Prosessering av apurinske/apyrimidinske (AP) seter i mammalske celler

Abstract The integrity of deoxyribonucleic acid (DNA) is continuously challenged by endogenous and exogenous DNA damaging agents. Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are amongst the most frequently formed lesions in cellular DNA and their repair is essential for genomic stability. AP sites in humans are processed and repaired mainly through base excision repair (BER), which is known to be initiated either by an AP endonuclease 1 (APE1) that incises 5’ to the AP site or by bifunctional DNA that incise 3’ to the AP site. In this study, the processing of AP sites in mammals was investigated by knockout mice models of endonuclease eight-like DNA glycosylases (Neil1,Neil2), as well as down-regulation of the bifuctional endonuclease three (NTH1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) in human epithelial cervical carcinoma (HeLa) S3 cells. The roles of bifunctional DNA glycosylases NEIL1, NEIL2 and NTH1 in AP site processing, using mouse and human whole-cell extracts, were examined by biochemical activity assays. Another aim of this study was to investigate whether an APE1-independent repair pathway involving NTH1 and TDP1 exist in mammals, since a recent study demonstrated such a mechanism in Schizosaccharomyces pombe where Tdp1 were shown to work downstream of Nth1. The results obtained in this study show that APE1 is responsible for the main incision activity, whereas the roles of NEIL1 and NEIL2 in AP site processing were demonstrated to be non-essential. The product of NTH1 incision was observed in both mice and HeLa S3 extracts, indicating that NTH1 is also involved in AP site processing, although to a lesser extent than APE1. Biochemical analysis using recombinant NTH1, also demonstrated poor ability to process an APE1-nicked DNA substrate compared to an intact AP site, indicating that NTH1 most likely work upstream of APE1, and do not compete with polymerase β downstream of APE1. The product of NTH1 was shown to be further processed by not only APE1, but also by TDP1 in both HeLa S3 cells and mice extracts, indicating that TDP1 is capable of working downstream of NTH1 in an APE1-independent manner and thus may serve as a back-up for APE1 in the repair of AP site. Sammendrag Integriteten av deoksyribonukleinsyre (DNA) utfordres kontinuerlig av endogene og eksogene forbindelser. Mutagene og cytotoksiske apurinske/apyrimidinske (AP)-seter er blant de mest forekommende DNA-skadene og deres reparasjon er essensiell for genomisk stabilitet. AP-seter i humane celler blir prosessert og reparert hovedsakelig gjennom baseeksisjonsreparasjon (BER), som antas å bli initiert av enten AP-endonuklease 1 (APE1) som kutter 5’ til AP setet, eller av en bifunksjonell DNA-glykosylase som kutter 3’ til AP setet. I dette studiet ble prosesseringen av AP seter i mammalske celler undersøkt ved bruk knockout musemodeller av endonuklease VIII-lik DNA-glykosylaser (Neil1,Neil2), samt ved nedregulering av den bifunksjonell DNA-glykosylasen endonuclease three (NTH1) og tyrosyl-DNA fosfodiesterase 1 (TDP1) i human kreftcellelinje (HeLa S3). Rollen av NEIL1, NEIL2 og NTH1 i prosesseringen av AP-seter ble undersøkt ved hjelp av biokjemiske aktivitetsanalyser. En annen hensikt med dette studiet var å undersøke om en APE1-uavhengig reparasjonsmekanisme som involverer NTH1 og TDP1 finnes i mammalske celler. Bakgrunnen for dette var en studie som nylig demonstrerte en tilsvarende reparasjonsmekanisme i Schizosaccharomyces pombe som tyder på at Tdp1 fungerer nedstrøms for Nth1. Resultatene fra dette studiet indikerer at APE1 er ansvarlig for mesteparten av kutteaktiviteten av AP seter, mens bidraget av NEIL1 og NEIL2 i AP-sete reparasjon, har mest sannsynlig ingen essensiell betydning i mammalske celler. Produktet av NTH1 aktiviteten ble også observert i både muse og HeLa S3 ekstrakter, noe som tyder på at NTH1 er involvert i AP-sete reparasjon, men i noe mindre grad enn APE1. Rekombinant NTH1 demonstrerte dårligere evne til å prosessere et APE1-kuttet DNA substrat enn et intakt AP-sete. Dette tyder på at NTH1 mest sannsynlig virker oppstrøms for APE1, og konkurrerer ikke med polymerase β nedstrøms for APE1. Produktet generert av NTH1, ble videre prosessert av ikke bare APE1, men også av TDP1 i både muse og HeLa S3 ekstrakter. Dette kan tyde på at TDP1 er i stand til å fungere nedstrøms for NTH1 på en APE1-uavhengig måte og kan derfor muligens fungere som en reserve for APE1 under AP-sete reparasjon.Rikshospitale

