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J. Biol. Chem., Vol. 279, Issue 34, 35788-35797, August 20, 2004
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From the
Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland and Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza, 2, I-20126 Milano, Italy
Received for publication, April 8, 2004 , and in revised form, May 28, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, members of the SR family of splicing factors, via its C-terminal alternating charge domain. This domain is also required for targeting CF Im68 to the nucleus. However, CF Im68 does not concentrate in splicing speckles but in foci that partially colocalize with paraspeckles, a subnuclear component in which other proteins involved in transcriptional control and RNA processing have been found. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Three major polypeptides of 25, 59, and 68 kDa and one minor polypeptide of 72 kDa copurify with CF Im activity from HeLa cell nuclear extract (10). Reconstitution of CF Im activity with recombinant proteins suggests that CF Im is a heterodimer consisting of the 25-kDa subunit and one of the larger polypeptides (11). All of the three larger proteins appear to be highly related in their amino acid sequence. Moreover, all of the CF Im subunits are only present in metazoan organisms. The primary sequence of the 25-kDa polypeptide contains a NUDIX motif (12). The amino acid composition of the 68-kDa protein has a domain organization that is reminiscent of spliceosomal SR proteins. Members of the SR family of splicing factors contain one or more N-terminal RNA recognition motifs (RRMs) that function in sequence-specific RNA binding and a C-terminal domain rich in alternating arginine and serine residues, referred to as RS domain, which is required for protein-protein interactions with other RS domains (13). In the 68-kDa protein, the RRM and the RS-like domain are separated by a region with high proline content (47%). SR proteins bound to specific RNA sequence elements are thought to recruit key splicing factors, thus enhancing the recognition of splice sites and controlling splice site selection in a concentration-dependent manner (for review see Ref. 14). Previous experiments have shown that preincubation of the RNA substrate with CF Im reduces the lag-phase of the cleavage reaction (11). This observation suggests that the binding of this factor to the pre-mRNA may be an early step in the assembly of the 3'-end-processing complex, such that CF Im could have a role similar to that of SR proteins in spliceosome assembly. Recent SELEX experiments identified a related set of sequences within the 3'-untranslated region of the pre-mRNA of its 68-kDa subunit to which CF Im preferentially binds (15).
In this report, we describe the analysis of the functional domains of the 25- and 68-kDa subunits of CF Im. To this end, we generated several deletion and point mutants of the two subunits. We expressed wild type and mutant proteins in heterologous systems and analyzed the purified proteins for protein-protein interactions and for RNA binding. We found that the 25-kDa subunit of CF Im interacts not only with PAP (16) but also with PABPN1. Although the amino acid composition of this subunit does not display a known RNA recognition motif, CF Im25 binds to RNA. Surprisingly, despite having an RRM, CF Im68 does not bind very strongly to RNA but requires its RRM for interaction with the 25-kDa protein instead. A yeast two-hybrid screen with the RS-like domain of CF Im68 identified members of the SR family of splicing factors, suggesting that CF Im may contribute to the coordination of splicing and 3'-end formation. Furthermore, we investigated the subcellular localization of CF Im. We found that CF Im subunits are localized to the nucleus and accumulate within a few bright foci that partially overlap with paraspeckles (17). Finally, we report that the RS-like domain is sufficient for targeting the protein to the nucleus.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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RS sense, 5'-AACAGTGGCATGCCTACATC-3'; RS antisense, 5'-CACCCAAGCTTCTACTCAATTCCATGAAGGCA-3'; ythSRs, 5'-CACCGGAATTCGAGTCCAAGTCTTATGG-3'; ythSRas, 5'-TAACCGCTCGAGCTAACGATGACGATATTC-3'; 5'-TraX, 5'-AGTCTAGAAGCGACAGCGGCGAGCA-3'; 3'-TraB, 5'-ACGGATCCTTAATAGCGACGAGGTGAGT-3'; 5'-9G8X, 5'-AGTCTAGATCGCGTTACGGGCGGTA-3'; and 3'-9G8B, 5'-ACGGATCCTCAGTCCATTCTTTCAGGA-C-3'.
