RESUMO
We previously performed a gene-trap screen in mouse cells with particular focus on clones in which the trapped protein-reporter fusions localise to compartments of the nucleus. Here we describe one such gene-trap line in which the fusion protein showed a unique, patchy distribution at the nuclear periphery. We have cloned the endogenous mouse and human cDNAs encoding the protein trapped in the F9/3D3 cell line. The predicted proteins (64 kDa) encoded by this novel gene are highly conserved and similar to an unpublished rat protein in sequence databases called p80 or lyric. The amino acid sequence of 3D3/lyric indicates that it may be a type-1b membrane protein with a single transmembrane domain (TMD). Antibodies against the endogenous protein recognise multiple isoforms, consistent with multiple 3D3/lyric mRNAs detected by Northern blot analysis. Subcellular fractionation and immunostaining show that 3D3/lyric is located not only principally in the endoplasmic reticulum (ER), but also in the nuclear envelope (NE), which is contiguous with this compartment. Furthermore, 3D3/lyric is also found in the nucleolus and is therefore a rare example of a protein that suggests a possible connection between this compartment and the endoplasmic reticulum.
Assuntos
Nucléolo Celular/química , Retículo Endoplasmático/química , Proteínas de Membrana/análise , Membrana Nuclear/química , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Humanos , Membranas Intracelulares/química , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Proteínas de Ligação a RNA , Ratos , Alinhamento de SequênciaRESUMO
A growing body of evidence supports the coordination of pre-mRNA processing and transcriptional regulation. We demonstrate here that mammalian PRP4 kinase (PRP4K) is associated with complexes involved in both of these processes. PRP4K is implicated in pre-mRNA splicing as the homologue of the Schizosaccharomyces pombe pre-mRNA splicing kinase Prp4p, and it is enriched in SC35-containing nuclear splicing speckles. RNA interference of Caenorhabditis elegans PRP4K indicates that it is essential in metazoans. In support of a role for PRP4K in pre-mRNA splicing, we identified PRP6, SWAP, and pinin as interacting proteins and demonstrated that PRP4K is a U5 snRNP-associated kinase. In addition, BRG1 and N-CoR, components of nuclear hormone coactivator and corepressor complexes, also interact with PRP4K. PRP4K coimmunoprecipitates with N-CoR, BRG1, pinin, and PRP6, and we present data suggesting that PRP6 and BRG1 are substrates of this kinase. Lastly, PRP4K, BRG1, and PRP6 can be purified as components of the N-CoR-2 complex, and affinity-purified PRP4K/N-CoR complexes exhibit deacetylase activity. We suggest that PRP4K is an essential kinase that, in association with the both U5 snRNP and N-CoR deacetylase complexes, demonstrates a possible coordination of pre-mRNA splicing with chromatin remodeling events involved in transcriptional regulation.
Assuntos
Proteínas de Drosophila , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/isolamento & purificação , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/isolamento & purificação , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas de Schizosaccharomyces pombe , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , DNA Helicases , Proteínas de Ligação a DNA , Genes de Helmintos , Histona Desacetilases/genética , Histona Desacetilases/isolamento & purificação , Histona Desacetilases/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Correpressor 1 de Receptor Nuclear , Proteínas Serina-Treonina Quinases/genética , Proteínas/metabolismo , Processamento Pós-Transcricional do RNA , Splicing de RNA , Fatores de Processamento de RNA , Proteínas de Ligação a RNA , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Schizosaccharomyces/enzimologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Bridging the gap between the number of gene sequences in databases and the number of gene products that have been functionally characterized in any way is a major challenge for biology. A key characteristic of proteins, which can begin to elucidate their possible functions, is their subcellular location. A number of experimental approaches can reveal the subcellular localization of proteins in mammalian cells. However, genome databases now contain predicted sequences for a large number of potentially novel proteins that have yet to be studied in any way, let alone have their subcellular localization determined. Here we ask whether using bioinformatics tools to analyse the sequence of proteins whose subnuclear localizations have been determined can reveal characteristics or signatures that might allow us to predict localization for novel protein sequences.