Processing of apurinic/apyrimidinic (AP) sites in mammalian cells

Abstract The integrity of deoxyribonucleic acid (DNA) is continuously challenged by endogenous and exogenous DNA damaging agents. Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are amongst the most frequently formed lesions in cellular DNA and their repair is essential for genomic stability. AP sites in humans are processed and repaired mainly through base excision repair (BER), which is known to be initiated either by an AP endonuclease 1 (APE1) that incises 5’ to the AP site or by bifunctional DNA that incise 3’ to the AP site. In this study, the processing of AP sites in mammals was investigated by knockout mice models of endonuclease eight-like DNA glycosylases (Neil1,Neil2), as well as down-regulation of the bifuctional endonuclease three (NTH1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) in human epithelial cervical carcinoma (HeLa) S3 cells. The roles of bifunctional DNA glycosylases NEIL1, NEIL2 and NTH1 in AP site processing, using mouse and human whole-cell extracts, were examined by biochemical activity assays. Another aim of this study was to investigate whether an APE1-independent repair pathway involving NTH1 and TDP1 exist in mammals, since a recent study demonstrated such a mechanism in Schizosaccharomyces pombe where Tdp1 were shown to work downstream of Nth1. The results obtained in this study show that APE1 is responsible for the main incision activity, whereas the roles of NEIL1 and NEIL2 in AP site processing were demonstrated to be non-essential. The product of NTH1 incision was observed in both mice and HeLa S3 extracts, indicating that NTH1 is also involved in AP site processing, although to a lesser extent than APE1. Biochemical analysis using recombinant NTH1, also demonstrated poor ability to process an APE1-nicked DNA substrate compared to an intact AP site, indicating that NTH1 most likely work upstream of APE1, and do not compete with polymerase β downstream of APE1. The product of NTH1 was shown to be further processed by not only APE1, but also by TDP1 in both HeLa S3 cells and mice extracts, indicating that TDP1 is capable of working downstream of NTH1 in an APE1-independent manner and thus may serve as a back-up for APE1 in the repair of AP site. Sammendrag Integriteten av deoksyribonukleinsyre (DNA) utfordres kontinuerlig av endogene og eksogene forbindelser. Mutagene og cytotoksiske apurinske/apyrimidinske (AP)-seter er blant de mest forekommende DNA-skadene og deres reparasjon er essensiell for genomisk stabilitet. AP-seter i humane celler blir prosessert og reparert hovedsakelig gjennom baseeksisjonsreparasjon (BER), som antas å bli initiert av enten AP-endonuklease 1 (APE1) som kutter 5’ til AP setet, eller av en bifunksjonell DNA-glykosylase som kutter 3’ til AP setet. I dette studiet ble prosesseringen av AP seter i mammalske celler undersøkt ved bruk knockout musemodeller av endonuklease VIII-lik DNA-glykosylaser (Neil1,Neil2), samt ved nedregulering av den bifunksjonell DNA-glykosylasen endonuclease three (NTH1) og tyrosyl-DNA fosfodiesterase 1 (TDP1) i human kreftcellelinje (HeLa S3). Rollen av NEIL1, NEIL2 og NTH1 i prosesseringen av AP-seter ble undersøkt ved hjelp av biokjemiske aktivitetsanalyser. En annen hensikt med dette studiet var å undersøke om en APE1-uavhengig reparasjonsmekanisme som involverer NTH1 og TDP1 finnes i mammalske celler. Bakgrunnen for dette var en studie som nylig demonstrerte en tilsvarende reparasjonsmekanisme i Schizosaccharomyces pombe som tyder på at Tdp1 fungerer nedstrøms for Nth1. Resultatene fra dette studiet indikerer at APE1 er ansvarlig for mesteparten av kutteaktiviteten av AP seter, mens bidraget av NEIL1 og NEIL2 i AP-sete reparasjon, har mest sannsynlig ingen essensiell betydning i mammalske celler. Produktet av NTH1 aktiviteten ble også observert i både muse og HeLa S3 ekstrakter, noe som tyder på at NTH1 er involvert i AP-sete reparasjon, men i noe mindre grad enn APE1. Rekombinant NTH1 demonstrerte dårligere evne til å prosessere et APE1-kuttet DNA substrat enn et intakt AP-sete. Dette tyder på at NTH1 mest sannsynlig virker oppstrøms for APE1, og konkurrerer ikke med polymerase β nedstrøms for APE1. Produktet generert av NTH1, ble videre prosessert av ikke bare APE1, men også av TDP1 i både muse og HeLa S3 ekstrakter. Dette kan tyde på at TDP1 er i stand til å fungere nedstrøms for NTH1 på en APE1-uavhengig måte og kan derfor muligens fungere som en reserve for APE1 under AP-sete reparasjon

Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform

Endonuclease V (ENDOV) is a ribonuclease with affinity for inosine which is the deamination product of adenosine. The genomes of most organisms, including human, encode ENDOV homologs, yet knowledge about in vivo functions and gene regulation is sparse. To contribute in this field, we analyzed mRNA and protein expression of human ENDOV (hENDOV). Analyses of public sequence databases revealed numerous hENDOV transcript variants suggesting extensive alternative splicing. Many of the transcripts lacked one or more exons corresponding to conserved regions of the ENDOV core domain, suggesting that these transcripts do not encode for active proteins. Three complete transcripts were found with open reading frames encoding 282, 308 and 309 amino acids, respectively. Recombinant hENDOV 308 and hENDOV 309 share the same cleavage activity as hENDOV 282 which is the variant that has been used in previous studies of hENDOV. However, hENDOV 309 binds inosine-containing RNA with stronger affinity than the other isoforms. Overexpressed GFP-fused isoforms were found in cytoplasm, nucleoli and arsenite induced stress granules in human cells as previously reported for hENDOV 282. RT-qPCR analysis of the 3’-termini showed that hENDOV 308 and hENDOV 309 transcripts are more abundant than hENDOV 282 transcripts in immortalized cell lines, but not in primary cells, suggesting that cells regulate hENDOV mRNA expression. In spite of the presence of all three full-length transcripts, mass spectrometry analyses identified peptides corresponding to the hENDOV 309 isoform only. This result suggests that further studies of human ENDOV should rather encompass the hENDOV 309 isoform

Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform

Endonuclease V (ENDOV) is a ribonuclease with affinity for inosine which is the deamination product of adenosine. The genomes of most organisms, including human, encode ENDOV homologs, yet knowledge about in vivo functions and gene regulation is sparse. To contribute in this field, we analyzed mRNA and protein expression of human ENDOV (hENDOV). Analyses of public sequence databases revealed numerous hENDOV transcript variants suggesting extensive alternative splicing. Many of the transcripts lacked one or more exons corresponding to conserved regions of the ENDOV core domain, suggesting that these transcripts do not encode for active proteins. Three complete transcripts were found with open reading frames encoding 282, 308 and 309 amino acids, respectively. Recombinant hENDOV 308 and hENDOV 309 share the same cleavage activity as hENDOV 282 which is the variant that has been used in previous studies of hENDOV. However, hENDOV 309 binds inosine-containing RNA with stronger affinity than the other isoforms. Overexpressed GFP-fused isoforms were found in cytoplasm, nucleoli and arsenite induced stress granules in human cells as previously reported for hENDOV 282. RT-qPCR analysis of the 3’-termini showed that hENDOV 308 and hENDOV 309 transcripts are more abundant than hENDOV 282 transcripts in immortalized cell lines, but not in primary cells, suggesting that cells regulate hENDOV mRNA expression. In spite of the presence of all three full-length transcripts, mass spectrometry analyses identified peptides corresponding to the hENDOV 309 isoform only. This result suggests that further studies of human ENDOV should rather encompass the hENDOV 309 isoform

