Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:A cap-binding protein complex mediating U snRNA export. 765 22

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.
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PMID:A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export. 860 13

In an attempt to further understand how nuclear events (such as gene expression, nuclear import/export, and cell cycle checkpoint control) might be subject to regulation by extracellular stimuli, we sought to identify nuclear activities under growth factor control. Using a sensitive photoaffinity labeling assay that measured [alpha-32P]GTP incorporation into nuclear proteins, we identified the 20-kDa subunit of the nuclear cap-binding complex (CBC) as a protein whose binding activity is greatly enhanced by the extracellular stimulation of serum-arrested cells. The CBC represents a 20- and 80-kDa heterodimer (the subunits independently referred to as CBP20 and CBP80, respectively) that binds the 7-methylguanosine cap on RNAs transcribed by RNA polymerase II. This binding facilitates precursor messenger RNA splicing and export. We have demonstrated that the [alpha-32P]GTP incorporation into CBP20 was correlated with an increased ability of the CBC to bind capped RNA and have used the [alpha-32P]GTP photoaffinity assay to characterize the activation of the CBC in response to growth factors. We show that the CBC is activated by heregulin in HeLa cells and by nerve growth factor in PC12 cells as well as during the G1/S phase of the cell cycle and when cells are stressed with UV irradiation. Additionally, we show that cap-dependent splicing of precursor mRNA, a functional outcome of CBC activation, can be catalyzed by growth factor addition to serum-arrested cells. Taken together, these data identify the CBC as a nuclear target for growth factor-coupled signal transduction and suggest novel mechanisms by which growth factors can influence gene expression and cell growth.
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PMID:The nuclear cap-binding complex is a novel target of growth factor receptor-coupled signal transduction. 993 12

Newly spliced mRNAs in mammalian cells are characterized by a complex of proteins at exon-exon junctions. This complex recruits Upf3 and Upf2, which function in nonsense-mediated mRNA decay (NMD). Both Upf proteins are detected on mRNA bound by the major nuclear cap-binding proteins CBP80/CBP20 but not mRNA bound by the major cytoplasmic cap-binding protein eIF4E. These and other data indicate that NMD targets CBP80-bound mRNA during a 'pioneer' round of translation, but whether nuclear eIF4E also binds nascent but dead-end transcripts is unclear. Here we provide evidence that nuclear CBP80 but not nuclear eIF4E is readily detected in association with intron-containing RNA and the C-terminal domain of RNA polymerase II. Consistent with this evidence, we demonstrate that RNPS1, Y14, SRm160, REF/Aly, TAP, Upf3X and Upf2 are detected in the nuclear fraction on CBP80-bound but not eIF4E-bound mRNA. Each of these proteins is also detected on CBP80-bound mRNA in the cytoplasmic fraction, indicating a presence on mRNA after export. The dynamics of mRNP composition before and after mRNA export are discussed.
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PMID:The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling. 1209 54

mRNAs are capped at their 5'-end by a unique cap structure containing N7-methyl guanine. Recognition of the cap structure is of paramount importance in some of the most central processes of gene expression as well as in some viral processes, such as priming of influenza virus transcription. The recent resolution of the structure of three evolutionary unrelated cap binding proteins, the vaccinia viral protein VP39, the eukaryotic translation factor eIF4E, and the nuclear cap-binding protein CBP20 showed that the recognition of the cap structure is achieved by the same general mechanism, i.e. by "sandwiching" of the N7-methyl guanine of the cap structure between two aromatic amino acid residues. The purpose of the present study was to test whether a similar cap recognition mechanism had independently evolved for the RNA polymerase of influenza virus. Combining in vivo and in vitro methods, we characterized two crucial aromatic amino acids, Phe363 and Phe404, in the PB2 subunit of the viral RNA polymerase that are essential for cap binding. The aromaticity of these two residues is conserved in influenza A, B, and C and even in the divergent Thogoto virus PB2 subunits. Thus, our results favor a similar mechanism of cap binding by the influenza RNA polymerase as in the evolutionary unrelated VP39, eIF4E, and CBP20.
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PMID:Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding. 1264 57

The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.
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PMID:Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex. 1604 98