Yeast Two-hybrid Screen—Yeast two-hybrid library screening and analysis were performed as described in the HybriZAP-2.1 two-hybrid protocol provided by Stratagene with the yeast strain YRG-2. The C-terminal charged domain of CF Im68 (aa 481–551) was amplified by PCR with the oligonucleotides ythSRs and ythSRas and then cloned into the EcoRI and SmaI restriction sites of the yeast vector pBD-Gal4. This construct was used to screen a pre-made HeLa cell cDNA library that was cloned into the yeast vector pAD-GAL4 (Stratagene). Reporter genes in the HybriZAP-2.1 vector system are -galactosidase (lacZ) and histidine (HIS3). The ability to express the reporter HIS3 gene was tested on SC plates lacking leucine, tryptophan, and histidine but containing 25 mM 3-aminotriazole. -Galactosidase activity was detected by filter lift assays (18). Library plasmids from positive colonies were recovered in Escherichia coli and sequenced.
Recombinant Protein Expression and Purification—For expression of recombinant GST fusion proteins in E. coli, the sequence of interest was cloned into the polylinker region of the pGDV expression plasmid (19). The coding region of CF Im25 was inserted into the NcoI and XhoI sites. To express fragments of the 25-kDa subunit of CF Im, the open reading frame was divided into three regions corresponding to amino acids 1–80, 81–160, and 161–227 that were amplified by PCR with the respective primers. The GST-25 mutant containing an internal deletion corresponding to amino acids 140–168 was obtained by digestion of the cDNA with Tth111I and Bsu36I, filling-in of the extremities, and religation. To obtain recombinant GST-U2AF subunits, the entire U2AF65 and U2AF35 open reading frames were amplified by PCR from a HeLa cDNA library with the oligonucleotides 5'-U2AF65 and 3'-U2AF65 and 5'-U2AF35 and 3'-U2AF35, respectively. The fragments were cloned into the BamHI and XhoI sites of pGEX vector (Amersham Biosciences).
The CF Im68 mutants RS, RRM, RRM/Pro, and Pro/RS were generated by PCR with the respective oligonucleotides, cloned into pGemTeasy (Promega), and sequenced by Centro Richerche Interdipartimentale Biotecnologie Innovative (Università di Padova). The fragments were excised with XmaI and BamHI and cloned into pGDV that had been linearized with the same enzymes.
E. coli BL21 (DE3) were transformed with these plasmids, and protein expression was induced with isopropyl-1-thio--D-galactopyranoside. Whereas GST-U2AF65, GST-25, and its deletion mutants were present in high amounts in the soluble fraction, soluble full-length GST-59 and GST-68 could neither be recovered from E. coli BL21 (DE3) pLysS nor from Epicurian Coli BL21 codon plus (Stratagene). In contrast, the expression of several mutant derivatives was successful. Although most of the induced proteins were in inclusion bodies, a significant amount was present in the soluble fraction. The proteins in the supernatant were bound to GST-Sepharose 4B (Amersham Biosciences) and purified according to the manufacturer's instructions.
Expression of wild type CF Im68 in baculovirus was achieved with the BAC-To-BAC baculovirus expression system (Invitrogen). The entire coding region together with 162 nucleotides of the 3'-untranslated region and eight additional adenosines was inserted into the EcoRI site of the baculovirus transfer vector pFASTBAC-Hta. Recombinant viruses were prepared by transforming the plasmid into DH10BAC-competent cells and inducing the formation of the recombinant bacmids. High molecular weight DNA was then prepared from selected E. coli clones, and 1–2 µg were used to transfect 1 x 106 Spodoptera frugiperda (Sf9) insect cells. After 2–3 days, the supernatant was recovered and 
1 ml of the virus was used to infect 2 x 106 cells that had been seeded on a 10-ml plate. The plates were again incubated at 27 °C for 3–4 days. At this stage, the cells were analyzed for protein expression by Western blot analysis. 2 ml of the resulting virus was added to a 30-ml suspension culture of Sf9 cells at a concentration of 1 x 106 cells/ml. After 4–5 days, cells were harvested, washed in PBS, and resuspended in approximately two packed volumes of lysis buffer (200 mM NaCl, 50 mM Tris, 10% glycerol, 0.02% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 0.7 µg/ml pepstatin, 0.4 µg/ml leupeptin). The lysate was centrifuged after incubation on ice for 30 min, and proteins were purified by Ni2+-nitrilotriacetic acid affinity chromatography.
