Role of Senataxin in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, uncurable neurodegenerative disorder characterized by the degradation of motor neurons leading to muscle impairment, failure, and death. Senataxin, encoded by the SETX gene, is a human helicase protein whose mutations have been linked with ALS onset, particularly in its juvenile ALS4 form. Using senataxin's yeast homolog Sen1 as a model for study, it is suggested that senataxin's N-terminus interacts with RNA polymerase II, whilst its C-terminus engages in helicase activity. Senataxin is heavily involved in transcription regulation, termination, and R-loop resolution, enabled by recruitment and interactions with enzymes such as ubiquitin protein ligase SAN1 and ribonuclease H (RNase H). Senataxin also engages in DNA damage response (DDR), primarily interacting with the exosome subunit Rrp45. The Sen1 mutation E1597K, alongside the L389S and R2136H gain-of-function mutations to senataxin, is shown to cause negative structural and thus functional effects to the protein, thus contributing to a disruption in WT functions, motor neuron (MN) degeneration, and the manifestation of ALS clinical symptoms. This review corroborates and summarizes published papers concerning the structure and function of senataxin as well as the effects of their mutations in ALS pathology in order to compile current knowledge and provide a reference for future research. The findings compiled in this review are indicative of the experimental and therapeutic potential of senataxin and its mutations as a target in future ALS treatment/cure discovery, with some potential therapeutic routes also being discussed in the review.

Sen1 architecture: RNA-DNA hybrid resolution, autoregulation, and insights into SETX inactivation in AOA2.

The senataxin (SETX, Sen1 in yeasts) RNA-DNA hybrid resolving helicase regulates multiple nuclear transactions, including DNA replication, transcription, and DNA repair, but the molecular basis for Sen1 activities is ill defined. Here, Sen1 cryoelectron microscopy (cryo-EM) reconstructions reveal an elongated inchworm-like architecture. Sen1 is composed of an amino terminal helical repeat Sen1 N-terminal (Sen1N) regulatory domain that is flexibly linked to its C-terminal SF1B helicase motor core (Sen1Hel) via an intrinsically disordered tether. In an autoinhibited state, the Sen1Sen1N domain regulates substrate engagement by promoting occlusion of the RNA substrate-binding cleft. The X-ray structure of an activated Sen1Hel engaging single-stranded RNA and ADP-SO4 shows that the enzyme encircles RNA and implicates a single-nucleotide power stroke in the Sen1 RNA translocation mechanism. Together, our data unveil dynamic protein-protein and protein-RNA interfaces underpinning helicase regulation and inactivation of human SETX activity by RNA-binding-deficient mutants in ataxia with oculomotor apraxia 2 neurodegenerative disease.

The Nrd1-Nab3-Sen1 transcription termination complex from a structural perspective.

A substantial part of living cells activity involves transcription regulation. The RNA polymerases responsible for this job need to know 'where/when' to start and stop in the genome, answers that may change throughout life and upon external stimuli. In Saccharomyces cerevisiae, RNA Pol II transcription termination can follow two different routes: the poly(A)-dependent one used for most of the mRNAs and the Nrd1/Nab3/Sen1 (NNS) pathway for non-coding RNAs (ncRNA). The NNS targets include snoRNAs and cryptic unstable transcripts (CUTs) generated by pervasive transcription. This review recapitulates the state of the art in structural biology and biophysics of the Nrd1, Nab3 and Sen1 components of the NNS complex, with special attention to their domain structures and interactions with peptide and RNA motifs, and their heterodimerization. This structural information is put into the context of the NNS termination mechanism together with possible prospects for evolution in the field.

Sen1 is a key regulator of transcription-driven conflicts.

Cellular homeostasis requires the coordination of several machineries concurrently engaged in the DNA. Wide-spread transcription can interfere with other processes, and transcription-replication conflicts (TRCs) threaten genome stability. The conserved Sen1 helicase not only terminates non-coding transcription but also interacts with the replisome and reportedly resolves genotoxic R-loops. Sen1 prevents genomic instability, but how this relates to its molecular functions remains unclear. We generated high-resolution, genome-wide maps of transcription-dependent conflicts and R-loops using a Sen1 mutant that has lost interaction with the replisome but is termination proficient. We show that, under physiological conditions, Sen1 removes RNA polymerase II at TRCs within genes and the rDNA and at sites of transcription-transcription conflicts, thus qualifying as a "key regulator of conflicts." We demonstrate that genomic stability is affected by Sen1 mutation only when in addition to its role at the replisome, the termination of non-coding transcription or R-loop removal are additionally compromised.

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast.

