scholarly journals A cap binding protein that may mediate nuclear export of RNA polymerase II-transcribed RNAs.

1992 ◽  
Vol 118 (6) ◽  
pp. 1287-1295 ◽  
Author(s):  
E Izaurralde ◽  
J Stepinski ◽  
E Darzynkiewicz ◽  
I W Mattaj
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Export ◽  
Binding Activity ◽  
Rna Transcripts ◽  
Recognition Specificity ◽  
Nuclear Extracts ◽  
Initiation Of Translation ◽  
U1 Snrna ◽  
Rna Export

It has previously been shown that efficient export of U1 snRNA or of microinjected, in vitro synthesized, RNA transcripts from the nucleus of Xenopus oocytes is facilitated by their monomethyl guanosine cap structures. Nuclear exit of these transcripts could be competitively inhibited by microinjection of an excess of a cap analog, the dinucleotide m7GpppG (Hamm, J., and I. W. Mattaj. 1990. Cell. 63:109-118). We have now analyzed the ability of several other related cap analogs to inhibit the export of U1 snRNA from the nucleus. The results define the recognition specificity of a factor(s) involved in RNA transport, and indicate that the cap binding activity (CBA) involved in RNA export is different from cap binding proteins (CBPs) involved in the initiation of translation. A CBP, whose specificity for different analogs correlates with the ability of the analogs to inhibit U1 snRNA export, is identified in nuclear extracts prepared from HeLa cells. We propose that this protein may have a role in the export of capped RNAs from the nucleus.

Sequence requirements for premature transcription arrest within the first intron of the mouse c-fos gene

1991 ◽  
Vol 11 (5) ◽  
pp. 2832-2841
Author(s):  
N Mechti ◽  
M Piechaczyk ◽  
J M Blanchard ◽  
P Jeanteur ◽  
B Lebleu
Keyword(s):  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Rna Transcripts ◽  
First Intron ◽  
Nuclear Extracts ◽  
Intron 1 ◽  
A Cell ◽  
Sequence Requirements

A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.

scholarly journals RanGTP-Binding Protein NXT1 Facilitates Nuclear Export of Different Classes of RNA In Vitro

2000 ◽  
Vol 20 (13) ◽  
pp. 4562-4571 ◽  
Author(s):  
Batool Ossareh-Nazari ◽  
Christèle Maison ◽  
Ben E. Black ◽  
Lyne Lévesque ◽  
Bryce M. Paschal ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Vitro Assay ◽  
U1 Snrna ◽  
Rna Export ◽  
Gtpase Ran ◽  
Export Signal

ABSTRACT To better characterize the mechanisms responsible for RNA export from the nucleus, we developed an in vitro assay based on the use of permeabilized HeLa cells. This new assay supports nuclear export of U1 snRNA, tRNA, and mRNA in an energy- and Xenopusextract-dependent manner. U1 snRNA export requires a 5′ monomethylated cap structure, the nuclear export signal receptor CRM1, and the small GTPase Ran. In contrast, mRNA export does not require the participation of CRM1. We show here that NXT1, an NTF2-related protein that binds directly to RanGTP, strongly stimulates export of U1 snRNA, tRNA, and mRNA. The ability of NXT1 to promote export is dependent on its capacity to bind RanGTP. These results support the emerging view that NXT1 is a general export factor, functioning on both CRM1-dependent and CRM1-independent pathways of RNA export.

scholarly journals Cell-cycle-specific transcription termination within the human histone H3.3 gene is correlated with specific protein–DNA interactions

1997 ◽  
Vol 69 (2) ◽  
pp. 101-110 ◽  
Author(s):  
ALAN TAYLOR ◽  
LIQUN ZHANG ◽  
JOHN HERRMANN ◽  
BEI WU ◽  
LARRY KEDES ◽  
...  
Keyword(s):  
Protein Binding ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Specific Protein ◽  
Binding Activity ◽  
Mobility Shift ◽  
Nuclear Extracts

