Nuclear targeting determinants of the far upstream element binding protein, a c-myc transcription factor

Abstract

FUSE binding protein (FBP) binds in vivo and in vitro with the single-stranded far upstream element (FUSE) upstream of the c-myc gene. In addition to its transcriptional role, FBP and its closely related siblings FBP2 (KSRP) and FBP3 have been reported to bind RNA and participate in various steps of RNA processing, transport or catabolism. To perform these diverse functions, FBP must traffic to different nuclear sites. To identify determinants of nuclear localization, full-length FBP or fragments thereof were fused to green fluorescent protein. Fluorescent-FBP localized in the nucleus in three patterns, diffuse, dots and spots. Each pattern was conferred by a distinct nuclear localization signal (NLS): a classical bipartite NLS in the N-terminal and two non-canonical signals, an α-helix in the third KH-motif of the nucleic acid binding domain and a tyrosine-rich motif in the C-terminal transcription activation domain. Upon treatment with the transcription inhibitor actinomycin D, FBP completely re-localized into dots, but did not exit from the nucleus. This is in contrast to many general RNA-binding proteins, which shuttle from the nucleus upon treatment with actinomycin D. Furthermore, FBP co-localized with transcription sites and with the general transcription factor TFIIH, but not with the splicing factor SC-35. Taken together, these data reveal complex intranuclear trafficking of FBP and support a transcriptional role for this protein.

INTRODUCTION

Increasing evidence indicates that many proteins are bi- or even multi-functional; sometimes the different roles played by a single molecule are logically connected, while other times the roles appear to be independent. Intrinsic to the reutilization of such proteins for different intracellular or extracellular processes is the capacity to deliver the right amount of each component to the correct location at the appropriate time (1,2). Even within a subcellular compartment such as the nucleus, a single protein may participate in several processes, and hence must be sublocalized to the correct place. Proteins larger than 20–40 kDa require nuclear localization signals (NLS) for import into the nucleus (3–6). Two canonical types of NLS are most frequently employed to direct the proteins required for replication, transcription and RNA processing and other nuclear processes to their appropriate targets. The first ‘core’ NLS is comprised of a hexapeptide including four or more arginine and lysine residues, flanked by acidic residues or the helix-breaking residues, proline and glycine (4,7). The second common NLS is constituted of two clusters of basic amino acids separated by ∼10 non-basic residues (4,7). Other sorts of NLSs are associated with ribosomal proteins and hnRNPs. For example the M9, KNS and HNS sequences target hnRNP A1, hnRNP K and HUR, respectively, to the nucleus (8–10).

The FUSE binding protein (FBP), named for its interaction with the far upstream element (FUSE) found upstream of the c-myc gene, has been dissected into three functional domains (11,12). Transactivation is conferred by the C-terminus of FBP (FBPC) through a tyrosine-activating sequence repeated three times. In contrast, the N-terminus (FBPN) represses transcription in cis and in trans. These effector domains of FBP interact directly with components of the transcription machinery in vivo and in vitro (13). The central DNA binding domain (FBPCD) is composed of four KH-motifs, each followed by an amphipathic helix. Replication defective adenoviruses expressing the central domain of FBP interfere directly with c-myc expression via the FUSE; similar viruses expressing antisense FBP also shut off c-myc transcription (14). Beyond a role in transcription, some reports suggest that FBP or its close relatives FBP2 (KSRP) and FBP3 may interact with certain RNA molecules and participate at various steps in transcription, or in RNA processing, transport or catabolism, in the nucleus or in the cytoplasm (15–18). If truly multi-functional, then FBP must traffic to assorted sites associated with different processes. To glean more insight into the potential for FBP to play multiple roles, a series of experiments were conducted to study the localization and targeting of FBP within intact cells.

MATERIALS AND METHODS

DNA constructs

An FBP cDNA containing the entire 644 amino acid open reading frame cloned into the mammalian expression vector pcDNA1 was digested by HindIII and then ligated into pEGFP-C2 (Clontech). Similarly, four deletion mutants of FBP were excised from pcDNA1FBPN, FBPcd, FBPC and FBPΔcd with HindIII and cloned into the GFP vector. Other progressive deletions for mapping NLSs were generated by digesting DNA fragments with appropriate restriction endonucleases. The point mutations in tyrosine motif YM3 were generated from their pcDNA1 parent (12).

