Members of the highly conserved serine/arginine-rich (SR) protein family are nuclear factors involved in splicing of metazoan mRNA precursors. In mammals, two nuclear import receptors, transportin (TRN)-SR1 and TRN-SR2, are responsible for targeting SR proteins to the nucleus. Distinctive features in the nuclear localization signal between Drosophila and mammalian SR proteins prompted an examination of the mechanism by which Drosophila SR proteins and their antagonist repressor splicing factor 1 (RSF1) are imported into nucleus. A Drosophila importin ß-family protein (Trn-SR), homologous to TRN-SR2, has been identified and characterized that specifically interacts with both SR proteins and RSF1. Drosophila Trn-SR has a broad localization in the cytoplasm and the nucleus, whereas an N-terminal deletion mutant colocalizes with SR proteins in nuclear speckles. Far Western experiments have established that the RS domain of SR proteins and the GRS domain of RSF1 are required for the direct interaction with Trn-SR, an interaction that can be modulated by phosphorylation. Using the yeast model system in which nuclear import of Drosophila SR proteins and RSF1 is impaired, it was demonstrated that complementation with Drosophila Trn-SR is sufficient to target these proteins to the nucleus. Together, the results imply that the mechanism by which SR proteins are imported to the nucleus is conserved between Drosophila and humans (Allemand, 2002).

Serine-arginine-rich proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. SR proteins have a modular structure that consists of one or two RNA recognition motifs (RRMs) and a C-terminal arginine-serine repeat of varying length (RS domain) (for reviews, see Manley, 1996; Graveley, 2000). Functionally, many of the SR proteins are able to bind several classes of specific RNA motifs known as exonic splicing elements, which play a key role in both alternative and constitutive splice site selection in several systems (for reviews, see Tacke, 1999; Blencowe, 2000). Some of the functions of SR proteins can be antagonized by RSF1, a splicing repressor isolated from Drosophila that also exhibits a modular organization with a N-terminal RRM-type RNA binding domain and a C-terminal part enriched in glycine (G), arginine (R), and serine (S) residues (GRS domain) (Labourier, 1999a; Labourier, 1999b). The RRM domain of these splicing factors mediates specific recognition of RNA sequences, whereas the RS or GRS domains are responsible for specific protein-protein interactions, which are instrumental for the assembly of the spliceosome (Allemand, 2002 and references therein).

SR proteins can be organized in the interphase nucleus in a characteristic speckled pattern, and also shuttle rapidly and continuously between the nucleus and the cytoplasm (Caceres, 1998). This distribution between cellular compartments is expected to alter the steady-state concentrations of SR proteins and thus affect the pattern of alternative splicing. Members of the importin ß (impß) family, termed transportin (TRN)-SR1 and TRN-SR2, have been shown to interact with human SR proteins (Kataoka, 1999; Lai, 2000; Lai, 2001). TRN-SR1 and TRN-SR2 have almost identical sequences except that TRN-SR1 contains additional unique regions in the central and C-terminal parts of the protein. Interaction between TRN-SR1/2 and SR proteins involves the RS domain and is abolished by RanGTP (Lai, 2000; Lai, 2001). Accordingly, a truncated TRN-SR2 that is defective in Ran binding, colocalizes with SR proteins in nuclear speckles (Lai, 2000). It is known that direct interaction of impß with specific nucleoporins mediates docking of the cargo complex to the cytoplasmic face of the nuclear pore complexes, whereas its interaction with RanGTP releases the cargo in the nucleus (Allemand, 2002 and references therein).

