Senataxin deficiency disrupts proteostasis through nucleolar ncRNA-driven protein aggregation.

Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.

Regulation of transcription patterns, poly(ADP-ribose), and RNA-DNA hybrids by the ATM protein kinase.

The ataxia telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response and also an important sensor of oxidative stress. Analysis of gene expression in ataxia-telangiectasia (A-T) patient brain tissue shows that large-scale transcriptional changes occur in patient cerebellum that correlate with the expression level and guanine-cytosine (GC) content of transcribed genes. In human neuron-like cells in culture, we map locations of poly(ADP-ribose) and RNA-DNA hybrid accumulation genome-wide with ATM inhibition and find that these marks also coincide with high transcription levels, active transcription histone marks, and high GC content. Antioxidant treatment reverses the accumulation of R-loops in transcribed regions, consistent with the central role of reactive oxygen species in promoting these lesions. Based on these results, we postulate that transcription-associated lesions accumulate in ATM-deficient cells and that the single-strand breaks and PARylation at these sites ultimately generate changes in transcription that compromise cerebellum function and lead to neurodegeneration over time in A-T patients.

A-T neurodegeneration and DNA damage-induced transcriptional stress.

Loss of the ATM protein kinase in humans results in Ataxia-telangiectasia, a disorder characterized by childhood-onset neurodegeneration of the cerebellum as well as cancer predisposition and immunodeficiency. Although many aspects of ATM function are well-understood, the mechanistic basis of the progressive cerebellar ataxia that occurs in patients is not. Here we review recent progress related to the role of ATM in neurons and the cerebellum that comes from many sources: animal models, post-mortem brain tissue samples, and human neurons in culture. These observations have revealed new insights into the consequences of ATM loss on DNA damage, gene expression, and immune signaling in the brain. Many results point to the importance of reactive oxygen species as well as single-strand DNA breaks in the progression of molecular events leading to neuronal dysfunction. In addition, innate immunity signaling pathways appear to play a critical role in ATM functions in microglia, responding to various forms of nucleic acid sensors and regulating survival of neurons and other cell types. Overall, the results lead to an updated view of transcriptional stress and DNA damage resulting from ATM loss that results in changes in gene expression as well as neuroinflammation that contribute to the cerebellar neurodegeneration observed in patients.

Regulation of transcription patterns, poly-ADP-ribose, and RNA-DNA hybrids by the ATM protein kinase.

The ATM protein kinase is a master regulator of the DNA damage response and also an important sensor of oxidative stress. Analysis of gene expression in Ataxia-telangiectasia patient brain tissue shows that large-scale transcriptional changes occur in patient cerebellum that correlate with expression level and GC content of transcribed genes. In human neuron-like cells in culture we map locations of poly-ADP-ribose and RNA-DNA hybrid accumulation genome-wide with ATM inhibition and find that these marks also coincide with high transcription levels, active transcription histone marks, and high GC content. Antioxidant treatment reverses the accumulation of R-loops in transcribed regions, consistent with the central role of ROS in promoting these lesions. Based on these results we postulate that transcription-associated lesions accumulate in ATM-deficient cells and that the single-strand breaks and PARylation at these sites ultimately generate changes in transcription that compromise cerebellum function and lead to neurodegeneration over time in A-T patients.

Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice.

The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5' strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site-a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate sites of MRN-dependent processing by identifying sites of CtIP association and by sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated most efficiently when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that both complexes bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.