Cells culture, transfections and fluorescence microscopy

HeLa cells were grown on cover slides overnight in Dulbecco’s modified minimal essential medium (DMEM) containing 10% fetal calf serum. Transient transfections were performed in HeLa cells using LipoFectamine plus (Life Technology) with 2 µg expression plasmids as described previously (19). Sixteen hours after transfection, cells were fixed in 4% paraformaldehyde and washed twice in PBS. The slides were mounted and stored at 4°C over several months. Green fluorescence images from green fluorescence protein (GFP) were photographed by Zeiss fluorescence microscopy.

Cell extract and western blotting

Cells were lysed with RIPA buffer (1% v/v Nonidet P-40, 1% w/v sodium deoxycholate, 0.1% w/v SDS, 0.15 M NaCl, 2 mM EDTA, 0.01 M sodium phosphate pH 7.2, 50 mM sodium fluoride, 0.2 mM sodium vanadate, 100 U/ml aprotinin) and incubated for 30 min on ice. Debris was removed by centrifugation (full speed in an Eppendorf 5415C microcentrifuge for 30 min at 13 000 r.p.m.) and the supernatant was analyzed by immunoblot with anti-GFP. The protein amounts of each sample were quantitated by both the Bio-Rad protein assay and the Coomassie Brilliant Blue staining of gels. Equal amounts of protein were separated by SDS–PAGE using 4–12% gradient gels (NuPAGE, Novex) and transferred onto nitrocellulose membranes. Membranes were incubated overnight at 4°C with 1:5000 primary monoclonal anti-GFP (Clontech), washed three times for 5 min with Tris-buffered saline containing 0.3% Tween 20 and incubated with 1:2500 diluted horseradish conjugated secondary antibodies for 1 h at room temperature. Membranes were washed as above and horseradish peroxidase was detected using enhanced chemiluminescence reagent (1.25 mM 3-aminophthalhydrazide, 0.2 mM coumaric acid, 0.3 mM hydrogen peroxide in 0.1 M Tris pH 8.5). Membranes were then exposed on Kodak X-Omat AR X-ray film (Eastman Kodak).

Actinomycin D treatment

HeLa cells were transfected as described (20). Sixteen hours after transfection, the cells were washed twice in PBS and re-incubated with pre-warmed DMEM with 5 µg/ml actinomycin D (Calbiochem) for 4–6 h prior to fixation for fluorescence photography.

Labeling of transcription sites and immunofluorescence procedures

Nascent RNA transcription sites were labeled based on previous procedures (21,22). Cells cultured on poly-l-lysine-coated glass-bottom chambers were used for immunostaining. All procedures were done at room temperature. The cells were transfected with the GFPFBP construct and cultured overnight. Nascent RNA transcription sites were labeled according to published procedures. Briefly, cells were washed with ice-cold TBS buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM MgCl₂) and further washed with glycerol buffer (20 mM Tris–HCl pH 7.4, 25% glycerol, 5 mM MgCl₂, 0.5 mM EGTA, 0.5 mM PMSF) for 10 min on ice. Cells on ice were permeabilized with 0.025% Triton X-100 in glycerol buffer (with 25 U/ml of RNasin; Promega) for 3 min and immediately incubated at room temperature for 30 min with nucleic acid synthesis buffer (50 mM Tris–HCl pH 7.4, 10 mM MgCl₂, 150 mM NaCl, 25% glycerol, 0.5 mM PMSF, 25 U/ml of RNasin, 1.8 mM ATP) supplemented with 0.5 mM CTP, GTP and BrUTP (Sigma Chemicals). After nucleotide incorporation, the cells were fixed with 4% freshly made paraformaldehyde in PBS on ice for 5 min, washed with ice-cold TBS-Tween buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.2 mM MgCl₂, 0.2% Tween 20), blocked with 5% goat serum and incubated with mouse anti-BrU (IgG, Roche) followed by Rhodamine-conjugated goat anti-mouse IgG (1:150; Roche) to detect transcription sites. All incubations were performed at room temperature.

