Transportin-Serine/Arginine rich: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
| Gene name - Transportin-Serine/Arginine
Synonyms - dTRN-SR
Cytological map position - 23A3--5
Function - signal transduction
Keywords - nucleo-cytoplasmic transport, SR-proteins
Symbol - Trn-SR
FlyBase ID: FBgn0031456
Genetic map position - 2L
Classification - Importin-ß
Cellular location - cytoplasmic and nuclear
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
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).
To examine the cellular distribution of Drosophila SR proteins and their antagonist RSF1, GFP was fused in frame to the amino terminus of each of dASF, d9G8, dSC35, Rbp1/SRp20, and RSF1 and the fusion proteins were transiently expressed in Drosophila S2 cells. All Drosophila SR fusion proteins, as well as RSF1and human SR proteins SF2/ASF (hASF/SF2) and SC35 (hSC35), were localized in the nucleus of S2 cells. In contrast to mammalian cells, where SR proteins colocalized with both speckles and diffuse pools of splicing factors excluding the nucleoli (Allemand, 2001), only a diffuse pattern was seen in S2 cells. Fusion proteins lacking the RS or GRS domain, like GFP alone, had a broader cellular distribution both in the nucleus and cytoplasm. In contrast, the GFP fusions that harbor the RS or the GRS domain alone displayed the same nuclear distribution as full-length fusion proteins, demonstrating that the GRS domain and the RS domain of all SR proteins from Drosophila act as nuclear localization signals. None of these fusions, however, were directed to nuclear speckles. Thus, the GRS domain of RSF1 and the RS domain of Drosophila SR proteins are necessary and sufficient for nuclear localization (Allemand, 2002).
To determine the specificity of binding of Trn-SR to target proteins, Far Western blotting was used. This method has been successfully applied to reveal specific interactions between members of the SR protein family and other splicing factor as well as for the study of interactions of yeast ribosomal proteins with their specific karyopherins. Proteins from HeLa and Drosophila Kc nuclear extracts were separated by SDS-PAGE gels, transferred to filters, renatured, and probed with 35S-labeled Trn-SR produced in rabbit reticulocyte lysate. In HeLa nuclear extracts, Trn-SR labeled predominantly protein species with apparent molecular weights of ~20, 30, 40, 55, 75, 85, and 130 kDa. Except for the 85- and 130-kDa proteins, which were only seen with Trn-SR, protein bands with identical mobilities also appeared when filters were probed with mAb 104, a mAb specific for the phosphoepitope present in a subset of SR proteins. These data suggested that the 20-, 30-, 40-, 55-, and 75-kDa bands are probably SRp20, SRp30, SRp40, SRp55, and SRp75, respectively. Based on the migration, the 85-kDa band is probably the recently identified serine-arginine-rich splicing regulatory protein, SRrp86, which contains a single N-terminal RRM and two C-terminal RS domains, but is not recognized by mAb 104. However, further studies are required to confirm this possibility. Binding of Trn-SR to human SR proteins correlated with their abundance in the extract, suggesting that Trn-SR has no preference for binding any of the SR proteins. There was, however, a weak signal at the level of SRp55 and SRp75, probably due to a less efficient renaturation than for the other SR proteins (Allemand, 2002).
Given that TRN-SR2 can directly interact with phosphorylated but not unphosphorylated SR proteins (Lai, 2001), tests were performed to see whether interactions between Trn-SR and proteins from HeLa extracts are sensitive to phosphorylation. HeLa nuclear extracts were subjected to phosphatase treatment before Far Western blotting. As expected, mAb 104 failed to detect any protein from treated extracts, implying that dephosphorylation of SR proteins was complete. In agreement with the fact that dephosphorylation of SR proteins alters their electrophoretic mobility, SR protein bands detected with 35S-labeled Trn-SR from treated extracts, had faster mobilities than those from untreated extracts. Dephosphorylated SR proteins, as well as the 85- and 130-kDa bands showed weaker binding signals, indicating that Trn-SR preferentially binds phosphorylated versions of these proteins (Allemand, 2002).
