Myc up-regulates formation of the mRNA methyl cap

2010 ◽  
Vol 38 (6) ◽  
pp. 1598-1601 ◽  
Author(s):  
Victoria H. Cowling
Keyword(s):  
Rna Polymerase Ii ◽  
Cell Transformation ◽  
Mrna Translation ◽  
Major Effect ◽  
Rna Modifications ◽  
Adenosylhomocysteine Hydrolase ◽  
Synthetic Lethal ◽  
Protein Encoding ◽  
Cell Growth And Proliferation ◽  
Capping Enzymes

The Myc proteins c-Myc and N-Myc are essential for development and tissue homoeostasis. They are up-regulated by growth factors and transmit the signal for cell growth and proliferation. Myc proteins are also prominent oncogenes in many human tumour types. Myc proteins regulate the transcription of protein-encoding mRNAs and the tRNAs and rRNA which mediate mRNA translation into protein. Myc proteins also up-regulate translation by increasing addition of the 7-methylguanosine cap (methyl cap) to the 5′ end of pre-mRNA. Addition of the methyl cap increases the rate at which transcripts are translated by directing RNA modifications and translation initiation. Myc induces methyl cap formation by promoting RNA polymerase II phosphorylation which recruits the capping enzymes to RNA, and by up-regulating the enzyme SAHH (S-adenosylhomocysteine hydrolase), which neutralizes the inhibitory by-product of methylation reactions. Myc-induced cap methylation is a major effect of Myc function, being necessary for activated protein synthesis, cell proliferation and cell transformation. Inhibition of cap methylation is synthetic lethal with elevated Myc protein expression, which indicates the potential for cap methylation to be a therapeutic target.

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 What’s all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain via mass spectrometry

2021 ◽  
Author(s):  
Blase Matthew LeBlanc ◽  
Rosamaria Yvette Moreno ◽  
Edwin Escobar ◽  
Mukesh Kumar Venkat Ramani ◽  
Jennifer S Brodbelt ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Phosphorylation State ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Protein Encoding ◽  
Small Nuclear Rnas ◽  
Rnap Ii ◽  
Carboxy Terminal ◽  
Encoding Genes

RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and...

scholarly journals Amino Acid Substitutions in Yeast TFIIF Confer Upstream Shifts in Transcription Initiation and Altered Interaction with RNA Polymerase II

2004 ◽  
Vol 24 (24) ◽  
pp. 10975-10985 ◽  
Author(s):  
Mohamed A. Ghazy ◽  
Seth A. Brodie ◽  
Michelle L. Ammerman ◽  
Lynn M. Ziegler ◽  
Alfred S. Ponticelli
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Primer Extension ◽  
Site Directed Mutagenesis ◽  
Protein Encoding ◽  
Transcription Factor Iif ◽  
Growth Properties ◽  
Encoding Genes

ABSTRACT Transcription factor IIF (TFIIF) is required for transcription of protein-encoding genes by eukaryotic RNA polymerase II. In contrast to numerous studies establishing a role for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae. In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed to probe the functional domains of the S. cerevisiae TFIIF subunits Tfg1 and Tfg2. Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo. Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfg1-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in vivo. Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II. Importantly, biochemical results demonstrated an altered interaction between mutant TFIIF protein and RNA polymerase II. These results provide direct evidence for the involvement of S. cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase II interaction is a determinant in this process.

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.

Dendritic Cell Adenosylhomocysteine Hydrolase-Like Protein 1 (AHCYL1) and Inositol Phospholipid Signaling Are Potential Targets for Modulating Their Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3215-3215
Author(s):  
Masato Kato ◽  
Brian Key ◽  
Adrian Carter ◽  
Nicola Z. Angel ◽  
Benjamine J. Cooper ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Calcium Release ◽  
Mrna Translation ◽  
Zebrafish Embryos ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Adenosylhomocysteine Hydrolase ◽  
Dc Function ◽  
Intracellular Calcium Release ◽  
Zebrafish Embryogenesis

