Project description:SETX (senataxin) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of amyotrophic lateral sclerosis (ALS) called ALS4, whereas recessive mutations are responsible for ataxia called ataxia with oculomotor apraxia type 2 (AOA2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed the effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX-depleted cells. Importantly, we discovered a strong connection between SETX and the macroautophagy/autophagy pathway, reflecting a direct effect on transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2.
Project description:Senataxin (SETX) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response, through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of ALS (amyotrophic lateral sclerosis) named ALS4, while recessive mutations are responsible for an ataxia called AOA2 (ataxia with oculomotor apraxia type 2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX depleted cells. Importantly, we discovered a strong connection between SETX and the autophagy pathway, reflecting a direct effect on the transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2.
Project description:Senataxin (SETX) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response, through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of ALS (amyotrophic lateral sclerosis) named ALS4, while recessive mutations are responsible for an ataxia called AOA2 (ataxia with oculomotor apraxia type 2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX depleted cells. Importantly, we discovered a strong connection between SETX and the autophagy pathway, reflecting a direct effect on the transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2.
Project description:Here we analysed the role of yeast Senataxin (Sen1) in coordinating replication with transcription and in protecting genome integrity. Senataxin is mutated in the two severe neurodegenerative diseases AOA2 and ALS4. We show that a fraction of Sen1/Senataxin DNA/RNA helicase associates with replication forks and protects the integrity of those fork encountering highly expressed RNAPII genes. sen1 mutants accumulate aberrant DNA structures and RNA-DNA hybrids while forks clash head-on with RNAPII transcription units and counteract recombinogenic events and accumulation of checkpoint signals. Nrd1, which acts togheter with Sen1 in trascription temination, is not recruited at replication forks. nrd1 mutants does not display replication defects, high genome instability and checkpoint activation observed in sen1 mutants The Sen1 function in replication can be therefore separable from its role in RNA processing. We propose a role for Sen1/Senataxin during chromosome replication in facilitating replisome progression across RNAPII transcribed genes thus preventing DNA-RNA hybrids accumulation when forks encounter nascent transcripts on the lagging strand template. Chip on chip analysis was carried out as described (Bermejo et al., 2011), employing anti-Flag monoclonal antibody M2 (Sigma-Aldrich) Labelled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.
Project description:Here we analysed the role of yeast Senataxin (Sen1) in coordinating replication with transcription and in protecting genome integrity. Senataxin is mutated in the two severe neurodegenerative diseases AOA2 and ALS4. We show that a fraction of Sen1/Senataxin DNA/RNA helicase associates with replication forks and protects the integrity of those fork encountering highly expressed RNAPII genes. sen1 mutants accumulate aberrant DNA structures and RNA-DNA hybrids while forks clash head-on with RNAPII transcription units and counteract recombinogenic events and accumulation of checkpoint signals. Nrd1, which acts togheter with Sen1 in trascription temination, is not recruited at replication forks. nrd1 mutants does not display replication defects, high genome instability and checkpoint activation observed in sen1 mutants The Sen1 function in replication can be therefore separable from its role in RNA processing. We propose a role for Sen1/Senataxin during chromosome replication in facilitating replisome progression across RNAPII transcribed genes thus preventing DNA-RNA hybrids accumulation when forks encounter nascent transcripts on the lagging strand template.
Project description:Senataxin, encoded by the SETX gene, contributes to multiple aspects of gene expression, including transcription and RNA processing. Mutations in SETX cause the recessive disorder ataxia with oculomotor apraxia type 2 (AOA2) and a dominant juvenile form of amyotrophic lateral sclerosis (ALS4). To assess the functional role of senataxin in disease, we examined differential gene expression in AOA2 patient fibroblasts, identifying a core set of genes showing altered expression by microarray and RNA-sequencing. To determine whether AOA2 and ALS4 mutations differentially affect gene expression, we overexpressed disease-specific SETX mutations in senataxin-haploinsufficient fibroblasts and observed changes in distinct sets of genes. This implicates mutation-specific alterations of senataxin function in disease pathogenesis and provides a novel example of allelic neurogenetic disorders with differing gene expression profiles. Weighted gene co-expression network analysis (WGCNA) demonstrated these senataxin-associated genes to be involved in both mutation-specific and shared functional gene networks. To assess this in vivo, we performed gene expression analysis on peripheral blood from members of 12 different AOA2 families and identified an AOA2-specific transcriptional signature. WGCNA identified two gene modules highly enriched for this transcriptional signature in the peripheral blood of all AOA2 patients studied. These modules were disease-specific and preserved in patient fibroblasts and in the cerebellum of Setx knockout mice demonstrating conservation across species and cell types, including neurons. These results identify novel genes and cellular pathways related to senataxin function in normal and disease states, and implicate alterations in gene expression as underlying the phenotypic differences between AOA2 and ALS4. Total RNA samples obtained from 1) an AOA2 patient and carrier fibroblast cell lines, 2) 2 biological replicates of haploinsufficient SETX fibroblast cell lines transfected with one of 4 different wild-type and mutant SETX constructs, 3) peripheral blood from 33 patients and carriers across 12 families, and 4) 2 tissues from 2 Setx knockout and 2 control mice were analyzed using expression microarray. This submission represents the microarray component of study.
Project description:Senataxin, encoded by the SETX gene, contributes to multiple aspects of gene expression, including transcription and RNA processing. Mutations in SETX cause the recessive disorder ataxia with oculomotor apraxia type 2 (AOA2) and a dominant juvenile form of amyotrophic lateral sclerosis (ALS4). To assess the functional role of senataxin in disease, we examined differential gene expression in AOA2 patient fibroblasts, identifying a core set of genes showing altered expression by microarray and RNA-sequencing. To determine whether AOA2 and ALS4 mutations differentially affect gene expression, we overexpressed disease-specific SETX mutations in senataxin-haploinsufficient fibroblasts and observed changes in distinct sets of genes. This implicates mutation-specific alterations of senataxin function in disease pathogenesis and provides a novel example of allelic neurogenetic disorders with differing gene expression profiles. Weighted gene co-expression network analysis (WGCNA) demonstrated these senataxin-associated genes to be involved in both mutation-specific and shared functional gene networks. To assess this in vivo, we performed gene expression analysis on peripheral blood from members of 12 different AOA2 families and identified an AOA2-specific transcriptional signature. WGCNA identified two gene modules highly enriched for this transcriptional signature in the peripheral blood of all AOA2 patients studied. These modules were disease-specific and preserved in patient fibroblasts and in the cerebellum of Setx knockout mice demonstrating conservation across species and cell types, including neurons. These results identify novel genes and cellular pathways related to senataxin function in normal and disease states, and implicate alterations in gene expression as underlying the phenotypic differences between AOA2 and ALS4. Total RNA samples obtained from 1) an AOA2 patient and carrier fibroblast cell lines, 2) 2 biological replicates of haploinsufficient SETX fibroblast cell lines transfected with one of 4 different wild-type and mutant SETX constructs, 3) peripheral blood from 33 patients and carriers across 12 families, and 4) 2 tissues from 2 Setx knockout and 2 control mice were analyzed using expression microarray. This submission represents the microarray component of study.