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Molecular and Cellular Biology, December 2003, p. 8405-8415, Vol. 23, No. 23
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.23.8405-8415.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
CP RNA-Binding Proteins
Departments of Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received 10 June 2003/ Returned for modification 22 July 2003/ Accepted 29 August 2003
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ABSTRACT |
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CPs comprise a subfamily of KH-domain-containing RNA-binding proteins with specificity for C-rich pyrimidine tracts. These proteins play pivotal roles in a broad spectrum of posttranscriptional events. The five major CP isoforms are encoded by four dispersed loci. Each isoform contains three repeats of the RNA-binding KH domain (KH1, KH2, and KH3) but lacks other identifiable motifs. To explore the complexity of their respective functions, we examined the subcellular localization of each CP isoform. Immunofluorescence studies revealed three distinct distributions: CP1 and CP2 are predominantly nuclear with specific enrichment of CP1 in nuclear speckles, CP3 and CP4 are restricted to the cytoplasm, and CP2-KL, an CP2 splice variant, is present at significant levels in both the nucleus and the cytoplasm. We mapped nuclear localization signals (NLSs) for CP isoforms. CP2 contains two functionally independent NLS. Both NLSs appear to be novel and were mapped to a 9-amino-acid segment between KH2 and KH3 (NLS I) and to a 12-amino-acid segment within KH3 (NLS II). NLS I is conserved in CP1, whereas NLS II is inactivated by two amino acid substitutions. Neither NLS is present in CP3 or CP4. Consistent with mapping studies, deletion of NLS I from CP1 blocks its nuclear accumulation, whereas NLS I and NLS II must both be inactivated to block nuclear accumulation of CP2. These data demonstrate an unexpected complexity in the compartmentalization of CP isoforms and identify two novel NLS that play roles in their respective distributions. This complexity of CP distribution is likely to contribute to the diverse functions mediated by this group of abundant RNA-binding proteins. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies from our laboratory and others have focused on the structures and actions of a subfamily of RNA-binding proteins, the CPs (31, 39). These proteins, also referred to as PCBPs (17) and hnRNP Es (34, 58), contain a triplication of the KH domain (43, 69). The 70-amino-acid KH domain comprises a triple-ß-sheet platform supporting three -helical segments (35, 36, 50, 51). Cocrystal structures reveal that the KH domain can interact in a highly specific manner with four to five contiguous bases in a target RNA (5, 27). Two KH domain subtypes have been identified: the type 1 KH domain (e.g., KH3 of hnRNP K) has a C-terminal ß extension, and the type 2 KH domain (e.g., ribosomal protein S3) contains an N-terminal ß extension (21). The KH domains in the CPs are type 1 (40). KH domains are often represented in proteins in multiple copies. Since each KH domain has the potential to independently interact with a target RNA sequence, the complexity and specificity of RNA interaction for these proteins can be quite high (66, 74; our unpublished data).
Our laboratory has focused on the role of CPs in mRNA stabilization. These studies have defined a cytosine (C)-rich cis-acting stability element within the 3' untranslated region (UTR) of the human (h) -globin mRNA that serves as a binding site for CP both in vitro and in vivo (28, 32, 83). Studies in our laboratory and others have identified closely related CP binding sites in the 3' UTRs of additional highly stable mRNAs (7, 10, 25, 61, 72, 86). In the case of the h-globin mRNA, the RNP "-complex" in the 3' UTR appears to represent a 1:1 interaction of CP with the C-rich motif (8), although there is evidence that additional proteins may also bind at this site (30, 63, 84, 85). These data suggest that the -complex may represent a widely distributed and general determinant of mRNA stabilization (25).