Immunoblotting of mAb 104 to nuclear extracts from Drosophila (Kc) cells revealed that SRp55/B52 is the prominent immunoreactive polypeptide, whereas 35S-labeled Trn-SR revealed several protein bands with molecular weights of ~26, 34, 55, 79, and 100 kDa. Detection of proteins corresponding to 26-79 kDa was strongly diminished after dephosphorylation, demonstrating that Trn-SR recognizes weakly unphosphorylated proteins. Consistent with this result, purified recombinant hSF2/ASF and dASF expressed in a baculovirus system (bSF2/ASF and bdASF), where the phosphorylation of recombinant proteins is expected to take place, bind Trn-SR more efficiently than unphosphorylated versions expressed in bacteria (eSF2/ASF and edASF). However, dephosphorylation did not alter the intensity of the 100-kDa band, whose identity is presently unknown. The 26-79-kDa protein bands have sizes consistent with those of recently identified SR proteins from Drosophila, and therefore are likely to be Drosophila SR proteins. The interaction between Trn-SR and Drosophila SR proteins is specific, because probing yeast S. cerevisiae whole-cell extracts with 35S-labeled Trn-SR did not reveal any reactive protein. Indeed, yeast S.cerevisiae does not have SR proteins and mAb 104 does not cross-react with any protein in yeast (Allemand, 2002).
To test whether another member of impß family was capable of interacting with SR proteins, a Far Western experiment was performed with Transportin (Trn), an ortholog to human TRN1 and S. cerevisiae Kap104p. TRN1 binds the M9 sequence of hnRNP A1 and is responsible for targeting this protein to the nucleus (Pollard, 1996), whereas Kap104p plays a role in the nuclear import of a yeast hnRNP-like protein Nab2p (Aitchison, 1996). The data clearly show that protein bands revealed by 35S-labeled Transportin are different from those detected by Trn-SR in HeLa and Kc extracts. In Drosophila extracts, for example, there were two sets of proteins that interact with Transportin, one at ~75 kDa, and others between 30 and 40 kDa. The latter proteins are probably Drosophila hnRNPs, Squid (SqdB and SqdS)/hp40, known to bind dTRN (Norvell, 1999). Unlike the interaction between Trn-SR and SR proteins, the interaction between Transportin and its target proteins is not sensitive to dephosphorylation. Thus, only Trn-SR, but not Transportin, binds Drosophila SR proteins (Allemand, 2002).
Far Western experiments demonstrate that Trn-SR does not bind proteins from yeast whole-cell extracts. This finding prompted use of yeast S. cerevisiae as a model system to examine the potential requirement of different SR proteins domains and eventually RSF1 for the interaction with Trn-SR. To this end, both wild-type and deletion mutants deleted in the RS domain (DeltaRS) of hSF2/ASF and Drosophila SR proteins and GRS domain (DeltaGRS) of RSF1 were expressed from a multicopy plasmid under control of the GAL10 promoter in yeast. The recombinant proteins contained an N-terminal HA-tag to facilitate their detection with an anti-HA antibody. Western blotting of total yeast extracts with an anti-HA antibody confirmed the expression of full-length and deletion mutants in a wild-type yeast strain. Probing duplicated filters with 35S-labeled Trn-SR revealed that Trn-SR bound efficiently to hSF2/ASF, dASF, dSC35, d9G8, Rbp1, and B52, but only weakly to RSF1. In sharp contrast, none of the mutant proteins deleted in the RS domain (hSF2/ASFDeltaRS, dASFDeltaRS, dSC35DeltaRS, d9G8DeltaRS, Rbp1DeltaRS, and B52DeltaRS) or GRS domain (RSF1DeltaGRS) were detected with 35S-labeled Trn-SR, showing that the RS domain is required for interaction between Trn-SR and SR proteins. The lack of recognition between Trn-SR and proteins deleted in the RS domain or GRS domain provides further evidence that the observed interaction between Trn-SR and the SR proteins or RSF1 is specific (Allemand, 2002).
