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).
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