Immunostaining

HeLa cells transfected with GFP–FBP plasmids were fixed with 4% paraformaldehyde in PBS for up to 20 min at room temperature and then washed twice with cold PBS. Immunocytochemistry was performed as described (23). The cells were then permeabilized by treatment with 80 mM HEPES pH 6.8, 5 mM MgCl₂ and 0.5% Triton X-100, for 5 min at room temperature. The blocking solution (PBS with 0.5% Triton X-100, 2% BSA) was added to the cells at 37°C for 30 min. The primary antibody (rabbit polyclonal anti-human p89, 1:00, Santa Cruz Biotechnology, Santa Cruz; mouse monoclonal anti-human SC-35, Sigma, St Louis) was added at a 1:1000 dilution to the blocking buffer, incubated for 1 h at 37°C and then washed twice in cold PBS. The secondary antibody conjugated with Rhodamine (Roche) was added directly to the cover slip and covered with a plastic membrane for 1 h at 37°C. After immunostaining, cells were stained for 20 min with diamidinophenylindole (DAPI, 1 µg/ml) and washed twice with PBS. Finally the slides were mounted and stored at –20°C for microscopic examination. Fluorescent images of the cells were obtained using a fluorescent microscope (Carl Zeiss).

RESULTS

Each of FBP’s three domains confers nuclear localization

Biochemically, FBP is enriched in the nuclear extracts. Immunostaining with affinity purified, polyclonal anti-FBP revealed localization of FBP in hundreds of dots throughout the nucleoplasm, against a background of homogeneous, diffuse nuclear staining (Fig. 1A). FBP staining of nucleoli and the cytoplasm was at the margin of detectability. To study the intracellular trafficking of FBP, a means was sought to eliminate potential interference from the immunologically cross-reacting FBP2 and FBP3. Although previous studies have demonstrated nuclear staining with affinity-purified anti-FBP, the relative contributions from each of the FBPs could not be directly assessed. To monitor uniquely the trafficking of FBP, a plasmid expressing a GFP–FBP fusion (GFPFBP) was transfected into HeLa cells and the sub-cellular localization of fluorescence was followed directly (24). GFPFBP fluorescence was tightly localized to the nucleus (Fig. 1A and B, panels 3 and 1–3, respectively). The pattern of GFPFBP fluorescence was essentially indistinguishable from the pattern of endogenous FBP immunostaining (Fig. 1A, compare panel 1 with panel 3). In contrast, GFP alone was distributed uniformly throughout the cell (data not shown). First, 80% of the cells showed a homogeneous distribution of FBP throughout the entire nucleoplasm (excluding the nucleolus) (Fig. 1A and B, panels 1 and 3 and panel 1, respectively; summarized in Fig. 1C). In other cells focal accumulations of FBP occurred. In ∼20% of the cells FBP was seen in hundreds of tiny, brightly fluorescent dots [Fig. 1B, panels 1 (arrow) and 2; summarized in Fig. 1C]. Third, in the nuclei of nearly 3% of the transfected cells, 20–40 coarsely fluorescent spots were observed (Fig. 1B, panel 4; summarized in Fig. 1C). Whether the dots and spots define distinct nuclear compartments is unknown; however, for heuristic purposes this classification is included. The intracellular distribution of FBP and GFPFBP were unperturbed by treatment with leptomycin B, an inhibitor of nuclear export (data not shown). So nuclear uptake of FBP is not coupled with delivery of nuclear cargoes to the cytoplasm. [Although these studies were conducted with HeLa cells, full-length FBP has been observed to be nuclear in several other cell-lines transfected with GFPFBP and anti-FBP stained only nuclei in a variety of normal and neoplastic tissue (L.He, A.Weber and D.Levens, unpublished observations)].

Figure 1.

