The integrated stress response induces R-loops and hinders replication fork progression

Choo, Josephine Ann Mun Yee; Schlösser, Denise; Manzini, Valentina; Magerhans, Anna; Dobbelstein, Matthias

doi:10.1038/s41419-020-2727-2

Cited by 9 publications

(6 citation statements)

References 59 publications

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“…DNA fiber analysis. Primary MM and UF cells were labeled with 50 μM IdU (Sigma-Aldrich I-7125) followed by 100 μM CldU (Sigma-Aldrich C-6891) for 30 min each as described previously 57 . UtSM and UtSM-RNaseH expressing cells were treated with 100 nM CCT254515 for 2 h in between Idu and Cldu treatments.…”

Section: Generation Of Stable Rnaseh-expressing Uterine Smooth Muscle...mentioning

confidence: 99%

Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids

Muralimanoharan

Shamby

Stansbury

et al. 2022

Sci Rep

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Uterine fibroid (UF) driver mutations in Mediator complex subunit 12 (MED12) trigger genomic instability and tumor development through unknown mechanisms. Herein, we show that MED12 mutations trigger aberrant R-loop-induced replication stress, suggesting a possible route to genomic instability and a novel therapeutic vulnerability in this dominant UF subclass. Immunohistochemical analyses of patient-matched tissue samples revealed that MED12 mutation-positive UFs, compared to MED12 mutation-negative UFs and myometrium, exhibited significantly higher levels of R-loops and activated markers of Ataxia Telangiectasia and Rad3-related (ATR) kinase-dependent replication stress signaling in situ. Single molecule DNA fiber analysis revealed that primary cells from MED12 mutation-positive UFs, compared to those from patient-matched MED12 mutation-negative UFs and myometrium, exhibited defects in replication fork dynamics, including reduced fork speeds, increased and decreased numbers of stalled and restarted forks, respectively, and increased asymmetrical bidirectional forks. Notably, these phenotypes were recapitulated and functionally linked in cultured uterine smooth muscle cells following chemical inhibition of Mediator-associated CDK8/19 kinase activity that is known to be disrupted by UF driver mutations in MED12. Thus, Mediator kinase inhibition triggered enhanced R-loop formation and replication stress leading to an S-phase cell cycle delay, phenotypes that were rescued by overexpression of the R-loop resolving enzyme RNaseH. Altogether, these findings reveal MED12-mutant UFs to be uniquely characterized by aberrant R-loop induced replication stress, suggesting a possible basis for genomic instability and new avenues for therapeutic intervention that involve the replication stress phenotype in this dominant UF subtype.

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Section: Generation Of Stable Rnaseh-expressing Uterine Smooth Muscle...mentioning

confidence: 99%

Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids

Muralimanoharan

Shamby

Stansbury

et al. 2022

Sci Rep

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“…We then sought to identify the mechanism underlying the elevated levels of RNA:DNA hybrids that we found in MM cells. Recently, it has been demonstrated that the Integrated Stress Response (ISR) induces R‐loops in U2OS cells (Choo et al , 2020). Since a peculiar characteristic of MM cells is a high Unfolded Protein Response (UPR) due to their abnormal production of immunoglobulins (Nikesitch et al , 2018), it is conceivable that this type of stress may contribute to the generation of R‐loops.…”

Section: Resultsmentioning

confidence: 99%

AATF/Che‐1 localizes to paraspeckles and suppresses R‐loops accumulation and interferon activation in Multiple Myeloma

