A novel set of nuclear localization signals determine distributions of the alphaCP RNA-binding proteins - PubMed (original) (raw)
A novel set of nuclear localization signals determine distributions of the alphaCP RNA-binding proteins
Alexander N Chkheidze et al. Mol Cell Biol. 2003 Dec.
Abstract
AlphaCPs 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 alphaCP 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 alphaCP isoform. Immunofluorescence studies revealed three distinct distributions: alphaCP1 and alphaCP2 are predominantly nuclear with specific enrichment of alphaCP1 in nuclear speckles, alphaCP3 and alphaCP4 are restricted to the cytoplasm, and alphaCP2-KL, an alphaCP2 splice variant, is present at significant levels in both the nucleus and the cytoplasm. We mapped nuclear localization signals (NLSs) for alphaCP isoforms. alphaCP2 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 alphaCP1, whereas NLS II is inactivated by two amino acid substitutions. Neither NLS is present in alphaCP3 or alphaCP4. Consistent with mapping studies, deletion of NLS I from alphaCP1 blocks its nuclear accumulation, whereas NLS I and NLS II must both be inactivated to block nuclear accumulation of alphaCP2. These data demonstrate an unexpected complexity in the compartmentalization of alphaCP isoforms and identify two novel NLS that play roles in their respective distributions. This complexity of alphaCP distribution is likely to contribute to the diverse functions mediated by this group of abundant RNA-binding proteins.
Figures
FIG. 1.
αCP isoforms and detection of endogenous αCP1, αCP2, and αCP2-KL. The five major αCP isoforms are shown on the left. The corresponding genetic loci and protein designations are noted. The position of each KH domain is indicated by a shaded box. Numbers above the protein diagrams indicate the sizes of each protein in amino acids. Alternatively spliced exons are shown for the αCP2-KL. Immunostainings of HeLa cells for endogenous αCP1, αCP2, and αCP2-KL are shown on the right. The antisera are specific for αCP1 (serum FF1), αCP2 (serum FF2), and αCP2 and αCP2-KL (serum FF3). Antibody-protein complexes were visualized with FITC-conjugated goat anti-rabbit secondary antibody and examined by fluorescence microscopy. In each case the immunostained image (FITC) was compared to the nuclear stain (DAPI [4′,6′-diamidino-2-phenylindole]) and the phase-contrast image.
FIG. 2.
Selective enrichment of αCP1 in nuclear speckles. (Top row) Localization of αCP1. HeLa cells were double immunostained with rabbit antiserum to αCP1 and mouse monoclonal antibody to SC-35. The αCP and SC35 immune complexes were detected with FITC-conjugated anti-rabbit and TX-conjugated anti-mouse antibodies, respectively, and then examined by confocal microscopy. Left panels show the αCP1 stain (FITC), middle panels show the SC35 stain (TX), and right panels show merges of the left and middle images. The yellow in the merge indicates colocalization of FITC and TX stains. (Bottom row) Localization of αCP2 (FF-2). Details are as described for the top row of images. The αCP2 is more diffusely distributed than αCP1 and does not appear to be enriched in speckles.
FIG. 3.
Expression and subcellular localization of αCP isoforms as fusion proteins in HeLa cells. (A) Schematic drawings of the Pk-αCP isoform fusion proteins. The chicken muscle Pk (black elipse) linked to an N-terminal myc epitope tag (open box) was fused in a continuous ORF corresponding to each of the indicated αCP isoforms (shaded boxes). The bipartite basic NLS of hnRNP K is denoted by the stippled oval. (B) Western analysis of fusion proteins expressed in HeLa cells. Expression vectors containing each of the indicated proteins (see panel A for schematic details) were transfected into HeLa cells, and extracts were harvested at 36 h. Proteins were analyzed by Western analysis with an anti-myc epitope antibody (monoclonal antibody 9E10). Molecular mass standards are indicated on the left in kilodaltons. (C) Intracellular distribution of the _myc_-Pk-αCP fusion proteins. At 36 h after transfection, HeLa cells were fixed and stained for immunofluorescence microscopy with anti-myc antibody (FITC). Cells in the field were visualized by phase-contrast imaging, and the nuclei were identified by DAPI staining.
FIG. 4.
