Molecular characterization of a novel, widespread nuclear protein that colocalizes with spliceosome components

Abstract

We report the identification and molecular characterization of a novel type of constitutive nuclear protein that is present in diverse vertebrate species, from Xenopus laevis to human. The cDNA-deduced amino acid sequence of the Xenopus protein defines a polypeptide of a calculated mass of 146.2 kDa and a isoelectric point of 6.8, with a conspicuous domain enriched in the dipeptide TP (threonine-proline) near its amino terminus. Immunolocalization studies in cultured cells and tissues sections of different origin revealed an exclusive nuclear localization of the protein. The protein is diffusely distributed in the nucleoplasm but concentrated in nuclear speckles, which represent a subnuclear compartment enriched in small nuclear ribonucleoprotein particles and other splicing factors, as confirmed by colocalization with certain splicing factors and Sm proteins. During mitosis, when transcription and splicing are downregulated, the protein is released from the nuclear speckles and transiently dispersed throughout the cytoplasm. Biochemical experiments have shown that the protein is recovered in a approximately 12S complex, and gel filtration studies confirm that the protein is part of a large particle. Immunoprecipitation and Western blot analysis of chromatographic fractions enriched in human U2 small nuclear ribonucleoprotein particles of distinct sizes (12S, 15S, and 17S), reflecting their variable association with splicing factors SF3a and SF3b, strongly suggests that the 146-kDa protein reported here is a constituent of the SF3b complex.

Figure 1

Isolation of a cDNA clone coding for a novel nuclear protein from X. laevis. (A) Schematic representation of the assembled pBT B2-Xen full-length cDNA and overlapping subclones (pBT B2.4; pBT B2.3; pBT B2.5) thereof. Partial restriction maps indicate the enzymes used for generating the full-length cDNA: S, SpeI; E, EcoRV; B, BamHI; H, HincII; X, XhoI; K, KpnI (see also MATERIALS and METHODS). Numbers give the nucleotide positions with reference to the assembled full-length cDNA. (B) Amino acid (aa) sequence deduced from cDNA clone pBT B2-Xen. The open reading frame comprises 1307 aa coding for a 146-kDa protein. An internal domain characterized by the presence of several TP-motifs (in bold letters) is denoted by a box (aa 208–512). A putative bipartite nuclear localization signal (NLS) is underlined, and sequences used for generating antibodies are marked by dotted lines.

Figure 2

Molecular characterization of the cDNA clone encoding the 146-kDa protein. (A) Identification of specific mRNAs by Northern blot analysis. Poly(A)⁺-RNA from Xenopus oocytes (Ov) and Xenopus A6 cells (A6) was separated in an agarose gel, transferred to a membrane, and probed with antisense cRNA derived from clone pBT B2-Xen. Note the specific reaction with a ∼4.4 kb RNA. RNA-size markers of 7.5, 4.4, 2.4, and 1.4 kb are indicated on the left (from top to bottom). (B) Coomassie blue staining of SDS-PAGE–separated rabbit reticulocyte lysates after in vitro transcription/translation in the absence (lane 1) or presence of the pBT B2-Xen template (lane 2). Lanes 3 and 4 show immunoprecipitates of translated protein in the presence or absence of antibody B2.4–1, respectively. R, reference proteins: 205, 116, 97.4, 66, 45, and 29 kDa (from top to bottom). (B′) Corresponding autoradiograph of translation products and immunoprecipitates. (C) Phase-contrast microscopy of cultured human hepatocellular carcinoma (PLC) cells used to determine the subcellular localization of the 146-kDa Xenopus protein carrying an amino-terminal myc tag in transfection experiments. (C′) Corresponding immunofluorescence using mAb 9E10 recognizing the myc tag. Bar, 20 μm

