Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor ß (TGF-ß), is reduced; that then leads to the activation of the TGF-ß pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins.

[A case of sporadic amyotrophic lateral sclerosis (ALS) with Senataxin (SETX) gene variant].

A 67-year-old man presented slowly progressive weakness of the extremities visited our hospital. Nerve conduction study showed axonal neuropathy and needle electromyography showed neurogenic changes with denervation findings in multiple limb muscles. While he was diagnosed as Probable amyotrophic lateral sclerosis (ALS), which is defined by the Awaji criteria for diagnosis of ALS, he did not develop either respiratory muscle paralysis or bulbar palsy, which are characteristic symptoms of sporadic ALS. Genetic testing revealed a novel gene variant in senataxin (SETX), the causative gene of ALS4. We could not make a definite diagnosis of ALS4 because he had no relatives who could perform genetic testing (segregation study). However, we considered the variant can be pathogenic because it was not previously reported and absent in at least 1,000 healthy control individuals, the variant site was highly conserved in mammals, and it may impair the function of senataxin protein (in silico analysis).

SUMOylated Senataxin functions in genome stability, RNA degradation, and stress granule disassembly, and is linked with inherited ataxia and motor neuron disease.

BACKGROUND: Senataxin (SETX) is a DNA/RNA helicase critical for neuron survival. SETX mutations underlie two inherited neurodegenerative diseases: Ataxia with Oculomotor Apraxia type 2 (AOA2) and Amyotrophic Lateral Sclerosis type 4 (ALS4). METHODS: This review examines SETX key cellular processes and we hypothesize that SETX requires SUMO posttranslational modification to function properly. RESULTS: SETX is localized to distinct foci during S-phase of the cell cycle, and these foci represent sites of DNA polymerase/RNA polymerase II (RNAP) collision, as they co-localize with DNA damage markers 53BP1 and H2AX. At such sites, SETX directs incomplete RNA transcripts to the nuclear exosome for degradation via interaction with exosome component 9 (Exosc9), a key component of the nuclear exosome. These processes require SETX SUMOylation. SETX was also recently localized within stress granules (SGs), and found to regulate SG disassembly, a process that similarly requires SUMOylation. CONCLUSION: SETX undergoes SUMO modification to function at S-phase foci in cycling cells to facilitate RNA degradation. SETX may regulate similar processes in non-dividing neurons at sites of RNAP II bidirectional self-collision. Finally, SUMOylation of SETX appears to be required for SG disassembly. This SETX function may be crucial for neuron survival, as altered SG dynamics are linked to ALS disease pathogenesis. In addition, AOA2 point mutations have been shown to block SETX SUMOylation. Such mutations induce an ataxia phenotype indistinguishable from those with SETX null mutation, underscoring the importance of this modification.

[A case of motor and sensory polyneuropathy and respiratory failure with novel heterozygous mutation of the senataxin gene].

The patient was a 29-year-old male. He took his first steps at two-and-a-half years old, but his physical strength deteriorated and he became non-ambulatory at 12 years old. He had respiratory failure at the age of 20, and finally underwent tracheostomy with invasive positive-pressure ventilation (TPPV). He showed distal dominant muscle weakness and atrophy, including the face. Spinal scoliosis was recognized. He had peripheral predominance of sensory disorders. Nerve conduction studies showed a decrease of compound muscle action potential and a reduction of motor nerve conduction velocity. Sensory nerve action potential was not evoked. In genetic analysis, c.23 C> T (p. T8M) heterozygous mutation was found in the senataxin gene (SETX). Although SETX is a causative gene of familial amyotrophic lateral sclerosis type 4 (ALS4), this case suggests that SETX mutation can also cause motor and sensory polyneuropathy.

Tight expression regulation of senataxin, linked to motor neuron disease and ataxia, is required to avert cell-cycle block and nucleolus disassembly.

