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These approaches have been extensively used in our laboratory for a number of cell lines of various origins and are particularly informative when used in primary cells that can be synchronized in quiescence, to avoid generation of replication stress-induced dsDNA breaks and consequent ATM activation through its canonical pathway. Second, we present a protocol for Fluorescence Recovery After Photobleaching (FRAP) of GFP-tagged splicing factors, a highly sensitive and reproducible readout to measure in living cells, the ATM influence on the spliceosome. First, we present an immunofluorescence-detection method that can be used to evaluate ATM activation by autophosphorylation, in fixed cells. This chapter describes the use of fluorescence microscopy methods that can be used to evaluate noncanonical ATM activation by transcription-blocking DNA damage. In turn, activated ATM signals to regulate spliceosome dynamics and AS genome wide. R-loops signal for noncanonical ATM activation which in quiescent cells occurs in absence of detectable dsDNA breaks. Displacement of spliceosomes from lesion-arrested RNA polymerases facilitates formation of R-loops between the nascent RNA and DNA adjacent to the transcription bubble. In response to stalling of RNAPII assembly of late-stage spliceosomes is disrupted resulting in increased splicing factor mobility.
Interestingly, ATM and the spliceosome influence each other’s activity in a reciprocal manner by a pathway that initiates when transcribing RNA polymerase II (RNAPII) encounters DNA lesions that prohibit forward translocation. ATM acts by modulating a variety of RNA metabolic pathways including nascent RNA splicing, a process catalyzed by the spliceosome. One of the master DDR coordinators, the Ataxia Telangiectasia Mutated (ATM) kinase, has a fundamental role in mediating DNA damage-induced changes in mRNA synthesis. DNA damage response (DDR) signaling pathways coordinate these responses and determine cellular fates in part, by transducing signals that modulate RNA metabolism. To ensure genomic integrity, cells employ mechanisms that convert signals generated by stochastic DNA damage into organized responses, including activation of repair systems, cell cycle checkpoints, and apoptotic mechanisms. Environmental genotoxins and metabolic byproducts generate DNA lesions that can cause genomic instability and disrupt tissue homeostasis.