The CPs involved in -complex assembly can represent several isoforms (8, 31, 39). Whether these various CP isoforms are performing unique functions or are redundant in their actions is not known. The functions mediated by CP RNP complexes are in fact quite diverse (for review, see reference 41). As mentioned above, assembly of the 3' UTR -complex in -globin mRNA mediates mRNA stabilization (32, 82, 83). This stabilization may reflect direct steric protection of the 3' UTR from endonucleolytic attack (79), and/or it may reflect protection of the poly(A) tail from rate-limiting decay via interactions in cis between the bound CP and the poly(A)-binding protein (30, 49, 78). CPs also mediate translational controls. An array of CP binding sites within the 3' UTR of the 15-lipoxygenase mRNA has been linked to developmentally regulated translational repression during erythroid maturation (56-58). In contrast, association of CP with the 5' UTR of the polio viral RNA serves as an enhancer of internal ribosome entry site-mediated translation (2, 3). CP binding within the 3' UTR has also been implicated in the activation of maternal mRNA translation in early embryonic development in Xenopus via control of cytoplasmic polyadenylation (59). Additional systems are reported to involve CP binding in the control of various aspects of mRNA expression (63, 84, 85; reviewed in reference 41). Thus, the targets and actions of the CPs are quite diverse and may reflect the actions of one or more of the defined CP isoforms.
CP isoforms are encoded by four unlinked loci in the human and mouse genomes: PCBP1, PCBP2, PCBP3, and PCBP4 (38, 39, 75) (see also Fig. 1, left). Each locus has been mapped, sequenced, and characterized for mRNA structure (38, 39). A total of five major CP isoforms have been identified in human or mouse tissues: CP1, CP2, CP3, CP4, and a major CP2 splice variant, CP2-KL, that differs from CP2 by the exclusion of a 31-amino-acid segment in the region between the KH2 and KH3 encoded by a single exon (exon 8a) (17, 38, 39). These proteins are broadly expressed in human and mouse tissues and demonstrate polyC-binding specificity (34, 38, 39; unpublished observations). CP1 and CP2 share the highest level of amino acid sequence similarity at 89% (75), CP3 is more divergent, and CP4 is the most distantly related (52% divergence from CP2 [39]). Each protein contains three similarly spaced KH domains; two KH repeats are located in the N terminus followed by a nonconserved region of variable length, and the third KH domain is located at the C terminus. Posttranslational modifications may regulate the binding of CPs to RNA. For example, phosphorylation of CP1 and CP2 results in a marked decrease in RNA-binding activity (34). An additional major determinant of CP isoform function may relate to subcellular localization. Although the defined roles of CP in mRNA stability and translational control suggest a cytoplasmic localization, prior attempts to sublocalize CPs did not resolve the question of whether CP is in fact nuclear or cytoplasmic (17, 18).
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In the present study each of the five major isoforms was individually assessed for subcellular localization, and the signals underlying their nuclear versus cytoplasmic distribution were defined. These data demonstrate an unexpected complexity in CP isoform compartmentalization based on a novel set of nuclear localization motifs.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CP2-KL, CP3, and CP4A, PCR-amplified fragments corresponding to the entire coding region of respective genes or subfragments were ligated into plasmid vectors as XhoI-XbaI fragments. Given the internal XbaI site for CP1 and CP2 and compatibility between XbaI and SpeI ends, CP1/2 full-length coding regions were cloned as XhoI-SpeI fragments into same parental vector. pB1005 (a gift from S. Smale, University of California at Los Angeles) (22), pGBD-CP1 and pGBD-CP2 (gifts from M. Kiledjian, Rutgers University) (30), and pET-CP3 and pET-CP4A were used as PCR templates to obtain a series of myc-tagged Pk-CP fusion plasmids: myc-Pk-CP2-KL, myc-Pk-CP1, myc-Pk-CP2, myc-Pk-CP3, and myc-Pk-CP4A, respectively. Deletion mutants of myc-Pk-CP1, myc-Pk-CP2, and myc-Pk-CP2/KL were generated by targeted PCR. The corresponding primers contained 5' XhoI site and a 3' XbaI site, and the amplified fragments after restriction nuclease digestion were inserted into myc-Pk. Deletion mutants contained the following CP amino acid sequences. For the N-terminal deletion series (Nd), mutants contained sequences from amino acids 17 to 331, 63 to 331, 101 to 331, 150 to 331, 257 to 331, and 306 to 331 of CP-KL (see Fig. 4A and B) and 284 to 356 of CP1 (see Fig. 7). For the C-terminal deletion series (Cd), mutants contained sequences from amino acids 1 to 17, 1 to 63, 1 to 101, 1 to 150, 1 to 257, and 1 to 306 of CP2-KL (see Fig. 4C and D) and 1 to 150 of CP1 (see Fig. 7). For the internal segment series (Is), mutants contained sequences from amino acids 150 to 256 and 257 to 306 of CP2-KL (see Fig. 5), 151 to 290, 151 to 228, 229 to 290, 270 to 290, 280 to 290, and 270 to 279 of CP-2 (see Fig. 6A and B), 291 to 339, 291 to 334, 318 to 339, 332 to 339, and 328 to 339 of CP-2 (see Fig. 6C and D), and 151 to 283 of CP-1 (see Fig. 7).
