scholarly journals Association of SARFH (sarcoma-associated RNA-binding fly homolog) with regions of chromatin transcribed by RNA polymerase II.

1995 ◽  
Vol 15 (8) ◽  
pp. 4562-4571 ◽  
Author(s):  
D Immanuel ◽  
H Zinszner ◽  
D Ron
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Polytene Chromosomes ◽  
Drosophila Embryo ◽  
Cdna Clones ◽  
Rna Pol Ii ◽  
Pol Ii

Many oncogenes associated with human sarcomas are composed of a fusion between transcription factors and the N-terminal portions of two similar RNA-binding proteins, TLS and EWS. Though the oncogenic fusion proteins lack the RNA-binding domain and do not bind RNA, the contribution from the N-terminal portion of the RNA-binding protein is essential for their transforming activity. TLS and EWS associate in vivo with RNA polymerase II (Pol II) transcripts. To learn more about the target gene specificity of this interaction, the localization of a Drosophila melanogaster protein that has extensive sequence identity to the C-terminal RNA-binding portions of TLS and EWS was studied in preparations of Drosophila polytene nuclei. cDNA clones encoding the full-length Drosophila TLS-EWS homolog, SARFH (stands for sarcoma-associated RNA-binding fly homolog), were isolated. Functional similarity to TLS and EWS was revealed by the association of SARFH with Pol II transcripts in mammalian cells and by the ability of SARFH to elicit homologous down-regulation of the levels of the mammalian proteins. The SARFH gene is expressed in the developing Drosophila embryo from the earliest stages of cellularization and is subsequently found in many cell types. In preparations of polytene chromosomes from salivary gland nuclei, SARFH antibodies recognize their target associated with the majority of active transcription units, revealed by colocalization with the phosphorylated form of RNA Pol II. We conclude that SARFH and, by homology, EWS and TLS participate in a function common to the expression of most genes transcribed by RNA Pol II.

scholarly journals P54nrb Forms a Heterodimer with PSP1 That Localizes to Paraspeckles in an RNA-dependent Manner

2005 ◽  
Vol 16 (11) ◽  
pp. 5304-5315 ◽  
Author(s):  
Archa H. Fox ◽  
Charles S. Bond ◽  
Angus I. Lamond
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Sequence Similarity ◽  
Dependent Manner ◽  
Rna Pol Ii ◽  
Nuclear Processes ◽  
Rna Polymerase Ii Transcription

P54nrb is a protein implicated in multiple nuclear processes whose specific functions may correlate with its presence at different nuclear locations. Here we characterize paraspeckles, a subnuclear domain containing p54nrb and other RNA-binding proteins including PSP1, a protein with sequence similarity to p54nrb that acts as a marker for paraspeckles. We show that PSP1 interacts in vivo with a subset of the total cellular pool of p54nrb. We map the domain within PSP1 that is mediating this interaction and show it is required for the correct localization of PSP1 to paraspeckles. This interaction is necessary but not sufficient for paraspeckle targeting by PSP1, which also requires an RRM capable of RNA binding. Blocking the reinitiation of RNA Pol II transcription at the end of mitosis with DRB prevents paraspeckle formation, which recommences after removal of DRB, indicating that paraspeckle formation is dependent on RNA Polymerase II transcription. Thus paraspeckles are the sites where a subset of the total cellular pool of p54nrb is targeted in a RNA Polymerase II-dependent manner.

scholarly journals A Yeast Heterogeneous Nuclear Ribonucleoprotein Complex Associated With RNA Polymerase II

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 557-571 ◽  
Author(s):  
Nicholas K Conrad ◽  
Scott M Wilson ◽  
Eric J Steinmetz ◽  
Meera Patturajan ◽  
David A Brow ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Temperature Sensitive ◽  
Yeast Two Hybrid ◽  
Pol Ii ◽  
Physical Interactions ◽  
Two Hybrid

Abstract Recent evidence suggests a role for the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) in pre-mRNA processing. The yeast NRD1 gene encodes an essential RNA-binding protein that shares homology with mammalian CTD-binding proteins and is thought to regulate mRNA abundance by binding to a specific cis-acting element. The present work demonstrates genetic and physical interactions among Nrd1p, the pol II CTD, Nab3p, and the CTD kinase CTDK-I. Previous studies have shown that Nrd1p associates with the CTD of pol II in yeast two-hybrid assays via its CTD-interaction domain (CID). We show that nrd1 temperature-sensitive alleles are synthetically lethal with truncation of the CTD to 9 or 10 repeats. Nab3p, a yeast hnRNP, is a high-copy suppressor of some nrd1 temperature-sensitive alleles, interacts with Nrd1p in a yeast two-hybrid assay, and coimmunoprecipitates with Nrd1p. Temperature-sensitive alleles of NAB3 are suppressed by deletion of CTK1, a kinase that has been shown to phosphorylate the CTD and increase elongation efficiency in vitro. This set of genetic and physical interactions suggests a role for yeast RNA-binding proteins in transcriptional regulation.

