Xrp1 governs the stress response program to spliceosome dysfunction

Unperturbed pre-mRNA splicing is crucial for proper expression of eukaryotic genes, but also maintenance of genome integrity. Loss of splicing factors is associated to DNA damage arising through different mechanisms, among which is the accumulation of mutagenic DNA/RNA hybrids (R-loops). RNase H1 recognizes and degrades the RNA moiety within R-loops, and is a commonly used marker for R-loops. Ecdysoneless protein was previously shown to participate in pre-mRNA splicing through its association with the U5 snRNP. In this study we have performed in vivo Targeted DamID using the Dam-RNase H1 construct to map the localization of R-loops throughout the Drosophila genome in control and Ecd-deficient Drosophila wing imaginal discs. Overall design: Low-level expression of UAS-LT3-Dam (noise control) and UAS-LT3-Dam-RNase H1 (binding signals) was performed in wing imaginal discs (WDs) of Drosophila third instar larvae in control animals and temperature-sensitive ecd1 mutants. Heat-sensitive tub-Gal80 prevented the expression of the Dam constructs at 25 °C at which the larval tissues were developing. The larvae were heat shocked at 29 °C for 24 hours prior to dissection on day 7 after egg laying. The heat shock simultaneously enables the transcription of the Dam constructs, and abolishes the function of the mutant ecd1 protein. The WDs were dissected, processed according to established Targeted DamID protocols and the derived material sequenced in three biological replicates.