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CP2 in Pk-CP2 were mutated by using a QuickChange kit (Stratagene) to S and G, respectively. myc-Pk-CP1/
NLSI, myc-Pk-CP2/NLSI, and myc-Pk-CP2/NLSI/mutNLSII were constructed by spliced-overlap extension by using myc-Pk-CP1, myc-Pk-CP2, and myc-Pk-CP2/mutNLSII as templates. Two segments of a corresponding template gene were PCR amplified independently and then fused in a subsequent reaction. The amplified fragments were inserted as XhoI-SpeI fragments into the myc-Pk vector.
Cell culture and transfection.
HeLa cells were cultured in Dulbecco modified Eagle medium (Gibco-BRL) supplemented with 10% fetal bovine serum, 100 µg of streptomycin per ml, and 100 U of penicillin per ml. Cells were seeded into Falcon 2 chamber tissue culture glass slides (Becton Dickinson Labware, Franklin Lakes, N.J.) at a density of 1 x 105 to 2 x 105 20 h prior to transfection. Transfection of cells was performed by calcium phosphate transfection (5 Prime
3 Prime, Inc., Boulder, Colo.). Medium containing the DNA mixture was removed 18 to 20 h after transfection and replaced with fresh medium. The time when the DNA was added was considered T0, and cells were fixed for immunofluoresence at 24 to 40 h.
Microscopy. Indirect immunofluorescence microscopy was carried out as described previously (9). After transfection, the cells were washed in 1x phosphate-buffered saline (PBS) and fixed in 1x PBS plus 3% paraformaldehyde (EM Science, Gibbstown, N.J.) for 30 min at room temperature. After fixation the cells were washed in PBS, permeabilized by the addition of 1x PBS-10% goat serum-1% Triton X-100 at room temperature for 10 min, and washed in 1x PBS. The cells were then blocked in antibody blocking-incubation buffer (ABB; 1x PBS, 10% goat serum, 0.1% Tween) for 60 min at room temperature prior to primary antibody incubation. Monoclonal anti-myc antibody 9E10 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) was then diluted 1:1,000 in ABB and applied to the cells at room temperature for 60 min. The cells were then washed and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) diluted at 1:100 in ABB for 60 min. The cells were then washed again, and glass slides were mounted with mounting medium (ProLong Antifade kit; Molecular Probes, Eugene, Oreg.) and sealed with nail polish.
For detection of endogenous CPs and for colocalization experiments, cells were fixed in 1x PBS plus 2% paraformaldehyde for 15 min at room temperature and incubated serially with anti-CP antibodies (FF1, FF2, and FF3) (8) and with a mouse monoclonal anti-SC35 antibody (1:150; Pharmingen, San Diego, Calif.), followed by incubation with FITC-conjugated goat anti-rabbit and Texas red (TX)-conjugated anti-mouse antibodies (1:100; Kirkegaard & Perry) in ABB for 60 min. Samples were examined by using a Leitz DMR microscope (Leica GmbH, Wetzlar, Germany), and images were captured by using a Hamamatsu color chilled 3CCD camera (model C5810; Hamamatsu Corp., Bridgewater, N.J.). Image analysis was performed by using Adobe Photoshop software (Adobe Systems, Inc., San Jose, Calif.). Optical sections were obtained by using a confocal laser scanning microscope (Leica). Fluoview was operated at excitation wavelengths of 488 nm (FITC) and 568 nm (TRITC [tetramethyl rhodamine isothiocyanate]) from an argon-krypton laser. Fluorescent signals of both fluorochromes were recorded simultaneously by two detectors.
