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

Abstract

The integrated stress response (ISR) allows cells to rapidly shut down most of their protein synthesis in response to protein misfolding, amino acid deficiency, or virus infection. These stresses trigger the phosphorylation of the translation initiation factor eIF2alpha, which prevents the initiation of translation. Here we show that triggering the ISR drastically reduces the progression of DNA replication forks within one hour, thus flanking the shutdown of protein synthesis with immediate inhibition of DNA synthesis. DNA replication is restored by compounds that inhibit eIF2alpha kinases or reactivate eIF2alpha. Mechanistically, the translational shutdown blocks histone synthesis, promoting the formation of DNA:RNA hybrids (R-loops) which interfere with DNA replication. Histone depletion alone induces R-loops and compromises DNA replication. Conversely, histone overexpression or R-loop removal by RNaseH1 each restores DNA replication in the context of ISR and histone depletion. In conclusion, the ISR rapidly stalls DNA synthesis through histone deficiency and R-loop formation. We propose that this shutdown mechanism prevents potentially detrimental DNA replication in the face of cellular stresses. SIGNIFICANCE The integrated stress response has long been explored regarding its immediate impact on protein synthesis. Translational shutdown represents an indispensable mechanism to prevent the toxicity of misfolded proteins and virus infections. Our results indicate that the shutdown mechanisms reach far beyond translation and immediately interfere with DNA synthesis as well. ISR depletes cells of new histones which induce accumulation of DNA:RNA hybrids. The impairment of DNA replication in this context supports cell survival during stress.Our work provides a link between the ISR and another subject of active research, i. e. the regulatory network of DNA replication forks.

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

“…TRiC is a chaperone complex that assists in protein folding [ 34 , 70 , 71 ] and has been reported to influence various cellular pathways including gene expression [ 72 ], cellular signalling [ 73 ], and protection against proteotoxic stress [ 74 ]. Interestingly, recent data indicate that activation of the integrated stress response, a signalling cascade that 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 [ 75 ]. 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 [ 76 ].…”

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

“…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.…”

“…It has recently been shown that cellular stress is capable of producing high levels of RNA:DNA hybrids (Choo et al , 2020). Therefore, the presence of a high quantity of these structures in MM patients could be produced by the constitutive UPR that distinguishes this pathology.…”

AATF/Che-1, a new component of paraspeckles, controls R-loops formation and Interferon activation in Multiple Myeloma

“…Remarkably, these findings concur with our finding that MM patients exhibit elevated levels of RNA:DNA hybrids compared to healthy controls, and this phenomenon appears to be related to disease progression, given that activation of IFN pathway is significantly found in ISS 3 and is markedly associated with a poor prognosis. It has recently been shown that cellular stress is capable of producing high levels of RNA:DNA hybrids (61). Therefore, the presence of a high quantity of these structures in MM patients could be produced by the constitutive UPR that distinguishes this pathology.…”

“…We then sought to identify the mechanism underlying the elevated levels of RNA:DNA hybrids that we found in MM. Recently, it has been demonstrated that the Integrated Stress Response (ISR) induces R-loops in U2OS cells (61). Since a peculiar characteristic of MM cells is a high Unfolded Protein Response (UPR) due to their abnormal production of immunoglobulins (62), it is conceivable that this type of stress may contribute to the generation of R-loops.…”