The 7SK small nuclear ribonucleoprotein links the cell responses to transcription and replication stress by promoting replication fork reversal and homologous recombination
Many genotoxic agents that block replication fork progression to cause DNA replication stress also block transcription, which results in transcription stress. The 7SK-small nuclear ribonucleoprotein (7SK-snRNP) regulates and promotes RNA polymerase II activity in response to transcription blocks. Individual 7SK-snRNP components HEXIM1, LARP7 and MEPCE are also reported to either facilitate or inhibit processes relevant to replication stress: conflicts between transcription and replication and homologous recombination (HR) at double-strand breaks (DSBs). Here, we investigate potential roles of 7SK-snRNP components in the response to replication stress-inducing agents such as camptothecin and hydroxyurea. We report that HEXIM1 and LARP7 promote replication fork slowing in response to agents that cause both replication- and transcription stress, in a manner consistent with their canonical 7SK-snRNP functions. Our data suggest that this role in fork slowing is mainly through facilitating RAD51-mediated replication fork reversal rather than transcription-replication conflicts. HEXIM1 and LARP7 promote RAD51 recruitment to replication-associated DSBs or post-replicative gaps under conditions of transcription stress such as induced by camptothecin or BET inhibitors, while to a lesser extent supporting HR at direct DSBs and not at all in absence of transcription stress. Our data support that LARP7 roles during replication stress are independent of its reported interaction with BRCA1 and both LARP7 and HEXIM1 promote survival of replication stress-inducing agents, especially camptothecin. 7SK-snRNP components are not recruited to stressed replication forks and RNA polymerase II inhibition phenocopies loss of these proteins. Taken together, our data support a model where 7SK-snRNP modulation of RNA polymerase II activity helps facilitate RAD51 function under transcription stress conditions, thereby linking the cell responses to transcription- and replication stress. Overall design: To investigate the impact of camptothecin (CPT) or hydroxyurea (HU) treatment, we used chromatin RNA-Seq to quantify nascent transcripts. Sequencing was performed on human U2OS osteosarcoma cells before and after 2 hours CPT and HU treatment. Parental U2OS cells before and after DMSO treatment were used as a control. We then performed gene expression profiling analysis using data obtained from Chromatin-RNA-Seq.