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PubMed1997

Interaction between the human nuclear cap-binding protein complex and hnRNP F.

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.

Amino Acid Sequence↗
PubMed1996

Importin provides a link between nuclear protein import and U snRNA export.

Importin-alpha mediates nuclear protein import by binding nuclear localization signals and importin-beta. We find approximately 30% of SRP1p, the yeast importin-alpha, in a nuclear complex with the Saccharomyces cerevisiae nuclear cap-binding protein complex (CBC). Similarly, a large fraction of Xenopus CBC is associated with importin-alpha in the nucleus. CBC promotes nuclear export of capped U snRNAs and shuttles between nucleus and cytoplasm. The CBC-importin-alpha complex binds specifically to capped RNA, suggesting that CBC might shuttle while bound to importin-alpha. Strikingly, importin-beta binding displaces the RNA from the CBC-importin-alpha complex. Thus, the commitment of CBC for nuclear reentry triggers the release of the export substrate into the cytoplasm. We provide evidence for a mechanism that ensures that importin-mediated RNA release is a specifically cytoplasmic event.

Amino Acid Sequence↗
PubMed1995

Identification of the factors that interact with NCBP, an 80 kDa nuclear cap binding protein.

It has been shown that the monomethylated cap structure plays important roles in pre-mRNA splicing and nuclear export of RNA. As a candidate for the factor involved in these nuclear events we have previously purified an 80 kDa nuclear cap binding protein (NCBP) from a HeLa cell nuclear extract and isolated its full-length cDNA. In this report, in order to obtain a clue to the cellular functions of NCBP, we attempted to identify a factor(s) that interacts with NCBP. Using the yeast two-hybrid system we isolated three clones from a HeLa cell cDNA library. We designated the proteins encoded by these clones NIPs (NCBP interacting proteins). NIP1 and NIP2 have an RNP consensus-type RNA binding domain, whereas NIP3 contains a unique domain of Arg-Glu or Lys-Glu dipeptide repeats. We also show that NCBP requires NIP1 for binding to the cap structure. Possible roles of NIPs in cap-dependent nuclear processes are discussed.

Amino Acid Sequence↗
PubMed1997

RNA movement between the nucleus and the cytoplasm.

The past year has seen significant advances in our understanding of the mechanism of RNA movement between the nucleus and the cytoplasm. The emerging view is that proteins bind to and escort RNAs to their proper subcellular location. The discovery of peptide signals that target nuclear export and the identification of novel protein mediators of RNA export are examples of significant recent discoveries.

Amino Acid Sequence↗
PubMed1995

Characterization of the in vivo phosphorylation sites of the mRNA.cap-binding complex proteins eukaryotic initiation factor-4E and p20 in Saccharomyces cerevisiae.

Eukaryotic translation is believed to be regulated via the phosphorylation of specific eukaryotic initiation factors (eIFs), including one of the cap-binding complex proteins, eIF-4E. We show that in the yeast Saccharomyces cerevisiae, both eIF-4E and another cap-binding complex protein, p20, are phosphoproteins. The major sites of phosphorylation of yeast eIF-4E are found to be located in the N-terminal region of its sequence (Ser2 and Ser15) and are thus in a different part of the protein from the main phosphorylation sites (Ser53 and Ser209) proposed previously for mammalian eIF-4E. The most likely sites of p20 phosphorylation are at Ser91 and/or Ser154. All of the major sites in the two yeast proteins are phosphorylated by casein kinase II in vitro. Casein kinase II phosphorylation of cap-complex proteins should therefore be considered as potentially involved in the control of yeast protein synthesis. Mutagenesis experiments revealed that yeast eIF-4E activity is not dependent on the presence of Ser2 or Ser15. On the other hand, we observed variations in the amount of (phosphorylated) p20 associated with the cap-binding complex as a function of cell growth conditions. Our results suggest that interactions of yeast eIF-4E with other phosphorylatable proteins, such as p20, could play a pivotal role in translational control.

Amino Acid Sequence↗
PubMed1996

The yeast splicing factor Mud13p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex.