scholarly journals Mechanism of RNA polymerase II stalling by DNA alkylation

2017 ◽  
Vol 114 (46) ◽  
pp. 12172-12177 ◽  
Author(s):  
Stefano Malvezzi ◽  
Lucas Farnung ◽  
Claudia M. N. Aloisi ◽  
Todor Angelov ◽  
Patrick Cramer ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Anticancer Agents ◽  
Excision Repair ◽  
Minor Groove ◽  
Dna Alkylation ◽  
Drug Induced ◽  
Rna Pol Ii ◽  
Pol Ii

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

scholarly journals Identification of cis Elements Directing Termination of Yeast Nonpolyadenylated snoRNA Transcripts

2004 ◽  
Vol 24 (14) ◽  
pp. 6241-6252 ◽  
Author(s):  
Kristina L. Carroll ◽  
Dennis A. Pradhan ◽  
Josh A. Granek ◽  
Neil D. Clarke ◽  
Jeffry L. Corden
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Large Degree ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Gel Mobility Shift

ABSTRACT RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.

scholarly journals Interaction of yeast RNA-binding proteins Nrd1 and Nab3 with RNA polymerase II terminator elements

RNA ◽  
2007 ◽  
Vol 13 (3) ◽  
pp. 361-373 ◽  
Author(s):  
K. L. Carroll ◽  
R. Ghirlando ◽  
J. M. Ames ◽  
J. L. Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins

scholarly journals cis- and trans-Acting Determinants of Transcription Termination by Yeast RNA Polymerase II

2006 ◽  
Vol 26 (7) ◽  
pp. 2688-2696 ◽  
Author(s):  
Eric J. Steinmetz ◽  
Sarah B. H. Ng ◽  
Joseph P. Cloute ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Nascent Transcript ◽  
Protein Coding ◽  
Pol Ii ◽  
Eukaryotic Genes ◽  
Discrete Surface ◽  
Cleavage And Polyadenylation ◽  
Selection For

ABSTRACT Most eukaryotic genes are transcribed by RNA polymerase II (Pol II), including those that produce mRNAs and many noncoding functional RNAs. Proper expression of these genes requires efficient termination by Pol II to avoid transcriptional interference and synthesis of extended, nonfunctional RNAs. We previously described a pathway for yeast Pol II termination that involves recognition of an element in the nascent transcript by the essential RNA-binding protein Nrd1. The Nrd1-dependent pathway appears to be used primarily for nonpolyadenylated transcripts, such as the small nuclear and small nucleolar RNAs (snoRNAs). mRNAs are thought to use a distinct pathway that is coupled to cleavage and polyadenylation of the transcript. Here we show that the terminator elements for two yeast snoRNA genes also direct polyadenylated 3′-end formation in the context of an mRNA 3′ untranslated region. A selection for cis-acting terminator readthrough mutations identified conserved features of these elements, some of which are similar to cleavage and polyadenylation signals. A selection for trans-acting mutations that induce readthrough of both a snoRNA and an mRNA terminator yielded mutations in the Rpb3 and Rpb11 subunits of Pol II that define a remarkably discrete surface on the trailing end of the enzyme. Our results suggest that, at least in budding yeast, protein-coding and noncoding Pol II-transcribed genes use similar mechanisms to direct termination and that the termination signal is transduced through the Rpb3/Rpb11 heterodimer.

scholarly journals RNA Polymerase II Accumulation in the Promoter-Proximal Region of the Dihydrofolate Reductase and γ-Actin Genes

2003 ◽  
Vol 23 (6) ◽  
pp. 1961-1967 ◽  
Author(s):  
Chonghui Cheng ◽  
Phillip A. Sharp
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dihydrofolate Reductase ◽  
Mammalian Cells ◽  
Proximal Region ◽  
Heptad Repeat ◽  
Pol Ii ◽  
Actin Genes ◽  
Carboxyl Terminal ◽  
Promoter Proximal Region

