RESUMO
U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is an essential component of the splicing machinery that is composed of two protein subunits, the 35 kDa U2AF(35) (U2AF1) and the 65 kDa U2AF(65) (U2AF2). U2AF interacts with various splicing factors within this machinery. Here we expand the list of mammalian splicing factors that are known to interact with U2AF(65) as well as the list of nuclear proteins not known to participate in splicing that interact with U2AF(65). Using a yeast two-hybrid system, we found fourteen U2AF(65)-interacting proteins. The validity of the screen was confirmed by identification of five known U2AF(65)-interacting proteins, including its heterodimeric partner, U2AF(35). In addition to binding these known partners, we found previously unrecognized U2AF(65) interactions with four splicing-related proteins (DDX39, SFRS3, SFRS18, SNRPA), two zinc finger proteins (ZFP809 and ZC3H11A), a U2AF(65) homolog (RBM39), and two other regulatory proteins (DAXX and SERBP1). We report which regions of U2AF(65) each of these proteins interacts with and we discuss their potential roles in regulation of pre-mRNA splicing, 3'-end mRNA processing, and U2AF(65) sub-nuclear localization. These findings suggest expanded roles for U2AF(65) in both splicing and non-splicing functions.
Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Splicing de RNA , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Animais , Sequência de Bases , DNA Complementar/genética , Feminino , Técnicas In Vitro , Camundongos , Proteínas Nucleares/genética , Gravidez , Ligação Proteica , Mapeamento de Interação de Proteínas , Precursores de RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/genética , Fator de Processamento U2AF , Técnicas do Sistema de Duplo-HíbridoRESUMO
Thioredoxin reductases (Txnrd) maintain intracellular redox homeostasis in most organisms. Metazoan Txnrds also participate in signal transduction. Mouse embryos homozygous for a targeted null mutation of the txnrd1 gene, encoding the cytosolic thioredoxin reductase, were viable at embryonic day 8.5 (E8.5) but not at E9.5. Histology revealed that txnrd1-/- cells were capable of proliferation and differentiation; however, mutant embryos were smaller than wild-type littermates and failed to gastrulate. In situ marker gene analyses indicated that primitive streak mesoderm did not form. Microarray analyses on E7.5 txnrd-/- and txnrd+/+ littermates showed similar mRNA levels for peroxiredoxins, glutathione reductases, mitochondrial Txnrd2, and most markers of cell proliferation. Conversely, mRNAs encoding sulfiredoxin, IGF-binding protein 1, carbonyl reductase 3, glutamate cysteine ligase, glutathione S-transferases, and metallothioneins were more abundant in mutants. Many gene expression responses mirrored those in thioredoxin reductase 1-null yeast; however, mice exhibited a novel response within the peroxiredoxin catalytic cycle. Thus, whereas yeast induce peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induced sulfiredoxin mRNA. In summary, Txnrd1 was required for correct patterning of the early embryo and progression to later development. Conserved responses to Txnrd1 disruption likely allowed proliferation and limited differentiation of the mutant embryo cells.