Deletion of Endonuclease V suppresses chemically induced hepatocellular carcinoma

Endonuclease V (EndoV) is a conserved inosine-specific ribonuclease with unknown biological function. Here, we present the first mouse model lacking EndoV, which is viable without visible abnormalities. We show that endogenous murine EndoV cleaves inosine-containing RNA in vitro, nevertheless a series of experiments fails to link an in vivo function to processing of such transcripts. As inosine levels and adenosine-to-inosine editing often are dysregulated in hepatocellular carcinoma (HCC), we chemically induced HCC in mice. All mice developed liver cancer, however, EndoV−/− tumors were significantly fewer and smaller than wild type tumors. Opposed to human HCC, adenosine deaminase mRNA expression and site-specific editing were unaltered in our model. Loss of EndoV did not affect editing levels in liver tumors, however mRNA expression of a selection of cancer related genes were reduced. Inosines are also found in certain tRNAs and tRNAs are cleaved during stress to produce signaling entities. tRNA fragmentation was dysregulated in EndoV−/− livers and apparently, inosine-independent. We speculate that the inosine-ribonuclease activity of EndoV is disabled in vivo, but RNA binding allowed to promote stabilization of transcripts or recruitment of proteins to fine-tune gene expression. The EndoV−/− tumor suppressive phenotype calls for related studies in human HCC

Deletion of Endonuclease V suppresses chemically induced hepatocellular carcinoma

Endonuclease V (EndoV) is a conserved inosinespecific ribonuclease with unknown biological function. Here, we present the first mouse model lacking EndoV, which is viable without visible abnormalities. We show that endogenous murine EndoV cleaves inosine-containing RNA in vitro, nevertheless a series of experiments fails to link an in vivo function to processing of such transcripts. As inosine levels and adenosine-to-inosine editing often are dysregulated in hepatocellular carcinoma (HCC), we chemically induced HCC in mice. All mice developed liver cancer, however, EndoV−/− tumors were significantly fewer and smaller than wild type tumors. Opposed to human HCC, adenosine deaminase mRNA expression and site-specific editing were unaltered in our model. Loss of EndoV did not affect editing levels in liver tumors, however mRNA expression of a selection of cancer related genes were reduced. Inosines are also found in certain tRNAs and tRNAs are cleaved during stress to produce signaling entities. tRNA fragmentation was dysregulated in EndoV−/− livers and apparently, inosine-independent. We speculate that the inosine-ribonuclease activity of EndoV is disabled in vivo, but RNA binding allowed to promote stabilization of transcripts or recruitment of proteins to finetune gene expression. The EndoV−/− tumor suppressive phenotype calls for related studies in human HCC

Deletion of Endonuclease V suppresses chemically induced hepatocellular carcinoma