The mechanism by which pre-mRNAs are initially recognized by the splicing machinery is not well understood. In the yeast system, commitment complexes are the earliest identified splicing complexes. They contain pre-mRNA, U1 snRNP, and the splicing factor Mud2p and probably correspond to the mammalian E complexes, which contain pre-mRNA, U1 snRNP, and the splicing factor U2AF. To identify other yeast commitment complex components, we have characterized mutant strains that are synthetic lethal with viable U1 snRNA mutations. We report here that MUD13 is a nonessential gene that encodes the yeast homolog of CBP20, the small subunit of the vertebrate nuclear cap-binding complex (CBC). Characterization of splicing in the delta-MUD13 strain and extract indicates that Mud13p is a yeast splicing factor and is the second identified non-snRNP commitment complex component. The observations also suggest that CBC interacts with other commitment complex components as well as with the substrate cap. Taken together with the accompanying results for a mammalian system, our data indicate that cap-binding proteins as well as the pre-mRNA cap contribute to early steps in spliceosome assembly.

Amino Acid Sequence↗
PubMed1996

A yeast cap binding protein complex (yCBC) acts at an early step in pre-mRNA splicing.

The function in splicing of a heterodimeric nuclear cap binding complex (yCBC) from the yeast Saccharomyces cerevisiae has been examined. Immunodepletion of splicing extracts with antibodies directed against one component of the complex, yCBP80, results in the efficient co-depletion of the second component, yCBP20, producing CBC-deficient splicing extract. This extract exhibits strongly reduced splicing efficiency and similar reductions in the assembly of both spliceosomes and of the earliest defined precursors to spliceosomes, commitment complexes. The addition of highly purified yCBC substantially restores these defects. These results, together with other data, suggest that CBCs play a highly conserved role in the recognition of pre-mRNA substrates at an early step in the splicing process.

Amino Acid Sequence↗
PubMed1996

Schizosaccharomyces pombe has a novel eukaryotic initiation factor 4F complex containing a cap-binding protein with the human eIF4E C-terminal motif KSGST.

Genetic and biochemical analyses were performed on the cytoplasmic cap-binding complex (eukaryotic initiation factor (eIF) 4F) of Schizosaccharomyces pombe. Genomic and cDNA sequencing of the S. pombe gene (tif1) encoding the cap-binding component eIF4E revealed the presence of two introns in a reading frame of 219 codons. The encoded sequence of 218 amino acids shows a greater degree of identity to the mammalian eIF4E sequence than does its counterpart from Saccharomyces cerevisiae. In particular, unlike its S. cerevisiae counterpart, S.pombe eIF4E has a C-terminal Ser209 within the motif KSGST that is a site of phosphorylation in hamster and rabbit eIF4E. Of relevance to its potential regulatory role, eIF4E was found to be encoded by an mRNA with a six-nucleotide leader and to be of low abundance in vivo. Cross-linking experiments identified S. pombe eIF4E as the major cap-binding protein while a further protein, p36, also showed cap-dependent binding. eIF4A was not associated with the cap-binding complex. While S. pombe eIF4E was shown capable of binding S. cerevisiae p20, an equivalent protein was absent from the eIF4F complex isolated from S. pombe cells. S. pombe 4F therefore shows a remarkable combination of structural and functional properties, some of which it shares with its higher and its lower eukaryotic counterparts.

Amino Acid Sequence↗
PubMed1997

A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E.

In the yeast Saccharomyces cerevisiae a small protein named p20 is found associated with translation initiation factor eIF4E, the mRNA cap-binding protein. We demonstrate here that p20 is a repressor of cap-dependent translation initiation. p20 shows amino acid sequence homology to a region of eIF4G, the large subunit of the cap-binding protein complex eIF4F, which carries the binding site for eIF4E. Both, eIF4G and p20 bind to eIF4E and compete with each other for binding to eIF4E. The eIF4E-p20 complex can bind to the cap structure and inhibit cap-dependent but not cap-independent translation initiation: the translation of a mRNA with the 67 nucleotide omega sequence of tobacco mosaic virus in its 5' untranslated region (which was previously shown to render translation cap-independent) is not inhibited by p20. Whereas the translation of the same mRNA lacking the omega sequence is strongly inhibited by p20. Disruption of CAF20, the gene encoding p20, stimulates the growth of yeast cells, overexpression of p20 causes slower growth of yeast cells. These results show that p20 is a regulator of eIF4E activity which represses cap-dependent initiation of translation by interfering with the interaction of eIF4E with eIF4G, e.g. the formation of the eIF4F-complex.