The RNA binding protein Npl3p that has been implicated in mRNA transport is one of the yeast proteins that resembles both hnRNP and SR proteins from mammalian cells. Curiously, the nuclear import receptor for Npl3p was identified as Mtr10p, the yeast relative of Trn-SR (Senger, 1998). Thus, it is possible that SR proteins in yeast use Mtr10p as a nuclear import receptor (Yeakley, 1999). Given that deletion of MTR10 results in cytoplasmic accumulation of Npl3p (Pemberton, 1997; Senger, 1998), the latter scenario predicts that in the absence of Mtr10p, SR proteins and potentially RSF1 should also accumulate in the cytoplasm. This hypothesis was tested by expressing GFP-SR fusion proteins in an MTR10 deletion mutant. To avoid overexpression of these fusion proteins, they were expressed using a centromeric plasmid without induction. The levels of expression were monitored by Western blot analysis with an anti-GFP antibody. When living cells were analyzed by fluorescence microscopy, GFP-SF2/ASF, GFP-dASF, GFP-dSC35, GFP-Rbp1, GFP-d9G8, GFP-RSF1, and GFP accumulated in the cytoplasm of the mtr10 mutant. Similar cytoplasmic localizations were observed in wild-type cells, indicating that the lack of Mtr10p was not responsible for the cytoplasmic accumulation of SR proteins. As expected, GFP-Npl3 also had a cytoplasmic distribution in the mtr10 mutant, whereas it had a nuclear localization in wild-type cells. As a control, the localization of Drosophila U1-70K fused to GFP was analyzed. dU1-70K, a specific protein associated with U1 snRNP, contains a classical nuclear localization signal, and as such its localization should not be affected by the MTR10 deletion. As expected, no inhibition of nuclear import of this protein was observed in either the mtr10 mutant or wild-type cells, again showing that Mtr10p could not replace Trn-SR for nuclear import of Drosophila SR proteins exogenously expressed in yeast. These results are also consistent with the idea that other nuclear import receptors, like Kap104, the functional homolog of Transportin, cannot replace Trn-SR to mediate nuclear localization of SR proteins in yeast. Moreover, none of the SR proteins expressed in yeast were able to bind Transportin, as revealed by Far Western (Allemand, 2002).
To test whether mislocalization of GFP-SR protein fusions in yeast could be corrected by Trn-SR, mtr10 mutants expressing Drosophila SR proteins, hSF2/ASF as well as RSF1 were transformed with plasmids expressing c-myc-tagged Trn-SR. Immunobloting with anti-c-myc antibodies revealed that similar levels of the c-myc-Trn-SR fusion were expressed. Expression of c-myc-Trn-SR in mtr10 mutant strains induces dramatic changes in the localization of GFP-Drosophila SR proteins, hSF2/ASF as well as RSF1, which exhibits an exclusive intranuclear location in the complemented strains. However, the distribution of GFP, dU1-70K and Npl3 remained unchanged. Taken together, these data show that Trn-SR specifically mediates the nuclear import of Drosophila SR proteins as well as their antagonist RSF1 (Allemand, 2002).
A transiently expressed TRN-SR2 mutant lacking the N-terminal 281-amino acids, DeltaN281, localizes predominantly in the nucleoplasm and accumulates in speckled domains in the nuclei of HeLa cells (Lai, 2000). To test whether deletion of the N-terminal domain of Trn-SR could similarly affect its cellular distribution, GFP was fused in frame to the N terminus of full-length Trn-SR or its truncated version lacking the first N-terminal 217-amino acids, DeltaN217, and transiently expressed fusion proteins in either HeLa cells or Drosophila S2 cells. In both cell lines, full-length Trn-SR fusion protein is detected both in the nucleus and the cytoplasm. In contrast, DeltaN217 is localized predominantly in the nucleus. Immunofluorescence with anti-SC35 antibodies clearly established that a Trn-SR mutant lacking the N-terminal region localizes to the nucleus of HeLa cells in a speckled pattern that coincides with the distribution of the SR protein SC35. Altogether, the results show that Trn-SR has the same localization properties as hTRN-SR2, suggesting that Trn-SR is the import receptor for Drosophila SR proteins (Allemand, 2002).
Three independent pathways of nuclear import have so far been identified in yeast, each mediated by cognate nuclear transport factors, or karyopherins. A new pathway to the nucleus has been characterized, mediated by Mtr10p, a protein first identified in a screen for strains defective in polyadenylated RNA export. Mtr10p is shown to be responsible for the nuclear import of the shuttling mRNA-binding protein Npl3p. A complex of Mtr10p and Npl3p was detected in cytosol, and deletion of Mtr10p was shown to lead to the mislocalization of nuclear Npl3p to the cytoplasm, correlating with a block in import. Mtr10p binds peptide repeat-containing nucleoporins and Ran, suggesting that this import pathway involves a docking step at the nuclear pore complex and is Ran dependent. This pathway of Npl3p import is distinct and does not appear to overlap with another known import pathway for an mRNA-binding protein. Thus, at least two parallel pathways function in the import of mRNA-binding proteins, suggesting the need for the coordination of these pathways (Pemberton, 1997).
MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85. Green fluorescent protein (GFP)-tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident. Purified Mtr10p forms a complex with Npl3p, an RNA-binding protein that shuttles in and out of the nucleus. In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)-containing protein is not. Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non-repetitive C-terminal tail. Mtr10p interacts in vitro with Gsp1p-GTP, but with low affinity. Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran-GTP plus RNA. This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran-GTP and newly synthesized mRNA (Senger, 1998).
The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. RS domain-bearing proteins do not utilize any of the known nuclear import receptors and a novel nuclear import receptor specific for SR proteins has been identified. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain-bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin ß/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family (Kataoka, 1999).
Mammalian SR proteins are currently thought to function in mRNA export as well as splicing. They contain multiple phosphorylated serine/arginine (RS/SR) dipeptides. Although SR domains can be phosphorylated by many kinases in vitro, the physiologically relevant kinase(s), and the role(s) of these modifications in vivo have remained unclear. Npl3 is a shuttling protein in budding yeast that is a substrate for the mammalian SR protein kinase, SRPK1, as well as the related yeast kinase, Sky1. Sky1p phosphorylates only one of Npl3p's eight SR/RS dipeptides. Mutation of the C-terminal RS to RA, or deletion of SKY1, results in the cytoplasmic accumulation of Npl3p. The redistribution of Npl3p is accompanied by its increased association with poly(A)+ RNA and decreased association with its import receptor, Mtr10p, in vivo. It is proposed that phosphorylation of Npl3p by the cytoplasmically localized Sky1p is required for efficient release of mRNA upon termination of export (Gilbert, 2001).
Telomerase is a ribonucleoprotein particle (RNP) involved in chromosome end replication, but its biogenesis is poorly understood. The RNA component of yeast telomerase (Tlc1) is synthesized as a polyadenylated precursor and then processed to a mature poly(A)- form. The karyopherin Mtr10p is required for the normal accumulation of mature Tlc1 and its proper localization to the nucleus. Neither TLC1 transcription nor the stability of poly(A)- Tlc1 is significantly affected in mtr10delta cells. Tlc1 is mostly nuclear in a wild-type background, and this localization is not affected by mutations in other telomerase components. Strikingly, in the absence of Mtr10p, Tlc1 is found dispersed throughout the entire cell. These results are compatible with two alternative models: (1) Mtr10p may import a cytoplasmic complex containing Tlc1 and perhaps other components of telomerase, and shuttling of Tlc1 from the nucleus to the cytoplasm and back may be necessary for the biogenesis of telomerase (the 'shuttling' model); (2) Mtr10p may be necessary for the nuclear import of some enzyme needed for the nuclear processing and maturation of Tlc1, and in the absence of this maturation, poly(A)+ Tlc1 is aberrantly exported to the cytoplasm (the 'processing enzyme' model) (Ferrezuelo, 2002).
Important progress in understanding messenger RNA export from the nucleus could be achieved by increasing the list of proteins that are involved in this process. Gbp2 has been identified as a novel shuttling RNA-binding protein in Saccharomyces cerevisiae. Nuclear import of Gbp2 is dependent on the receptor Mtr10 and the serine/arginine-specific protein kinase Sky1. Deletion of the genes encoding both of these proteins or disruption of two of the arginine/serine repeats each shifts the steady-state localization of Gbp2 to the cytoplasm. Interestingly, deletion of MTR10 only also causes an increase in poly(A)(+) RNA binding by Gbp2, suggesting a role of Mtr10 in the dissociation of Gbp2 from mRNA in the cytoplasm. The nuclear export of Gbp2 is always coupled to mRNA export and is dependent on continuous RNA polymerase II transcription and mRNA-export factors. Although GBP2 is not essential for normal cell growth, overexpression of this gene is toxic and causes a nuclear retention of bulk poly(A)(+) RNA. Together, these findings clearly show an involvement of Gbp2 in mRNA transport. In addition, as a non-essential protein, Gbp2 also has the interesting potential to be spatially or temporally regulated (Windgassen, 2003).
