are a conserved family of pre-mRNA splicing factors that are concerned with the execution of constitutive splicing and alternative splice site choice in plants and animals. They can modulate AS by effecting splice site choice in a concentration and phosphorylation-dependent manner, potentially regulating tissue-specific, developmental-regulated and stress responsive AS in plants and animals (Duque, 2011).
SR proteins consist of a N-terminal domain which contains one or two RNA recognition motifs (RRMs) which bind specific RNA sequences (the SRE), and a downstream C-terminal region which contains a sequence greater than 50 amino acids long that is rich in serine and arginine (the RS domain) that recruits other proteins or in some cases contacts the pre-mRNA branch point; they can also contain signals for cellular localization (Caceres, Screaton, & Krainer, 1998; J. C. Long & Caceres, 2009; H. Shen & Green, 2006). According to standard nomenclature in mammals the RS domain has 40% RS content with consecutive RS or SR repeats, whereas in plants the RS domain has a RS content of 20% RS or SR dipeptides (Barta, Kalyna, & Reddy, 2010; Manley & Krainer, 2010). Barta et al 2010 have proposed that in Arabidopsis the SR proteins can be divided into six sub families (Figure 2). The SR subfamily has four members and consists of two RRM domains, the second of which contains the evolutionary conserved SWQDLKD motif followed by a SR domain featuring characteristic SR dipeptides; the RSZ subfamily contains one Zinc knuckle and comprises three members; the SC subfamily consisting of a single RRM domain followed by a SR domain (one member in Arabidopsis); the SCL subfamily which is very similar to the SC subfamily but contains a N-terminal charged extension (four members in Arabidopsis); the RS2Z subfamily which contains two Zinc knuckles with an additional SP-rich region following the RS domain (two members in Arabidopsis)
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and the RS subfamily that consists of two RRM domains followed by a RS domain (four Arabidopsis members) (Barta et al., 2010). Using this nomenclature two previously classified SR proteins SR45 and SR45a are no longer classed as SR proteins, which is consistent with their mammalian homologues that have been declassified as SR proteins by Manley and Krainer (Barta et al., 2010; Manley & Krainer, 2010).
Figure 2. Domain Architecture of the Arabidopsis SR Protein Subfamilies. The SR subfamily proteins contain an evolutionary conserved SWQDLKD motif in their second RRM with a downstream RS domain featuring characteristic SR dipeptides. The RSZ subfamily consists of SR proteins that contain one Zinc knuckle. The SC subfamily proteins have a single RRM followed by a downstream RS domain. The plant-specific SCL subfamily (SC35-like) proteins are similar to the SC subfamily but have an N-terminal charged extension. The proteins of the plant-specific RS2Z subfamily possess two Zinc knuckles and have an additional SP-rich region following the RS domain. The plant-specific RS subfamily proteins contain two RRMs (without the SWQDLKD motif) followed by the RS domain rich in RS dipeptides. Red oval represent RRM domain, purple ovals represent RRM domain with the SWQDLKD motif, blue rectangles represent RS domain consisting of SR dipeptides, yellow circles represent Zinc knuckles, turquoise rectangles represent RS domain, purple rectangle represent N-terminal charged extension and green rectangles represent a SP-rich region; adapted from Barta et al 2010.
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Flowering plants contain the largest number of SR proteins amongst eukaryotes, 18 in Arabidopsis (Barta et al., 2010), compared to seven in Caenorhabditis elegans (Longman, Johnstone, & Caceres, 2000), and 12 in humans (Manley & Krainer, 2010). This is thought to be due to genome amplification, particularly inter-chromosomal duplication events, with at least 12 Arabidopsis genes that encode SR proteins being located on duplicated regions. Most of the duplicated genes display different
spatiotemporal expression patterns indicating functional diversification (Kalyna & Barta, 2004).
