Project 10. Gene paralogues, RNA processing and R-loops.

RNA splicing alterations lead to R-loop accumulation, inducing replication stress and chromosome fragility, potentially contributing to pathological states, such as neurodegeneration. Despite intricate RNA processing across higher eukaryotic organs, knowledge about cell-specific R-loop roles during cellular differentiation is limited. Our previous work uncovered the core RNA spliceosome subunit SNRPB and its paralogue SNRPN as targets of a broad regulatory network for dosage-sensitive genes. While spliceosome components are typically ubiquitously expressed and function as housekeeping genes, SNRPN stands out by replacing SNRPB in specific tissues, like neurons or heart. Despite their high sequence similarity, our preliminary data indicate intrinsic differences. We hypothesize that these differences confer resilience, enabling responses to environmental changes, a crucial attribute for neurons, which constantly adapt to stimuli. Notably, previous studies on SNRPN/B and splicing rely heavily on long-term interventions like chronic knock-outs or RNA interference, providing limited insights into immediate cellular responses after splicing disruption. Additionally, the link between RNA splicing and R-loops is mainly explored in cancer cells (e.g., U2OS, HeLa), leaving primary cells and differentiation roles less understood.

To address these gaps, our project centers on spliceosome subunits in primary cells (male and female mouse embryonic stem cells, differentiated neurons) and leveraging on degrons, which allow acute perturbations. This approach allows us to probe alternative splicing dynamics in response to environmental cues and its interplay with R-loops (see Figure 1).

Figure 1. Graphical representation of the research proposal addressing the connection between splicing paralogues, tissue-specificity and R-loops.

Our research aims to elucidate:

  1. How do R-loops connected to splicing differ in male versus female mouse ES cells versus in vitro-generated cortical neurons?
  2. What are the dynamics and temporal progression after the loss of the spliceosome (accumulation of mis-spliced transcripts, other RNA processing defects, the accumulation of R-loops)?
  3. Do splicing associated R-loops enable the rapid responses (i.e. change in splicing pattern) upon a change in environmental conditions (e.g. growth factor stimulation)?

This project will be pursued in collaboration with the Basilicata-group at the University Medical Center (UMC) Mainz.

In our experiments, we will make use of existing gRNA/CRISPR constructs, mES cell gene targeting and the creation of swap-alleles and the dTag technology, which we have established in the lab. With this approach, we hope to gain insights into the interplay of splicing and gene paralogues on the R-loops in primary cells.