Figure 3

Identification of the 146-kDa protein in different cell culture lines and nuclear fractions from Xenopus oocytes. (A) Coomassie blue-stained total cellular proteins. Cell lines shown are: X. laevis kidney epithelial cells, line A6 (lane 1); chicken embryonic fibroblasts line CEF (lane 2); rat kangaroo cells, PtK2 (lane 3); embryonal mouse cells of line 3T3-L1 (lane 4); rat vascular smooth muscle-derived cells of line RV (lane 5); bovine kidney epithelial cell line MDBK (lane 6); human primary liver carcinoma cells of line PLC (lane 7); and human cervical adenocarcinoma cells of line HeLa (lane 8). (A′) Corresponding autoradiogram showing immunochemiluminescence detection of the antigenic polypeptide using antibody B2.4–1. A shorter exposure of lane 1 is presented because of the very strong reaction of the antibodies generated against the Xenopus protein. The comparable weak reaction in lane 3 might be due to partial degradation of the protein. (B) Coomassie blue staining of various nuclear fractions of Xenopus oocytes separated by SDS-PAGE. Total mass-isolated nuclei (lane 1); proteins of the LSP, HSP, and HSS fractions of fractionated oocyte nuclei (lanes 2–4); and Xenopus egg extract (lane 5). (B′) Corresponding immunoblot probed with antibody B2.4–1, which specifically reacts with the 146-kDa protein present in all fractions analyzed. (B") Probing of a parallel immunoblot with mAB No-185 directed against the well characterized nucleolar protein NO38 to ascertain the fractionation procedure. Reference proteins (R) are the same as in Figure 2.

Figure 4

Immunoprecipitation of the 146-kDa protein with antibody B2.4–1. The following fractions were separated by SDS-PAGE and stained with Coomassie blue. (A) Proteins of Xenopus egg extract before immunoprecipitation (lane 1); immunoprecipitate obtained from Xenopus egg extract (lane 2); immunoprecipitate after incubation with PBS as a negative control (lane 3); proteins of the egg extract that bind nonspecifically to protein G-Sepharose in the absence of antibodies (lane 4); immunoprecipitate obtained with antibodies against the nucleolar protein NO29. (B) Proteins of Xenopus A6 cell lysates before immunoprecipitation (lane 1); immunoprecipitate obtained from A6-lysates (lane 2). The 146-kDa protein (indicated by arrows) is immunoprecipitated in Coomassie blue-visible amounts (panel A, lane 2, and panel B, lane 2). The nature of the slightly smaller polypeptides (∼110–130 kDa) visible in the immunoprecipitates is unknown. R, reference proteins as shown in Figure 2.

Figure 5

Analysis of the native state of the 146-kDa protein by sucrose gradient centrifugation and gel filtration. (A) Cell lysates of Xenopus A6 cells were fractionated after sucrose gradient centrifugation, separated by SDS-PAGE, and stained with Coomassie blue. Fraction numbers are indicated on top of the lanes (the top of the gradient is on the left). Bars indicate the peak positions of the reference proteins bovine serum albumin (4.3S; fraction 3), catalase (11.3S; fraction 7), and thyroglobulin (16.5S; fraction 11). R, reference proteins are the same as in Figure 2. (A′) Corresponding immunoblot using antibody B2.4–1. The 146-kDa protein is recovered in fraction 8 with a sedimentation coefficient of ∼12S. (B) A similar study was performed with a Xenopus egg extract. Coomassie blue staining of the resulting protein fractions after sucrose gradient centrifugation. (B′) Corresponding autoradiogram showing immunochemical detection of the 146-kDa protein in fractions 7–10. The bulk of the protein is recovered in fraction 8, which corresponds to a mean S value of 12.5. (C) Proteins from Xenopus A6 cell lysates were fractionated by gel filtration, separated by SDS-PAGE, and stained with Coomassie blue. Bars on top of the lanes indicate the peak positions of the cofractionated reference proteins: dextran blue (M_app 2,000,000; fraction 2), thyroglobulin (M_app 669,000; fraction 12), ferritin (M_app 440,000; fraction 17), and catalase (M_app 232,000; fraction 24). (C′) Corresponding immunoblot with antibody B2.4–1. The 146-kDa protein is detectable in fraction 5 and fractions 8–10 (main peaks are denoted by arrows), corresponding to M_app 1,400,000 and 1,000,000, respectively.