GST Pull-down Experiments—0.5–1 µg of purified GST-25 protein was incubated with 100 µg of either HeLa cell nuclear extract or whole cell extracts prepared from transfected HeLa or human embryonic kidney 293 cells for 1 h at 4 °C. 50 µl of glutathione-Sepharose beads then were added and incubated for an additional 2 h. The resin was washed three times with 1 ml of IPP150. Proteins were eluted with 2x Laemmli buffer and separated by 12% SDS-PAGE. Proteins then were blotted onto nitrocellulose, and the membrane was probed with anti-CF Im68 polyclonal antibodies or anti-T7 monoclonal antibody (Novagen).
[35S]Methionine (Amersham Biosciences) was used to label proteins in a transcription/translation system (Promega). Purified GST or GST fusion proteins (500 ng) were incubated with in vitro translated proteins in 30 µl of total reaction volume. Volumes were adjusted with GST buffer (75 mM KCl, 50 mM Tris-HCl, pH 7.9, 10% glycerol, 10 nM reduced glutathione, 0.01% Nonidet P-40, 1 mM dithiothreitol, 50 µg/ml bovine serum albumin). After incubation for 1 h at 4 °C, 25 µl of glutathione-Sepharose beads were added and the volume was adjusted to 500 µl with 1x PBS, 0.01% Nonidet P-40. Incubation was continued for 1 h, and the resin was washed three times with IPP150. Proteins were eluted from the beads by the addition of 2x Laemmli buffer, separated by 10% SDS-PAGE, and visualized by autoradiography.
CF Im subunits were cloned into the BlueScript vector (KS, Stratagene), which allows in vitro transcription/translation with the TNT-coupled reticulocyte lysate system (Promega). The open reading frame of the 25-kDa subunit was cloned into BamHI and EcoRI, respectively (pBS25). The open reading frames of CF Im59 and CF Im68 were inserted into the EcoRI site of BlueScript (Stratagene). The CF Im68 mutant that carries two point mutations in the RNP2 motif (G86V,N87V) was generated with two consecutive PCR reactions with primers BclI (5'-end), Bsu36I (3'-end), RNP2 sense, and RNP2 antisense. Two internal BclI and Bsu36I restriction sites were used to substitute the wild type sequence with the PCR fragment carrying the mutations.
The SR mutant was generated by PCR with pBS68 and primers that contained the SphI (RS sense) and HindIII (RS antisense) restriction sites. The amplified fragment was inserted into pBS68 that had been digested with SphI and HindIII and resulted in a C-terminally truncated version lacking aa 482–551 of the full-length protein.
RNA Binding Assays—GST pull-down experiments were adapted from Dichtl and Keller (19). Approximately, 500 ng of GST fusion protein were incubated with 30–100 fmol of labeled RNA under cleavage conditions (20) for 30 min at 30 °C. GST-Sepharose was added and incubated for 1 h at 4 °C. The beads were washed extensively with 1x PBS, 0.01% Nonidet P-40 and resuspended in the proteinase K mixture. After 30 min, the supernatant was transferred to a fresh tube and the RNA was precipitated with ethanol and resolved on 6% denaturing polyacrylamide gels.
For UV cross-linking experiments, 20-µl reactions were set up on ice as follows. To 8 µl of premixture (2 mM dithiothreitol, 0.01% Nonidet P-40, 20 mM creatine phosphate, 2% polyethylene glycol, 0.4 mM cordycepin, 5 units/µl RNA Guard, 0.025 µg/µl creatine kinase, 1.5 mM MgCl2), either 1 pmol of CF Im25/68 and CF Im68, 5 pmol of CF Im25, or 4 pmol of purified CF Im was incubated with 200 fmol of labeled L3 pre-mRNA. These proteins had been expressed in E. coli and assembled in vitro as described previously (11). 75 mM potassium acetate and 200 fmol of RNA substrate (resuspended in water) were added, and volumes were adjusted with buffer E. Reactions were incubated for 10 min at room temperature and exposed to UV light (2 x 250 mJ, UV Stratalinker 1800, Stratagene) in a microtiter plate at room temperature. Samples were treated with 5 ng/µl RNase A (Sigma) for 1 h at 37 °C, and proteins were separated by SDS-PAGE and exposed on x-ray film.