Messenger RNAs are transported to the cytoplasm bound to several shuttling mRNA-binding proteins. Hrb1, a novel component of the transported ribonucleoprotein complex in Saccharomyces cerevisiae, has been characterized. The protein is similar to the other two serine/arginine (SR)-type proteins in yeast, Gbp2 and Npl3. Hrb1 is nuclear at steady state and its import is mediated by the karyopherin Mtr10. Hrb1 binds to poly(A)+ RNA in vivo and its binding is significantly increased in MTR10 mutants, suggesting a role for Mtr10 in dissociating Hrb1 from the mRNAs. Interestingly, by comparing the export requirements of all three SR proteins similarities were found but also striking differences. While the export of all three proteins is dependent on the export of mRNAs in general, since no transport is observed in mutants defective in transcription (rpb1-1) or mRNA export (mex67-5), specific requirements were found for components of the THO complex, involved in transcription elongation. While both Hrb1 and Gbp2 depend on Mft1 and Hpr1 for their nuclear export, Npl3 is exported independently of both proteins. These findings suggest that Hrb1 and Gbp2, but not Npl3, might be loaded onto the growing mRNA via the THO complex components Mtf1 and Hrp1 (Hacker, 2004)
A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. This study describes the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, evidence is provided that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP (Windgassen, 2004).
Serine/arginine-rich proteins are mainly involved in the splicing of precursor mRNA. RS domains are also found in proteins that have influence on other aspects of gene expression. Proteins that contain an RS domain are often located in the speckled domains of the nucleus. The RS domain derived from a human papillomavirus E2 transcriptional activator can target a heterologous protein to the nucleus, as it does in many other SR proteins, but is insufficient for localization in speckles. By using E2 as a bait in a yeast two-hybrid screen, a human importin-ß family protein that is homologous to yeast Mtr10p and almost identical to human transportin-SR was identified. This transportin-SR2 (TRN-SR2) protein can interact with several cellular SR proteins. More importantly, TRN-SR2 can directly interact with phosphorylated, but not unphosphorylated, RS domains. Finally, an indirect immunofluoresence study revealed that a transiently expressed TRN-SR2 mutant lacking the N-terminal region becomes localized to the nucleus in a speckled pattern that coincides with the distribution of the SR protein SC35. Thus, these results likely reflect a role of TRN-SR2 in the cellular trafficking of phosphorylated SR proteins (Lai, 2000).
Serine/arginine-rich proteins are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine/serine (RS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. An importin ß family protein, transportin-SR2 (TRN-SR2) specifically interacts with phosphorylated RS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SR proteins. Using in vitro import assays, it has been shown that nuclear import of SR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, it was found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, it was shown that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles in vitro. Thus, TRN-SR2 may perhaps escort SR protein cargoes to nuclear subdomains (Lai, 2001).
Alternative splicing of precursor mRNA is often regulated by SR proteins and hnRNPs, and varying their concentration in the nucleus can be a mechanism for controlling splice site selection. To understand the nucleocytoplasmic transport mechanism of splicing regulators is of key importance. SR proteins are delivered to the nucleus by TRN-SRs, importin ß-like nuclear transporters. A non-SR protein, RNA-binding motif protein 4 (RBM4), has been identified as a novel substrate of TRN-SR2. TRN-SR2 interacts specifically with RBM4 in a Ran-sensitive manner. TRN-SR2 indeed mediates the nuclear import of a recombinant protein containing the RBM4 C-terminal domain. This domain serves as a signal for both nuclear import and export, and for nuclear speckle targeting. Finally, both in vivo and in vitro splicing analyses demonstrate that RBM4 not only modulates alternative pre-mRNA splicing but also acts antagonistically to authentic SR proteins in splice site and exon selection. Thus, a novel splicing regulator with opposite activities to SR proteins shares an identical import pathway with SR proteins to the nucleus (Lai, 2003).
SR proteins and related RS domain-containing polypeptides are an important class of splicing regulators in higher eukaryotic cells. The RS domain facilitates nuclear import of SR proteins and mediates protein-protein interactions during spliceosome assembly; both functions appear to subject to regulation by phosphorylation. Previous studies have identified two nuclear import receptors for SR proteins, transportin-SR1 and transportin-SR2. Transportin-SR1 and transportin-SR2 are the alternatively spliced products of the same gene and transportin-SR2 is the predominant transcript in most cells and tissues examined. While both receptors import typical SR proteins in a phosphorylation-dependent manner, they differentially import the RS domain-containing splicing regulators hTra2alpha and hTra2ß in different phosphorylation states. It is suggested that differential regulation of nuclear import may serve as a mechanism for homeostasis of RS domain-containing splicing factors and regulators in the nucleus and for selective cellular responses to signaling (Yun, 2003).
Search PubMed for articles about Drosophila Transportin-Serine/Arginine rich
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date revised: 12 January 2018
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