Periodic Reporting for period 4 - TRANSDAM (The transcription-related DNA damage response)
Reporting period: 2020-09-01 to 2022-02-28
Our cells respond to DNA damage, such as that induced by exposure to UV-irradiation, but the response has remained enigmatic despite decades of study. The effect of such damage on transcription of genes is particularly complex. At the local level, bulky DNA lesions cause stalling of the RNA polymerase (RNAPII), resulting in a block to transcript production. Damage-stalled RNAPII functions as a 'molecular beacon' that triggers rapid repair, so-called transcription-coupled repair, the process whereby DNA damage in active genes is preferentially removed. Importantly, however, UV-damage also affects transcription genome-wide, so that even genes that are not damaged temporarily cease to be active. mRNA translation and protein stability is affected as well. It was the aim of our ERC-funded research to better understand the cellular pathways and molecular mechanisms that underlie these disparate responses. Several important findings were made during the ERC grant period (see details in “Summary of the major achievements since the start of the action”). For example, we made the ground-breaking discovery that the reason for the general downregulation of gene transcription after UV-irradiation is that the enzyme that reads the genes, RNAPII, is degraded in response to stalling at DNA lesions. Likewise, we found that both the protein gene-products and a stable non-coding RNA, differentially expressed from the same gene after UV-irradiation, regulate the cell’s response to UV damage. All in all, the results uncovered in this action have provided significant new insight into the basic mechanisms governing the response to UV-induced DNA damage in human cells. A number of human diseases are caused by defects in correctly responding to such damage, and our results has also helped shed new light on the disease mechanisms and hopefully provided new avenues for how to successfully treat them.
In order to facilitate identification of new factors and mechanisms involved in this response, we have performed several distinct biological screens in parallel (proteomic and genomic screens). This was complemented by characterization of transcription and mRNA splicing after UV damage by various genome-wide techniques. Our 'multi-omic' approach explores the same process from various angles and places less emphasis on hits from an individual screen and instead focuses on factors that score in several screens. This has resulted in the identification of several new factors and unexplored mechanisms. We now investigate the function of several of these factors by the use of a multi-disciplinary approach, including biochemical and cell biological approaches as well as proteomics and genomics. Some of these studies have already been published. For example, we have shown that, at the level of gene expression, the UV damage response is associated with a shift from expression of long messenger RNAs (mRNAs) to shorter RNA transcript isoforms, due to alternative RNA splicing/termination (Williamson et al., Cell 2017; 168, p843-855). Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ASCC3 transcript isoform of which the 3’-noncoding region, rather than an encoded protein, is critical for transcription recovery after DNA damage. This study provides the first clear example of a gene that can encode both a protein (via its mRNA) and a regulatory, non-coding, RNA. Among several other important advances, we have also been studying transcriptional termination further, and the role of the SCAF4 and SCAF8 proteins in particular (Gregersen et al., Cell 2019; 177, 1797–1813). These proteins turn out to be the first examples of eukaryotic mRNA anti-terminator proteins. Likewise, a another recently published study on the connection between transcription stress and DNA damage showed that the well-known transcription elongation factor TFIIS is important to counteract transcription stress, R-loops and genome instability (Zatreanu et al., Molecular Cell 2019; 76, 57-69).
Our work has uncovered several important new ways in which cells respond to UV damage, both at the level of gene transcription, and at the level of mRNA translation, protein modification, and stability. The ERC funding has allowed us to stay at the forefront of the field and our results have had great impact on the general understanding of the cellular DNA damage response, and the response to UV-induced DNA damage in particular.