The mechanisms regulating the cellular localization of SR proteins are determined, at least in part, by the phosphorylation status of the RS domain. Remarkably, TRN-SR2 has a strong preference for phosphorylated RS domains and mediates nuclear import of phosphorylated, but not unphosphorylated, SR proteins (Lai, 2000; Lai, 2001). Phosphorylation of serine residues in the RS domain releases these factors from storage/assembly loci (nuclear speckles) and recruits them to the sites of active transcription (Misteli, 1998). Furthermore, overexpression of either SR protein kinase (SRPK) or Clk/Sty, a prototypical kinase with dual specificity, capable of phosphorylating tyrosines as well as serines and threonines, causes cytoplasmic accumulation of SR proteins (Gui, 1994; Colwill, 1996). The Drosophila homolog of human SF2/ASF (dASF), which lacks phosphorylation sites for Drosophila SRPK (dSRPK) in the RS domain, is unable to shuttle between the nucleus and the cytoplasm, although it is imported to the nucleus (Allemand, 2001). Because several Drosophila SR proteins have distinctive features in their RS domain compared with their human ortholog proteins (Allemand, 2001), it is unknown whether the phosphorylation-mediated cellular localization is conserved between the two species. It is also unknown whether the GRS domain of RSF1, like the RS domain, mediates import of RSF1 to the nucleus. This study demonstrates that both the RS domain of Drosophila SR proteins and the GRS domain of RSF1 serve as unique nuclear localization signals. A Drosophila impß family protein (Trn-SR) homologous to human TRN-SR2 that specifically interacts with both human and Drosophila SR proteins as well as RSF1 has been identified, and it serves as the nuclear import receptor for many SR proteins and their antagonist RSF1 in Drosophila (Allemand, 2002).

Thus Drosophila Trn-SR, homologous to human TRN-SR2 is a nuclear import receptor for both Drosophila SR proteins and their antagonist RSF1. The extensive conservation between vertebrate and Drosophila Trn-SR supports the hypothesis that an important event in the regulation of splicing takes place at the level of nuclear uptake of SR proteins and their antagonist. Considering that the SR- and RSF1-related proteins can affect splice site selection in a concentration-dependent manner, the regulation of this nuclear traffic of splicing factors may play an important role in the regulation of alternative splicing. Previous studies have shown that protein kinases that modify the RS domain of SR proteins may contribute to their spatial and temporal regulation, as well as to the modulation of their activity. In this context, it is significant that phosphorylation of these factors alters their interaction with Trn-SR. Using far Western experiments, this study was able to show that both human and Drosophila SR proteins interact with Trn-SR in a phosphorylation-dependent manner. Like the mammalian TRN-SR2, Trn-SR preferentially associates with phosphorylated SR proteins. Thus, these results demonstrate the high degree of evolutionary conservation of function of the transportin pathway and corroborate previous work in mammalian cells showing that phosphorylation of the RS domain is an important determinant in the nuclear uptake of SR proteins (Yeakley, 1999; Lai, 2000; Lai, 2001; Allemand, 2002 and references therein).

Although Trn-SR2 was shown to interact with only SR proteins, this study provides the first evidence that the Drosophila homolog Trn-SR is able to bind both SR proteins and the splicing repressor RSF1. The most prominent feature of the GRS domain of RSF1, that mediates interaction with Trn-SR, is its abundance in Gly, Ser, and Arg, but its primary sequence is not significantly similar to the RS domain of the SR protein. The observed interaction of Trn-SR with the GRS domain strengthens the idea that Trn-SR recognizes its import substrates not only via a primary sequence but also by secondary and/or tertiary structural features. Consistent with this idea, the RS domains of individual vertebrate SR proteins are conserved only in terms of their overall composition and the presence of many consecutive RS or SR dipeptides, whereas several Drosophila SR proteins have a glycine hinge region between the RNA binding domain [with one or two RRM(s)] and the RS repeats. Significantly, the Drosophila homologue of hSF2/ASF, dASF, which lacks the RS repeats and instead has a G-rich region at the RS domain (Allemand, 2001), interact with Trn-SR and is efficiently imported in yeast complemented with Drosophila Trn-SR. Furthermore, although phosphorylation of the RS domain by SRPK1 has been shown to be critical for efficient nuclear import of SR fusion proteins via TRN-SR2, this does not seem to apply for Trn-SR. RSF1 and dASF, for instance, which are not substrates for SRPK kinases from different organisms (Allemand, 2001), interacts with Trn-SR in a RS- or GRS-domain-dependent manner and are efficiently imported both in S2 and yeast cells, provided that the cells contained Trn-SR. This result is consistent with recent reports showing that phosphorylation by SRPKs promotes shuttling of SR proteins between the nucleus and cytoplasm (Allemand, 2001; Gilbert, 2001). Thus, the mechanism by which Trn-SR recognizes its target proteins is of considerable interest and remains to be clarified. The yeast system may be useful for analyzing the phosphorylation sites of RS or GRS domains at different levels of SR kinases and for examining whether differentially phosphorylated RS or GRS domains exhibit different affinities to Trn-SR (Allemand, 2002).

