Multiomic Analysis of the UV-Induced DNA Damage Response

Boeing, Stefan; Williamson, Laura; Encheva, Vesela; Gori, Ilaria; Saunders, Rebecca E.; Instrell, Rachael; Aygün, Ozan; Rodríguez‐Martínez, Marta; Weems, Juston; Kelly, Gavin; Conaway, Joan Weliky; Conaway, Ronald C.; Stewart, Aengus; Howell, Michael; Snijders, Ambrosius P.; Svejstrup, Jesper Q.

doi:10.1016/j.celrep.2016.04.047

Cited by 178 publications

(196 citation statements)

References 51 publications

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“…Gene expression profiling by 4sU-Seq also revealed that changes in newly synthesized RNA upon IR exposure are extensive, indicating that the transcriptional response is global not only upon UV irradiation (Boeing et al 2016) but also upon DSB formation. Nevertheless, to establish effective DDR, a timely expression of subset of transcripts that code for DNA repair, signaling, cell cycle, and cell survival factors, is necessary.…”

Section: Ddx54 In Genotoxic Stress Responsementioning

confidence: 99%

DDX54 regulates transcriptome dynamics during DNA damage response

et al. 2017

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The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A) + RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by proteinprotein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.

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Section: Ddx54 In Genotoxic Stress Responsementioning

confidence: 99%

DDX54 regulates transcriptome dynamics during DNA damage response

et al. 2017

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“…3). Interestingly, a growing number of studies have reported data to suggest that (i) UV irradiation preferentially inhibits elongation, rather than transcription initiation [28][29][30]38 , (ii) P-TEFb and NELF are important regulators of UVresponse 41,54,69 and (iii) although elongation gradually decelerates due to the encounter of Pol II with DNA lesions, significant initiation/early elongation activity (assessed by nRNA-seq) is observed in the first thousand bases of actively transcribed regions 28,29,38 , a characteristic that has also been used for the identification of active TSSs genome-wide after UV 30 . These features are consistent with our finding that new Pol II-hypo molecules are constantly recruited to PICs post-UV (see Fig.…”

Section: Discussionmentioning

confidence: 99%

Continuous transcription initiation guarantees robust repair of transcribed genes and regulatory regions in response to DNA damage

Liakos

Konstantopoulos

Lavigne³

et al. 2019

Preprint

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Inhibition of RNA synthesis caused by DNA damage-impaired RNA polymerase II (Pol II) elongation is found to conceal a local increase in de novo transcription, slowly progressing from Transcription Start Sites (TSSs) to gene ends. Although associated with accelerated repair of Pol II-encountered lesions and limited mutagenesis, it is still unclear how this mechanism is maintained during recovery from genotoxic stress. Here we uncover a surprising widespread gain in chromatin accessibility and preservation of the active histone mark H3K27ac after UV-irradiation. We show that the concomitant increase in Pol II release from promoter-proximal pause (PPP) sites of most active genes, PROMoter uPstream Transcripts (PROMPTs) and enhancer RNAs (eRNAs) favors unrestrained initiation, as demonstrated by the synthesis of short nascent RNAs, including TSSassociated RNAs (start-RNAs). In accordance, drug-inhibition of the transition into elongation replenished the post-UV reduced levels of pre-initiating pol II at TSSs. Continuous engagement of new Pol II thus ensures maximal transcription-driven DNA repair of active genes and non-coding regulatory loci. Together, our results reveal an unanticipated layer regulating the UV-triggered transcriptional-response and provide physiologically relevant traction to the emerging concept that transcription initiation rate is determined by pol II pauserelease dynamics.

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“…Only six proteins were found to be ubiquitylated exclusively in ring stages, of which three are rhoptry proteins. Our bioinformatics analysis below is extrapolated from these qualitative label free experiments since our efforts to apply quantitative mass spectrometry approaches based on isotope labelling such as TMT or dimethyl labelling [27] were not compatible with the relatively low yield of modified peptides.…”

Section: The P Falciparum Ubiquitome: Distribution Of Ubiquitylationmentioning

confidence: 99%

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

Encheva

Green

Lasonder

et al. 2019

Preprint

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Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage.These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasite's elaboration of a new food vacuole.

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Multiomic Analysis of the UV-Induced DNA Damage Response

Cited by 178 publications

References 51 publications

DDX54 regulates transcriptome dynamics during DNA damage response

DDX54 regulates transcriptome dynamics during DNA damage response

Continuous transcription initiation guarantees robust repair of transcribed genes and regulatory regions in response to DNA damage

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

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