Volume 6, Issue 3, Pages 673-682 (September 2000) Pre-mRNA Splicing Imprints mRNA in the Nucleus with a Novel RNA-Binding Protein that Persists in the Cytoplasm  Naoyuki Kataoka, Jeongsik Yong, V.Narry Kim, Francisco Velazquez, Robert A. Perkinson, Fan Wang, Gideon Dreyfuss  Molecular Cell  Volume 6, Issue 3, Pages 673-682 (September 2000) DOI: 10.1016/S1097-2765(00)00065-4

Figure 1 Amino Acid Sequence of Y14 and Alignment with Putative Homologs in Several Species Y14 and proteins from mouse (AL022712), Xenopus laevis, D. melanogaster (AC006074), C.elegans (Z32683), and S. pombe (AL021813) were aligned using ClustalW alignment. Identical residues are indicated by dark shading, and similar residues are shown by light shading. The positions of RNP2 and RNP1 consensus motifs of the RNA binding domain are indicated by bars. Molecular Cell 2000 6, 673-682DOI: (10.1016/S1097-2765(00)00065-4)

Figure 2 Specificity of Anti-Y14 Antibodies and Immunolocalization of the Y14 Protein (A) Western blotting on HeLa cell total lysate. The band corresponding to Y14 is indicated by an arrow. (B) The anti-Y14 antibody can specifically precipitate Y14. Anti-hnRNP C1 (4F4), anti-Y14 (4C4), and control antibody (SP2/0) were immobilized on protein G sepharose and incubated with in vitro translated 35S-labeled hnRNP C1 and Y14. An aliquot equivalent to 10% of the amount of hnRNP C1 and Y14 included in the reaction is shown in the lanes marked translation. (C) Subcellular localization of Y14 in HeLa cells. Hela cells grown on the coverslips were fixed, permeabilized, and stained with anti-Y14, 4C4. Molecular Cell 2000 6, 673-682DOI: (10.1016/S1097-2765(00)00065-4)

Figure 3 Immunoprecipitation of RNAs from In Vitro Splicing Reactions (A–E) The indicated 32P-labeled pre-mRNAs and mRNAs were incubated with HeLa cell nuclear extracts under splicing conditions, followed by immunoprecipitation with the indicated antibodies. Recovered RNAs were separated by polyacrylamide gel electrophoresis. The structure of immunoprecipitated RNAs is schematically shown at the right of each panel. Boxes and line represent exons and intron, respectively. Lanes marked total contain 10% of input RNA. Molecular Cell 2000 6, 673-682DOI: (10.1016/S1097-2765(00)00065-4)

Figure 4 Immunoprecipitation of RNAs from Xenopus Oocyte Nucleus and Cytoplasm Immunoprecipitations and RNA analyses were performed as described in Figure 3. N and C represent nucleus and cytoplasm, respectively. (A) 32P-labeled Ad2 pre-mRNA was injected in oocyte nuclei. RNA was immunoprecipitated from the nuclear and the cytoplasmic fractions with anti-Y14 (4C4), anti-Xenopus (x)hnRNP C1 (10C1), and SP2/0. (B) 32P-labeled Ad2 and DHFR mRNAs were injected into oocyte nuclei, and following incubation RNAs were immunoprecipitated as described in (A). (C) Ad2 pre-mRNA, Ad2 mRNA, and DHFR mRNA was injected into oocyte cytoplasm, and following incubation the cytoplasmic fraction was used for RNA immunoprecipitation. Molecular Cell 2000 6, 673-682DOI: (10.1016/S1097-2765(00)00065-4)

Figure 5 Y14 Associates with pre-mRNAs or mRNAs after Removal of a Single Intron Immunoprecipitations and RNA analyses were performed as described in Figure 3 and Figure 4. N and C represent nucleus and cytoplasm, respectively. 32P-labeled CDC-derived pre-mRNA comprising exons 13–15 and the intervening sequences as found in the native pre-mRNA was injected into oocyte nuclei. RNA was immunoprecipitated from the nuclear and the cytoplasmic fractions with anti-Y14 (4C4), anti-Xenopus (x)hnRNP C1 (10C1), and SP2/0. The designation of the products and the splicing intermediates is based on previous characterization (Ohno et al. 1987). Molecular Cell 2000 6, 673-682DOI: (10.1016/S1097-2765(00)00065-4)

Figure 6 Y14 Is in a Novel RNP Complex Different from General hnRNP Complexes (A) Immunoprecipitations were performed from 35S-labeled HeLa cell nucleoplasm with anti-hnRNP A1 (4B10), anti-hnRNP (4F4), and anti-Y14 (4C4) antibodies followed by SDS-PAGE. Proteins specific or enriched in Y14 complex are shown by closed circles. Both hnRNP A1, C1, and Y14 are indicated. (B) Immunoprecipitations were performed using the indicated antibodies and subjected to Western blot analyses. The lane marked as nucleoplasm contains 3% of input fraction. SP2/0 was used as a negative control. Molecular Cell 2000 6, 673-682DOI: (10.1016/S1097-2765(00)00065-4)

Figure 7 Y14 Shuttles Rapidly between the Nucleus and the Cytoplasm Expression vectors encoding hnRNP A1, C1, and the given NPc fusion proteins were transfected into HeLa cells. After expression of the transfected cDNAs, the cells were fused with mouse NIH3T3 cells to form heterokaryones and incubated in media containing 100 μg/ml cycloheximide for 1 hr. The cells were then fixed and stained for immunofluorescence microscopy with mAb 9E10 (anti-myc tag: panel anti-myc) to localize the proteins, and Hoechst 33258 (panel Hoechst), which differentiates the human and mouse nuclei within the heterokaryon (arrows identify the mouse nuclei). The panels marked Phase show the phase-contrast image of the hetrokaryons; the cytoplasmic edge is highlighted by a broken line. Molecular Cell 2000 6, 673-682DOI: (10.1016/S1097-2765(00)00065-4)

Figure 8 A Schematic Representation of Two Different Nuclear RNP Complexes The new Y14-containing nuclear RNP complex and general hnRNP complex are discussed in the text. Molecular Cell 2000 6, 673-682DOI: (10.1016/S1097-2765(00)00065-4)