FBP possesses multiple, non-overlapping NLSs. (A) The intranuclear pattern of the GFPFBP chimera parallels endogenous FBP. Panel 1, fluorescence micrographs of endogenous FBP stained with the FBP-specific antibody and FITC-second antibody revealing homogenous and dotted nuclear patterns in HeLa cells. A cell with a pattern of dots is marked with an arrowhead. Panel 2, the same cells were also stained with DAPI to illustrate that FBP staining is confined to the nucleus. Panel 3, HeLa cells were seeded on the coverslip in 6-well plates and grown overnight to 75% confluency and then transfected with the GFPFBP-plasmid. An enlarged GFPFBP-expressing cell is shown in the inset to visualize better the fine dots and to facilitate comparison with panel 1. Panel 4, FBP was immunostained with Rhodamin conjugated secondary antibody. Panel 5, merging the green and red reveals co-trafficking of transfected and endogenous FBP. (B) Wild-type FBP fused with GFP has several phenotypes, homogenous (panel 1) and focal accumulations ranging from fine dots (panel 2) to coarser spots (panel 3) while each subdomain FBPN (panel 5), FBPCD (panel 6), FBPC (panel 7) has its own NLS conferring distinct intranuclear patterns. GFP alone is uniformly distributed within the cell (data not shown). (C) The different patterns shown in (B) were quantitated. Dots and spots were counted separately in case this assignation distinguishes between nuclear subcompartments. Striped boxes, solid boxes and ellipses indicate α-helix, KH-repeats and tyrosine-repeats, respectively. Hom, homogenous; speck, spots; N, nuclear; C, cytoplasmic; N + C, dispersed throughout the cell. For wild-type GFPFBP, 500 cells were counted and classified into three different nuclear phenotypes, 81.4% were homogeneously distributed, 16.8% dotted and 1.8% were in spots. Both FBPN and FBPC were totally in nuclei. But only two-thirds of GFP-FBPCD distributed into nuclei while 28.8% formed a perinuclear ring structure surrounding the nuclear membrane. (D) GFP immunoblot was performed to verify and quantitate GFP fusion protein expression. Arrowheads indicate GFP chimera. An arrow marks a non- specific band (NS).

In order to determine which sequences in FBP localize it to the nucleus, each of its functional domains, FBPN, FBPCD and FBPC as well as FBPΔCD lacking the DNA-binding central domain (CD), were fused with GFP by cloning into pEGFP and transfected into HeLa cells. Surprisingly each of these non-overlapping protein fragments was efficiently directed into the nucleus. Both the N-terminal repression domain (Fig. 1B, panel 5; summarized in Fig. 1C) and the C-terminal activation domain (Fig. 1B, panel 7; summarized in Fig. 1C) were almost entirely localized to the nucleus as was the protein deleted of the DNA-binding domain (Fig. 1B, panel 4; summarized in Fig. 1C). The nucleic acid binding central domain, FBPCD, was uniformly distributed in the nucleoplasm, excepting the nucleolus, but was also found in coarse agglomerations in the cytoplasm (Fig. 1B, panel 6; summarized in Fig. 1C). Thus, each domain of FBP possessed a nuclear import signal capable of functioning autonomously. Immunoblot analysis confirmed the sizes and levels of expression of the chimeric proteins (Fig. 1D). Therefore, the capacity or lack thereof for nuclear trafficking was an intrinsic property of the engineered fusion-proteins and not due to spurious truncation.

FBPN contains a classical bipartite NLS

Inspection of the FBP amino acid sequence revealed a candidate bipartite NLS within its N-terminal repression domain (25). To test whether this segment of FBP was responsible for nuclear import, GFP–FBPN derivatives were prepared. Successive deletions of FBPN from residue 106 toward the N-terminus were examined. Deletions until residue 92 were fully localized to the nucleus, whereas deletions to or beyond residue 53 were completely impaired for this activity (Fig. 2A, panels 1–4; summarized in Fig. 2B). The candidate NLS was indeed located between residues 53 and 92. To prove that this bipartite NLS-like sequence was sufficient to target nuclear transport, residues 63–78 comprising only this region were fused to GFP and were found to be sufficient to drive GFP into the nucleus (summarized in Fig. 2B). This classical bi-partite NLS is conserved in FBP2 (KSRP), but is lacking in FBP3 (Fig. 2D). The expression levels and sizes of the fusion proteins were confirmed using SDS–PAGE and immunoblot (Fig. 2C).

Figure 2.

FBPN contains a functional bipartite NLS. (A) Segments of FBPN were fused to GFP, transfected and assessed for nuclear localization; panel 1, residues 1–92 fused to GFP; panel 2, residues 1–53 fused to GFP; panel 3, residues 1–36 fused to GFP; panel 4, residues NLS 63–78 fused to GFP. (B) Summary of transfected GFPFBPN derivatives and quantitation of trafficking. Amino acids 63–78 efficiently traffic into nuclei. (C) GFP immunoblot was performed to verify and quantitate GFP fusion protein expression. Arrowheads indicate GFP chimera. A non-specific band (NS) is marked by an arrow. (D) This bipartite NLS is conserved in FBP and FBP2, but not FBP3.