Figure 6

Immunolocalization studies in cultured cells from different species. Phase contrast micrographs are shown in A–J and the corresponding immunofluorescence micrographs in A′–J′. (A and A′) Xenopus kidney epithelial cells, line A6. (B, and B′) Bovine mammary gland-derived cells, line BMGE+H. (C and C′) Embryonal mouse line 3T3-L1. (D and D′) Human primary liver carcinoma line PLC. (E–J and E′–J′) Distribution of the 146-kDa protein during mitosis in rat kangaroo cells, PtK2; (E and E′) interphase; (F and F′) prophase; (G and G′) metaphase; (H and H′) late metaphase; (I and I′) anaphase; (J and J′) telophase. Bar, 20 μm

Figure 7

Immunofluorescence microscopy of the 146-kDa protein in different tissues and X. laevis erythrocytes. (A and B) Phase contrast micrographs of frozen sections through Xenopus intestine and human esophagus, respectively. (A′ and B′) Corresponding immunofluorescence micrographs with antibody B2.4–1. (C) Phase contrast micrograph of X. laevis erythrocytes in a blood smear preparation. (C′) Corresponding immunofluorescence micrograph with antibody B2.4–1. Bar, 20 μm (in A and C); 40 μm (B)

Figure 8

Laser scanning confocal microscopy of double-labeling experiments. The distribution of the 146-kDa protein (A, D, G, and J; antibody B2.4–1) was compared with that of snRNP-specific (Sm) proteins (B and E; mAbY12), the protein splicing factor SF3a66 (panel H; mAb66), and the coiled body-specific protein coilin (panel K; anticoilin). The corresponding overlays are shown in panels C, F, I, and L. The cells shown in panels D–F were treated for 4 h with AMD (5 μg/ml). Bar, 10 μm

Figure 9

Identification of the 146-kDa protein in fractions of HeLa cell nuclei enriched in splicing factors and U2 snRNPs. (A) Coomassie blue-stained gel after SDS-PAGE of total nuclear extract (lane 1) and fractions enriched in splicing factors SF3a and SF3b (lane 2), SF3a (lane 3), SF3b (lane 4), and fractions enriched in U2 snRNPs of different composition: 17S, i.e., U2 snRNP associated with SF3a and SF3b (lane 5); 15S, containing U2 snRNP associated with SF3b (lane 6); and 12S, U2 snRNP lacking SF3a and SF3b (lane 7). R, reference proteins as shown in Figure 2. (B) Corresponding immunoblot with antibody B2.4–1 directed against the 146-kDa protein. (C) In parallel, a second filter was probed with mAb 66 directed against the 66-kDa component of SF3a.

Figure 10

Immunoprecipitation of U2 snRNP particles with antibody B2.4–1. (A) Coomassie blue-stained SDS gel after immunoprecipitation of the 17S, 15S, and 12S U2 snRNP fractions in the absence (−) or presence (+) of antibody B2.4–1. Arrows indicate the 146-kDa protein. R, reference proteins as shown in Figure 2. (B) Immunoblot of a parallel SDS gel incubated with antibody B2.4–1. (C) After stripping of the membrane the same blot was incubated with mAb 66. The arrowhead indicates SF3a66. (D) A Northern blot of snRNAs immunoprecipitated from the 17S, 15S, and 12S U2 snRNP fractions (IP, input) in the absence of antibody (−) or in the presence of antibody B2.4–1 (146) or mAb 66 (66) was probed with a radiolabeled antisense U2 snRNA transcript. The input corresponds to the amount of fraction used for the immunoprecipitations.