Abstract Endonuclease V (EndoV) is a conserved inosine-specific ribonuclease with unknown biological function. Here, we present the first mouse model lacking EndoV, which is viable without visible abnormalities. We show that endogenous murine EndoV cleaves inosine-containing RNA in vitro, nevertheless a series of experiments fails to link an in vivo function to processing of such transcripts. As inosine levels and adenosine-to-inosine editing often are dysregulated in hepatocellular carcinoma (HCC), we chemically induced HCC in mice. All mice developed liver cancer, however, EndoV−/− tumors were significantly fewer and smaller than wild type tumors. Opposed to human HCC, adenosine deaminase mRNA expression and site-specific editing were unaltered in our model. Loss of EndoV did not affect editing levels in liver tumors, however mRNA expression of a selection of cancer related genes were reduced. Inosines are also found in certain tRNAs and tRNAs are cleaved during stress to produce signaling entities. tRNA fragmentation was dysregulated in EndoV−/− livers and apparently, inosine-independent. We speculate that the inosine-ribonuclease activity of EndoV is disabled in vivo, but RNA binding allowed to promote stabilization of transcripts or recruitment of proteins to fine-tune gene expression. The EndoV−/− tumor suppressive phenotype calls for related studies in human HCC

Endonuclease v regulates atherosclerosis through c-c motif chemokine ligand 2-mediated monocyte infiltration

BACKGROUND: In cardiovascular diseases, atherosclerotic disorder are the most frequent and important with respect to morbidity and mortality. Inflammation mediated by immune cells is central in all parts of the atherosclerotic progress, and further understanding of the underlying mechanisms is needed. Growing evidence suggests that deamination of adenosine-toinosine in RNA is crucial for a correct immune response; nevertheless, the role of adenosine-to-inosine RNA editing in atherogenesis has barely been studied. Several proteins have affinity for inosines in RNA, one being ENDOV (endonuclease V), which binds and cleaves RNA at inosines. Data on ENDOV in atherosclerosis are lacking. METHODS AND RESULTS: Quantitative polymerase chain reaction on ENDOV mRNA showed an increased level in human carotid atherosclerotic plaques compared with control veins. Inosine-ribonuclease activity as measured by an enzyme activity assay is detected in immune cells relevant for the atherosclerotic process. Abolishing EndoV in atherogenic apolipoprotein E-deficient (ApoE−/−) mice reduces the atherosclerotic plaque burden, both in size and lipid content. In addition, in a brain stroke model, mice without ENDOV suffer less damage than control mice. Finally, lack of EndoV reduces the recruitment of monocytes to atherosclerotic lesions in atherogenic ApoE−/− mice. CONCLUSIONS: ENDOV is upregulated in human atherosclerotic lesions, and data from mice suggest that ENDOV promotes atherogenesis by enhancing the monocyte recruitment into the atherosclerotic lesion, potentially by increasing the effect of CCL2 activation on these cell

Endonuclease v regulates atherosclerosis through c-c motif chemokine ligand 2-mediated monocyte infiltration

Background In cardiovascular diseases, atherosclerotic disorder are the most frequent and important with respect to morbidity and mortality. Inflammation mediated by immune cells is central in all parts of the atherosclerotic progress, and further understanding of the underlying mechanisms is needed. Growing evidence suggests that deamination of adenosine‐to‐inosine in RNA is crucial for a correct immune response; nevertheless, the role of adenosine‐to‐inosine RNA editing in atherogenesis has barely been studied. Several proteins have affinity for inosines in RNA, one being ENDOV (endonuclease V), which binds and cleaves RNA at inosines. Data on ENDOV in atherosclerosis are lacking. Methods and Results Quantitative polymerase chain reaction on ENDOV mRNA showed an increased level in human carotid atherosclerotic plaques compared with control veins. Inosine‐ribonuclease activity as measured by an enzyme activity assay is detected in immune cells relevant for the atherosclerotic process. Abolishing EndoV in atherogenic apolipoprotein E‐deficient (ApoE−/−) mice reduces the atherosclerotic plaque burden, both in size and lipid content. In addition, in a brain stroke model, mice without ENDOV suffer less damage than control mice. Finally, lack of EndoV reduces the recruitment of monocytes to atherosclerotic lesions in atherogenic ApoE−/− mice. Conclusions ENDOV is upregulated in human atherosclerotic lesions, and data from mice suggest that ENDOV promotes atherogenesis by enhancing the monocyte recruitment into the atherosclerotic lesion, potentially by increasing the effect of CCL2 activation on these cells