Another striking result of this analysis is the finding that the RS domain of all SR proteins tested is sufficient to trigger nuclear localization in S2 cells, whereas in mammalian cells Drosophila SR proteins containing two RRMs, such as SF2/ASF, dASF and B52/SRp55, do not require the RS domain for proper nuclear localization (Allemand, 2001). It is therefore possible that in mammalian cells, there are at least two nuclear import pathways for SR proteins: RS-dependent and RS-independent. This could explain why mammalian cells contain two transportins, TRN-SR1 and TRN-SR2, mediating nuclear import of SR proteins. Given that TRN-SR2 is capable of targeting phosphorylated but not unphosphorylated SR proteins to the nucleus (Lai, 2001) and that TRN-SR1 does not seem to have such a requirement (Kataoka, 1999), it is tempting to speculate that TRN-SR1 is responsible for the import of SR proteins regardless of phosphorylation and/or presence or absence of the RS domain. In contrast, Drosophila cells only have one transportin for both SR proteins and RSF1, which do not have a classical RS domain (Allemand, 2002).

During initial steps of the spliceosome assembly, the RS or GRS domain of SR proteins and RSF1 are used for a variety of protein-protein interactions, which play crucial roles in splice site selection. These domains can also mediate a specific interaction with Trn-SR. Far Western experiments demonstrate that Trn-SR has the ability to interact with multiple RS-containing proteins from two highly divergent species, Drosophila and human. However, only a weak binding of Trn-SR to human SRp75 was observed when this protein was abundant. A similar result was also obtained with TRN-SR1 (Kataoka, 1999), suggesting that SRp75 either has a different receptor or is difficult to renature under the conditions used for Far Western analysis. The latter possibility is favored because Far Western experiments performed with labeled SR proteins also show weaker binding to SRp75 compared with other SR proteins (Allemand, 2002 and references therein).

Thus, this work paves the way toward a molecular genetic analysis of the biological role of Trn-SR, which may allow the elucidation of factors modifying the activity of SR proteins. The interaction between Trn-SR and target proteins is so robust that it can be used to screen expression libraries with ³⁵S-labeled Trn-SR. Among potential candidates, are the 100- and the 85-kDa proteins identified from Drosophila Kc and HeLa nuclear extracts, respectively. Given that SR proteins and their antagonist RSF1 are specific targets for Trn-SR, novel factors may be identified that regulate splice site selection by regulating the effects of some SR proteins. Further studies designed to determine whether the 85-kDa band corresponds to SRrp86, which has such a function in splicing, should confirm this prediction (Allemand, 2002).

GENE STRUCTURE

cDNA clone length - 3316 bp

Bases in 5' UTR - 118

Exons - 5

Bases in 3' UTR - 399

PROTEIN STRUCTURE

Amino Acids - 932

Structural Domains

The RS domains of human SR proteins interact directly with two different nuclear import receptors, TRN-SR1 (Kataoka, 1999) and TRN-SR2 (Lai, 2000). A BLAST homology search with full-length TRN-SR1 and 2 in the fly database (BDGP database) revealed a 914-amino acid protein that bears significant homology to these nuclear import receptors (39/59% identity vs. similarity) and was therefore named Trn-SR. Because TRN-SR1 has additional amino acids both in the middle and the C terminus, Drosophila Trn-SR was more similar to hTRN-SR2 than hTRN-SR1. Both Trn-SR2 and Trn-SR have significant similarity with the Saccharomyces cerevisiae protein Mtr10p (Kadowaki, 1994; 25% / 44% identity vs. similarity for Trn-SR), a nuclear import receptor for the mRNA-binding protein Npl3p (Pemberton, 1997; Senger, 1998; Allemand, 2002 and references therein).

Comparisons of the cDNA and genomic sequences reveal that Drosophila Trn-SR is a single copy gene composed of six exons spaced by five introns. Thus, unlike mammals, D. melanogaster seems to have only one nuclear import receptor, Trn-SR, which is probably responsible for the nuclear localization of all SR proteins (Allemand, 2002).

date revised: 10 June 2005

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