N7-methylguanosine methylation of tRNAs regulates survival to stress in cancer.

Tumour progression and therapy tolerance are highly regulated and complex processes largely dependent on the plasticity of cancer cells and their capacity to respond to stress. The higher plasticity of cancer cells highlights the need for identifying targetable molecular pathways that challenge cancer cell survival. Here, we show that N7-guanosine methylation (m7G) of tRNAs, mediated by METTL1, regulates survival to stress conditions in cancer cells. Mechanistically, we find that m7G in tRNAs protects them from stress-induced cleavage and processing into 5' tRNA fragments. Our analyses reveal that the loss of tRNA m7G methylation activates stress response pathways, sensitising cancer cells to stress. Furthermore, we find that the loss of METTL1 reduces tumour growth and increases cytotoxic stress in vivo. Our study uncovers the role of m7G methylation of tRNAs in stress responses and highlights the potential of targeting METTL1 to sensitise cancer cells to chemotherapy.

ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair.

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

Preparation of a radiobiology beam line at the 18 MeV proton cyclotron facility at CNA.

Proton therapy has gained interest in recent years due to its excellent clinical outcomes. However, the lack of accurate biological data, especially in the Bragg peak region of clinical beams, makes it difficult to implement biophysically optimized treatment plans in clinical practice. In this context, low energy proton accelerator facilities provide the perfect environment to collect good radiobiological data, as they can produce high LET beams with narrow energy distributions. This study presents the radiobiology beam line that has been designed at the 18 MeV proton cyclotron facility at the National Centre of Accelerators (CNA, Seville, Spain), to perform irradiations of mono-layer cell cultures. To ensure that all the cells receive the same dose with a suitable dose rate, low beam intensities and broad and homogeneous beam profiles are necessary. To do so, at the CNA an unfocused beam has been used, broadened with a 500 µm thick aluminium scattering foil. Homogeneous dose profiles, with deviations lower than 10% have been obtained over a circular surface of 35 mm diameter for an incident average energy of 12.8 MeV. Further, a Monte Carlo simulation of the beam line has been developed with Geant4, and benchmarked towards experimental measurements, with differences generally below 1%. Once validated, the code has been used, together with an ionization chamber, for dosimetry studies, to characterize the beam and monitor the dose. Finally, cultures of Human Bone Osteosarcoma cells (U2OS) have been successfully irradiated at the radiobiology beam line, investigating the effects of radiation in terms of DNA damage induction.

The Helicase PIF1 Facilitates Resection over Sequences Prone to Forming G4 Structures.

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpression.

Differential effect of the overexpression of Rad2/XPG family endonucleases on genome integrity in yeast and human cells.

Eukaryotic cells possess several DNA endonucleases that are necessary to complete different steps in DNA metabolism. Rad2/XPG and Rad27/FEN1 belong to a group of evolutionary conserved proteins that constitute the Rad2 family. Given the important roles carried out by these nucleases in DNA repair and their capacity to create DNA breaks, we have investigated the effect that in vivo imbalance of these nucleases and others of the family have on genome integrity and cell proliferation. We show that overexpression of these nucleases causes genetic instability in both yeast and human cells. Interestingly, the type of recombination event and DNA damage induced suggest specific modes and timing of action of each nuclease that are beyond their known DNA repair function and are critical to preserve genome integrity. In addition to identifying new sources of genome instability, a hallmark of cancer cells, this study provides new genetic tools for studies of genome dynamics.

Liposome-based immunotherapy against autoimmune diseases: therapeutic effect on multiple sclerosis.

AIM: Based on the ability of apoptosis to induce immunological tolerance, liposomes were generated mimicking apoptotic cells, and they arrest autoimmunity in Type 1 diabetes. Our aim was to validate the immunotherapy in other autoimmune disease: multiple sclerosis. MATERIALS & METHODS: Phosphatidylserine-rich liposomes were loaded with disease-specific autoantigen. Therapeutic capability of liposomes was assessed in vitro and in vivo. RESULTS: Liposomes induced a tolerogenic phenotype in dendritic cells, and arrested autoimmunity, thus decreasing the incidence, delaying the onset and reducing the severity of experimental disease, correlating with an increase in a probably regulatory CD25+ FoxP3- CD4+ T-cell subset. CONCLUSION: This is the first work that confirms phosphatidylserine-liposomes as a powerful tool to arrest multiple sclerosis, demonstrating its relevance for clinical application.

Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice.

DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks.

New tools to study DNA double-strand break repair pathway choice.

A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB) repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences.

Sem1 is a functional component of the nuclear pore complex-associated messenger RNA export machinery.

The evolutionarily conserved protein Sem1/Dss1 is a subunit of the regulatory particle (RP) of the proteasome, and, in mammalian cells, binds the tumor suppressor protein BRCA2. Here, we describe a new function for yeast Sem1. We show that sem1 mutants are impaired in messenger RNA (mRNA) export and transcription elongation, and induce strong transcription-associated hyper-recombination phenotypes. Importantly, Sem1, independent of the RP, is functionally linked to the mRNA export pathway. Biochemical analyses revealed that, in addition to the RP, Sem1 coenriches with components of two other multisubunit complexes: the nuclear pore complex (NPC)-associated TREX-2 complex that is required for transcription-coupled mRNA export, and the COP9 signalosome, which is involved in deneddylation. Notably, targeting of Thp1, a TREX-2 component, to the NPC is perturbed in a sem1 mutant. These findings reveal an unexpected nonproteasomal function of Sem1 in mRNA export and in prevention of transcription-associated genome instability. Thus, Sem1 is a versatile protein that might stabilize multiple protein complexes involved in diverse pathways.

Tho1, a novel hnRNP, and Sub2 provide alternative pathways for mRNP biogenesis in yeast THO mutants.

THO is a protein complex that functions in cotranscriptional mRNP formation. Yeast THO1 and SUB2 (Saccharomyces cerevisiae) were identified as multicopy suppressors of the expression defects of the hpr1Delta mutant of THO. Here we show that multicopy THO1 suppresses the mRNA accumulation and export defects and the hyperrecombination phenotype of THO mutants but not those of sub2Delta, thp1Delta, or spt4Delta. Similarly, Sub2 overexpression suppresses the RNA export defect of hpr1Delta. Tho1 is a conserved RNA binding nuclear protein that specifically binds to transcribed chromatin in a THO- and RNA-dependent manner and genetically interacts with the shuttling hnRNP Nab2. The ability of Tho1 to suppress hpr1Delta resides in its C-terminal half, which contains the RNA binding activity and is located after a SAP/SAF (scaffold-associated protein/scaffold-associated factor) domain. Altogether, these results suggest that Tho1 is an hnRNP that, similarly to Sub2, assembles onto the nascent mRNA during transcription and participates in mRNP biogenesis and export. Overexpression of Tho1 or Sub2 may provide alternative ways for mRNP formation and export in the absence of a functional THO complex.

Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation.

THO/TREX is a conserved eukaryotic complex formed by the core THO complex plus proteins involved in mRNA metabolism and export such as Sub2 and Yra1. Mutations in any of the THO/TREX structural genes cause pleiotropic phenotypes such as transcription impairment, increased transcription-associated recombination, and mRNA export defects. To assay the relevance of THO/TREX complex in transcription, we performed in vitro transcription elongation assays in mutant cell extracts using supercoiled DNA templates containing two G-less cassettes. With these assays, we demonstrate that hpr1delta, tho2delta, and mft1delta mutants of the THO complex and sub2 mutants show significant reductions in the efficiency of transcription elongation. The mRNA expression defect of hpr1delta mutants was not due to an increase in mRNA decay, as determined by mRNA half-life measurements and mRNA time course accumulation experiments in the absence of Rrp6p exoribonuclease. This work demonstrates that THO and Sub2 are required for efficient transcription elongation, providing further evidence for the coupling between transcription and mRNA metabolism and export.