Yra1-bound RNA-DNA hybrids cause orientation-independent transcription-replication collisions and telomere instability.

R loops are an important source of genome instability, largely due to their negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and required for mRNA export, but its excess causes lethality and genome instability. Here, we show that, in addition to ssDNA and ssRNA, Yra1 binds RNA-DNA hybrids in vitro and, when artificially overexpressed, can be recruited to chromatin in an RNA-DNA hybrid-dependent manner, stabilizing R loops and converting them into replication obstacles in vivo. Importantly, an excess of Yra1 increases R-loop-mediated genome instability caused by transcription-replication collisions regardless of whether they are codirectional or head-on. It also induces telomere shortening in telomerase-negative cells and accelerates senescence, consistent with a defect in telomere replication. Our results indicate that RNA-DNA hybrids form transiently in cells regardless of replication and, after stabilization by excess Yra1, compromise genome integrity, in agreement with a two-step model of R-loop-mediated genome instability. This work opens new perspectives to understand transcription-associated genome instability in repair-deficient cells, including tumoral cells.

Expression of human RECQL5 in Saccharomyces cerevisiae causes transcription defects and transcription-associated genome instability.

RECQL5 is a member of the conserved RecQ family of DNA helicases involved in the maintenance of genome stability that is specifically found in higher eukaryotes and associates with the elongating RNA polymerase II. To expand our understanding of its function we expressed human RECQL5 in the yeast Saccharomyces cerevisiae, which does not have a RECQL5 ortholog. We found that RECQL5 expression leads to cell growth inhibition, increased genotoxic sensitivity and transcription-associated hyperrecombination. Chromatin immunoprecipitation and transcriptomic analysis of yeast cells expressing human RECQL5 shows that this is recruited to transcribed genes and although it causes only a weak impact on gene expression, in particular at G + C-rich genes, it leads to a transcription termination defect detected as readthrough transcription. The data indicate that the interaction between RNAPII and RECQL5 is conserved from yeast to humans. Unexpectedly, however, the RECQL5-ID mutant, previously shown to have reduced the association with RNAPII in vitro, associates with the transcribing polymerase in cells. As a result, expression of RECQL5-ID leads to similar although weaker phenotypes than wild-type RECQL5 that could be transcription-mediated. Altogether, the data suggests that RECQL5 has the intrinsic ability to function in transcription-dependent and independent genome dynamics in S. cerevisiae.

MicroRNA-27a-3p targets FoxO signalling to induce tumour-like phenotypes in bile duct cells.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a heterogeneous and lethal malignancy, the molecular origins of which remain poorly understood. MicroRNAs (miRs) target diverse signalling pathways, functioning as potent epigenetic regulators of transcriptional output. We aimed to characterise miRNome dysregulation in CCA, including its impact on transcriptome homeostasis and cell behaviour. METHODS: Small RNA sequencing was performed on 119 resected CCAs, 63 surrounding liver tissues, and 22 normal livers. High-throughput miR mimic screens were performed in three primary human cholangiocyte cultures. Integration of patient transcriptomes and miRseq together with miR screening data identified an oncogenic miR for characterization. MiR-mRNA interactions were investigated by a luciferase assay. MiR-CRISPR knockout cells were generated and phenotypically characterized in vitro (proliferation, migration, colony, mitochondrial function, glycolysis) and in vivo using subcutaneous xenografts. RESULTS: In total, 13% (140/1,049) of detected miRs were differentially expressed between CCA and surrounding liver tissues, including 135 that were upregulated in tumours. CCA tissues were characterised by higher miRNome heterogeneity and miR biogenesis pathway expression. Unsupervised hierarchical clustering of tumour miRNomes identified three subgroups, including distal CCA-enriched and IDH1 mutant-enriched subgroups. High-throughput screening of miR mimics uncovered 71 miRs that consistently increased proliferation of three primary cholangiocyte models and were upregulated in CCA tissues regardless of anatomical location, among which only miR-27a-3p had consistently increased expression and activity in several cohorts. FoxO signalling was predominantly downregulated by miR-27a-3p in CCA, partially through targeting of FOXO1. MiR-27a knockout increased FOXO1 levels in vitro and in vivo, impeding tumour behaviour and growth. CONCLUSIONS: The miRNomes of CCA tissues are highly remodelled, impacting transcriptome homeostasis in part through regulation of transcription factors like FOXO1. MiR-27a-3p arises as an oncogenic vulnerability in CCA. IMPACT AND IMPLICATIONS: Cholangiocarcinogenesis entails extensive cellular reprogramming driven by genetic and non-genetic alterations, but the functional roles of these non-genetic events remain poorly understood. By unveiling global miRNA upregulation in patient tumours and their functional ability to increase proliferation of cholangiocytes, these small non-coding RNAs are implicated as critical non-genetic alterations promoting biliary tumour initiation. These findings identify possible mechanisms for transcriptome rewiring during transformation, with potential implications for patient stratification.

Integrative molecular characterisation of gallbladder cancer reveals micro-environment-associated subtypes.