et al. 2022

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Several kinds of stress promote the formation of three-stranded RNA:DNA hybrids called R-loops. Insufficient clearance of these structures promotes genomic instability and DNA damage, which ultimately contribute to the establishment of cancer phenotypes. Paraspeckle assemblies participate in R-loop resolution and preserve genome stability, however, the main determinants of this mechanism are still unknown. This study finds that in Multiple Myeloma (MM), AATF/Che-1 (Che-1), an RNA-binding protein fundamental to transcription regulation, interacts with paraspeckles via the lncRNA NEAT1_2 (NEAT1) and directly localizes on R-loops. We systematically show that depletion of Che-1 produces a marked accumulation of RNA:DNA hybrids. We provide evidence that such failure to resolve Rloops causes sustained activation of a systemic inflammatory response characterized by an interferon (IFN) gene expression signature. Furthermore, elevated levels of R-loops and of mRNA for paraspeckle genes in patient cells are linearly correlated with Multiple Myeloma progression. Moreover, increased interferon gene expression signature in patients is associated with markedly poor prognosis. Taken together, our study indicates that Che-1/NEAT1 cooperation prevents excessive inflammatory signaling in Multiple Myeloma by facilitating the clearance of R-loops. Further studies on different cancer types are needed to test if this mechanism is ubiquitously conserved and fundamental for cell homeostasis.

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“…TriC is a chaperone complex that assists in protein folding (Knowlton et al, 2021; Martín-Cófreces et al, 2021; Yam et al, 2008) and which has been reported to influence various cellular pathways including gene expression (Shaheen et al, 2021), cellular signalling (Weng et al, 2021), and protection against proteotoxic stress (Llamas et al, 2021). Interestingly, recent data indicate that activation of the integrated stress response, a cellular signalling cascade which responds to protein misfolding, leads to inhibition of histone gene synthesis and consequent formation of R-loops that are known to inhibit DNA RF progression (Choo et al, 2020). Curiously however, published data also show that inhibition of RF progression caused by lack of histone synthesis is not associated with dramatic elevation of RPA-ssDNA (Mejlvang et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

A genome-wide screen identifies SCAI as a modulator of the UV-induced replicative stress response in human cells

Lemay¹,

St-Hilaire²,

Gezzar-Dandashi³

et al. 2022

Preprint

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Helix-destabilizing DNA lesions induced by environmental mutagens such as UV light cause genomic instability by strongly blocking the progression of DNA replication forks (RF). At blocked RF, single-stranded DNA (ssDNA) accumulates and is rapidly bound by Replication Protein A (RPA) complexes. Such stretches of RPA-ssDNA constitute platforms for recruitment/activation of critical factors that promote DNA synthesis restart. However, during periods of severe replicative stress, RPA availability may become limiting due to inordinate sequestration of this multifunctional complex on ssDNA, thereby negatively impacting multiple vital RPA-dependent processes. Here, we performed a genome-wide screen to identify factors which restrict the accumulation of RPA-ssDNA during UV-induced replicative stress. While this approach revealed some expected “hits” acting in pathways such as nucleotide excision repair, translesion DNA synthesis, and the intra-S phase checkpoint, it also identifed SCAI, whose role in the replicative stress response was previously unappreciated. Upon UV exposure, SCAI knock-down caused elevated accumulation of RPA-ssDNA during S phase, accompanied by reduced cell survival and compromised RF progression. These effects were independent of the previously reported role of SCAI in 53BP1-dependent DNA double-strand break repair. We also found that SCAI colocalized with stalled RF, and that its depletion promoted nascent DNA degradation. Finally, we (i) provide evidence that EXO1 is the major nuclease underlying ssDNA formation and consequent DNA replication defects in SCAI knockout cells and, consistent with this, (ii) demonstrate that SCAI inhibits EXO1 activity on a ssDNA gap in vitro . Taken together, our data establish SCAI as a novel regulator of the replicative stress response in human cells.

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The integrated stress response induces R-loops and hinders replication fork progression

Cited by 9 publications

References 59 publications

Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids

Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids

AATF/Che‐1 localizes to paraspeckles and suppresses R‐loops accumulation and interferon activation in Multiple Myeloma

A genome-wide screen identifies SCAI as a modulator of the UV-induced replicative stress response in human cells

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