SR proteins interact with RNA through the RRM domain, providing binding specificity, the RS domain aids in spliceosomal assembly via binding of other spliceosomal proteins (Reddy et al., 2013). In mammalian cells during early spliceosome assembly the binding of U1 snRNP to the 5ʹSS is mediated by the SR protein SRSF1, via direct interaction with the RS motif of the U1-70K component of the U1 snRNP in a phosphorylation-dependent manner (Cho et al., 2011). This SR protein along with SC35 has been implicated in the bridging of the 5ʹ and 3ʹ splice sites via interactions with U1-70K and the 35Kd subunit of the splicing factor U2AF (U2AF35) (J. Y. Wu & Maniatis, 1993). SR proteins have also been implicated in the incorporation of the tri snRNP complex (U4/U6.U5 snRNP) into the spliceosome (Roscigno & Garcia-Blanco, 1995). It is thought that similar interactions between SR proteins, RNA and spliceosomal components occur in plant systems. Lorković et al
2004 showed in immune-precipitation experiments the Arabidopsis homologue of SRSF1 (AtSR34) and AT-U1-70K (the Arabidopsis version of the U1-70K component of the U1 snRNP) protein co-expressed in protoplast showing a clear interaction
(Lorković, Lopato, Pexa, Lehner, & Barta, 2004). However Golovkin and Reddy et al
1999 showed that four other Arabidopsis SR proteins (At-SCL33, At-SRZ21, At-SRZ22, and the SR-like protein SR45) interact with the Arabidopsis U1 snRNP 70-kDa
protein, and a Clk/Sty protein kinase (AFC-2) from Arabidopsis phosphorylated all four of these SR proteins (Golovkin & Reddy, 1999). This indicates that the
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stages of spliceosome assembly, and intron recognition in plants is likely to be different.
1.2.2.2.2 hnRNP proteins
Traditionally SR proteins were thought of as activators of splicing with hnRNP proteins acting as repressors by binding to the pre-mRNA preventing other splicing factors from accessing the binding site (B.-B. Wang & Brendel, 2004b). However it is now thought that the type of action may be more context dependent (Wachter et al., 2012). For example, in mice, the hnRNP-like proteins belonging to the neuro- oncological neural antigen (NOVA) family recognise an YCAY motif that can act as either an ESS if it is in an exon preceding an AS exon or as an ISS if it is in an intron following an AS exon (Ule et al., 2006).
The RNA regulatory map appears to be highly conserved between insects and
mammals, for example the Drosophila melanogaster ortholog of mammalian NOVA1 and NOVA2, Pasilla (PS), has regions enriched for YCAY repeats upstream and within PS-repressed exons and downstream from PS-activated exons, consistent with the location of these motifs near NOVA-regulated exons in mammals. The target gene orthologs regulated by PS and NOVA1/2 are almost entirely non-overlapping potentially indicating that the regulatory codes of individual RNA-binding proteins may be conserved between species but the regulatory modules controlled by these proteins are evolving (Brooks et al., 2011).
The Polypyrimidine tract-binding (PTB) proteins are probably the most intensively studied hnRNP proteins, with homologues found between the mammalian system and plants. In HeLa cells, Lin and Patton et al 1995 showed that PTB effects the splicing of α-tropomysin (α-TM) (C. H. Lin & Patton, 1995). The second and third exon of α-TM are mutually exclusive and PTB inhibits the splicing that joins the first and third exon (1-3 splicing) with U2AF antagonising this splicing. In contrast, SR proteins activate the splicing that joins the first and second exon (1-2 splicing) demonstrating how hnRNP and SR proteins can work together to produce AS
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isoforms (C. H. Lin & Patton, 1995). In Arabidopsis there are three homologues of the human PTB (AtPTB1, AtPTB2 and AtPTB3) (B.-B. Wang & Brendel, 2004b). Simpson et al 2014 showed using a protoplast transient expression system that the Arabidopsis proteins AtPTB1 and AtPTB2 reduced the inclusion/splicing of a novel potato
invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing; the co-expression of NpU2AF65 alleviated this repression, indicating that PTB and U2AF also compete for pre-mRNA binding in plants (Simpson
et al., 2014). This indicates the mechanisms of activation and repression of splice
sites by hnRNP may be conserved between mammals and plants.
Plants are thought to contain more hnRNP proteins than humans; Wang and Brendel
et al 2004 computationally identified 35 other potential hnRNP proteins in
Arabidopsis (not including the PTB hnRNP proteins) that have human homologues and 17 plant-specific potential plant hnRNP proteins, compared to 20 hnRNPs found in humans (identified as hnRNP A to hnRNP U) (B.-B. Wang & Brendel, 2004b).