scholarly journals NCBP3 positively impacts mRNA biogenesis

2020 ◽  
Vol 48 (18) ◽  
pp. 10413-10427 ◽  
Author(s):  
Yuhui Dou ◽  
Isabelle Barbosa ◽  
Hua Jiang ◽  
Claudia Iasillo ◽  
Kelly R Molloy ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Export ◽  
Rna Binding ◽  
Rna Degradation ◽  
Exon Junction Complex ◽  
Protein Protein Interaction ◽  
Cap Binding Complex ◽  
Interaction Screening

Abstract The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5′cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein–protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting mRNA expression.

scholarly journals NCBP3 is a productive mRNP component

2020 ◽  
Author(s):  
Yuhui Dou ◽  
Isabelle Barbosa ◽  
Hua Jiang ◽  
Claudia Iasillo ◽  
Kelly R. Molloy ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Export ◽  
Rna Binding ◽  
Rna Degradation ◽  
Exon Junction Complex ◽  
Protein Protein Interaction ◽  
Cap Binding Complex ◽  
Interaction Screening

AbstractThe nuclear Cap Binding Complex (CBC), consisting of Nuclear Cap Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5’cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein-protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting the productive fate of mRNA.

Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro

1992 ◽  
Vol 12 (7) ◽  
pp. 3165-3175
Author(s):  
M Bennett ◽  
S Piñol-Roma ◽  
D Staknis ◽  
G Dreyfuss ◽  
R Reed
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Packaging ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Hnrnp Proteins ◽  
Heterogeneous Nuclear Ribonucleoproteins

We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events.

scholarly journals Formation of a Tap/NXF1 Homotypic Complex Is Mediated through the Amino-Terminal Domain of Tap and Enhances Interaction with Nucleoporins

2008 ◽  
Vol 19 (1) ◽  
pp. 327-338 ◽  
Author(s):  
Leah H. Matzat ◽  
Stephen Berberoglu ◽  
Lyne Lévesque
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Direct Interaction ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Multimeric Complex ◽  
Rna Export ◽  
Amino Terminal Domain

Nuclear export of mRNAs is mediated by the Tap/Nxt1 pathway. Tap moves its RNA cargo through the nuclear pore complex by direct interaction with nucleoporin phenylalanine-glycine repeats. This interaction is strengthened by the formation of a Tap/Nxt1 heterodimer. We now present evidence that Tap can form a multimeric complex with itself and with other members of the NXF family. We also show that the homotypic Tap complex can interact with both Nxt1 and nucleoporins in vitro. The region mediating this oligomerization is localized to the first 187 amino acids of Tap, which overlaps with its RNA-binding domain. Removal of this domain greatly reduces the ability of Tap to bind nucleoporins in vitro and in vivo. This is the first report showing that the Tap amino terminus modulates the interaction of Tap with nucleoporins. We speculate that this mechanism has a regulatory role for RNA export independent of RNA binding.

scholarly journals In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation

2013 ◽  
Vol 203 (6) ◽  
pp. 971-984 ◽  
Author(s):  
Roland G. Heym ◽  
Dennis Zimmermann ◽  
Franziska T. Edelmann ◽  
Lars Israel ◽  
Zeynep Ökten ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Messenger Rna ◽  
Rna Binding ◽  
Long Distance ◽  
Long Distance Transport ◽  
In Vitro Reconstitution ◽  
Localization Elements ◽  
Transport Complex

The assembly and composition of ribonucleic acid (RNA)–transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.

scholarly journals Multimerization of human cytomegalovirus regulatory protein UL69 via a domain that is conserved within its herpesvirus homologues

2007 ◽  
Vol 88 (2) ◽  
pp. 405-410 ◽  
Author(s):  
Peter Lischka ◽  
Marco Thomas ◽  
Zsolt Toth ◽  
Regina Mueller ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Nuclear Export ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Transcriptional Level ◽  
Homologous Proteins ◽  
Interaction Domain ◽  
Self Association

The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that has counterparts in all herpesviruses. Some of these proteins have been shown to function primarily at the post-transcriptional level in promoting nuclear export of viral transcripts. Consistently, this group has reported recently that pUL69 is an RNA-binding, nucleocytoplasmic shuttling protein that facilitates the cytoplasmic accumulation of unspliced mRNA via its interaction with the cellular mRNA export factor UAP56. Evidence has been presented to suggest that some of the pUL69 homologues self-interact and function in vivo as multimers. Herein, the possibility of pUL69 self-association was examined and it has been demonstrated that pUL69 can interact with itself in vitro and in vivo in order to form high-molecular-mass complexes. The self-interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses, suggesting that multimerization is a conserved feature of this protein family.

scholarly journals Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro.