Amino Acid Sequence↗
PubMed1997

Mutations in Saccharomyces cerevisiae that block meiotic prophase chromosome metabolism and confer cell cycle arrest at pachytene identify two new meiosis-specific genes SAE1 and SAE3.

Two new meiosis-specific genes, SAE1 and SAE3, have been identified in a screen for mutations that confer an intermediate block in meiotic prophase. Such mutations confer a block to spore formation that is circumvented by addition of a mutation that eliminates meiotic recombination initiation and other aspects of chromosome metabolism, i.e., spo11. We show that sae1-1 and sae3-1 mutations each confer a distinct defect in meiotic recombination. sae1-1 produces recombinants but very slowly and ultimately to less than half the wild-type level; sae3-1 makes persistent hyper-resected meiotic double-strand breaks and has a severe defect in formation of recombinants. Both mutants arrest at the pachytene stage of meiotic prophase, sae1-1 temporarily and sae3-1 permanently. The phenotypes conferred by sae3-1 are similar to those conferred by mutation of the yeast RecA homologue DMC1, suggesting that SAE3 and DMC1 act at the same step(s) of chromosome metabolism. These results provide further evidence that intermediate blocks to prophase chromosome metabolism cause cell-cycle arrest. SAE1 encodes a 208-residue protein homologous to vertebrate mRNA cap-binding protein 20. SAE3 corresponds to a meiosis-specific RNA encoding an unusually short open reading frame of 50 codons.

Amino Acid Sequence↗
PubMed1996

A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export.

In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5' domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, and the retention of CBC on the RNP during translocation through the NPC supports its proposed involvement in RNA export.

Animals↗
PubMed1995

A cap-binding protein complex mediating U snRNA export.

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.

Amino Acid Sequence↗
PubMedDate not supplied

Small nuclear RNA associated proteins: autoantigens in connective tissue diseases.

Small nuclear ribonucleoproteins (smRNPs) are complexes of uridylic acid (U)-rich snRNAs with at least 7 core proteins and a varying number of specific proteins. Most of these proteins act as targets for autoantibodies in autoimmune patients. In the last two years, a number of cDNAs coding for these antigens have been cloned and some specific features of the deduced amino acid sequences of these proteins are discussed. The recombinant antigens, produced in high yields by the microorganism, can be used very efficiently in qualitative and quantitative ELISA tests. It is to be expected that the availability of rather large amounts of pure antigen will be of great help in elucidating the question of why these autoantibodies are generated and what their relation to the disease might be.

Autoantibodies↗
PubMed1996

A nuclear cap-binding complex facilitates association of U1 snRNP with the cap-proximal 5' splice site.

The mechanism by which intron-containing RNAs are recognized by the splicing machinery is only partly understood. A nuclear cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts, has previously been shown to play a role in pre-mRNA splicing. Using a combination of splicing complex and psoralen cross-linking analysis we demonstrate that CBC is required for efficient recognition of the 5' splice site by U1 snRNP during formation of E (early) complex on a pre-mRNA containing a single intron. However, in a pre-mRNA containing two introns, CBC is not required for splicing of the cap distal intron. In this case, the presence of an intact polypyrimidine tract in the cap-proximal intron renders splicing of the cap-distal intron independent of CBC. These results support models in which the splice sites in a pre-mRNA are originally recognized by interactions spanning exons. The defects in splicing and U1 snRNP binding caused by CBC depletion can be specifically reversed by recombinant CBC. In summary, efficient recognition of the cap-proximal 5' splice site by U1 snRNP is facilitated by CBC in what may be one of the earliest steps in pre-mRNA recognition. Data in Colot et al. (this issue) indicate that this function of CBC is conserved in humans and yeast.