Abstract Recipient and donor dendritic cells (DC) initiate acute graft versus host disease after allogeneic hematopoietic stem cell transplantation and activated DC predict for severe disease. Using a differential display technique, we isolated the cDNA for an adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), a novel intracellular protein with ~50% protein identity to adenosylhomocysteine hydrolase (AHCY) and showed that it was upregulated in activated human DC (Dekker et al. Immunogenetics.2002;53:993). AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor (IP3R), suggesting that AHCYL1 is involved in intracellular calcium release (Ando et al. J Biol Chem.2003; 278:10602). Given that intracellular calcium levels control DC function, we reasoned that AHCYL1 was a potential target for modulating DC function. Therefore, we sought functional data using the zebrafish model. We identified two zebrafish AHCYL1 orthologs (zAHCYL1A and B) by bioinformatics and reverse transcriptase-polymerase chain reaction (RT-PCR). Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative RT-PCR showed that zAHCYL1A and B mRNA expression was regulated differently to the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligos against zAHCYL1A and B into zebrafish embryos inhibited zAHCYL1A and B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies, that were similar to those obtained by depleting intracellular calcium with thapsigargin. The injection of hAHCY had little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in zebrafish embryogenesis by modulating IP3R function and consequent intracellular calcium release. It also suggests that blocking AHCYL1 in DC may have a significant effect on DC function, which might, ultimately, be exploited therapeutically. We have generated AHCYL1 gene deleted mice and these will be used to explore these questions further.

scholarly journals The DHX33 RNA Helicase Promotes mRNA Translation Initiation

2015 ◽  
Vol 35 (17) ◽  
pp. 2918-2931 ◽  
Author(s):  
Yandong Zhang ◽  
Jin You ◽  
Xingshun Wang ◽  
Jason Weber
Keyword(s):  
Translation Initiation ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Mrna Translation ◽  
Rna Helicase ◽  
Rna Helicases ◽  
80S Ribosome ◽  
Protein Levels ◽  
Cell Growth And Proliferation ◽  
Rna Complexes

DEAD/DEAH box RNA helicases play essential roles in numerous RNA metabolic processes, such as mRNA translation, pre-mRNA splicing, ribosome biogenesis, and double-stranded RNA sensing. Herein we show that a recently characterized DEAD/DEAH box RNA helicase, DHX33, promotes mRNA translation initiation. We isolated intact DHX33 protein/RNA complexes in cells and identified several ribosomal proteins, translation factors, and mRNAs. Reduction of DHX33 protein levels markedly reduced polyribosome formation and caused the global inhibition of mRNA translation that was rescued with wild-type DHX33 but not helicase-defective DHX33. Moreover, we observed an accumulation of mRNA complexes with the 80S ribosome in the absence of functional DHX33, consistent with a stalling in initiation, and DHX33 more preferentially promoted structured mRNA translation. We conclude that DHX33 functions to promote elongation-competent 80S ribosome assembly at the late stage of mRNA translation initiation. Our results reveal a newly recognized function of DHX33 in mRNA translation initiation, further solidifying its central role in promoting cell growth and proliferation.

scholarly journals Two distinct HIRA-dependent pathways handle H3.3 de novo deposition and recycling during transcription

2019 ◽  
Author(s):  
Júlia Torné ◽  
Dominique Ray-Gallet ◽  
Ekaterina Boyarchuk ◽  
Mickaël Garnier ◽  
Antoine Coulon ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activity ◽  
De Novo ◽  
Major Effect ◽  
Chromatin States ◽  
Epigenetic Information ◽  
Chromatin Integrity ◽  
Local Enrichment ◽  
Fine Tune

ABSTRACTThe packaging of DNA into nucleosomes represents a challenge for transcription. Nucleosome disruption and histone eviction enables RNA Polymerase II progression through DNA, a process that compromises chromatin integrity and the maintenance of epigenetic information. Here, we used the imaging SNAP-tag system to distinguish new and old histones and monitor chromatin re-assembly coupled to transcription in cells. First, we uncovered a loss of both old variants H3.1 and H3.3 that depends on transcriptional activity, with a major effect on H3.3. Focusing on transcriptionally active domains, we revealed a local enrichment in H3.3 with dynamics involving both new H3.3 incorporation and old H3.3 retention. Mechanistically, we demonstrate that the HIRA chaperone is critical to handle both new and old H3.3, and showed that this implicates different pathways. The de novo H3.3 deposition depends strictly on HIRA trimerization as well as its partner UBN1 while ASF1 interaction with HIRA can be bypassed. In contrast, the recycling of H3.3 requires HIRA but proceeds independently of UBN1 or HIRA trimerization and shows an absolute dependency on ASF1-HIRA interaction. Therefore, we propose a model where HIRA can coordinate these distinct pathways for old H3.3 recycling and new H3.3 deposition during transcription to fine-tune chromatin states.

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