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Results for “Nuclear Cap-Binding Protein Complex”

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PubMed1998

Internal and overall motions of the translation factor eIF4E: cap binding and insertion in a CHAPS detergent micelle.

The mRNA cap-binding protein eIF4E is the limiting factor in the eIF4F translation initiation complex, which mediates the binding of the 40S ribosome to the mRNA. 15N relaxation studies have been used to characterize the backbone dynamics of deuterated eIF4E in a CHAPS micelle for the apoprotein, the m7GDP-bound form, and the dinucleotide (m7GpppA)-bound form, as well as for CHAPS-free eIF4E. Large differences in overall correlation time between the CHAPS-free form (11.8 ns) and samples containing different concentrations of CHAPS (15.9-19.4 ns) indicate that eIF4E is embedded in a large micelle in the presence of CHAPS, with a total molecular weight in the range of 40-60 kDa. CHAPS seems to restrict the mobility of the a2-b3 and a4-b5 loops which are thought to be embedded in the micelle. No significant changes in overall mobility were seen between the m7 GDP-bound form, the m7GpppA-bound form, and the apoprotein. Amide hydrogen exchange data indicate the presence of slowly exchanging amides in two surface-exposed helices (a2 and a4), as well as the a4-b5 loop, indicating protection by the CHAPS micelle. The micelle covers the convex side of the protein away from the cap-binding site.

Cholic Acids↗

PubMed2001

Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m(7) guanosine cap.

In eukaryotes the majority of mRNAs have an m(7)G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3' end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure.

3T3 Cells↗

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↗

PubMed1997

The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain.

Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four- to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.

Adenosine Triphosphatases↗

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↗

PubMed2001

An mRNA cap binding protein, ABH1, modulates early abscisic acid signal transduction in Arabidopsis.

The plant hormone abscisic acid (ABA) regulates important stress and developmental responses. We have isolated a recessive ABA hypersensitive mutant, abh1, that shows hormone specificity to ABA. ABH1 encodes the Arabidopsis homolog of a nuclear mRNA cap binding protein and functions in a heterodimeric complex to bind the mRNA cap structure. DNA chip analyses show that only a few transcripts are down-regulated in abh1, several of which are implicated in ABA signaling. Consistent with these results, abh1 plants show ABA-hypersensitive stomatal closing and reduced wilting during drought. Interestingly, ABA-hypersensitive cytosolic calcium increases in abh1 guard cells demonstrate amplification of early ABA signaling. Thus, ABH1 represents a modulator of ABA signaling proposed to function by transcript alteration of early ABA signaling elements.

Abscisic Acid↗

PubMed2001

Crystal structure of the human nuclear cap binding complex.

The heterodimeric nuclear cap binding complex (CBC) binds to 5'-capped polymerase II transcripts. It enhances the efficiency of several mRNA maturation steps and is essential for U snRNA nuclear export in multicellular eukaryotes. The 2A crystal structure of human CBC shows that the large subunit, CBP80, comprises three domains, each containing consecutive helical hairpins and resembling the so-called MIF4G domain found in several other proteins involved in RNA metabolism. The small subunit, CPB20, has an RNP fold and associates with the second and third domains of CBP80. Site-directed mutagenesis revealed 4 residues of CBP20 which are critical for cap binding. A model for cap binding is proposed based on these results and the known mode of binding of RNA to RNP domains.

Amino Acid Sequence↗

PubMed1999

The role of the 5' untranslated region of an mRNA in translation regulation during development.

Cap-dependent ribosomal scanning occurs on the majority of cellular 5' UTRs. This process is severely hampered on long 5' UTRs, containing AUGs and secondary structure. These characteristics are often found in mRNAs encoding regulatory proteins like proto-oncogenes, growth factors, their receptors, and homeodomain proteins. A number of these mRNAs use an alternative mechanism of translation initiation, involving an internal ribosomal entry site (IRES). Cellular mRNAs containing a complex 5' UTR or an IRES share an intriguing characteristic: their translational efficiency can be very specifically regulated by their 5' UTR, providing post-transcriptional regulation. During embryonic development, the 5' UTRs of Antp. Ubx RAR beta 2 c-mos and c-myc regulate protein expression in a spatio-temporal manner. Translation initiation on a number of growth factor RNAs (IGFII, PDGF2, TGF beta, FGF-2, and VEGF) is specifically regulated during differentiation, growth, and stress. Furthermore, 5' UTR activity, mutations in the 5' UTR, or the occurrence of alternative 5' UTRs have been implicated in the progression of various forms of cancer. The mechanisms involved in 5' UTR mediated control are not well understood. Binding of trans-acting factors could mediate translation stimulation or repression. Furthermore, the precise localization of upstream AUGs and the activity of the cap-binding initiation factor 4E are suggested to be important for translation regulation of these mRNAs. This review focuses on 5' UTRs whose activity is regulated, the processes during which this regulation occurs, and as far as known the mechanisms involved.