ABSTRACT The carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) can be phosphorylated at serine 2 (Ser-2) and serine 5 (Ser-5) of the CTD heptad repeat YSPTSPS, and this phosphorylation is important in coupling transcription to RNA processing, including 5′ capping, splicing, and polyadenylation. The mammalian endogenous dihydrofolate reductase and γ-actin genes have been used to study the association of Pol II with different regions of transcribed genes (promoter-proximal compared to distal regions) and the phosphorylation status of its CTD. For both genes, Pol II is more concentrated in the promoter-proximal regions than in the interior regions. Moreover, different phosphorylation forms of Pol II are associated with distinct regions. Ser-5 phosphorylation of Pol II is concentrated near the promoter, while Ser-2 phosphorylation is observed throughout the gene. These results suggest that the accumulation of paused Pol II in promoter-proximal regions may be a common feature of gene regulation in mammalian cells.

scholarly journals The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II

2021 ◽  
Author(s):  
Julie Carnesecchi ◽  
Panagiotis Boumpas ◽  
Patrick van Nierop y Sanchez ◽  
Katrin Domsch ◽  
Hugo Daniel Pinto ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Control Cell ◽  
Active Form ◽  
Pol Ii ◽  
Rna Interaction ◽  
Drosophila Cells

Transcription Factors (TFs) play a pivotal role in cell fate decision by coordinating distinct gene expression programs. Although most TFs act at the DNA regulatory layer, few TFs can bind RNA and modulate mRNA splicing. Yet, the mechanistic cues underlying TFs function in splicing remain elusive. Focusing on the Drosophila Hox TF Ultrabithorax (Ubx), our work shed light on a novel layer of Ubx function at the RNA level. Transcriptome and genome-wide binding profiles in embryonic mesoderm and Drosophila cells indicate that Ubx regulates mRNA expression and splicing to promote distinct functions in defined cellular contexts. Ubx modulates splicing via its DNA-binding domain, the Homeodomain (HD). Our results demonstrate a new RNA-binding ability of Ubx in cells and in vitro. Notably, the N51 amino acid of the HD, which mediates Ubx-DNA interaction, is non-essential for Ubx-RNA interaction in vitro but is required in vivo. We find that the N51 amino acid is necessary to mediate interaction between Ubx and the active form of the RNA Polymerase II (Pol II S2Phos) in Drosophila cells. By combining molecular and imaging approaches, our results reveal that Ubx mediates elongation-coupled splicing via a dynamic interplay with active Pol II and chromatin binding. Overall, our work uncovered a novel role of the Hox TFs at the mRNA regulatory layer. This could be an essential function for other classes of TFs to control cell diversity.

scholarly journals Artificial Recruitment of TFIID, but Not RNA Polymerase II Holoenzyme, Activates Transcription in Mammalian Cells

2000 ◽  
Vol 20 (12) ◽  
pp. 4350-4358 ◽  
Author(s):  
David R. Dorris ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Pol Ii ◽  
Activation Domains ◽  
Synergistic Activation ◽  
The One ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain. Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells. Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters. In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains. In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter. Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters. Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme.

scholarly journals FUS is sequestered in nuclear aggregates in ALS patient fibroblasts

2014 ◽  
Vol 25 (17) ◽  
pp. 2571-2578 ◽  
Author(s):  
Jacob C. Schwartz ◽  
Elaine R. Podell ◽  
Steve S. W. Han ◽  
James D. Berry ◽  
Kevin C. Eggan ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Loss Of Function ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Transcription Start Sites ◽  
Sirna Knockdown ◽  
Als Patients ◽  
Interfering Rna ◽  
Fus Protein

Mutations in the RNA-binding protein FUS have been shown to cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We investigate whether mutant FUS protein in ALS patient–derived fibroblasts affects normal FUS functions in the nucleus. We investigated fibroblasts from two ALS patients possessing different FUS mutations and a normal control. Fibroblasts from these patients have their nuclear FUS protein trapped in SDS-resistant aggregates. Genome-wide analysis reveals an inappropriate accumulation of Ser-2 phosphorylation on RNA polymerase II (RNA Pol II) near the transcription start sites of 625 genes for ALS patient cells and after small interfering RNA (siRNA) knockdown of FUS in normal fibroblasts. Furthermore, both the presence of mutant FUS protein and siRNA knockdown of wild-type FUS correlate with altered distribution of RNA Pol II within fibroblast nuclei. A loss of FUS function in orchestrating Ser-2 phosphorylation of the CTD of RNA Pol II is detectable in ALS patient–derived fibroblasts expressing mutant FUS protein, even when the FUS protein remains largely nuclear. A likely explanation for this loss of function is the aggregation of FUS protein in nuclei. Thus our results suggest a specific mechanism by which mutant FUS can have biological consequences other than by the formation of cytoplasmic aggregates.

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