The FBP central domain contains an atypical NLS

The nucleic acid binding central domain of FBP supported several patterns of fluorescence when fused with GFP. Almost two-thirds of the GFP–FBPCD expressing cells displayed homogeneous nuclear staining with relative sparing of the nucleolus (Fig. 3A, panel 1). Almost one-third of the cells developed intensely fluorescent coarsely beaded accumulations of GFP–FBPCD forming a peri-nuclear cuff (Fig. 3A, panel 1). The remaining cells were diffusely fluorescent either throughout or just in the cytoplasm (summarized in Fig. 3B).

Figure 3.

The nuclear localization signal in the FBP central domain (FBPCD) resides in a single α-helix (amino acids 328–348). (A) Fluorescence micrographs of GFPFBPCD derivatives shown in (B). The II→PP mutations positioned at amino acids 338, 339 in the fourth α-helix abolished its nuclear targeting. (B) Summary of transfected GFPFBPCD derivatives and quantitation of trafficking. (C) GFP immunoblot was performed to verify and quantitate GFP fusion protein expression. Arrowheads indicate GFP chimera. An arrow marks a non-specific band (NS). (D) Similar α-helices including a conserved isoleucine dyad are found in all FBP family members.

GFP–FBPCD was less efficiently targeted to the nucleus than GFP fusions with full-length FBP or either effector domain. Conceivably, the nuclear localization of FBPCD might be due to diffusion of FBP throughout the cell with nuclear retention due to entrapment by nucleic acids. If occurring, such passive retention would require functional DNA or RNA binding by GFP–FBPCD. A series of C-terminal truncations of GFP–FBPCD (residue 480) were expressed in HeLa cells to determine which FBPCD sequences allowed nuclear retention. Deletion through KH-repeat 4 (residue 416) did not impair nuclear localization (Fig. 3A, panel 2; summarized in Fig. 3B). Deletion from the C-terminus of FBPCD to residue 381 still allowed nuclear import of FBPCD, but removal of just seven more residues abrogated nuclear uptake (Fig. 3A, panels 3 and 4; summarized in Fig. 3B). N-terminal truncations of FBPCD (from residue 107 to 274) revealed that KH-repeats 1 and 2 were dispensable for nuclear localization (Fig. 3A, panel 6; summarized in Fig. 3B). N-terminal deletions, which extended through KH-repeat 3 preserved nuclear targeting until encroaching upon α-helix 4 (Fig. 3A, panel 5; summarized in Fig. 3B). Thus delimited by N- and C-terminal deletions, residues 366–386 encompassing α-helix 4 were tested and found to confer the same pattern of nuclear targeting seen with the entire central domain (Fig. 3A, panel 7; summarized in Fig. 3B). Because this same protein fragment lacked nucleic acid binding activity, the nuclear accumulation of FBPCD was not due to passive ensnarement of the protein by nuclear nucleic acids. Mutation of isoleucines 378 and 379, conserved among all the FBPs (Fig. 3D), to helix-destabilizing prolines completely abolished the NLS activity of α-helix 4 (Fig. 3A, panel 8; summarized in Fig. 3B). Thus, an NLS is completely embedded within a structural element of KH-repeat 3. Similar, but not identical α-helices occur in FBP2 and FBP3. Immunoblot analysis verified the synthesis of GFP fusion proteins (Fig. 3C). Hence, FBPCD, like FBPN, contains a short segment directing import into the nucleus.