5' Untranslated Regions↗

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↗

PubMed1997

Participation of the nuclear cap binding complex in pre-mRNA 3' processing.

Communication between the 5' and 3' ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5' end is bound by the nuclear cap binding complex (CBC). This RNA-protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3' end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5' and 3' ends of the pre-mRNA during processing is mediated by the physical association of the CBC/cap complex with 3' processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of "closed-loops," due to interactions between factors bound at their 5' and 3' ends.

Cell Nucleus↗

PubMed1998

Snurportin1, an m3G-cap-specific nuclear import receptor with a novel domain structure.

The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5'-2,2,7-terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G-cap but not m7G-cap structures. Snurportin1 enhances the m3G-capdependent nuclear import of U snRNPs in both Xenopus laevis oocytes and digitonin-permeabilized HeLa cells, demonstrating that it functions as an snRNP-specific nuclear import receptor. Interestingly, solely the m3G-cap and not the Sm core NLS appears to be recognized by snurportin1, indicating that at least two distinct import receptors interact with the complex snRNP NLS. Snurportin1 represents a novel nuclear import receptor which contains an N-terminal importin beta binding (IBB) domain, essential for function, and a C-terminal m3G-cap-binding region with no structural similarity to the arm repeat domain of importin alpha.

Amino Acid Sequence↗

PubMed2000

A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5' cap-binding protein, eIF4E.

The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in the control of cell growth. eIF4E binds to the mRNA 5' cap structure m(7)GpppN (where N is any nucleotide), and promotes ribosome binding to the mRNA in the cytoplasm. However, a fraction of eIF4E localizes to the nucleus. Here we describe the cloning and functional characterization of a new eIF4E-binding protein, referred to as 4E-T (eIF4E-Transporter). We demonstrate that 4E-T is a nucleocytoplasmic shuttling protein that contains an eIF4E-binding site, one bipartite nuclear localization signal and two leucine-rich nuclear export signals. eIF4E forms a complex with the importin alphabeta heterodimer only in the presence of 4E-T. Overexpression of wild-type 4E-T, but not of a mutant defective for eIF4E binding, causes the nuclear accumulation of HA-eIF4E in cells treated with leptomycin B. Taken together, these results demonstrate that the novel nucleocytoplasmic shuttling protein 4E-T mediates the nuclear import of eIF4E via the importin alphabeta pathway by a piggy-back mechanism.

Amino Acid Sequence↗

PubMed2001

Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation.

eIF5 stimulates the GTPase activity of eIF2 bound to Met-tRNA(i)(Met), and its C-terminal domain (eIF5-CTD) bridges interaction between eIF2 and eIF3/eIF1 in a multifactor complex containing Met-tRNA(i)(Met). The tif5-7A mutation in eIF5-CTD, which destabilizes the multifactor complex in vivo, reduced the binding of Met-tRNA(i)(Met) and mRNA to 40S subunits in vitro. Interestingly, eIF5-CTD bound simultaneously to the eIF4G subunit of the cap-binding complex and the NIP1 subunit of eIF3. These interactions may enhance association of eIF4G with eIF3 to promote mRNA binding to the ribosome. In vivo, tif5-7A eliminated eIF5 as a stable component of the pre-initiation complex and led to accumulation of 48S complexes containing eIF2; thus, conversion of 48S to 80S complexes is the rate-limiting defect in this mutant. We propose that eIF5-CTD stimulates binding of Met-tRNA(i)(Met) and mRNA to 40S subunits through interactions with eIF2, eIF3 and eIF4G; however, its most important function is to anchor eIF5 to other components of the 48S complex in a manner required to couple GTP hydrolysis to AUG recognition during the scanning phase of initiation.

Codon, Initiator↗

PubMed2000

Inhibition of mRNA turnover in yeast by an xrn1 mutation enhances the requirement for eIF4E binding to eIF4G and for proper capping of transcripts by Ceg1p.