Cell Culture and Transfections—HeLa and human embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and transfected with 1 µg of plasmid DNA/60-mm dish containing 60% confluent cells with the calcium phosphate method. The epitope-tagged 9G8 and hTra2 expression plasmids were constructed by amplifying the cDNAs isolated in the yeast two-hybrid screen with specific primers and subcloning the resulting PCR products as a XbaI-BamHI fragment into pCGTHCFFLT7 expression vector (21). Amplification was performed with Amplitaq Gold (Roche Applied Science) and oligonucleotides (5'-9G8X, 3'-9G8B, 5'-TraX, and 3'-TraB) purchased from MWG. Construction of pCG-SRp20 was described previously (22).
Indirect Immunofluorescence—Cells were either plated on glass coverslips and transfected or distributed after transfection on polylysinecoated coverslips. 24 h after transfection, cells were washed with PBS, fixed with 3% paraformaldehyde, and permeabilized with 0.2% Triton X-100 for 5 min on ice. Cells were incubated 1 h at 37 °C with the primary antibody, washed three times with PBS containing 2% bovine serum albumin, and again incubated for 1 h at 37 °C with the appropriate secondary antibody.
For localization of endogenous CF Im, polyclonal antibodies raised against CF Im68 and CF Im25 (11) were used at a 1:500 and 1:200 dilution, respectively. Colocalization studies of GFP fusion proteins and endogenous SRp20 protein were performed with a mouse monoclonal antibody (Zymed Laboratories Inc.) used as a 1:100 dilution and detected with a Texas Red-conjugated anti-mouse IgG (Molecular Probes, 1:1000) secondary antibody. SC35 was detected with a mouse monoclonal antibody (Sigma, 1:2000), and endogenous CPSF was detected with a mouse monoclonal antibody (3). Secondary antibodies were Alexa 488-conjugated anti-mouse IgG or Alexa 594-conjugated anti-rabbit IgG (Molecular Probes) used as a 1:1000 dilution.
GFP expression plasmids were constructed by cloning DNA fragments encoding full-length and mutant CF Im68 into pEGFP-C1 plasmid (Clontech). GFP-68 was obtained by digesting pBS68 with BamHI and SalI and inserting the two fragments that are generated (BamHI/SalI and SalI/SalI) into pEGFP-C1 linearized with BglII and SalI. GFP-68 deletion mutants were excised from the corresponding GST constructs with XmaI and BamHI (filled) and cloned into pEGFP-C1 linearized with XmaI and XbaI (filled).
Confocal Fluorescence Microscopy—Fluorescence microscopy of fixed cells was carried out with a Nikon YFL microscope equipped with a x60, 1.4 oil Plan-Apochromat objective or a Leica DM IRE2 confocal microscope equipped with an argon/krypton laser (488 nm) to excite GFP fluorescence and a helium/neon laser (543 nm) to excite the Alexa 594 fluorescence and a x63, 1.4 oil HCX Plan-Apochromat objective. For double-labeling experiments, images from the same focal plane were sequentially recorded in different channels and superimposed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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CF Im25 Interacts with PAP and PABPN1—The binding of CPSF and CstF to conserved sequence elements in the pre-mRNA located upstream and downstream of the poly(A) site mediates specific recognition of the polyadenylation site. As has been shown previously, the association of CF Im with the substrate leads to a faster assembly of the 3'-end-processing complex (11). Therefore, CF Im may help to recruit CPSF and CstF to the pre-mRNA. To test this hypothesis, we tested in GST pull-down experiments for the direct interaction of CF Im with CPSF and CstF. However, our results with GST-25 suggest only weak association with in vitro transcribed/translated CstF50, CstF77, and CPSF160 and no detectable interaction with the other CstF and CPSF subunits (results not shown). Therefore, if the recruitment of CstF and CPSF is facilitated by CF Im, this does not appear to be mediated by a direct interaction with the smallest subunit of CF Im.