In vitro studies using highly purified calf thymus RNA polymerase II and a fragment spanning the first intron of H3.3 as template DNA have demonstrated the existence of a strong transcription termination site consisting of thymidine stretches. In this study, nuclear run-on experiments have been performed to assess the extent to which transcription elongation is blocked in vivo using DNA probes corresponding to regions 5′ and 3′ of the in vitro termination sites. These studies suggest that H3.3 expression is stimulated following the inhibition of DNA synthesis through the elimination of the transcription elongation block. Interestingly, both the in vivo and in vitro experiments have revealed that the transcriptional block/termination sites are positioned immediately downstream of a 73 bp region that has been over 90% conserved between the chicken and human H3.3 genes. The extreme conservation of this intronic region suggests a possible role in maintaining cis-acting function. Electrophoretic mobility shift experiments show that HeLa cell nuclear extracts contain protein factors that bind specifically to the region of transcription elongation block. Furthermore, we demonstrate a correlation between the protein binding activity and the transcriptional block in cells that have been either arrested at the initiation of S phase or were replication-interrupted by hydroxyurea. DNA footprinting experiments indicate that the region of protein binding is at the 3′ end of the conserved region and overlaps with one of the three in-vitro-mapped termination sites.

scholarly journals Sequence requirements for premature transcription arrest within the first intron of the mouse c-fos gene.

1991 ◽  
Vol 11 (5) ◽  
pp. 2832-2841 ◽  
Author(s):  
N Mechti ◽  
M Piechaczyk ◽  
J M Blanchard ◽  
P Jeanteur ◽  
B Lebleu
Keyword(s):  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Rna Transcripts ◽  
First Intron ◽  
Nuclear Extracts ◽  
Intron 1 ◽  
A Cell ◽  
Sequence Requirements

A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.

scholarly journals RNA polymerase II transcription factors H4TF-1 and H4TF-2 require metal to bind specific DNA sequences.

1987 ◽  
Vol 7 (12) ◽  
pp. 4582-4584 ◽  
Author(s):  
L Dailey ◽  
S B Roberts ◽  
N Heintz
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Chelating Agents ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Rna Polymerase Ii Transcription

Specific DNA-binding and in vitro transcription activities of H4TF-1 and H4TF-2 are inactivated by chelating agents. Binding activity is restored by addition of Zn2+, and H4TF-2 is also reactivated by Fe2+. In contrast, preformed factor-DNA complexes are resistant to chelators. Therefore, metal ions are a required component of the H4TF-1 and H4TF-2 DNA-binding domains.

scholarly journals Repression of TFIIH Transcriptional Activity and TFIIH-Associated cdk7 Kinase Activity at Mitosis

1998 ◽  
Vol 18 (3) ◽  
pp. 1467-1476 ◽  
Author(s):  
John J. Long ◽  
Anne Leresche ◽  
Richard W. Kriwacki ◽  
Joel M. Gottesfeld
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Kinase Inhibitor ◽  
Large Subunit ◽  
Cyclin B ◽  
Viral Gene ◽  
Gene Promoters ◽  
Nuclear Extracts ◽  
Carboxy Terminal

ABSTRACT Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21 Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis.

scholarly journals Analysis of Cellular Factors That Mediate Nuclear Export of RNAs Bearing the Mason-Pfizer Monkey Virus Constitutive Transport Element

2000 ◽  
Vol 74 (13) ◽  
pp. 5863-5871 ◽  
Author(s):  
Yibin Kang ◽  
Hal P. Bogerd ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Export ◽  
Biological Activities ◽  
Critical Role ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Rna Molecules ◽  
Constitutive Transport Element ◽  
Rna Export

ABSTRACT There is now convincing evidence that the human Tap protein plays a critical role in mediating the nuclear export of mRNAs that contain the Mason-Pfizer monkey virus constitutive transport element (CTE) and significant evidence that Tap also participates in global poly(A)+ RNA export. Previously, we had mapped carboxy-terminal sequences in Tap that serve as an essential nucleocytoplasmic shuttling domain, while others had defined an overlapping Tap sequence that can bind to the FG repeat domains of certain nucleoporins. Here, we demonstrate that these two biological activities are functionally correlated. Specifically, mutations in Tap that block nucleoporin binding also block both nucleocytoplasmic shuttling and the Tap-dependent nuclear export of CTE-containing RNAs. In contrast, mutations that do not inhibit nucleoporin binding also fail to affect Tap shuttling. Together, these data indicate that Tap belongs to a novel class of RNA export factors that can target bound RNA molecules directly to the nuclear pore without the assistance of an importin β-like cofactor. In addition to nucleoporins, Tap has also been proposed to interact with a cellular cofactor termed p15. Although we were able to confirm that Tap can indeed bind p15 specifically both in vivo and in vitro, a mutation in Tap that blocked p15 binding only modestly inhibited CTE-dependent nuclear RNA export. However, p15 did significantly enhance the affinity of Tap for the CTE in vitro and readily formed a ternary complex with Tap on the CTE. This result suggests that p15 may play a significant role in the recruitment of the Tap nuclear export factor to target RNA molecules in vivo.