Gel electrophoresis and immunoblotting. HeLa cells were electroporated with 40 µg of plasmid DNA per 100-mm petri dish. Resuspended cells were harvested 32 h posttransfection by scraping in 0.5 ml of Laemmli sodium dodecyl sulfate sample buffer. Samples were analyzed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis. Monoclonal anti-myc antibody 9E10 (Santa Cruz Biotechnology) at a 1:1,000 dilution and horseradish peroxidase-conjugated anti-mouse antibody (Boehringer Mannheim Corp., Indianapolis, Ind.) at a 1:5,000 dilution were used to detect fusion proteins, and the complexes were visualized with an enhanced chemiluminescence substrate (SuperSignal Substrate; Pierce, Rockford, Ill.).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CP proteins are present in both nucleus and cytoplasm.
Initial studies of CP subcellular localization focused on the most abundantly expressed isoforms: CP1, CP2, and CP2-KL (Fig. 1). HeLa cells were stained with antisera specific to CP1 and to CP2 (antisera FF1 and FF2, respectively) and a third antibody raised against an epitope common to CP2 and CP-2KL but absent from CP1 (antisera FF3) (8). The specificities of these antisera were confirmed by Western blotting (8). In previous studies we demonstrated that the first two antisera, when used against mouse and human cell extracts, recognized a single band (CP-1 and CP-2, respectively), whereas the third antiserum recognized a band comigrating with CP-2 and an additional, smaller band. The size of this additional band was consistent with that of CP2-KL. The first two antisera (FF1 and FF2) revealed exclusive nuclear localization of CP1 and CP2. In contrast, the third antisera (FF3) revealed dual nuclear and cytoplasmic staining. Since full-length CP2 is exclusively nuclear (FF2 staining), this dual-staining pattern with the FF3 antibody indicates that at least a portion of the CP2-KL is cytoplasmic (see below for further analysis). Direct analyses of endogenous CP3 and CP4 isoforms were similarly attempted. However, in these two cases, the isoform-specific antisera failed to give unambiguous signals. The distributions of these proteins were addressed by detection of epitope-tagged fusion proteins (see below). We conclude from these data that the major isoforms of CP have distinct subcellular distributions.
CP1 is enriched in nuclear speckles.
The intracellular localization of endogenous CP1 and CP2 were further characterized by confocal microscopy (Fig. 2). Staining with the CP1-specific antibody revealed that the nuclear protein was concentrated in clusters (Fig. 2, upper panel). These sites were consistent in size and number with nuclear speckles. To confirm this assignment, cells were double immunostained for CP1 and splicing factor SC35 (14, 15, 73). The SC35 immunostaining revealed the same distribution as the CP1 stain, although it was somewhat more focused. A merge of the two signals clearly demonstrated significant colocalization of CP1 and SC35 in nuclear speckles. Confocal analysis with antisera specific to CP2 (FF2) confirmed the nuclear-restricted pattern but, in contrast to the CP1, the CP2 staining was particulate and diffuse rather than speckled. Merging the CP2 and SC35 stains failed to demonstrate significant enrichment of CP2 in the speckles (Fig. 2, lower panel). Thus, confocal analysis confirmed the nuclear localization of CP1 and CP2 and revealed a selective concentration of CP1 in nuclear speckles.
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CP isoforms reveals three distribution patterns.