An interaction between CF Im25 and PAP has been described previously (16). The authors isolated the 25-kDa subunit of CF Im in a yeast two-hybrid screen with the C-terminal domain of mouse PAP (aa 472–739) as a bait. In addition to PAP, we found that GST-25 interacts with PABPN1 (Fig. 2A, lane 8). A deletion of the C-terminal 226 residues of PAP did not affect its binding to GST-25 (Fig. 2A, PAPC, lane 5). Incubation of GST-25 with HeLa cell nuclear extract and subsequent Western blot analysis confirmed the interaction of GST-25 with PAP and PABPN1 (results not shown).
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The N-terminal RRM of CF Im68 Is Sufficient for the Interaction with CF Im25—To identify functional domains of the 68-kDa subunit of CF Im, we took a similar approach to that used for the characterization of CF Im25. Several CF Im68 deletion and point mutants were generated and cloned into a bacterial expression vector (Fig. 3A). Unfortunately, neither the full-length 68-kDa protein nor any of the mutants containing the proline-rich domain within the central region, with the exception of the Pro/RS fragment, could be efficiently expressed as GST fusion proteins in E. coli. In vitro translated CF Im25 was found to associate with both GST-68RRM and GST-68RRM/RS (Fig. 3B, lanes 3 and 5) but not with the GST-68RS or GST-68Pro/RS fragments (Fig. 3B, lanes 4 and 6). These results were confirmed by pull-down assays in which recombinant GST-25 was incubated with in vitro translated wild type or mutant CF Im68. As shown in Fig. 3C, both wild type CF Im68 and a C-terminal truncation lacking the RS-like domain are efficiently precipitated by GST-25 (Fig. 3C, lanes 3 and 6 respectively). Instead, the substitution of two amino acids within the RNP2 motif of the RRM of CF Im68 (G87V,N88V) abolished interaction with the 25-kDa subunit (Fig. 3C, lane 9). These results suggest that the interaction between CF Im68 and CF Im25 is mediated by the RRM and, in particular, by the RNP2 motif within the 68-kDa protein.
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In a similar way, we also mapped the RNA binding region of the 68-kDa polypeptide. As shown in Fig. 4C, the N-terminal fragment containing the RRM did not significantly bind the RNA (lanes 3–6). In contrast, both the C-terminal RS-like domain and the RRM/RS fusion polypeptides were able to efficiently pull-down the substrate (lanes 7–14). This probably reflects an ionic interaction between the positively charged RS-like domain and the RNA.
Synergistic RNA binding of CF Im25 and CF Im68 was confirmed by RNA pull-down assays with the same GST-68 proteins used before and in the presence of increasing amounts of the recombinant histidine-tagged 25-kDa subunit (Fig. 4D, His-CF Im25). The addition of the 25-kDa subunit increased the amount of RNA precipitated by the GST-RRM/RS protein (Fig. 4D, lanes 13–16) and enabled the GST-RRM fragment to pulldown RNA (Fig. 4D, lanes 3–6). This could be explained by the concomitant interaction of the 25-kDa polypeptide with the RNA and the GST-tagged RRM fragment of the 68-kDa protein. The addition of CF Im25 to the RS-like domain of CF Im68 did not increase the amount of precipitated RNA (Fig. 4D, lanes 8–11), most probably because the 25-kDa protein cannot interact with GST-68RS. Taken together, these results suggest that the 25-kDa protein and one of the larger subunits cooperate to recognize the CF Im binding sites on the pre-mRNA.
The RS-like Domain of CF Im68 Associates with SR Proteins—The C-terminal portion of CF Im68 consists of RD-, RE-, and RS-dipeptide repeats up to a total length of 60 amino acids. Similar alternating charge domains consisting of RS repeats have been described to occur in a family of splicing factors termed SR proteins. The RS domain has been shown to promote protein-protein interactions, direct subcellular localization, and, in certain situations, nucleocytoplasmic shuttling. To isolate proteins that interact with the C terminus of CF Im68, a yeast two-hybrid screen was performed with a fragment encoding amino acids 481–551 (called 68RS) as bait. A HeLa cell cDNA library was screened, and an analysis of positive colonies revealed among the strongest interactors some known members of the SR protein family, namely hTra-2 (23, 24), SRp20 (also named X16) (25), and 9G8 (26).