Mapping nuclear import signals in αCP2-KL (A) N-terminal deletion set. The three KH domains are denoted by shaded boxes; numbers above the protein body indicate its size and the positions of KH domains in amino acids. The N and C terminus of each expressed protein is indicated along with its name. The positions of the KH domains are shown for reference. Other symbols are as defined in Fig. 3. (B) Subcellular localization of the αCP-KL Nd proteins. Representative micrographs of the immunofluorescence analyses with anti-myc are shown. The identity of each expressed protein is indicated below the frame. (C) C-terminal deletion set. Details are as described for panel A. (D) Subcellular localization of the αCP-KL Cd proteins. Details are as described for panel B.
FIG. 5.
αCP-2KL contains two independent NLSs. Schematics of two internal segments of the αCP2-KL fused with _myc_-Pk are shown. Immunofluorescence micrographs show the subcellular localization of the indicated αCP2-KL fusion proteins expressed in transfected HeLa cells.
FIG. 6.
Fine mapping of NLS I and NLS II. (A) Fine-mapping NLS I. Schematics of αCP2 and six subsegments assessed for NLS function. The termini of each fragment are indicated (numbers refer to the positions in the full-length αCP2). The hatch-marked box depicts a position of the additional 31-amino-acid intra-KH2/KH3 segment lacking in αCP2-KL. Each segment was fused in frame with _myc_-Pk prior to expression in HeLa cells. (B) Immunofluorescence analysis of NLS I activity. The frames show the subcellular localization of the αCP2 variants illustrated in panel A. Representative micrographs of the immunofluorescence analysis are shown. (C) Fine-mapping NLS II. Details are as described for panel A. (D) Immunofluorescence analysis of NLS II activity. Details are as described for panel B. The last inset, αCP1/[320-331], shows the subcellular localization of a _myc_-Pk fusion to the region of αCP1 corresponding to the minimal NLS II (amino acids 328 to 339) segment of αCP2 (see Fig. 7B for alignment).
FIG. 7.
Sequence alignments of major αCP isoforms at NLS I and NLS II. (A) Alignment of the sequences corresponding to NLS I. This alignment shows perfect conservation between αCP1 and αCP2. There is no region presented in αCP3 or αCP4 that has significant homology to this sequence. (B) Alignments of the sequences corresponding to NLS II. These alignments reveal divergence among αCP2, the other αCP isoforms, and hnRNP K. Conserved amino acids are shaded.
FIG. 8.
NLS activity in αCP1 is restricted to the region between KH2 and KH3. (A) Division of αCP1 into three segments for NLS mapping. The termini of each segment are noted. (B) Immunofluorescence analysis of the αCP1 subregions. _myc_-Pk-tagged αCP1 subregions were expressed in HeLa cells, and the subcellular distribution of each protein was determined. Representative micrographs of the immunofluorescence analysis are shown.
FIG. 9.
Nuclear localization of the full-length αCP1 and αCP2 proteins is dependent on defined set of novel NLSs. (A) Schematic of wild-type _myc_-Pk-αCP1 and the mutant lacking NLS I (_myc_-Pk-αCP1/ΔNLSI). The cross-hatched box represents NLS I. The primary sequence of NLS I is shown. (B) Immunofluorescence micrographs showing the subcellular localization of wild-type and NLSI-deleted αCP1 proteins. Plasmids encoding _myc_-Pk-αCP1 and _myc_-Pk-αCP1/ΔNLSI proteins were transfected into HeLa cells, and the subcellular distribution of the proteins was determined. Representative micrographs of the immunofluorescence analysis are shown. (C) Schematic of wild-type αCP2 and the mutants lacking one or both of NLS I and NLS II. A schematic of wild-type _myc_-Pk-αCP2 and three derivative mutants in which NLS I, NLS II, or NLS I plus NLS II were inactivated (_myc_-Pk-αCP2/ΔNLSI, _myc_-Pk-αCP2/mutNLSII, and _myc_-Pk-αCP2/ΔNLSI/mutNLSII, respectively) is shown. The two cross-hatched boxes represent NLS I and NLS II. The position of deleted NLS I is indicated by the gap, and the boxes with ovals depict the mutated NLS II. (D) Immunofluorescence micrographs showing the subcellular localization of the wild-type αCP2 and the derivative NLS mutants. Plasmids encoding _myc_-Pk-tagged αCP2, _myc_-Pk-αCP2/ΔNLSI, _myc_-Pk-αCP2/mutNLSII, and _myc_-Pk-αCP2/ΔNLSI/mutNLSII were transfected into HeLa cells, and the subcellular distribution of the proteins was determined. Representative micrographs of the immunofluorescence analysis are shown.
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