Anja-Katrin Bielinsky

Chimeric RNAs are a class of molecules defined as RNA transcripts containing genetic information for multiple genes. While their expression patterns across tissue types and disease states have been well documented, the biological function of these transcripts remains poorly understood. In this study, we developed an integrative prediction pipeline to establish an atlas of 569,640 chimeric RNAs across 1019 cancer cell lines. Leveraging this resource, we performed a systematic functional dependency screen by integrating our predictions with perturbation datasets from The Cancer Dependency Map project. This approach moves beyond prediction to identifying high-confidence functional chimeric RNAs essential for cellular processes, including a subset of recurrent chimeric transcripts generated by cis - splicing of adjacent genes. We demonstrate that depletion of selected candidate chimeras disrupts cell cycle progression and cellular fitness and that these transcripts are frequently dysregulated in primary breast tumors. Together, these results demonstrate that chimeric RNAs constitute a previously underappreciated layer of gene expression, expanding the functional genome beyond conventional genetics.

DNA lesions can stall replication, generating ssDNA gaps that require filling to maintain genome stability. The E3 ubiquitin ligase RFWD3/FANCW targets RPA and PCNA to regulate replication dynamics and damage tolerance, but its roles in these processes and influence on cell mutability remain incompletely understood. Here, we show that RFWD3 prevents the accumulation of ssDNA gaps in UV-irradiated cells through its E3 ligase activity and interaction with RPA. Mutational signature analysis implicates RFWD3 in the spontaneous mutability of human cells and reveals its involvement in 6-4 photoproducts tolerance. Accordingly, photorepair of 6-4 photoproducts, but not of cyclobutane pyrimidine dimers, alleviates gap accumulation in RFWD3-depleted cells. RFWD3 KO has limited impact on PCNA ubiquitylation, but its depletion sharply decreases UV-induced RPA ubiquitylation. Moreover, cells expressing an RPA mutant that cannot be ubiquitylated accumulate ssDNA gaps epistatically with RFWD3 loss. We propose that RFWD3-dependent RPA ubiquitylation regulates post-replicative ssDNA gaps to promote helix-distorting lesion tolerance.

The use of programmable nucleases has transformed genome editing and functional genomics. Clustered regularly interspaced palindromic repeats (CRISPR)/CRIS

Accurate and efficient DNA replication constitutes the most effective safeguard against genome instability. Numerous aspects of replication initiation, elongation, and termination are tightly regulated by post-translational modifications. In this review, we summarize recent advances in elucidating pathways regulated by ubiquitin and the small ubiquitin-like modifier, SUMO, and compare insights gained in yeast with those obtained in vertebrate systems. These reversible modifications play critical roles in both DNA replication and replication-coupled repair processes. When active replisomes encounter obstacles such as nucleotide depletion, DNA secondary structures, or base lesions that impede fork progression, multiple genome surveillance pathways are activated to coordinate the replication stress response. Stalled replication forks undergo remodeling and reversal, thereby stabilizing the fork and facilitating replication restart. In parallel, diverse tolerance mechanisms have evolved to enable lesion bypass or replication traverse, which transiently alters the replication machinery yet permits continuation of DNA synthesis. At the core of these processes are the DNA damage tolerance and Fanconi anemia pathways, whose components collaborate to prevent under-replication during S phase and beyond. Furthermore, ubiquitin and SUMO signaling act synergistically through the activity of SUMO-targeted ubiquitin ligases. These enzymes sequester damaged replication forks at the nuclear periphery and promote recombination-mediated restart under stringent spatiotemporal control of the replication checkpoint. Failure of these mechanisms forces the cell to engage in a final, "do-or-die" attempt to initiate DNA synthesis during mitosis, a process that is also orchestrated by ubiquitin signaling.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited skin disorder characterized by fragile skin, blistering, and chronic wounds. Keratinocytes, the primary cells in the epidermis, are directly affected by persistent injury in RDEB, contributing to chronic inflammation. High mobility group box 1 (HMGB1) is elevated in the serum of individuals with RDEB. However, its role in keratinocyte inflammation remains unclear. Here, we report an increase in HMGB1 expression in keratinocytes at chronic wound sites compared to matched non-wounded skin from an RDEB individual, suggesting a potential link to local inflammation. Pharmacological inhibition of HMGB1 using inflachromene reduced lipopolysaccharide (LPS)-induced inflammatory responses in a keratinocyte cell line, supporting a role for keratinocyte-specific HMGB1 in inflammation. Surprisingly, deletion of HMGB1 alone or together with its paralogue HMGB2 did not suppress the inflammatory response to LPS. Furthermore, inflachromene still reduced inflammation in these knockout cells. This unexpected discrepancy between genetic deletion and pharmacologic inhibition points to a more complex role for HMGB1 or off-target effects of the compound. These findings suggest that HMGB1 may contribute to inflammation in keratinocytes, but its exact function needs further investigation.

RAD18 is a conserved eukaryotic E3 ubiquitin ligase that promotes genome stability through multiple pathways. One of these is gap-filling DNA synthesis at active replication forks and in post-replicative DNA. RAD18 also regulates homologous recombination (HR) repair of DNA breaks; however, the current literature describing the contribution of RAD18 to HR in mammalian systems has not reached a consensus. To investigate this, we examined three independent RAD18-null human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e., HCT116 mutants were hyper-recombinogenic (hyper-rec). Interestingly, these phenotypes were linked to RAD18’s role in PCNA K164 ubiquitination, as HCT116 PCNA^K164R/+ mutants were also hyper-rec, consistent with previous studies in rad18^−/− and pcna^K164R avian DT40 cells. Importantly, the knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. We propose that the hierarchy of post-replicative repair and HR, intrinsic to each cell type, dictates whether RAD18 is required for suppression of hyper-recombination and that this function is linked to PCNA K164 ubiquitination.

Homologous recombination (HR) and translesion synthesis (TLS) promote gap-filling DNA synthesis to complete genome replication. One factor involved in both pathways is RAD18, an E3 ubiquitin ligase. Although RAD18’s role in promoting TLS through the ubiquitination of PCNA at lysine 164 (K164) is well established, its requirement for HR-based mechanisms is currently less clear. To assess this, we inactivated RAD18 in three human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e . HCT116 mutants were hyper-recombinogenic (hyper-rec). Loss of RAD18 also impaired TLS activity in HCT116 cells, but unexpectedly, did not reduce clonogenic survival. Interestingly, these phenotypes appear linked to PCNA K164 ubiquitination, as HCT116 PCNA ^K164R/+ mutants were also hyper-rec and showed reduced TLS activity, consistent with previous studies in rad18 ^-/- or pcna ^K164R avian DT40 mutant cells. Importantly, knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. Taken together, these data suggest that the roles of human RAD18 in directing distinct gap-filling DNA synthesis pathways varies depending on cell type and that these functions are linked to PCNA ubiquitination.