BACKGROUND & AIMS: Gallbladder cancer (GBC) is the most common type of biliary tract cancer, but the molecular mechanisms involved in gallbladder carcinogenesis remain poorly understood. In this study, we applied integrative genomics approaches to characterise GBC and explore molecular subtypes associated with patient survival. METHODS: We profiled the mutational landscape of GBC tumours (whole-exome sequencing on 92, targeted sequencing on 98, in total 190 patients). In a subset (n = 45), we interrogated the matched transcriptomes, DNA methylomes, and somatic copy number alterations. We explored molecular subtypes identified through clustering tumours by genes whose expression was associated with survival in 47 tumours and validated subtypes on 34 publicly available GBC cases. RESULTS: Exome analysis revealed TP53 was the most mutated gene. The overall mutation rate was low (median 0.82 Mut/Mb). APOBEC-mediated mutational signatures were more common in tumours with higher mutational burden. Aflatoxin-related signatures tended to be highly clonal (present in ≥50% of cancer cells). Transcriptome-wide survival association analysis revealed a 95-gene signature that stratified all GBC patients into 3 subtypes that suggested an association with overall survival post-resection. The 2 poor-survival subtypes were associated with adverse clinicopathologic features (advanced stage, pN1, pM1), immunosuppressive micro-environments (myeloid-derived suppressor cell accumulation, extensive desmoplasia, hypoxia) and T cell dysfunction, whereas the good-survival subtype showed the opposite features. CONCLUSION: These data suggest that the tumour micro-environment and immune profiles could play an important role in gallbladder carcinogenesis and should be evaluated in future clinical studies, along with mutational profiles. LAY SUMMARY: Gallbladder cancer is highly fatal, and its causes are poorly understood. We evaluated gallbladder tumours to see if there were differences between tumours in genetic information such as DNA and RNA. We found evidence of aflatoxin exposure in these tumours, and immune cells surrounding the tumours were associated with survival.

The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage.

RNA-DNA hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1's helicase activity plays a crucial role in responding to persistent RNA-DNA hybrids.

RNA polymerase II contributes to preventing transcription-mediated replication fork stalls.

Transcription is a major contributor to genome instability. A main cause of transcription-associated instability relies on the capacity of transcription to stall replication. However, we know little of the possible role, if any, of the RNA polymerase (RNAP) in this process. Here, we analyzed 4 specific yeast RNAPII mutants that show different phenotypes of genetic instability including hyper-recombination, DNA damage sensitivity and/or a strong dependency on double-strand break repair functions for viability. Three specific alleles of the RNAPII core, rpb1-1, rpb1-S751F and rpb9∆, cause a defect in replication fork progression, compensated for by additional origin firing, as the main action responsible for instability. The transcription elongation defects of rpb1-S751F and rpb9∆ plus our observation that rpb1-1 causes RNAPII retention on chromatin suggest that RNAPII could participate in facilitating fork progression upon a transcription-replication encounter. Our results imply that the RNAPII or ancillary factors actively help prevent transcription-associated genome instability.

Unraveling the actin cytoskeleton in the malignant transformation of cholangiocyte biology.

Correct actin cytoskeleton organization is vital in the liver organ homeostasis and disease control. Rearrangements of the actin cytoskeleton may play a vital role in the bile duct cells cholangiocytes. An abnormal actin network leads to aberrant cell morphology, deregulated signaling networks and ultimately triggering the development of cholangiocarcinoma (CCA) and paving the route for cancer cell dissemination (metastasis). In this review, we will outline alterations of the actin cytoskeleton and the potential role of this dynamic network in initiating CCA, as well as regulating the course of this malignancy. Actin rearrangements not only occur because of signaling pathways, but also regulate and modify cellular signaling. This emphasizes the importance of the actin cytoskeleton itself as cause for aberrant signaling and in promoting tumorigenic phenotypes. We will highlight the impact of aberrant signaling networks on the actin cytoskeleton and its rearrangement as potential cause for CCA. Often, these exact mechanisms in CCA are limited understood and still must be elucidated. Indeed, focusing future research on how actin affects and regulates other signaling pathways may provide more insights into the mechanisms of CCA development, progression, and metastasis. Moreover, manipulation of the actin cytoskeleton organization highlights the potential for a novel therapeutic area.

Harmful DNA:RNA hybrids are formed in cis and in a Rad51-independent manner.

DNA:RNA hybrids constitute a well-known source of recombinogenic DNA damage. The current literature is in agreement with DNA:RNA hybrids being produced co-transcriptionally by the invasion of the nascent RNA molecule produced in cis with its DNA template. However, it has also been suggested that recombinogenic DNA:RNA hybrids could be facilitated by the invasion of RNA molecules produced in trans in a Rad51-mediated reaction. Here, we tested the possibility that such DNA:RNA hybrids constitute a source of recombinogenic DNA damage taking advantage of Rad51-independent single-strand annealing (SSA) assays in the yeast Saccharomyces cerevisiae. For this, we used new constructs designed to induce expression of mRNA transcripts in trans with respect to the SSA system. We show that unscheduled and recombinogenic DNA:RNA hybrids that trigger the SSA event are formed in cis during transcription and in a Rad51-independent manner. We found no evidence that such hybrids form in trans and in a Rad51-dependent manner.

Epigenome Remodeling in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) comprises a heterogeneous collection of malignancies arising within the biliary tract, characterized by late diagnosis, innate chemoresistance, and abysmal prognosis. Sequencing data have uncovered recurrent mutations in diverse epigenetic regulators, implicating epigenetic destabilization at the root of these tumors. However, few studies have characterized biliary tumor epigenomes. In this Opinion article, we argue that an epigenome-oriented approach to CCA could establish diverse interconnections between many key aspects of research on this disease, including molecular heterogeneity, diverse cells of origin, and prominent tumor microenvironments. Moreover, we discuss plausible causes of epigenome dysregulation in biliary tumors, including genetic, epigenetic, metabolic, microenvironmental, and physiological factors. Lastly, we assess the translational potential of epigenomics in CCA to uncover robust biomarkers and therapeutic opportunities for this growing group of patients with limited treatment options.