Localization of CP1 and CP2 to the nucleus was unexpected since these proteins lacked identifiable nuclear import signals (53). In addition, prior studies had identified poly(C)-binding activity in cytosolic extracts (31, 80). Thus, it was likely that the cytosolic poly(C)-binding activity might reflect CP2-KL, CP3, and/or CP4 content. To explore these possibilities, full-length open reading frames (ORFs) encoding each of the five major CP isoforms were isolated and fused in frame to the ORF encoding the 55-kDa Pk and an N-terminal myc epitope tag (Fig. 3A). The high molecular weight of the myc-Pk-CPs fusion proteins prevents nuclear entry by passive diffusion; their nuclear sublocalization should accurately reflect active transport. Control constructs included the myc-Pk tag alone and the myc-Pk tag fused to a known NLS. The resultant cDNAs were expressed in transfected HeLa cells. Cell extracts were analyzed by immunoblotting to confirm size, amount, and immunoreactivity of each expressed fusion protein (Fig. 3B). Localization of each fusion protein was then determined by immunostaining with a myc epitope monoclonal antibody (Fig. 3C). myc-Pk was appropriately restricted to the cytoplasm and was efficiently targeted to the nucleus when fused to the 18-amino-acid bipartite basic NLS from hnRNP K (Pk-NLS; Fig. 3C) (64). The recombinant myc-Pk-tagged CP1 and CP2 proteins were both confined to the nucleus. This was consistent with the analysis of the native proteins (Fig. 1 and 2). Analysis of the epitope-tagged CP2-KL demonstrated that this isoform is present in both the nucleus and the cytoplasm. In contrast, the CP3 and CP4 fusion proteins were both confined to the cytoplasm. These data confirmed and extended the analysis of endogenous proteins and demonstrated three distinct patterns of nuclear/cytoplasmic compartmentalization: CP1 and CP2 are predominantly nuclear, CP2-KL is both nuclear and cyoplasmic, and CP3 and CP4 are cytoplasmic.
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CP1, CP2, and CP2-KL isoforms are all present in the nucleus. However, inspection of the primary sequences failed to reveal matches with the bipartite-basic or simian virus 40 large T-type NLS (11), the A1 M9 domain (45, 67), or other NLSs reported at lower frequencies (53). The sequence determinant(s) responsible for the nuclear localization therefore appeared to represent novel structures and were mapped by a functional assay (12, 13, 45). CP2-KL was used for initial mapping since its presence in both compartments made it the most sensitive to perturbations in sorting signals. Sets of nested N-terminal and C-terminal deletions were constructed for CP2-KL; the termini of the deletions were designed to coincide with the termini of the KH domains (Fig. 4A and C). The first N-terminal deletion [Nd(17-331)] eliminated much of the cytoplasmic accumulation seen in the native CP2-KL but maintained the nuclear accumulation (Fig. 4B; compare with Fig. 3C). Each of the next four deletions maintained the accentuated nuclear accumulation of the fusion protein. Remarkably, nuclear localization was entirely lost subsequent to the sixth and most extensive deletion, Nd(306-331). These data suggested that there is an NLS encoded within the C-terminal 74 amino acids [Nd(257-331)]. A potential role of the extreme N terminus of CP2 in nuclear export remains to be rigorously tested.
NLS activity was next mapped by using a reciprocal set of C-terminal deletions (Cd) of the myc-Pk-CP2-KL fusion protein (Fig. 4C and D). The first two deletions failed to alter the balance of nuclear versus cytoplasmic accumulation of the CP2-KL [Cd(1-306) and Cd(1-257)]. However, the more extensively truncated proteins localized exclusively in the cytoplasm [Cd(1-150), Cd(1-101), Cd(1-63), and Cd(1-17)], indicating that there is an NLS activity located between amino acids 150 and 257.
The combined results of the N- and C-terminal truncation sets could not be reconciled on the basis of a single NLS. The most parsimonious model comprised two separate NLSs: one located in the region between KH2 and a second located within KH3 itself. This model of two distinct NLSs was tested.
Identification of two independent NLS motifs in CP2.
