RESUMO
Cellular functions of tumor suppressor proteins can be mediated by protein-protein interactions. Using p53 as a probe to screen an expression library, a cDNA encoding a 250 kDa protein was isolated. Recombinant forms of this protein, designated PACT, bind to wild type p53 while two different mutations abolish this interaction. PACT protein can also interfere with p53 specific DNA binding. PACT contains a serine/arginine (SR) rich region and a C' terminal lysine rich domain. The 250 kDa PACT protein can be precipitated from cell lysates by a method specific for SR proteins. snRNPs can be co-immunoprecipitated from cells with anti-PACT antibodies. These antibodies stain cell nuclei in a speckled pattern reminiscent of the distribution of known splicing factors. Recently, RBQ1, a truncated human homologue of PACT was identified by virtue of Rb binding. We show that RBQ1 is truncated as a result of a possible mutational event. PACT can interact with both cellular Rb and p53.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA/metabolismo , Proteínas de Ligação a RNA , Testículo/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Clonagem Molecular , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Mutação , Proteína do Retinoblastoma/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
Nuclear RNA transcripts of split genes and their splicing products, as well as the general population of nuclear polyadenylated RNA are packaged in multi-component large nuclear ribonucleoprotein (lnRNP) particles. These lnRNP particles, which sediment at the 200 S region in sucrose gradients, contain all U small nuclear RNPs required for precursor messenger RNA (pre-mRNA) splicing and several protein splicing factors, including U2AF and the SR proteins. Electron microscopy of lnRNP particles revealed a large compact structure of 50 nm in diameter. In this study we employed automated computed tomography from electron micrographs for the three-dimensional (3D) image reconstruction of individual lnRNP particles isolated from mammalian cells nuclei and negatively stained. For each particle, a tilt series of 71 images was collected by direct digital recording of the images on a CCD camera attached to a computer controlled TEM facility. The 3D image was reconstructed according to the back projection principle. For rendering, real time display and comparison of the reconstructed particles, interactive computer graphics was employed. The reconstructed 3D images show a compact structure composed of four major subunits connected to each other. Comparison of the reconstructed lnRNP particles revealed morphological similarity of the individual particles, as well as similarity among the sub-structures. Based on these observations we propose a model for the packaging of nuclear pre-mRNAs in lnRNP particles where each substructure represents a functional unit. This model is compatible with the requirements for alternative splicing in multi-intronic pre-mRNAs, and with the fact that the splicing of multi-intronic pre-mRNAs does not occur in a sequential manner.