Genetic and cellular sensitivity of Caenorhabditis elegans to the chemotherapeutic agent cisplatin

Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the pro-apoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1.Instituto de Salud Carlos III PI15/00895 PI16/01898European Regional Development Fund/FEDERNetherlands Organization for Scientific Research 711.014.005Sociedad Española de Oncología MédicaMinisterio de Economía y Competitividad BFU2007-67123 BFU2015-64408-PEuropean Social Fund BFU2015-64408-

Genetic and cellular sensitivity of Caenorhabditis elegans to the chemotherapeutic agent cisplatin

Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the proapoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1. This article has an associated First Person interview with the first author of the paper

Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis

Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability

Unraveling the functional roles of sftb-1/SF3B1 and prpf-4/PRPF4B in Caenorhabditis elegans splicing and human disease

Caenorhabditis elegans provides a powerful experimental system for understanding fundamental questions in biomedical research, including the consequences of pre-mRNA splicing alterations. Here, we have applied CRISPR and RNA-sequencing technologies to investigate the functions of two evolutionary conserved splicing factors that have been linked to disease in different ways. We have established that cancer-related mutations in sftb-1 cause splicing alterations in C. elegans. The use of distinct alleles has uncovered novel synthetic lethal interactions between sftb-1 mutations and further perturbations in the U2 snRNP, while holding the potential to be expanded to other biological processes. Moreover, we have humanized an SFTB-1 domain to sensitize C. elegans to the splicing modulators pladienolide B and herboxidiene. Finally, we have initiated the functional characterization of the splicing regulatory kinase prpf-4 in this nematode by studying its dynamic subcellular location pattern and the requirement of its kinase activity during C. elegans development.El nematode Caenorhabditis elegans és un sistema experimental que pot ajudar a resoldre qüestions fonamentals en recerca biomèdica, incloses les conseqüències d’alteracions en el processament de pre-ARN missatgers o splicing. En aquest treball, hem aplicat les tècniques de CRISPR i seqüenciació d’ARN per a investigar les funcions de dues proteïnes involucrades en splicing, les quals s’han conservat al llarg de l’evolució i s’han associat a diverses malalties de formes diferents. Hem observat que mutacions en el gen sftb-1 relacionades amb càncer alteren el procés de splicing en C. elegans. Mitjançant l’ús de diferents al·lels, hem descobert noves interaccions de letalitat sintètica entre mutacions en el gen sftb-1 i alteracions addicionals en el complex U2 snRNP. El sistema té el potencial de ser utilitzat per a identificar altres interaccions amb diferents processos biològics. A més, hem humanitzat un domini de la proteïna SFTB-1 per sensibilitzar els cucs als fàrmacs pladienolide B i herboxidiene, que actuen com a moduladors de l’splicing. Finalment, hem iniciat la caracterització funcional de la quinasa reguladora del procés de splicing prpf-4 en aquest nematode mitjançant l’estudi de la seva localització subcel·lular dinàmica i del requeriment de la seva activitat quinasa durant el desenvolupament de C. elegans

Unraveling the functional roles of sftb-1/SF3B1 and prpf-4/PRPF4B in Caenorhabditis elegans splicing and human disease

Caenorhabditis elegans provides a powerful experimental system for understanding fundamental questions in biomedical research, including the consequences of pre-mRNA splicing alterations. Here, we have applied CRISPR and RNA-sequencing technologies to investigate the functions of two evolutionary conserved splicing factors that have been linked to disease in different ways. We have established that cancer-related mutations in sftb-1 cause splicing alterations in C. elegans. The use of distinct alleles has uncovered novel synthetic lethal interactions between sftb-1 mutations and further perturbations in the U2 snRNP, while holding the potential to be expanded to other biological processes. Moreover, we have humanized an SFTB-1 domain to sensitize C. elegans to the splicing modulators pladienolide B and herboxidiene. Finally, we have initiated the functional characterization of the splicing regulatory kinase prpf-4 in this nematode by studying its dynamic subcellular location pattern and the requirement of its kinase activity during C. elegans development.El nematode Caenorhabditis elegans és un sistema experimental que pot ajudar a resoldre qüestions fonamentals en recerca biomèdica, incloses les conseqüències d’alteracions en el processament de pre-ARN missatgers o splicing. En aquest treball, hem aplicat les tècniques de CRISPR i seqüenciació d’ARN per a investigar les funcions de dues proteïnes involucrades en splicing, les quals s’han conservat al llarg de l’evolució i s’han associat a diverses malalties de formes diferents. Hem observat que mutacions en el gen sftb-1 relacionades amb càncer alteren el procés de splicing en C. elegans. Mitjançant l’ús de diferents al·lels, hem descobert noves interaccions de letalitat sintètica entre mutacions en el gen sftb-1 i alteracions addicionals en el complex U2 snRNP. El sistema té el potencial de ser utilitzat per a identificar altres interaccions amb diferents processos biològics. A més, hem humanitzat un domini de la proteïna SFTB-1 per sensibilitzar els cucs als fàrmacs pladienolide B i herboxidiene, que actuen com a moduladors de l’splicing. Finalment, hem iniciat la caracterització funcional de la quinasa reguladora del procés de splicing prpf-4 en aquest nematode mitjançant l’estudi de la seva localització subcel·lular dinàmica i del requeriment de la seva activitat quinasa durant el desenvolupament de C. elegans