The tyrosine-rich transcription activation motifs of FBPC are required for its nuclear localization

To identify the sequence elements that direct FBPC to the nucleus, a series of C-terminal deletions between residues 449 and 644 of FBP were fused with GFP and each was tested for nuclear localization. Deletion to residue 576 resulted in a 95% loss of NLS activity (Fig. 4A, panel 2; summarized in Fig. 4B). The deletion to residue 531 retained just 0.4% NLS activity (Fig. 4A, panel 3; summarized in Fig. 4B) and beyond residue 495 NLS activity was completely lost (summarized in Fig. 4B). Deletion to residue 576 removed the last two of the three tyrosine-rich motifs, YM1, 2 and 3. These motifs individually or in combination have proved to be potent transcriptional transactivators (12). Only constructs with all three YM motifs were efficiently targeted to the nucleus. Fusion proteins bearing wild-type YM3 retained some capacity for nuclear localization, but the same mutations which disabled transcription activation by YM3 eliminated even this residual activity (Fig. 4A, panels 8–10; summarized in Fig. 4B). YM1 and 2 were ineffective at directing nuclear fluorescence when fused with GFP (Fig. 4A, panels 6 and 7; summarized in Fig. 4B). Appropriate expression of all protein chimeras was confirmed (Fig. 4C).

Figure 4.

Tyrosine-rich transcription activating repeats (YM1–3) are required to localize FBPC into nuclei. (A) Deleting YM1–3 progressively impairs nuclear localization. Proteins with YM1–3 efficiently localize to the nucleus, panels 1 and 4. Proteins with two (panel 5) or one tyrosine-rich motifs (panels 2, 6, 7 and 8) localize to the nucleus less well (3). Compared with YM1 or YM2 (panels 6 and 7), YM3 most strongly traffics to the nucleus (panel 8). The same mutations that disable YM3 transcription activation (12) abrogate nuclear localization (panel 8 versus panels 9 and 10). (B) Summary and quantitation of impaired nuclear localization with progressive deletion or mutation of FBPC. (C) Expression of GFPFBPC deletion constructs in HeLa cells was monitored by GFP immunoblot. The location of each expressed protein was pointed by arrowhead. A non-specific band is unmarked. (D) Conservation of YM1–3 through FBP family. YM3 persistently exists among FBP family, which implied it is an essential role in FBP family function. FBP2 duplicatesYM2, whereas FBP3 loses YM1 motifs.

Inhibiting transcription prompts an intranuclear redistribution of FBP

The known functional activities resident in FBP (11–14) and the experiments reported above, support a nuclear role for FBP. But some workers have proposed a cytoplasmic role for FBP in the degradation of GAP-43 mRNA and other KH-motif family members are zip-code binding proteins which help to deliver particular mRNAs to appropriate intra-cytoplasmic sites (18,26). Furthermore the prototype for the KH family, which includes FBP, is hnRNP K, itself a bi- or multi-functional protein shuttling between the nucleus and cytoplasm and regulating processes in both compartments (9,27–30). Inhibition of transcription often reduces the nuclear pool of hnRNA and results in the failure of nuclear uptake or re-localization of hnRNP proteins to the cytoplasm (31,32). hnRNP K is primarily nuclear in actively transcribing cells but contains a shuttling domain which can drive reporter proteins into the cytoplasm in the absence of transcription (9). To test if FBP similarly re-localizes after inhibiting transcription, GFPFBP-transfected cells were treated with actinomycin D. No re-partitioning of GFPFBP to the cytoplasm was seen. Surprisingly, a dramatic intranuclear re-distribution occurred as the diffuse nuclear fluorescence seen in 80% of untreated cells coalesced into hundreds of fine intranuclear dots after treatment in all cells (Fig. 5A, panel 2; summarized in Fig. 5B). Prior to treatment just 20% of the cells demonstrated this multi-punctate pattern. FBPΔCD responded identically to actinomycin D treatment as did GFPFBP (Fig. 5A, panel 3; summarized in Fig. 5B).

Figure 5.

Transcription determines sub-nuclear compartmentalization of FBP. (A) Fluorescence micrographs of FBP or its subdomains before (Fig. 1B) and after transcription inhibition with actinomycin D. Following addition of actinomycin D, FBP (panel 2) and FBPΔCD (panel 3) completely re-localized into ‘dots’. FBPN remained diffusely nuclear (panel 4). More surprisingly, FBPCD was totally locked out of nuclei (panel 5). FBPC partially diffused into cytoplasm (panel 6). (B) The summary and quantitation of re-distribution of FBP and derivatives following treatment with actinomycin D.

The C-terminal activation domain, FBPC, was distributed diffusely and in dots throughout the nucleus, prior to actinomycin D treatment. After treatment approximately one-half to one-quarter of the fluorescent material within each compartment escaped into the cytoplasm (Fig. 5A, panel 6; summarized in Fig. 5B). The fluorescent pattern of GFPFBPN was not changed by actinomycin D treatment (Fig. 5A, panel 4; summarized in Fig. 5B).