Based on the results of the N-terminal and C-terminal CP2-KL deletion studies, the regions between KH2 and KH3 [Is(150-256)] and the region encompassing KH3 through the C terminus of the CP2-KL protein [Is(257-306)] were individually tested for NLS function (Fig. 5). The segments from amino acids 150 to 256 and amino acids 257 to 306 each individually resulted in nuclear localization of the fused myc-Pk reporter. These data confirmed that CP2-KL contains at least two separable and independent NLSs: NLS I, located in the region between KH2 and KH3, and NLS II, located within the third KH domain.
Since neither NLS I nor NLS II contained a recognizable nuclear localization motif, we mapped the region between KH2 and KH3 at a higher resolution. To maximize the information from these studies, the mapping was done on CP2 that contains the additional 31-amino-acid intra-KH2/KH3 segment lacking in CP2-KL. A nested set of six subfragments of this region was generated (Fig. 6A). Each fragment was fused to the myc-Pk ORF and expressed in HeLa cells. As shown in Fig. 6B, a 10-amino-acid fragment located just N terminal to KH3 domain (i.e., from amino acids 270 to 279) was sufficient for NLS function. In a similar fashion, a set of six nested fragments representing the C terminus of the CP2 KH3 domain were tested for NLS function (Fig. 6C). The expression patterns of these fusion proteins were sufficient to localize NLS II activity to a 12-amino-acid fragment at the C terminus of the KH3 domain (Fig. 6D). Thus, two short peptides derived from CP2 could independently target a fusion protein to the nucleus.
Selective conservation of NLS I in CP1.
The sequences corresponding to the two NLS in CP2 were compared to the other CP isoforms. The sequence of NLSI in CP2 is fully conserved in CP1 (Fig. 7A). In contrast, the region corresponding to the CP2 NLS II contained two divergent residues in CP1, and even greater divergence was noted for the cytoplasmic CP3 and CP4 (Fig. 7B). The functional conservation of NLS motifs in CP1 was tested. CP1 was divided into three segments (Fig. 8A). Each segment was fused to myc-Pk, and the distribution of the expressed protein was determined in transfected cells (Fig. 8B). The fragment encompassing the region between KH2 and KH3, Is(151-283), directed recombinant protein transport to the nucleus. In contrast, the fragment encompassing KH3 and the C terminus, Nd(284-356), lacked NLS function. To clarify the functional impact of these two amino acid substitutions on NLS II function, the CP1 peptide corresponding to NLS II was directly tested by fusing it to the myc-Pk reporter. The expressed fusion protein was limited to the cytoplasm (Fig. 6C and D). Similarly, the corresponding regions of CP3, CP4, and hnRNP K (Fig. 7B) were all incapable of directing myc-Pk reporter to nucleus (data not shown). These data demonstrate that CP1 carries a single NLS (NLS I) and that CP2 and CP2-KL contain two independently functioning NLSs (NLS I and NLS II). In contrast, CP3 and CP4 lack effective NLS determinants.
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CP1 and CP2 proteins (Fig. 9). NLS I (amino acids 262 to 271) was deleted from CP1 (Fig. 9A). The resulting myc-Pk fusion protein, CP1/NLS I, was restricted to the cytoplasm of transfected HeLa cells (Fig. 9B). Thus, deletion of NLS I resulted in the loss of nuclear accumulation.