RNA export factor (REF) is a component of the exon junction complex (EJC) that is deposited on mRNA in a splicing-dependent manner, and targets spliced mRNA for export. In this study, analysis of the RNA-binding protein complexes revealed that REF associates with beta-globin mRNA at the region other than the EJC deposition site. Comparison between RNA polymerase II and T7 transcription and further analysis showed that the deposition of REF apart from the EJC is dependent on the 5' cap structure, but not splicing. Excess amounts of m(7)GpppG cap analog reduced REF binding to intronless mRNA, and a co-immunoprecipitation experiment revealed that REF interacts with the cap-binding protein CBP20. The export of Cy3-labeled intronless beta-globin mRNA from nuclei of HeLa cells was enhanced by co-injection of CBP20 and REF. Thus, REF recruited by CBP20 may play a stimulatory role to export the capped intronless mRNAs.
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PMID:The interaction between cap-binding complex and RNA export factor is required for intronless mRNA export. 1736 67

MicroRNAs (miRNAs) are 21 nt RNAs that regulate many biological processes in plants by mediating translational inhibition or cleavage of target transcripts. Arabidopsis mutants defective in miRNA biogenesis have overlapping and highly pleiotropic phenotypes including serrated leaves and ABA hypersensitivity. Recent evidence indicates that miRNA genes are transcribed by RNA polymerase II (Pol II). Since Pol II transcripts are capped, we hypothesized that CBP (cap-binding protein) 20 and 80 may bind to capped primary miRNA (pri-miRNA) transcripts and play a role in their processing. Here, we show that cbp20 and cbp80 mutants have reduced miRNA levels and increased pri-miRNA levels. Co-immunoprecipitation experiments revealed that pri-miRNAs 159, 166, 168 and 172 could be associated with CBP20 and CBP80. We found that CBP20 and CBP80 are stabilized by ABA by a post-translational mechanism, and these proteins are needed for ABA induction of miR159 during seed germination. The lack of miR159 accumulation in ABA-treated seeds of cbp20/80 mutants leads to increased MYB33 and MYB101 transcript levels, and presumably higher levels of these positive regulators result in ABA hypersensitivity. Genetic and molecular analyses show that CBP20 and 80 have overlapping function in the same developmental pathway as SE and HYL1. Our results identify new components in miRNA biogenesis.
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PMID:Two cap-binding proteins CBP20 and CBP80 are involved in processing primary MicroRNAs. 1882 88

The cap-binding protein complex (CBC) binds to the caps of all RNA polymerase II transcripts, and plays an important role in RNA metabolism. We characterized interactions, localization and nuclear-cytoplasmic transport of two subunits of the Arabidopsis thaliana cap-binding protein complex (AtCBC): AtCBP20 and AtCBP80. Using CFP/YFP-tagged proteins, we show that transport of AtCBC from the cytoplasm to the nucleus in the plant cell is different from that described in other eukaryotic cells. We show that the smaller subunit of the complex, AtCBP20, plays a crucial role in the nuclear import of AtCBC. The C-terminal part of AtCBP20 contains two functionally independent nuclear localization signals (NLSs). At least one of these two NLSs is required for the import of CBC into the plant nucleus. The interaction between the A. thaliana CBP20 and CBP80 was also analyzed in detail, using the yeast two-hybrid system and fluorescence resonance energy transfer (FRET) assays. The N-terminal part of AtCBP20 is essential for interaction with AtCBP80. Furthermore, AtCBP80 is important for the protein stability of the smaller subunit of CBC. Based on these data, we propose a model for the nuclear-cytoplasmic trafficking of the CBC complex in plants.
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PMID:The Arabidopsis CBP20 targets the cap-binding complex to the nucleus, and is stabilized by CBP80. 1945 42

The nuclear cap-binding complex (CBC) is a heterodimer composed of CBP20 and CBP80 subunits and has roles in the biogenesis of messenger RNAs (mRNAs), small nuclear RNAs (snRNAs) and microRNAs. CBP20 is a phylogenetically conserved protein that interacts with the 7-methyl guanosine (m7G) cap added to the 5' end of all RNA polymerase II transcripts. CBP80 ensures high affinity binding of the cap by CBP20 and provides a platform for interactions with other factors. Here we characterize an alternative splice variant of CBP20, termed CBP20S. The CBP20S transcript has an in-frame deletion, leading to the translation of a protein lacking most of the RNA recognition motif (RRM). We show that CBP20S is conserved among mammalian species and is expressed in human cell lines and bone marrow cells. Unlike the full-length CBP20, CBP20S does not bind CBP80 or the m7G cap. Nevertheless, CBP20S does bind mRNA, is localized to an active transcription site and redistributed to nucleolar caps upon transcription inhibition. Our results suggest that this novel form CBP20S plays a role in transcription and/or RNA processing independent of CBP80 or the cap.
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PMID:Binding properties and dynamic localization of an alternative isoform of the cap-binding complex subunit CBP20. 2132 24


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