2-Hydroxy-2‘-deoxyadenosine 5‘-Triphosphate Enhances A·T → C·G Mutations Caused by 8-Hydroxy-2‘-deoxyguanosine 5‘-Triphosphate by Suppressing Its Degradation upon Replication in a HeLa Extract†

Biochemistry ◽  
2007 ◽  
Vol 46 (22) ◽  
pp. 6639-6646 ◽  
Author(s):  
Kazuya Satou ◽  
Hiroshi Kasai ◽  
Chikahide Masutani ◽  
Fumio Hanaoka ◽  
Hideyoshi Harashima ◽  
...  
Keyword(s):  
Hela Extract

scholarly journals The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream.

1997 ◽  
Vol 17 (8) ◽  
pp. 4667-4676 ◽  
Author(s):  
R C Chan ◽  
D L Black
Keyword(s):  
Splice Site ◽  
Binding Protein ◽  
Cell Extract ◽  
Neural Cell ◽  
Splicing Regulation ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Splice Site Sequence ◽  
Hela Extract

The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. We previously identified conserved intronic elements flanking N1 that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. We now show that these upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds.

scholarly journals Mutations induced by 2-hydroxy-dATP during in vitro replication with a HeLa extract

2003 ◽  
Vol 3 (1) ◽  
pp. 325-326
Author(s):  
K. Satou ◽  
H. Harashima ◽  
H. Kamiya
Keyword(s):  
In Vitro Replication ◽  
Hela Extract

scholarly journals Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro.

1995 ◽  
Vol 15 (11) ◽  
pp. 6377-6385 ◽  
Author(s):  
R C Chan ◽  
D L Black
Keyword(s):  
Hela Cell ◽  
Regulatory Sequence ◽  
Upstream Regulatory Sequence ◽  
Nuclear Extracts ◽  
Repressor Proteins ◽  
Sequence Requirements ◽  
Hela Extract

The neuron-specific N1 exon of the mouse c-src transcript is normally skipped in nonneuronal cells. In this study, we examined the sequence requirements for the exclusion of this exon in nonneuronal HeLa cell nuclear extracts. We found that the repression of the N1 exon is mediated by specific intron sequences that flank the N1 exon. Mutagenesis experiments identified conserved CUCUCU elements within these intron regions that are required for the repression of N1 splicing. The addition of an RNA competitor containing the upstream regulatory sequence to the HeLa extract induced splicing of the intron downstream of N1, indicating that the competitor sequence binds to splicing repressor proteins. The similarities between this mechanism for src splicing repression and the repression of other regulated exons point to a common role of exon-spanning interactions in splicing repression.

scholarly journals m7G cap-eIF4E interaction stimulates polysome formation by enhancing first-round initiation kinetics

2021 ◽  
Author(s):  
Lexi Sun ◽  
Anthony Gaba ◽  
Hongyun Wang ◽  
Xiaohui Qu
Keyword(s):  
Single Molecule ◽  
Wheat Germ ◽  
Initiation Factor ◽  
Translation Efficiency ◽  
Eukaryotic Initiation Factor 4E ◽  
Formation Kinetics ◽  
Kinetics Of ◽  
Hela Extract ◽  
Dependent Mechanism

Translation in eukaryotic cells occurs predominantly through a 7-methylguanosine (m7G) cap-dependent mechanism. m7G cap interactions with eukaryotic initiation factor 4E (eIF4E) facilitates 43S recruitment to the mRNA 5' end and enhances the translation efficiency of mRNA. However, it remains poorly understood how m7G cap-eIF4E interactions affect polysome formation kinetics. Here, we examine the role of the m7G cap in polysome formation by utilizing a single-molecule approach to track individual ribosomes during active translation. Translation was monitored in wheat germ extract with capped and uncapped synthetic mRNAs and in HeLa extract with purified human eIF4E titration. The presence of the m7G cap and the supplementation of eIF4E to eIF4E-deficient extract enhanced the kinetics of the first initiation event of polysomes. Subsequent to the first initiation event, efficient polysome-forming initiation events occurred independent of mRNA m7G capping status and eIF4E concentration. Our results indicate that m7G cap-eIF4E interactions in wheat germ and HeLa extracts promote polysome formation by enhancing first-round initiation kinetics. The dynamics of individual translation events on polysomal mRNAs suggest that first-round initiation events activate mRNAs for efficient subsequent rounds of polysome-forming initiation.

scholarly journals Induction of Substitution and Deletion Mutations by 2-Hydroxyadenine during Replication in a HeLa Extract

2006 ◽  
Vol 28 (3) ◽  
pp. 92-96
Author(s):  
Kazuya Satou ◽  
Hiroshi Kasai ◽  
Hideyoshi Harashima ◽  
Hiroyuki Kamiya
Keyword(s):  
Deletion Mutations ◽  
Hela Extract
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