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CP2, they were inactivated separately and in combination (Fig. 9C). myc-Pk-CP2/NLSI encoded an CP2 lacking the NLS I (residues 270 to 279). myc-Pk-CP2/mutNLSII contains two amino acid substitutions at positions 331 (T-S) and 332 (D-G) that inactivate NLS II function (Fig. 8). The third recombinant, myc-Pk-CP2/NLSI/mutNLSII, lacks both NLSs. Each protein was expressed in HeLa cells (Fig. 9D). As expected, myc-Pk-CP2 localized exclusively in the nucleus. Deletion of NLS I (myc-Pk-CP2/NLSI) resulted in some redistribution of the protein to the cytoplasm, but it was not sufficient in the context of the otherwise-intact CP2 to fully block nuclear localization. Mutation of NLS II alone (myc-Pk-CP2/mutNLSII) also caused some redistribution of the protein to the cytoplasm, although the majority of the protein remained in the nucleus. In contrast, the combined inactivation of both NLS I and NLS II (myc-Pk-CP2/NLSI/mutNLSII) resulted in exclusive redistribution of CP2 to the cytoplasm. These observations indicate that the single NLS I is both necessary and sufficient for the nuclear localization of CP1, whereas two distinct NLSs are present in CP2 and appear to play redundant roles in protein transport. |
DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CPs comprise a highly abundant subset of RNA-binding proteins. The four dispersed PCBP loci encode five distinct CP isoforms. These proteins play prominent roles in posttranscriptional control of cellular and viral mRNAs. To what extent CP isoforms overlap in functions and/or mediate specific controls is now under study. As a step in this direction, we have defined the intracellular localizations of five major CP isoforms. In the process of these studies, we have identified a novel set of NLSs that contribute their respective distributions.
CP1 and CP2 localize predominantly to the nucleus. This conclusion is based on immunofluorescence analysis of endogenous proteins with isoform-specific antisera, as well as epitope-tagged fusion proteins expressed in cells. The nuclear localization of these two CP isoforms can be compared to two prior data sets. In one study, mCBP (mouse homolog of CP2) appeared to be predominantly nuclear (17), whereas a second study reported cytoplasmic localization of PCBP1 (CP1) and PCBP2 (CP2) (18). Our present data agree with the first study. To try to clarify the discrepancy with the second cited study, we repeated analyses by using the fixation technique, cells, and staining protocols specified in that study, including the use of the same antibody preparations (gifts from R. Andino). Despite these efforts, we were unable to resolve the conflicting data since CP1 and CP2 were consistently detected only in the nuclear compartment.
Although both CP1 and CP2 are localized to the nucleus, their respective distributions are not identical. CP1 is selectively concentrated in nuclear speckles, whereas CP2 is more diffusely distributed (Fig. 2). Speckles, or interchromatin granule clusters (IGCs) (reviewed in references 33 and 70), represent sites at which splicing factors are concentrated prior to assembly on newly synthesized transcripts (26, 29, 48). Recently, Mintz et al. biochemically purified IGCs. Consistent with the present study, CP1 was specifically identified as one of 75 IGC-associated proteins (47). Association of other CP isoforms with IGCs was not observed. On the other hand, Funke et al. presented evidence for in vivo interaction between CP2-KL and splicing factor 9G8 (17). We were not able to detect convincing data that support a selective enrichment of CP2/CP2-KL in speckles. Although it is possible that the interaction with 9G8 occurs outside of speckles, further studies are needed to clarify this issue. The localization of CP1 to nuclear speckles raises the possibility that this particular CP isoform has a role in pre-mRNA splicing. This function is supported by the recent finding that interference with nuclear CP activity via targeted RNA decoys blocks efficient splicing of the h-globin transcript (D. Eastmond, J. Kong, and S. A. Liebhaber, unpublished data). Thus, nuclear localization and the function of CPs is supported by a number of lines of evidence and is strengthened by the current data set.
The structural basis for the distinct subcellular compartmentalization of the various CP isoforms is undoubtedly complex. The present study focuses on determinants of CP nuclear localization. Two functional NLSs were identified in CP2, and one of these is conserved in CP1. Remarkably, the newly identified NLS I and NLS II have no apparent similarity to each other and bear little or no resemblance to NLSs described in the literature. Specifically, they lack similarity to either the bipartite-basic type or the simian virus 40 large T-type NLSs or to shuttling signals found in hnRNP A1 (45, 67), hnRNP K (46), HuR (16, 62), and human immunodeficiency virus type 1 Rev (44, 71). Protein basic local alignment search tool (BLAST) (1) searches optimized for searching for small sequences (i.e., 1, "search for short nearly exact matches," matrix PAM30; gap penalties: existence, 9; extension, 1) failed to identify other proteins with significant primary sequence homology. Although it is unlikely that these two NLSs are unique to the CPs, they do appear to be uncommon. The presence of two independent NLSs in one protein is not unprecedented. As an example, hnRNP K contains a classical NLS at the N-terminal end and KNS (i.e., hnRNP K nuclear shuttling domain) between the KH2 and KH3 RNA-binding domains (46). It is interesting that hnRNP K does not contain the two NLSs in the closely related CP1 and CP2 (Fig. 7).