Trade-offs between life-history traits in a coldwater fish in the Mediterranean Sea: the case of blue whiting Micromesistius poutassou

16 pages, 4 figures, 5 tables, supporting information https://doi.org/10.1111/jfb.13993A combination of traditional and emerging methodologies was used to assess the trade-offs between several life-history traits (linked to reproduction and condition) and parasitism in a commercially-exploited cold-water species, blue whiting Micromesistius poutassou, in the Mediterranean Sea. The use of histological and gravimetric methods revealed conflicting evidence as to the fecundity type (indeterminate or determinate) of this species. Moreover, there seem to be condition-mediated compensations between egg quality and egg quantity. The effects of parasitism on reproduction and condition are species-specific and occur mainly at high intensities of infection; they include a lower batch fecundity (affecting reproductive potential), a higher hepatosomatic index and a higher spleen-somatic index. Considering the fact that larger fish spawn more eggs and that the minimum landing size is lower than the size at maturity, these results may have implications for the future management of M. poutassou stocks in the Mediterranean Sea. Local environmental conditions may account for geographical differences regarding infection in M. poutassou. Altogether, the results support the idea that the complex trade-offs between parasitism, reproduction and condition need to be considered in order to understand the status of cold-water species such as M. poutassouPeer reviewe

Genetic and cellular sensitivity of to the chemotherapeutic agent cisplatin

Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties, namely the onset of resistance to the drug and the cytotoxic effect in patients. Here, we used Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause of cisplatin-induced infertility. RNA sequencing data indicate that cisplatin triggers a systemic stress response, in which DAF-16/FOXO and SKN-1/NRF2, two conserved transcription factors, are key regulators. We determined that inhibition of the DNA damage-induced apoptotic pathway does not confer cisplatin protection to the animal. However, mutants for the pro-apoptotic BH3-only gene ced-13 are sensitive to cisplatin, suggesting a protective role of the intrinsic apoptotic pathway. Finally, we demonstrated that our system can also be used to identify mutations providing resistance to cisplatin and therefore potential biomarkers of innate cisplatin-refractory patients. We show that mutants for the redox regulator trxr-1, ortholog of the mammalian thioredoxin reductase 1 TRXR1, display cisplatin resistance. By CRISPR/Cas9, we determined that such resistance relies on the presence of the single selenocysteine residue in TRXR-1.This article has an associated First Person interview with the first author of the paper

Modeling of autosomal-dominant retinitis pigmentosa in Caenorhabditis elegans uncovers a nexus between global impaired functioning of certain splicing factors and cell type-specific apoptosis

Retinitis pigmentosa (RP) is a rare genetic disease that causes gradual blindness through retinal degeneration. Intriguingly, seven of the 24 genes identified as responsible for the autosomal-dominant form (adRP) are ubiquitous spliceosome components whose impairment causes disease only in the retina. The fact that these proteins are essential in all organisms hampers genetic, genomic, and physiological studies, but we addressed these difficulties by using RNAi in Caenorhabditis elegans. Our study of worm phenotypes produced by RNAi of splicing-related adRP (s-adRP) genes functionally distinguishes between components of U4 and U5 snRNP complexes, because knockdown of U5 proteins produces a stronger phenotype. RNA-seq analyses of worms where s-adRP genes were partially inactivated by RNAi, revealed mild intron retention in developing animals but not in adults, suggesting a positive correlation between intron retention and transcriptional activity. interestingly, RNAi of s-adRP genes produces an increase in the expression of atl-1 (homolog of human ATR), which is normally activated in response to replicative stress and certain DNA-damaging agents. The up-regulation of atl-1 correlates with the ectopic expression of the pro-apoptotic gene egl-1 and apoptosis in hypodermal cells, which produce the cuticle, but not in other cell types. Our model in C. elegans resembles s-adRP in two aspects: The phenotype caused by global knockdown of s-adRP genes is cell type-specific and associated with high transcriptional activity. Finally, along with a reduced production of mature transcripts, we propose a model in which the retina-specific cell death in s-adRP patients can be induced through genomic instability