FBPCD, possessing the weakest NLS of FBP’s functional domains, partly localized to the cytoplasm prior to actinomycin D treatment, but the bulk of the protein remained nuclear. Upon blocking RNA synthesis, however, nuclear enrichment of FBPCD was lost as the molecule became either entirely delocalized throughout the cell or accumulated in a cytoplasmic, peri-nuclear halo (Fig. 5A, panel 5; summarized in Fig. 5B). So the NLS activities resident within each domain of FBP were differentially coupled with ongoing transcription. The preservation of the resident NLS activities of full-length FBP, FBPN and FBPC despite transcription inhibition indicated that the nuclear localization of FBP was not due to adventitious association with nuclear RNA.

FBP co-localizes with nascent transcripts and the transcription machinery

If FBP’s N-terminal transcriptional repression and C-terminal activation domains engage the transcription machinery in vivo, then at least some of the time FBP should traffic to transcription sites and interact with the transcription machinery. To visualize transcription sites in vivo, the GFPFBP-transfected cells were permeabilized and incubated with BrUTP. Subsequent staining with anti-bromo-uridine revealed co-localization of FBP and newly synthesized nascent RNA molecules (Fig. 6, top row). Because in vitro transcription experiments have indicated that FBP’s transactivation domain operates through TFIIH (J.Liu, personal communication), experiments were conducted to determine whether this in vitro observation has an in vivo correlate. Therefore, GFPFBP-transfected cells were stained with anti-TFIIH. In fact, FBP co-localized with TFIIH using a polyclonal antibody recognizing p89/XPB, the largest subunit of this multifunctional complex (Fig. 6, middle row). TFIIH staining was seen in hundreds of microdots peppered throughout the nucleus, a pattern commonly observed using antibodies directed against components of the transcription machinery. In contrast, FBP did not co-localize in nuclear speckles with SC-35, a protein commonly used as a marker associated with RNA processing components (33).

Figure 6.

FBP co-localizes with both transcription sites and general transcription factor TFIIH, but not with splicing factor SC-35. HeLa cells, transfected with GFPFBP (middle panel) were permeabilized and transcriptionally pulse-labeled with 5-bromo-2-uridine-5-triphosphate and then immunostained for sites of BrUTP incorporation (red, top row). The merged pictures of GFPFBP fluorescence and transcription sites show extensive co-localization (right, top row). GFPFBP transfected cells were also immunostained for the p89/XPB subunit of TFIIH (red, middle row), again revealing co-localization (right, middle row). However, GFPFBP localized to different sites than SC35 (red, bottom row).

DISCUSSION

The complex routes traveled by many nuclear proteins must be delimited by functional constraints. Biochemical and genetic characterization of a protein’s physiological role provide a guide to understand its intracellular travels. Without such a map, even the highly regulated flow of important molecules may be disguised or dismissed. The dynamic compartmentalization displayed by many transcription factors only makes sense within the contexts of the upstream pathways regulating factor activity and of the biology of the downstream target genes. NFκB, glucocorticoid receptor, β-catenin, SMADs, SREBP and many other transcription factors most of the time and in most cells localize away from their sites of operation (34–38). Similarly, components of the splicing machinery traffic from cytoplasmic sites of synthesis, to assembly points, and once nuclear, a variety of RNA-binding proteins commute from storage depots to target RNA processing centers (8,32,39,40). Observation of these molecules as markers or indicators of intracellular transport mechanisms without consideration of functional context could lead to incomplete or even misleading conclusions concerning their function and regulation. From promoter activity, to splicing, mRNA transport and mRNA degradation, FBP and its close relatives FBP2 and FBP3 have been proposed to participate in all stages of mRNA synthesis and catabolism (11,12,14,16–18). What then is the significance of the intranuclear distribution and multiple NLSs in FBP within the context of FBP function?