To verify these interactions in vivo, we used GST-68RS in pull-down assays with total extracts of human cells transfected with plasmids encoding cDNAs for hTra-2, SRp20, and 9G8 (Fig. 5A). All of the constructs encoded proteins with a bacteriophage T7 gene 10 (T7) epitope tag at their N termini, allowing the detection of the exogenous proteins with a monoclonal antibody that recognizes this epitope (see "Experimental Procedures") (21). Western blot analysis revealed that a significant fraction of hTra-2, SRp20, and 9G8 was found to associate with the RS-like domain of CF Im68 (Fig. 5, lane 3 of each panel). However, none of those SR proteins was detected if incubated with GST (lane 1), GST-25 (lane 2), GST-RRM68 (lane 4), or GST-U2AF65 (lane 5), indicating that the interaction is specific for the RS-like domain of CF Im68. The interaction with hTra-2, SRp20, and 9G8 was confirmed in vitro by GST-pull down experiments with in vitro transcribed/translated proteins (results not shown). These results indicate that CF Im68 interacts directly with a specific subset of SR proteins, raising the intriguing possibility that CF Im contributes to the definition of the last exon during the processing of intron-containing pre-mRNAs.
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Because of the similarity of CF Im large subunits and SR proteins, we wanted to determine their subcellular localization. Immunofluorescence microscopy with polyclonal antisera raised against the 25- and the 68-kDa CF Im subunits (11) revealed that endogenous CF Im is localized in the nucleoplasm with the exclusion of the nucleoli, with a granular distribution, and with concentration in few discrete foci (Fig. 6, c and c'). To confirm the staining pattern observed with the antibodies, we transiently expressed both CF Im subunits fused to the GFP. A portion of the transfected cell population expressed the GFP fusion proteins at very high levels and showed aberrant localization patterns. Therefore, we selected cells that expressed low levels of the GFP fusion proteins. In these cells, indirect immunofluorescence showed that, 20–24 h after transfection, the transiently expressed GFP-68 and GFP-25 localized exclusively to the nucleus with a diffuse pattern and, in addition, within a few distinct foci, in agreement with the localization observed with the specific antibodies (Fig. 6, b and b'). To determine whether these foci corresponded to cleavage bodies, we performed double-labeling experiments with a monoclonal antibody that specifically recognizes the 100-kDa subunit of CPSF (3). However, Fig. 7A shows that the foci detected with anti-CF Im68 do not correspond to cleavage bodies visualized by anti-CPSF100. This unexpected result prompted us to assay markers for known subnuclear compartments to clarify the nature of the CF Im foci. We used antibodies specific for p80 coilin to detect Cajal bodies (34) and for SC-35 to detect splicing speckles (35, 36). However, GFP-68 was neither localized to Cajal bodies (Fig. 7B, panels a–a'') nor to speckles (Fig. 7B, panels b–b'') but was always localized in close proximity to both of them. The observation that GFP-68 foci were adjacent to speckles prompted us to colocalize GFP-68 and the recently identified PSP1 protein (17). This protein accumulates in punctate subnuclear structures that are frequently localized adjacent to SC35 speckles and, therefore, are termed paraspeckles. Fig. 7B (panels c–c'') shows that GFP-68 and PSP1, at least partially, colocalize in paraspeckles.
