scholarly journals An RNA Polymerase II Complex Containing Capping Enzymes and Viral Oncoproteins

IUBMB Life ◽  
2000 ◽  
Vol 50 (2) ◽  
pp. 125-129
Author(s):  
Maria Eugenia Cabrejos ◽  
Edio Maldonado
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Viral Oncoproteins ◽  
Capping Enzymes

An RNA Polymerase II Complex Containing Capping Enzymes and Viral Oncoproteins

IUBMB Life ◽  
2000 ◽  
Vol 50 (2) ◽  
pp. 125-129
Author(s):  
Maria Eugenia Cabrejos ◽  
Edio Maldonado
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Viral Oncoproteins ◽  
Capping Enzymes

scholarly journals An N-Terminal Region of Lassa Virus L Protein Plays a Critical Role in Transcription but Not Replication of the Virus Genome

2009 ◽  
Vol 84 (4) ◽  
pp. 1934-1944 ◽  
Author(s):  
Michaela Lelke ◽  
Linda Brunotte ◽  
Carola Busch ◽  
Stephan Günther
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Scale ◽  
Critical Role ◽  
Terminal Region ◽  
Lassa Virus ◽  
Mrna Synthesis ◽  
L Protein ◽  
Mrna Pool ◽  
Capping Enzymes

ABSTRACT The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. N- and C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In the present study, we have employed a large-scale mutagenesis approach to map functionally relevant residues in these regions. The main targets were acidic (Asp and Glu) and basic residues (Lys and Arg) known to form catalytic and binding sites of capping enzymes and endoribonucleases. A total of 149 different mutants were generated and tested in the Lassa virus replicon system. Nearly 25% of evolutionarily highly conserved acidic and basic side chains were dispensable for function of L protein in the replicon context. The vast majority of the remaining mutants had defects in both transcription and replication. Seven residues (Asp-89, Glu-102, Asp-119, Lys-122, Asp-129, Glu-180, and Arg-185) were selectively important for mRNA synthesis. The phenotype was particularly pronounced for Asp-89, Glu-102, and Asp-129, which were indispensable for transcription but could be replaced by a variety of amino acid residues without affecting genome replication. Bioinformatics disclosed the remote similarity of this region to type IIs endonucleases. The mutagenesis was complemented by experiments with the RNA polymerase II inhibitor α-amanitin, demonstrating dependence of viral transcription from the cellular mRNA pool. In conclusion, this paper describes an N-terminal region in L protein being important for mRNA, but not genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.

scholarly journals Apoptosis and Autophagy Induction in Mammalian Cells by Small Interfering RNA Knockdown of mRNA Capping Enzymes

2008 ◽  
Vol 28 (19) ◽  
pp. 5829-5836 ◽  
Author(s):  
Chun Chu ◽  
Aaron J. Shatkin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Proteolytic Processing ◽  
Tunel Staining ◽  
Interfering Rna ◽  
Capping Enzymes

ABSTRACT Addition of a 5′ cap to RNA polymerase II transcripts, the first step of pre-mRNA processing in eukaryotes from yeasts to mammals, is catalyzed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine-N-7)methyltransferase. The effects of knockdown of these capping enzymes in mammalian cells were investigated using T7 RNA polymerase-synthesized small interfering RNA and also a lentivirus-based inducible, short hairpin RNA system. Decreasing either guanylyltransferase or methyltransferase resulted in caspase-3 activation and elevated terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining characteristic of apoptosis. Induction of apoptosis was independent of p53 tumor suppressor but dependent on BAK or BAX. In addition, levels of the BH3 family member Bim increased, while Mcl-1 and Bik levels remained unchanged during apoptosis. In contrast to capping enzyme knockdown, apoptosis induced by cycloheximide inhibition of protein synthesis required BAK but not BAX. Both Bim and Mcl-1 levels decreased in cycloheximide-induced apoptosis while Bik levels were unchanged, suggesting that apoptosis in siRNA-treated cells is not a direct consequence of loss of mRNA translation. siRNA-treated BAK−/− BAX−/− double-knockout mouse embryonic fibroblasts failed to activate capase-3 or increase TUNEL staining but instead exhibited autophagy, as demonstrated by proteolytic processing of microtubule-associated protein 1 light chain 3 (LC3) and translocation of transfected green fluorescent protein-LC3 from the nucleus to punctate cytoplasmic structures.

scholarly journals RNA Polymerase II C-Terminal Domain Phosphorylation Patterns in Caenorhabditis elegans Operons, Polycistronic Gene Clusters with Only One Promoter