scholarly journals Multifunctional Roles for the N-Terminal Basic Motif of Alfalfa mosaic virus Coat Protein: Nucleolar/Cytoplasmic Shuttling, Modulation of RNA-Binding Activity, and Virion Formation

2012 ◽  
Vol 25 (8) ◽  
pp. 1093-1103 ◽  
Author(s):  
Mari Carmen Herranz ◽  
Vicente Pallas ◽  
Frederic Aparicio
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Nuclear Export ◽  
Rna Binding ◽  
Nuclear Export Signal ◽  
Alfalfa Mosaic Virus ◽  
Binding Activity ◽  
Systemic Movement ◽  
Virion Formation

In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation.

Identification of two transcription factors that bind to specific elements in the promoter of the adenovirus early-region 4

1988 ◽  
Vol 8 (3) ◽  
pp. 1290-1300
Author(s):  
H Watanabe ◽  
T Imai ◽  
P A Sharp ◽  
H Handa
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Base Pairs ◽  
Early Region ◽  
Dnase I Footprinting ◽  
Nuclear Extracts ◽  
Early Region 4

Two kinds of trans-acting factors that regulate transcription from the promoter of the adenovirus early-region 4 (E4) have been identified by reconstituting nuclear extracts of HeLa cells. They were designated E4TF1 and E4TF3 for E4 transcription factors. These factors were responsible for efficient and accurate transcription in vitro from the E4 promoter, as were another transcription factor, designated E4TF2, and a crude fraction containing endogenous RNA polymerase II. E4TF1 stimulated transcription from the E4 promoter but not from the major late promoter or the E4 mutant promoter lacking the E4TF1-binding site. Footprint analysis of E4TF1 revealed that it binds to a specific region, residing between 132 and 152 base pairs upstream from the initiation site of the E4 mRNA. E4TF3 also regulated transcription from the E4 promoter. E4TF3 protected four ca. 20-base-pair regions in a DNase I footprinting assay. They were located around 40, 160, 230, and 260 base pairs upstream from the initiation site of E4 mRNA. Specific inhibition of E4 transcription was observed by addition of DNA fragments covering one of the E4TF1- and E4TF3-binding sites to in vitro transcription assays. These results suggest that both E4TF1 and E4TF3 regulate E4 transcription by binding to the specific upstream elements in the E4 promoter. These factors may be involved in the E1A transactivation of E4 transcription.

scholarly journals EWS, but Not EWS-FLI-1, Is Associated with Both TFIID and RNA Polymerase II: Interactions between Two Members of the TET Family, EWS and hTAFII68, and Subunits of TFIID and RNA Polymerase II Complexes

1998 ◽  
Vol 18 (3) ◽  
pp. 1489-1497 ◽  
Author(s):  
Anne Bertolotti ◽  
Thomas Melot ◽  
Joël Acker ◽  
Marc Vigneron ◽  
Olivier Delattre ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromosomal Translocation ◽  
Binding Studies ◽  
Pol Ii ◽  
Nuclear Extracts ◽  
Vitro Binding ◽  
Tfiid Complex

ABSTRACT The t(11;22) chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminus-encoding region of theEWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI-1 gene. As the function of the protein encoded by the EWS gene remains unknown, we investigated the putative role of EWS in RNA polymerase II (Pol II) transcription by comparing its activity with that of its structural homolog, hTAFII68. We demonstrate that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs). In vitro binding studies revealed that both EWS and hTAFII68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAFII68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAFII68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAFII68 have been identified, confirming the association with the polymerase. In contrast to EWS, the tumorigenic EWS–FLI-1 fusion protein is not associated with either TFIID or Pol II in Ewing cell nuclear extracts. These observations suggest that EWS and EWS–FLI-1 may play different roles in Pol II transcription.

Sign in / Sign up

Export Citation Format

Share Document