The Senataxin (SETX) protein exhibits strong sequence conservation with the helicase domain of the yeast protein Sen1p, and recessive SETX mutations cause a severe ataxia, known as Ataxia with Oculomotor Apraxia type 2, while dominant SETX mutations cause Amyotrophic Lateral Sclerosis type 4. SETX is a very low abundance protein, and its expression is tightly regulated, such that large increases in mRNA levels fail to significantly increase protein levels. Despite this, transient transfection in cell culture can boost SETX protein levels on an individual cell basis. Here we found that over-expression of normal SETX, but not enzymatically-dead SETX, is associated with S-phase cell-cycle arrest in HEK293A cells. As SETX interacts with the nuclear exosome to ensure degradation of incomplete RNA transcripts, and SETX localizes to sites of collision between the DNA replication machinery and the RNAP II complex, altered dosage or aberrant function of SETX may impede this process to promote S-phase cell-cycle arrest. Because neurons are enriched for long transcripts with additional antisense regulatory transcription, collisions of RNAP II complexes may occur in such post-mitotic cells, underscoring a role for SETX in maintaining neuron homeostasis.

Finding the Right Blend: Interplay Between Structure and Sodium Ion Conductivity in the System Na5AlS4-Na4SiS4.

The rational design of high performance sodium solid electrolytes is one of the key challenges in modern battery research. In this work, we identify new sodium ion conductors in the substitution series Na5-x Al1-x Si x S4 (0 ≤ x ≤ 1), which are entirely based on earth-abundant elements. These compounds exhibit conductivities ranging from 1.64 · 10-7 for Na4SiS4 to 2.04 · 10-5 for Na8.5(AlS4)0.5(SiS4)1.5 (x = 0.75). We determined the crystal structures of the Na+-ion conductors Na4SiS4 as well as hitherto unknown Na5AlS4 and Na9(AlS4)(SiS4). Na+-ion conduction pathways were calculated by bond valence energy landscape (BVEL) calculations for all new structures highlighting the influence of the local coordination symmetry of sodium ions on the energy landscape within this family. Our findings show that the interplay of charge carrier concentration and low site symmetry of sodium ions can enhance the conductivity by several orders of magnitude.

High-throughput sequencing revealed a novel SETX mutation in a Hungarian patient with amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the degeneration of the motor neurons. To date, 126 genes have been implicated in ALS. Therefore, the heterogenous genetic background of ALS requires comprehensive genetic investigative approaches. METHODS: In this study, DNA from 28 Hungarian ALS patients was subjected to targeted high-throughput sequencing of the coding regions of three Mendelian ALS genes: FUS, SETX, and C9ORF72. RESULTS: A novel heterozygous missense mutation (c.791A>G, p.N264S) of the SETX gene was identified in a female patient presenting an atypical ALS phenotype, including adult onset and lower motor neuron impairment. No further mutations were detected in the other Mendelian ALS genes investigated. CONCLUSION: Our study contributes to the understanding of the genetic and phenotypic diversity of motor neuron diseases (MNDs). Our results also suggest that the elucidation of the genetic background of MNDs requires a complex approach, including the screening of both Mendelian and non-Mendelian genes.

SETX sumoylation: A link between DNA damage and RNA surveillance disrupted in AOA2.

Senataxin (SETX) is a putative RNA:DNA helicase that is mutated in two distinct juvenile neurological disorders, AOA2 and ALS4. SETX is involved in the response to oxidative stress and is suggested to resolve R loops formed at transcription termination sites or at sites of collisions between the transcription and replication machineries. R loops are hybrids between RNA and DNA that are believed to lead to DNA damage and genomic instability. We discovered that Rrp45, a core component of the exosome, is a SETX-interacting protein and that the interaction depends on modification of SETX by sumoylation. Importantly, we showed that AOA2 but not ALS4 mutations prevented both SETX sumoylation and the Rrp45 interaction. We also found that upon replication stress induction, SETX and Rrp45 co-localize in nuclear foci that constitute sites of R-loop formation generated by transcription and replication machinery collisions. We suggest that SETX links transcription, DNA damage and RNA surveillance, and discuss here how this link can be relevant to AOA2 disease.