Although most KH-proteins such as hnRNP K, ZBP1, Vg1 RBP, FMRP, SAM68, etc., participate in, or have been proposed to participate in, nuclear processes, especially RNA processing, they are also believed to be involved with cytoplasmic functions such as translation, control of RNA transport, signal trandsuction, etc. (28,41–46). In contrast, in our studies FBP virtually always abides within the nucleus. So if FBP proves to be multi-functional, its primary function is likely to be nuclear. FBP has three independent and non-overlapping segments directing nuclear import, one within each of its functional domain. At least two of these determinants of nuclear localization are enmeshed with functional modules of FBP activity; one motif is contained within the nucleic-acid binding KH-repeat 3, while another overlaps the transcription-activating tyrosine-rich C-terminus. To a first approximation, the pattern of immunostaining exhibited by native FBP appears to be a composite created by superimposing the activities of each FBP-NLS. Full-length FBP is mostly found homogeneously dispersed and partly focused in hundreds of dots; less frequently it is concentrated in fewer spots. This pattern mostly closely parallels the NLS activities found in the N- and C-termini of FBP. Therefore, all three NLSs seem to operate at least occasionally and appear not to be latent activities. The different intranuclear patterns programmed by each FBP NLS may indicate FBP functions or is stored at multiple nuclear sites.

Four arguments relate the nuclear sublocalization of FBP. First, the N- and C-terminal NLSs occur in domains also possessing transcription effector function in vivo and in vitro. Second, FBP localizes at sites of transcription. Third, FBP co-localizes with TFIIH, an essential general transcription factor. Fourth, inhibition of transcription did not propel endogenous FBP to cytoplasmic destinations. Thus, FBP is dissimilar from most hnRNP molecules which shuttle, with their final RNA cargoes, to the cytoplasm and remain extra-nuclear when RNA synthesis, and hence nuclear re-uptake, is inhibited (32).

FBP and FBP2/KSRP have been associated in vitro with splicing complexes, so perhaps under particular conditions, FBP participates in RNA processing. In fact FBP binds tightly with the FBP interacting repressor (FIR). FIR regulates the activity of general transcription factor TFIIH to repress activated transcription. But alternatively spliced forms of FIR have been reported to bind to RNA and to interact with the splicing machinery (47). The relative roles of FBP and FIR in transcription, RNA-processing or other intranuclear processes requires further illumination.

The bipartite NLS in FBP’s N-terminus is standard, the NLSs in the central and C-terminal domains are non-canonical. The non-standard NLS might be imported using standard or non-standard nuclear receptors; or alternatively their nuclear uptake may be controlled through partner proteins imported using conventional mechanisms. The NLS in the central domain neatly resides in a single α-helix; alone this NLS is not over-powering and in some cells, FBPCD is cytoplasmic. Whether nuclear uptake is less efficient in these cells or if the enhanced cytoplasmic retention is due to partner proteins is not known. It is possible that partitioning of FBPCD between the nucleus and cytoplasm is influenced by interactions with the FIR, which binds with the FBPCD (13). Such interactions might serve either to retard or promote nuclear transport.

The NLS in the C-terminus (FBPC) does not map neatly to a discrete sequence; rather the efficiency of nuclear localization becomes progressively diminished as more and more of this domain is deleted. FBPC NLS activity is most impaired by deletions or mutations, which encroach upon the third tyrosine-rich activating motif, YM3. As this domain binds with TFIIH (J.Liu, personal communication), the distribution of FBPC may be coupled with the trafficking of TFIIH, a nine-subunit complex associated with transcription, DNA-repair and cell-cycle regulation (48).

The data indicate that FBP NLSs operate in parallel and not in series. Therefore, the nuclear address of FBP is not hierarchical. FBP is not first delivered to the nucleus with a typical NLS, then sorted and sub-localized. Rather parallel nuclear import pathways seem to draw upon a common pool of FBP. Therefore the intranuclear distribution of FBP must be determined by the total amount of FBP and the relative rate constants governing exchange between compartments. Interfering with the equilibrium or steady-state flux of FBP between sites might uncouple, but not necessarily disrupt the relevant pathways. The ability of FBP NLSs to operate in isolation from each other indicates that FBP is unlikely to flow or carry cargo in an obligatory order to sequential nuclear stations. If acting in series, then removal of any FBP NLSs would improperly localize FBP and disrupt the formation and regulation of downstream complexes. As functional and genetic studies of FBP progress, the logic of the sub-cellular trafficking of FBP promises to reveal connections between specific gene transcription and other cellular processes.