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N, panel b') or carried amino acid substitutions in the RNP2 of the RRM (results not shown) were concentrated in the nucleus (excluding the nucleoli), similarly to the wild type protein (Fig. 6). All of the deletion mutants lacking the C-terminal RS-like domain localized to the cytoplasm (Fig. 8, 68RS46, panel c'; RRM and RRM/Pro, data not shown). Interestingly, a domain search with PSORT (37) reveals the presence of a putative nuclear localization signal (RRHR) located 46 amino acids upstream of the stop codon. Consistent with this prediction, the deletion of this portion of the charged domain was sufficient to prevent nuclear import of the truncated protein (Fig. 8, 68RS46, panel c'). The requirement for the RS domain for nuclear localization was confirmed by the cellular distribution of a mutant protein in which the N-terminal RRM is fused directly to the RS-like domain (Fig. 8, RRM/RS, panel d'). This protein was found both in the cytoplasm and in the nucleus. Furthermore, the fusion of the RS domain to GFP is sufficient to direct nuclear import of the chimeric protein (Fig. 8, 68RS, panel e'). These results indicate that the C-terminal charged domain may contain one or more nuclear localization signals. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The primary sequence of CF Im68 reveals the presence of two known sequence motifs: an N-terminal RRM of the RNP type and a C-terminal alternating charge domain enriched in RS, RD, and RE repeats that resembles the RS domain of spliceosomal SR proteins. Our results indicate that the RRM of CF Im68 is primarily engaged in protein-protein interaction with the 25-kDa subunit of CF Im. Although RRMs are generally considered nucleic acid binding domains, several reports have implicated them in protein-protein interactions. For example, the U2 small nuclear RNP-specific protein U2B'' was found to interact with U2A' via its RRM (38). Recently, the crystal structure of the complex between the Drosophila melanogaster Y14 and Mago proteins was determined (39). Y14 and Mago associate with spliced mRNAs and are components of the exon junction complex. Similar to CF Im25, Mago does not reveal recognizable motifs. Whereas the amino acid sequence of Y14 predicts a canonical RRM, the structure of the complex reveals that Y14 RRM is engaged in protein recognition rather than in RNA binding. A similar interaction occurs between the two subunits of the splicing factor U2AF. Human U2AF is a heterodimer composed of a 65-kDa large subunit (U2AF65) and a 35-kDa small subunit (U2AF35). It was shown that the atypical RRM of U2AF35 interacts with the proline-rich region of U2AF65 (40). In addition, U2AF65 was found to bind to the human splicing factor SF1 via its noncanonical third RRM (41). As in the case of U2AF where both subunits work in concert to recognize weak polypyrimidine tracts and promote U2 small nuclear RNP binding, we suspect that the 25-kDa protein and one of the larger subunits of CF Im cooperate to recognize the CF Im binding sites on the pre-mRNA and thus promote assembly of the 3'-end-processing complex.
The isolation of three SR proteins in a yeast two-hybrid screen with the RS-like domain of CF Im68 provides new insight into the coordination of splicing and 3'-end formation. Recognition of the 3'-terminal exons has been postulated to involve an interaction of splicing and polyadenylation factors, and a growing number of reports support this view (42–50). Because the RS domain of SR proteins mediates protein-protein interactions that are important for spliceosome assembly (51), it has been suggested that CF Im68 could interact via its C-terminal alternating charge domain with one or more SR proteins and/or SR-related splicing factors (for review see Ref. 51). Two recent reports identified CF Im as a component of purified spliceosomes (52, 53). SRp20 and 9G8 that were identified in our screen belong to the family of SR proteins that function in the constitutive splicing reaction and also as alternative splicing regulators (25, 54). Moreover, SRp20 has been shown to mediate the recognition of an alternative 3'-terminal exon by affecting the efficiency of CstF binding to the pre-mRNA (55).