Base Sequence↗
PubMed1987

Monoclonal antibody-aided characterization of cellular p220 in uninfected and poliovirus-infected HeLa cells: subcellular distribution and identification of conformers.

A monoclonal antibody directed against the Mr-220,000 subunit (p220) of the mRNA cap-binding complex has been prepared and used to analyze the sucrose gradient sedimentation and subcellular location of p220 and its poliovirus-induced cleavage products. The antibody reacted with p220 on immunoblots of cell lysates from uninfected cells, but only with several smaller polypeptides, the p220 cleavage products, in cell lysates from poliovirus-infected cells. The sedimentation of p220 antigens from uninfected or infected cells was analyzed by immunoblot and by enzyme-linked immunosorbent assay (ELISA) of sucrose gradient fractions. The results indicate that antibody reactivity was partially influenced by antigen conformation. Major forms of intact p220 and cleaved p220 were identified by immunoblot, and these had similar sedimentation properties. ELISA analysis of the same gradient fractions detected only uncleaved p220; p220 cleavage products were not recognized. Furthermore, the antibody recognized two forms of native uncleaved p220, one of which appeared to bind antibody with greater affinity. This result suggested the existence of conformational variants of p220. The differential reactivity of the antibody for cleaved versus uncleaved p220 served as a useful control during indirect immunofluorescence analysis to determine the subcellular distribution of p220 antigens. The distribution of p220 in uninfected cells was mainly cytoplasmic, but some nuclear antigens were also apparent. After poliovirus infection only the nuclear pattern remained. Disappearance of the cytoplasmic pattern confirmed the inability of the antibody to react with native p220 cleavage products. The cytoplasmic pattern also disappeared after human rhinovirus 14 infection, but not after mengovirus infection, results which correlated with the ability of human rhinovirus 14 and the inability of mengovirus to induce the cleavage of p220. The results demonstrate that p220 is not likely to be associated with the cytoskeleton and hint at the possibility of a partially nuclear location.

Amino Acid Sequence↗
PubMed1994

A nuclear cap binding protein complex involved in pre-mRNA splicing.

A cap-binding protein complex (CBC) present in the nuclei of HeLa cells has been characterized. Purified CBC consists of two previously identified proteins, CBP80 and CBP20. These proteins are shown to cofractionate to apparent homogeneity and to be coimmunoprecipitable with anti-CBP80 antibodies. Analysis of the inhibition of pre-mRNA splicing in vitro and in vivo by chemically modified analogs of the cap structure, and of the binding of these analogs to CBC in vitro, suggests a role for the complex in splicing. Extracts immunodepleted of CBC do not efficiently splice an adenoviral pre-mRNA owing to blockage of an early step in splicing complex formation. CBC may therefore play a role in pre-mRNA recognition.

Amino Acid Sequence↗
PubMed1992

A fraction of the mRNA 5' cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus.

The 5' cap structure m7GpppN (where N is any nucleotide) is a ubiquitous feature of cellular eukaryotic mRNAs. The cap is multifunctional as it is involved in translation, nucleocytoplasmic transport, splicing, and stabilization of mRNA against 5' exonucleolytic degradation. The cap binding protein, eukaryotic initiation factor 4E (eIF-4E), is a translation initiation factor that binds to the cap structure and is part of a complex (eIF-4F) that promotes mRNA binding to ribosomes. Overexpression of eIF-4E in fibroblasts results in cell transformation. To test the hypothesis that some of the biological effects of eIF-4E might be effected by a nuclear function, we determined the cellular distribution of eIF-4E. By means of indirect immunofluorescence experiments using polyclonal and monoclonal antibodies against eIF-4E as well as transfected epitope-tagged eIF-4E, we demonstrate that a fraction of eIF-4E localizes to the nucleus. These results suggest that eIF-4E is also involved in a nuclear function.

Amino Acid Sequence↗