NLSs can be grouped on the basis of both transport pathways and structure. A subset of NLSs described in the literature demonstrates transcription-dependent transport activity (45, 67, 81). To determine whether the NLSs found in CP1 and CP2 fall in this functional category, we transfected cells with myc-Pk-tagged CPs and then treated the cells with either actinomycin D or 5,6-dichlororibofuranosylbenzimidazole (DRB). These treatments failed to result in any change in intracellular localization of the endogenous or epitope-tagged CP1 or CP2 (data not shown). In addition, the identified NLS I and NLS II sequences do not match to the previously described importin- and importin-ß recognition signals (23, 60, 76). It remains to be determined whether the apparently novel NLSs of CP1 and CP2 represent noncanonical binding sites of the currently known transport receptors, or whether they interact with novel factors of the intracellular transport.
The distribution of CP2-KL in both nuclear and cytoplasmic compartments is of particular interest because this protein comprises the major CP isoform in certain cell types (8, 38, 39). The structure of the nuclear CP2 differs from that of CP2-KL by the inclusion of a 31-amino-acid alternatively spliced segment located between the KH2 and KH3 RNA-binding domains (see Fig. 1). It is formally possible that this 31-amino-acid segment encodes an additional NLS. However, this appears unlikely since this isolated segment is unable to localize a fused myc-Pk to the nucleus (Fig. 6B). A more plausible model would be that this segment encodes a nuclear retention signal that anchors CP2 in the nucleus. The presence of a nuclear retention signal in hnRNP C proteins establishes a precedent for this model (52).
Visual examination of the CP amino acid sequences reveals a candidate N-terminal Leu-rich motif between that may serve a direct nuclear export function. Although we observed that its deletion shifted CP2-KL localization to the nuclear compartment (Fig. 4A and B), the direct role of this sequence in intracellular trafficking of CP proteins remains to be fully tested. The presence of nuclear export signal and NLS motifs in CP1 and CP2 would support a shuttling activity of these proteins (see below).
The colocalization of NLS II with the KH3 RNA-binding domain of CP2 suggests a possible interplay between RNA-binding domain and subcellular transport. CP2 may initially bind to target mRNAs in the nucleus during transcription and/or processing. An initial association of CPs with target mRNAs in the nucleus is supported by the recent finding that expression of RNA decoys to CP selectively in the nucleus results in a block in both nuclear (splicing) and cytoplasmic (translation and stabilization) activities (40; Eastmond et al., unpublished). Binding of CP to a target mRNA in the nucleus may block NLS II which is located within an RNA-binding (KH) domain. This might facilitate redistribution of the CP-RNP complex to the cytoplasm because both NLS I and NLS II are needed for efficient nuclear localization (Fig. 9). Once in the cytoplasm, the cytoplasmic function(s) of the CP2-RNP complex can be achieved; the CP2 may then dissociate from the cytoplasmic mRNA. The newly exposed NLS II, in conjunction with NLS I, could then mediate efficient reimportation of CP2/2-KL to the nucleus. A similar model of RNP cycling via reversible NLS function has been suggested for HIV Rev (23). The proposed reversible RNA association of CPs with target mRNAs may be further facilitated by posttranscriptional modifications (34). CP may thus bind to a target mRNA in the nucleus, facilitate mRNP export, have an impact on cytoplasmic control over mRNA stability and/or translation, and then return to the nucleus. Given the present delineation of CP localization and underlying NLS motifs, these models can now be further explored.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grant RO1 HL65449 and by the generosity of the Doris Duke Foundation.
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FOOTNOTES |
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