ABSTRACT
hnRNP F was identified in a screen for proteins that interact with human CBP80 and CBP20, the components of the nuclear cap-binding complex (CBC). In vitro interaction studies showed that hnRNP F can bind to both CBP20 and CBP80 individually. hnRNP F and CBC bind independently to RNA, but hnRNP F binds preferentially to CBC-RNA complexes rather than to naked RNA. The hnRNP H protein, which is 78% identical to hnRNP F and also interacts with both CBP80 and CBP20 in vitro, does not discriminate between naked RNA and CBC-RNA complexes, showing that this effect is specific. Depletion of hnRNP F from HeLa cell nuclear extract decreases the efficiency of pre-mRNA splicing, a defect which can be partially compensated by addition of recombinant hnRNP F. Thus, hnRNP F is required for efficient pre-mRNA splicing in vitro and may participate in the effect of CBC on pre-mRNA splicing.
Subject(s)
Cross-Linking Reagents/metabolism , RNA Caps/metabolism , RNA, Heterogeneous Nuclear/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Amino Acid Sequence , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Heterogeneous-Nuclear Ribonucleoprotein Group F-H , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Macromolecular Substances , Molecular Sequence Data , RNA Cap-Binding ProteinsABSTRACT
Cap structures are added cotranscriptionally to all RNA polymerase II transcripts. They affect several processes including RNA stability, pre-messenger RNA splicing, RNA export from the nucleus and translation initiation. The effect of the cap on translation is mediated by the initiation factor eIF-4F, whereas the effect on pre-mRNA splicing involves a nuclear complex (CBC) composed of two cap binding proteins, CBP80 and CBP20. A role for CBC in the nuclear export of capped RNAs has also been proposed. We report here the characterization of human and Xenopus CBP20s. Antibodies against recombinant CBP20 prevent interaction of CBC with capped RNAs in vitro. Following microinjection into Xenopus oocytes, the antibodies inhibit both pre-mRNA splicing and export of U small nuclear RNAs to the cytoplasm. These results demonstrate that CBC mediates the effect of the cap structure in U snRNA export, and provide direct evidence for the involvement of a cellular RNA-binding factor in the transport of RNA to the cytoplasm.
Subject(s)
RNA Caps , RNA, Small Nuclear/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Antibodies/immunology , Biological Transport , Cell Nucleus/metabolism , Cytoplasm/metabolism , HeLa Cells , Humans , Molecular Sequence Data , RNA Cap-Binding Proteins , RNA-Binding Proteins/immunology , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , XenopusSubject(s)
Chromosomes, Human, Pair 14/genetics , DNA-Binding Proteins/genetics , RNA-Binding Proteins/genetics , Ribonucleoproteins/genetics , Animals , Chromosome Mapping , DNA, Complementary , DNA-Binding Proteins/metabolism , Heterogeneous-Nuclear Ribonucleoproteins , Humans , In Situ Hybridization, Fluorescence , Polypyrimidine Tract-Binding Protein , RNA Precursors/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolismABSTRACT
The yeast nucleolar protein-encoding gene NSR1 was isolated by low-stringency screening of a yeast genomic library with the human heterogeneous nuclear ribonucleoprotein type A1 (hnRNP A1) cDNA probe, and was mapped to chromosome VII. RNA abundance was determined and the transcription start point and polyadenylation site were mapped. A comparison between the Nsr1 and hnRNP A1 proteins, based on homopolymer RNA binding to their structural domains in vitro, revealed a striking biochemical similarity. When the N-terminal, lysine- and arginine-rich domain of Nsr1 was removed, the truncated protein behaved similarly to hnRNP A1; furthermore, the two RRM (RNA recognition motif) domains of Nsr1 behaved in the same manner as the two RRM domains of hnRNP A1. The biochemical data, therefore, would support the hypothesis that the two RRM domains in hnRNP A1 and Nsr1 interact with RNA in a similar manner in both mammalian and yeast cells, respectively.
Subject(s)
Fungal Proteins/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Nuclear Proteins , RNA-Binding Proteins/genetics , Ribonucleoproteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Base Sequence , Chromosome Mapping , Cloning, Molecular , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Gene Expression , Genes, Fungal/genetics , Heterogeneous Nuclear Ribonucleoprotein A1 , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Molecular Sequence Data , Polyribonucleotides/metabolism , RNA, Fungal/biosynthesis , RNA, Messenger/biosynthesis , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Deletion/physiology , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
The regulation of chloride conductance was investigated in the T84 human colon carcinoma cell line by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium. The permeable cAMP analog 8-Br-cAMP (100 microM) and the calcium ionophore ionomycin (1 microM) activate a chloride conductance. A prolonged (4 h) preincubation of cells with phorbol 12-myristate 13-acetate (100 nM) or with the diacylglycerol analog 1-oleoyl-2-acetyl-glycerol (100 microM): (i) down-modulates to almost zero the protein kinase C activity in the membranes; (ii) inhibits the activation of the chloride conductance mediated by 8-Br-cAMP but not by calcium; (iii) reduces the mRNA without changing the expression of the protein product of the cystic fibrosis gene. The data suggest that PKC is essential for the activation of the cAMP-dependent chloride conductance in T84 cells.
Subject(s)
Chlorides/metabolism , Cyclic AMP/pharmacology , Cystic Fibrosis/metabolism , Membrane Proteins/metabolism , Protein Kinase C/metabolism , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Biological Transport , Chloride Channels , Colonic Neoplasms , Cystic Fibrosis Transmembrane Conductance Regulator , Diglycerides/pharmacology , Down-Regulation , Electric Conductivity , Fluorescence , Fluorescent Dyes , Humans , Ionomycin/pharmacology , Membrane Proteins/drug effects , Protein Kinases/metabolism , Quinolinium Compounds , RNA, Messenger/biosynthesis , Tetradecanoylphorbol Acetate/pharmacology , Tumor Cells, CulturedABSTRACT
A human supernumerary minichromosome (MC), found in a newborn baby and sorted on a fluorescence-activated cell sorter (FACS-440) has been previously described [Ferretti et al., Cytotechnology 1 (1987) 7-12]. We report here on the construction of a library of EcoRI fragments in the phage lambda gtWES.lambda B', starting from 7.5 ng of MC DNA, and describe the isolation of single-copy DNA clones from the library in a two-step procedure. We employed in situ hybridization to unambiguously select the clones specific for the MC, and used three of these clones to demonstrate that it originated from chromosome 9.