hTra2 is one of the two homologues of the alternative splicing regulator of the D. melanogaster sex determination cascade. It binds to purine-rich exonic splicing enhancers (56) and was reported to promote exon 7 inclusion of the "survival of motor neuron 2" mRNA in vivo (57). Therefore, our results suggest that CF Im68 through the interaction with a specific subset of SR proteins could participate in the definition of the last exon and in the choice between alternative 3'-terminal exons. It is interesting to note that both CF Im68 and CF Im25 but not the 59-kDa polypeptide have been found in purified spliceosomes (52, 53). So far, we have been unable to demonstrate an interaction of CF Im59 with SR proteins, albeit this CF Im subunit also possesses an RS-like C-terminal domain.2
The large subunit of CF Im has a modular domain organization consisting of an N-terminal RRM, a proline-rich central region, and a C-terminal RS-like domain. Our results suggest that each of these domains contributes to the correct intracellular distribution of the protein. In particular, the RS-like domain is a major determinant for CF Im68 nuclear localization since fusion of this domain to GFP results in exclusive nuclear distribution. In contrast, the RRM does not appear to contain any nuclear targeting signal. Fusion of this region to GFP results in a diffuse distribution throughout the cell, similar to that of GFP alone. However, when the RRM is fused to the RS-like domain, the resulting polypeptide is predominantly nuclear but cytoplasmic as well. We do not know at present whether the nuclear and cytoplasmic distributions observed with this mutant protein represent incomplete nuclear import and/or incomplete retention of the protein in the nucleus. The cytoplasmic and nuclear distributions of the RRM/RS mutant could be explained by the absence of the proline-rich region that may contain a weak nuclear localization signal that is required for complete nuclear targeting. This explanation is consistent with the observation that a mutant lacking the entire RS-like domain is still partially nuclear (results not shown). Alternatively, the central proline-rich part of CF Im68 could contain a nuclear retention signal (NRS). The first NRS has been identified in human RNP C1, a non-shuttling human RNP that contains a proline-rich region, clusters of basic residues, potential phosphorylation sites for casein kinase II and protein kinase C, and a potential glycosylation site (58). Recently, NRSs have been mapped in two non-shuttling SR proteins, SC35 and SRp40 (33). The only apparent similarity in the primary sequence between the NRS regions of SC35 and human RNP C1 is within the proline-rich region. It remains to be established whether CF Im68 contains an NRS and if it is a shuttling protein.
Immunofluorescence localization studies showed that SR proteins are organized in the interphase nucleus in a characteristic speckled pattern and that their RS domains are required for targeting proteins to these structures (22, 59, 60). The presence of an RS-like domain in CF Im68 prompted us to test whether this factor localizes to splicing speckles. Localization of CF Im subunits, both with specific antibodies and as GFP fusions, showed that CF Im is distributed throughout the nucleoplasm excluding the nucleoli and, in addition, concentrates in a few discrete foci. Double-labeling experiments with specific antibodies directed against marker proteins of known subnuclear compartments revealed that CF Im foci are often located in close proximity to both Cajal bodies and splicing speckles that do not correspond to cleavage bodies. This distribution is very similar to the behavior of the PSP1, a recently described protein of unknown function (17). This protein was first identified by mass spectrometry as a nucleolar component. However, localization studies detected it in a novel type of subnuclear bodies termed paraspeckles because they are often found close to splicing speckles (17).
Only three other proteins have been reported to localize in paraspeckles in addition to PSP1. They are PSP2, p54nrb, and the polypyrimidine tract-binding protein-associated splicing factor (PSF). PSF was first identified as a factor that associates with polypyrimidine tract-binding protein and was shown to be required at an early step in spliceosome assembly (61). PSF was also identified as a component of a small nuclear RNP-free complex (SF-A) that contains U1 small nuclear RNP-specific protein A and is implicated in 3'-end processing (27). PSF and p54nrb share considerable sequence homology and have been shown to bind the C-terminal domain of the largest subunit of RNA polymerase II (32). Therefore, PSF and p54nrb might participate in coupling transcription to pre-mRNA splicing. The identification of CF Im68 as an additional component of paraspeckles suggests a possible function for these structures in the transcription and processing of pre-mRNAs.
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¶ Both authors contributed equally to the work.
|| To whom correspondence should be addressed: Dept. of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza, 2, I-20126 Milano, Italy. Tel.: 39-02-6448-3352; Fax: 39-02-6448-3569; E-mail: silvia.barabino{at}unimib.it.
1 The abbreviations used are: pre-mRNA, mRNA precursor; CPSF, cleavage and polyadenylation specificity factor; CstF, cleavage stimulation factor; CF Im, cleavage factor I; CF IIm, cleavage factor IIm; PAP, poly(A) polymerase; PABPN1, nuclear poly(A)-binding protein; RRMs, RNA recognition motifs; GST, glutathione S-transferase; GFP, green fluorescent protein; PSP1, paraspeckle protein 1; NRS, nuclear retention signal; PSF, polypyrimidine tract-binding protein-associated splicing factor; PBS, phosphate-buffered saline; aa, amino acid; RNP, ribonuclear particle.
2 S. Dettwiler, C. Aringhieri, S. Cardinale, W. Keller, and S. M. L. Barabino, unpublished observations.
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