A05: Regulation of splicing fidelity in response to DNA double-strand breaks

Faithful repair of DNA double-strand breaks (DSBs) is essential for preserving genomic integrity and for ensuring cellular and organismal homeostasis. Cells respond to DSBs by initiating a complex set of signaling pathways collectively called the DSB response. These pathways not only orchestrate the repair of the DNA lesion but also influence nearly all facets of cellular activity, including gene expression. Indeed, the occurrence of DSBs induces comprehensive alterations in gene transcription and promotes alternative splicing events throughout the genome. Yet, whether these profound changes solely reflect the cell’s adaptation to the presence of a highly detrimental DNA lesion, or if they signify a more general loss of splicing fidelity, remains uncertain. In addition, DSBs also cause changes in the transcriptional landscape locally around the break site that include both the silencing of ongoing transcription in the vicinity of the break and the promotor-independent activation of transcription to and from the break. A key consequence of these events is the accumulation of aborted RNA transcripts and of damage-induced long non-coding RNAs. In addition, we recently made the intriguing observation that individual spliceosome core and accessory factors display distinct accumulation or exclusion patterns at damaged chromatin, suggesting that the splicing machinery and, as such, RNA maturation, are differentially regulated at DNA lesions. While there is a growing consensus that DNA damage-associated RNAs are critical for efficient DNA repair, it is currently unclear how the presence of a DSB affects the splicing fidelity of the transcripts that are generated in cis of the break. The overarching goal of this research proposal is to understand how DSBs affect splicing fidelity locally at DSB lesions and globally across the genome. We will address the following specific questions: (1) How do alterations in local chromatin structure influence the dynamics of the spliceosome at DSBs? (2) Is the spliceosome required for RNA processing at break sites? If so, how does this dependency intersect with signaling processes and repair accuracy? (3) What is the effect of DSBs on splicing fidelity throughout the entire genome? By addressing these questions, our research aims to provide a comprehensive understanding of how DSBs influence splicing accuracy both globally and in the immediate vicinity of the DNA lesion.