2010 ◽  
Vol 30 (15) ◽  
pp. 3887-3893 ◽  
Author(s):  
Alfonso Garrido-Lecca ◽  
Thomas Blumenthal
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Clusters ◽  
Serine Phosphorylation ◽  
Heptad Repeat ◽  
Processing Enzymes ◽  
Terminal Domain ◽  
Additional Support ◽  
Capping Enzymes

ABSTRACT The heptad repeat of the RNA polymerase II (RNAPII) C-terminal domain is phosphorylated at serine 5 near gene 5′ ends and serine 2 near 3′ ends in order to recruit pre-mRNA processing factors. Ser-5(P) is associated with gene 5′ ends to recruit capping enzymes, whereas Ser-2(P) is associated with gene 3′ ends to recruit cleavage and polyadenylation factors. In the gene clusters called operons in Caenorhabditis elegans, there is generally only a single promoter, but each gene in the operon forms a 3′ end by the usual mechanism. Although downstream operon genes have 5′ ends, they receive their caps by trans splicing rather than by capping enzymes. Thus, they are predicted to not need Ser-5 phosphorylation. Here we show by RNAPII chromatin immunoprecipitation (ChIP) that internal operon gene 5′ ends do indeed lack Ser-5(P) peaks. In contrast, Ser-2(P) peaks occur at each mRNA 3′ end, where the 3′-end formation machinery binds. These results provide additional support for the idea that the serine phosphorylation of the C-terminal domain (CTD) serves to bring RNA-processing enzymes to the transcription complex. Furthermore, these results provide a novel demonstration that genes in operons are cotranscribed from a single upstream promoter.

scholarly journals 5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II

1997 ◽  
Vol 11 (24) ◽  
pp. 3306-3318 ◽  
Author(s):  
S. McCracken ◽  
N. Fong ◽  
E. Rosonina ◽  
K. Yankulov ◽  
G. Brothers ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Capping Enzymes

scholarly journals Separable Functions of the Fission Yeast Spt5 Carboxyl-Terminal Domain (CTD) in Capping Enzyme Binding and Transcription Elongation Overlap with Those of the RNA Polymerase II CTD

2010 ◽  
Vol 30 (10) ◽  
pp. 2353-2364 ◽  
Author(s):  
Susanne Schneider ◽  
Yi Pei ◽  
Stewart Shuman ◽  
Beate Schwer
Keyword(s):  
Rna Polymerase ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Pol Ii ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Capping Enzyme ◽  
Carboxyl Terminal ◽  
Capping Enzymes

ABSTRACT An interaction network connecting mRNA capping enzymes, the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD), elongation factor Spt5, and the Cdk7 and Cdk9 protein kinases is thought to comprise a transcription elongation checkpoint. A crux of this network is Spt5, which regulates early transcription elongation and has an imputed role in pre-mRNA processing via its physical association with capping enzymes. Schizosaccharomyces pombe Spt5 has a distinctive CTD composed of tandem nonapeptide repeats of the consensus sequence 1TPAWNSGSK9. The Spt5 CTD binds the capping enzymes and is a substrate for threonine phosphorylation by the Cdk9 kinase. Here we report that deletion of the S. pombe Spt5 CTD results in slow growth and aberrant cell morphology. The severity of the spt5-ΔCTD phenotype is exacerbated by truncation of the Pol II CTD and ameliorated by overexpression of the capping enzymes RNA triphosphatase and RNA guanylyltransferase. These results suggest that the Spt5 and Pol II CTDs play functionally overlapping roles in capping enzyme recruitment. We probed structure-activity relations of the Spt5 CTD by alanine scanning of the consensus nonapeptide. The T1A change abolished CTD phosphorylation by Cdk9 but did not affect CTD binding to the capping enzymes. The T1A and P2A mutations elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects and conferred sensitivity to growth inhibition by 6-azauracil that was exacerbated by partial truncations of the Pol II CTD. The T1A phenotypes were rescued by a phosphomimetic T1E change but not by capping enzyme overexpression. These results imply a positive role for Spt5 CTD phosphorylation in Pol Il transcription elongation in fission yeast, distinct from its capping enzyme interactions. Viability of yeast cells bearing both Spt5 CTD T1A and Pol II CTD S2A mutations heralds that the Cdk9 kinase has an essential target other than Spt5 and Pol II CTD-Ser2.

Nutrient-regulated gene expression in eukaryotes

2006 ◽  
Vol 73 ◽  
pp. 85-96 ◽  
Author(s):  
Richard J. Reece ◽  
Laila Beynon ◽  
Stacey Holden ◽  
Amanda D. Hughes ◽  
Karine Rébora ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Direct Interaction ◽  
Signalling Pathways ◽  
A Cell ◽  
One Step ◽  
Higher Eukaryotes ◽  
Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

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