1992 ◽  
Vol 12 (7) ◽  
pp. 3165-3175 ◽  
Author(s):  
M Bennett ◽  
S Piñol-Roma ◽  
D Staknis ◽  
G Dreyfuss ◽  
R Reed
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Packaging ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Hnrnp Proteins ◽  
Heterogeneous Nuclear Ribonucleoproteins

We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events.

scholarly journals Evidence that Negative Elongation Factor Represses Transcription Elongation through Binding to a DRB Sensitivity-Inducing Factor/RNA Polymerase II Complex and RNA

2002 ◽  
Vol 22 (9) ◽  
pp. 2918-2927 ◽  
Author(s):  
Yuki Yamaguchi ◽  
Naoto Inukai ◽  
Takashi Narita ◽  
Tadashi Wada ◽  
Hiroshi Handa
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Interactions ◽  
Rna Binding ◽  
Elongation Factor ◽  
Protein Protein Interactions ◽  
Negative Elongation Factor ◽  
Nascent Transcripts

ABSTRACT Negative elongation factor (NELF) is a human transcription factor complex that cooperates with DRB sensitivity-inducing factor (DSIF)/hSpt4-hSpt5 to repress elongation by RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, including NELF-A, a candidate gene product for Wolf-Hirschhorn syndrome, and NELF-E, a putative RNA-binding protein with arginine-aspartic acid (RD) dipeptide repeats. Here we report several important findings regarding the DSIF/NELF-dependent elongation control. First, we have established an effective method for purifying the active NELF complex using an epitope-tagging technique. Second, the five polypeptides each are important and together are sufficient for its function in vitro. Third, NELF does not bind to either DSIF or RNAPII alone but does bind to the preformed DSIF/RNAPII complex. Fourth, NELF-E has a functional RNA-binding domain, whose mutations impair transcription repression without affecting known protein-protein interactions. Taken together, we propose that NELF causes RNAPII pausing through binding to the DSIF/RNAPII complex and to nascent transcripts. These results also have implications for how DSIF and NELF are regulated in a gene-specific manner in vivo.

scholarly journals Molecular functions of nuclear actin in transcription

2006 ◽  
Vol 172 (7) ◽  
pp. 967-971 ◽  
Author(s):  
Piergiorgio Percipalle ◽  
Neus Visa
Keyword(s):  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
In Vivo Studies ◽  
Macromolecular Assemblies ◽  
In Vivo Experiments ◽  
Specific Subset

Actin is not only a major cytoskeletal component in all eukaryotic cells but also a nuclear protein that plays a role in gene transcription. We put together data from in vitro and in vivo experiments that begin to provide insights into the molecular mechanisms by which actin functions in transcription. Recent studies performed in vitro have suggested that actin, in direct contact with the transcription apparatus, is required in an early step of transcription that is common to all three eukaryotic RNA polymerases. In addition, there is evidence from in vivo studies that actin is involved in the transcription elongation of class II genes. In this case, actin is bound to a specific subset of premessenger RNA binding proteins, and the actin–messenger RNP complex may constitute a molecular platform for recruitment of histone-modifying enzymes. We discuss a general model for actin in RNA polymerase II transcription whereby actin works as a conformational switch in conjunction with specific adaptors to facilitate the remodeling of large macromolecular assemblies at the promoter and along the active gene.

scholarly journals eEF1A Is a Novel Component of the Mammalian Nuclear Protein Export Machinery

2008 ◽  
Vol 19 (12) ◽  
pp. 5296-5308 ◽  
Author(s):  
Mireille Khacho ◽  
Karim Mekhail ◽  
Karine Pilon-Larose ◽  
Arnim Pause ◽  
Jocelyn Côté ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Point Mutations ◽  
Nuclear Retention ◽  
Von Hippel Lindau ◽  
Trna Species

The cytoplasmic translation factor eEF1A has been implicated in the nuclear export of tRNA species in lower eukaryotes. Here we demonstrate that eEF1A plays a central role in nuclear export of proteins in mammalian cells. TD-NEM (transcription-dependent nuclear export motif), a newly characterized nuclear export signal, mediates efficient nuclear export of several proteins including the von Hippel-Lindau (VHL) tumor suppressor and the poly(A)-binding protein (PABP1) in a manner that is dependent on ongoing RNA polymerase II (RNA PolII)-dependent transcription. eEF1A interacts specifically with TD-NEM of VHL and PABP1 and disrupting this interaction, by point mutations of key TD-NEM residues or treatment with actinomycin D, an inhibitor of RNA PolII-dependent transcription, prevents assembly and nuclear export. siRNA-induced knockdown or antibody-mediated depletion of eEF1A prevents in vivo and in vitro nuclear export of TD-NEM–containing proteins. Nuclear retention experiments and inhibition of the Exportin-5 pathway suggest that eEF1A stimulates nuclear export of proteins from the cytoplasmic side of the nuclear envelope, without entering the nucleus. Together, these data identify a role for eEF1A, a cytoplasmic mediator of tRNA export in yeast, in the nuclear export of proteins in mammalian cells. These results also provide a link between the translational apparatus and subcellular trafficking machinery demonstrating that these two central pathways in basic metabolism can act cooperatively.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

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