Association of hnRNP S1 Proteins C2 and D2 with Vimentin Intermediate Filaments.

Abstract

Keywords: S I protein/hnRNP/intermediate filament/microtubule/micro filament/vimentin ABSTRACT. SI proteins A-D are hnRNPproteins which were originally isolated from cell nuclei of various tissues, by selective extraction at pH 4.9 from the supernatants of nuclei mildly treated with DNase I or RNase A. In the present study, a hybridoma was isolated which produced a monoclonal antibody that reacted specifically with SI proteins C2 and D2. Whenthe antibody was used in indirect immunofluorescence staining of cultured cells, it stained, in addition to the nuclei, the cytoskeleton-like fibrous structures in the cytoplasm. Wedemonstrate that the cytoskeletal filaments are vimentin intermediate filaments. This is the first report on the hnRNP protein-association with cytoskeleton, and will help to clarify cytoplasmic mRNA localization as well as cytoplasmic distribution of hnRNPproteins.SI proteins constitute a group of nuclear proteins (8) present at sites sensitive to RNaseas well as DNase,and can be extracted at pH4.9 from the reaction supernatant of nuclei mildly treated with either enzyme (6, 9 (44.5); Dl (41.5), and D2 (39.5). They were found in all rat tissues examined, and in cells from various species including mammals, bird (chicken), fish (carp), amphibian (frog) (8 and unpublished), and echinoderm (starfish) (2). A polyclonal antibody was raised in a rabbit against rat liver SI protein B reacted with the proteins B2, Cl and Dl (7,14). Withthis antiserum, the SI proteins were detected in the extranucleolar nucleoplasm, localizing in the euchromatin bordering the heterochromatic areas (14), where most of the RNApolymerase II transcription takes place (3). In a molecular cloning from a rat liver CDNA expression library using the polyclonal antibody, a clone was isolated that encoded a new class of RNA-binding protein, Sl-1, of 852 amino acid residues (10). Although the Sl-1 protein did not coincide with the SI proteins, the Sl-1 and B2, Cl, and Dl were similar in that they possess the same or similar epitope structures as well as the RNA-bindingactivities. A hybridoma that produces a monoclonal antibody specific for the SI proteins C2 and D2 was isolated.With the mono-and polyclonal antibodies, all the SI proteins (C2 and D2, and B2, Cl and Dl) were demonstrated to occur in the nucleus as a soluble form as well as a chromatin-bound form. By sedimentation and immunoprecipitation analyses of the soluble fraction from rat liver nuclei, the antibody-reacting SI proteins were demonstrated to be in association with RNA-protein complexes of heterogeneous sizes, with S-values up to 200 S or more. Four regions of C2 and D2 were microsequenced, and each region gave the identical amino acid sequence between C2 and D2, indicating that they are homologous proteins (probably resulting from alternative splicing). Moreover, these peptide sequences were identical to those contained by the human type A/B hnRNP protein (12). Thus, the SI proteins C2 and D2 are identical or closely related to the type A/B hnRNPprotein that has tw...

Association of hnRNP S1 proteins with vimentin intermediate filaments in migrating cells

“…A hybridoma producing monoclonal antibody McAb 351, which is highly specific for S1 proteins C2 and D2, has been isolated. Cell staining with this antibody showed that they occur not only in the nucleus but also in the cytoplasm often in association with VFs in cultured cells (Tsugawa et al, 1997). In this study, we verified the association of S1 proteins with VFs, and investigated its biological significance.…”

“…Indirect immunofluorescence staining with antibody McAb 351, specific for S1 proteins C2 and D2, shows localization of these proteins on vimentin filaments (VFs) as well as in the nuclei of VF-containing cells such as HaK cells and HeLa cells (Tsugawa et al, 1997). S1 proteins on VFs (S1 fibers) were similarly observed in ARL rat liver epithelial cells (Fig.…”

“…No vimentin was precipitated without the antibody or with control antibodies irrelevant to S1 proteins. As McAb 351 does not recognize vimentin (Tsugawa et al, 1997), the results verified that S1 proteins form complexes with vimentin.…”

“…Each solution was made in phosphate-buffered saline (PBS) and between each step, cells were washed three or four times with PBS. Primary antibodies were mouse monoclonal antibody against vimentin (V9, 1:150 dilution, IT), rabbit (Cappel or Medak) or goat (RDI or Chemicon) polyclonal antibody against vimentin (1:50-200 dilution), and mouse monoclonal antibody McAb 351 against rat S1 proteins C2 and D2 (1:100 dilution) (Tsugawa et al, 1997). Secondary antibodies (1:30-200 dilution) were FITC-conjugated goat or rabbit anti-mouse IgG antibodies (Biosource International or Zymed), rhodamine-conjugated goat anti-rabbit IgG (AP156R, 1:50 dilution, Chemicon International) or TR-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology).…”

“…Reference S1 proteins were prepared from rat liver nuclei as described (Inoue et al, 1983;Tsugawa et al, 1997). ARL cells were solubilized in a standard SDS sample buffer (SDS and 2-mercaptoethanol were at 2% and 1% respectively) and the lysates sonicated and heated at 95°C for 5 minutes.…”