Null mutants of XRN1, encoding the major cytoplasmic exoribonuclease in yeast, are viable but accumulate decapped, deadenylated transcripts. A screen for mutations synthetic lethal with xrn1Delta identified a mutation in CDC33, encoding eIF4E. This mutation (glutamate to glycine at position 72) affected a highly conserved residue involved in interaction with eIF4G. Synthetic lethality between xrn1 and cdc33 was not relieved by high-copy expression of eIF4G or by disruption of the yeast eIF4E binding protein Caf20p. High-copy expression of a mutant eIF4G defective for eIF4E binding resulted in a dominant negative phenotype in an xrn1 mutant, indicating the importance of this interaction in an xrn1 mutant. Another allele of CDC33, cdc33-1, along with mutations in CEG1, encoding the nuclear guanylyltransferase, were also synthetic lethal with xrn1Delta, whereas mutations in PRT1, encoding a subunit of eIF3, were not. Mutations in CDC33, CEG1, PRT1, PAB1, and TIF4631, encoding eIF4G1, have been shown to lead to destabilization of mRNAs. Although such destabilization in cdc33, ceg1, and pab1 mutants can be partially suppressed by an xrn1 mutation, we observed synthetic lethality between xrn1 and either cdc33 or ceg1 and no suppression of the inviability of a pab1 null mutation by xrn1Delta. Thus, the inhibition of mRNA turnover by blocking Xrn1p function does not suppress the lethality of defects upstream in the turnover pathway but it does enhance the requirement for (7)mG caps and for proper formation of the eIF4E/eIF4G cap recognition complex.

Cell Nucleus↗

PubMed1996

Nucleocytoplasmic transport.

Active transport of proteins and RNAs between the nucleus and cytoplasm is a major process in eukaryotic cells. Recently, factors that recognize transport substrates and mediate nuclear import or export have been characterized, revealing interactions that target substrates to the nuclear pore complexes, through which translocation occurs. Translocation requires energy, and for the import process this energy is at least partly consumed by the action of the small guanosine triphosphatase Ran. In the first half of the review, some of the well-established general background information on nucleocytoplasmic transport is discussed. The second half describes recent information on the mechanistic details of nuclear import and export as well as major unresolved issues such as how directionality is conferred on either import or export. The whole review is slanted toward discussion of metazoan cells.

Amino Acid Sequence↗

PubMed2000

The role of nuclear cap binding protein Cbc1p of yeast in mRNA termination and degradation.

The cyc1-512 mutation in Saccharomyces cerevisiae causes a 90% reduction in the level of iso-1-cytochrome c because of the lack of a proper 3'-end-forming signal, resulting in low levels of eight aberrantly long cyc1-512 mRNAs which differ in length at their 3' termini. cyc1-512 can be suppressed by deletion of either of the nonessential genes CBC1 and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex. The upf1-Delta deletion suppressed the cyc1-512 defect by diminishing degradation of the longer subset of cyc1-512 mRNAs, suggesting that downstream elements or structures occurred in the extended 3' region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations. On the other hand, suppression of cyc1-512 defects by cbc1-Delta occurred by two different mechanisms. The levels of the shorter cyc1-512 transcripts were enhanced in the cbc1-Delta mutants by promoting 3'-end formation at otherwise-weak sites, whereas the levels of the longer cyc1-512 transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation. Furthermore, cbc1-Delta greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1 strain which is defective in mRNA export. We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei.

Fungal Proteins↗

PubMed2002

The SMN complex is associated with snRNPs throughout their cytoplasmic assembly pathway.

The common neurodegenerative disease spinal muscular atrophy is caused by reduced levels of the survival of motor neurons (SMN) protein. SMN associates with several proteins (Gemin2 to Gemin6) to form a large complex which is found both in the cytoplasm and in the nucleus. The SMN complex functions in the assembly and metabolism of several RNPs, including spliceosomal snRNPs. The snRNP core assembly takes place in the cytoplasm from Sm proteins and newly exported snRNAs. Here, we identify three distinct cytoplasmic SMN complexes, each representing a defined intermediate in the snRNP biogenesis pathway. We show that the SMN complex associates with newly exported snRNAs containing the nonphosphorylated form of the snRNA export factor PHAX. The second SMN complex identified contains assembled Sm cores and m(3)G-capped snRNAs. Finally, the SMN complex is associated with a preimport complex containing m(3)G-capped snRNP cores bound to the snRNP nuclear import mediator snurportin1. Thus, the SMN complex is associated with snRNPs during the entire process of their biogenesis in the cytoplasm and may have multiple functions throughout this process.

Cell Line↗