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3' untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3' end formation factors, which revealed 3'UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3'UTRs. We show that the anti-cancer drug cordycepin, 3' deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3' end processing and controlled by the availability of 3' end factors, nucleotide levels and chromatin landscape.

Data from crosslinking and analysis of cDNAs (CRAC) of Nab3 in yeast cells expressing a circular ncRNA decoy.

Pervasive transcription originating from the ubiquitous activity of RNA Polymerase II (RNAPII) generates a vast mass of non-coding RNAs (ncRNAs) that represent a potential harm to gene expression. In the compact genome of the yeast Saccharomyces cerevisiae, the main genomewide safeguard against pervasive ncRNAs is the Nrd1-Nab3-Sen1 (NNS) complex, composed of two RNA-binding proteins (Nrd1 and Nab3) and the helicase Sen1. The NNS complex directs transcription termination of ncRNA genes and promotes the rapid degradation of pervasive transcripts from yeast nuclei through its physical and functional coupling to the nuclear RNA exosome. We have recently shown that inhibition of the exosome in yeast cells leads to the accumulation of ncRNAs complexed with Nab3 and Nrd1, decreasing recycling of these termination factors to sites of transcription and inducing global termination defects at NNS targets. Consistent with the notion that ncRNAs out-titrate Nab3 and Nrd1 termination factors, we have shown that a similar genomewide termination impairment could be achieved by expressing a circular RNA decoy containing a Nab3 binding target [1]. In relation to this previous research article, here we expand our observations on the effect of the circular RNA decoy on NNS termination. We aimed at verifying that the Nab3 binding sequence present on the decoy is indeed efficiently sequestering Nab3 as intended by design, leading to the expected decrease of Nab3 binding on NNS targets. We employed the crosslinking and cDNA analysis protocol (CRAC) on yeast cells expressing the circular ncRNA decoy or a control construct. We present data from high-resolution genomewide RNA binding of Nab3 in three independent biological replicates of these S.cerevisiae cells, normalized by spiked-in S.pombe lysates. These data allow the useful assessment of the extent of co-transcriptional binding decrease of Nab3 by decoy ncRNA titration and will be valuable for further analyses of NNS targeting mechanisms.

Degradation of Non-coding RNAs Promotes Recycling of Termination Factors at Sites of Transcription.

A large share of the non-coding transcriptome in yeast is controlled by the Nrd1-Nab3-Sen1 (NNS) complex, which promotes transcription termination of non-coding RNA (ncRNA) genes, and by the nuclear exosome, which limits the steady-state levels of the transcripts produced. How unconstrained ncRNA levels affect RNA metabolism and gene expression are long-standing and important questions. Here, we show that degradation of ncRNAs by the exosome is required for freeing Nrd1 and Nab3 from the released transcript after termination. In exosome mutants, these factors are sequestered by ncRNAs and cannot be efficiently recycled to sites of transcription, inducing termination defects at NNS targets. ncRNA-dependent, genome-wide termination defects can be recapitulated by the expression of a degradation-resistant, circular RNA containing a natural NNS target in exosome-proficient cells. Our results have important implications for the mechanism of termination, the general impact of ncRNAs abundance, and the importance of nuclear ncRNA degradation.

GmVTL1a is an iron transporter on the symbiosome membrane of soybean with an important role in nitrogen fixation.

Legumes establish symbiotic relationships with soil bacteria (rhizobia), housed in nodules on roots. The plant supplies carbon substrates and other nutrients to the bacteria in exchange for fixed nitrogen. The exchange occurs across a plant-derived symbiosome membrane (SM), which encloses rhizobia to form a symbiosome. Iron supplied by the plant is crucial for rhizobial enzyme nitrogenase that catalyses nitrogen fixation, but the SM iron transporter has not been identified. We use yeast complementation, real-time PCR and proteomics to study putative soybean (Glycine max) iron transporters GmVTL1a and GmVTL1b and have characterized the role of GmVTL1a using complementation in plant mutants, hairy root transformation and microscopy. GmVTL1a and GmVTL1b are members of the vacuolar iron transporter family and homologous to Lotus japonicus SEN1 (LjSEN1), which is essential for nitrogen fixation. GmVTL1a expression is enhanced in nodule infected cells and both proteins are localized to the SM. GmVTL1a transports iron in yeast and restores nitrogen fixation when expressed in the Ljsen1 mutant. Three GmVTL1a amino acid substitutions that block nitrogen fixation in Ljsen1 plants reduce iron transport in yeast. We conclude GmVTL1a is responsible for transport of iron across the SM to bacteroids and plays a crucial role in the nitrogen-fixing symbiosis.

Senataxin Ortholog Sen1 Limits DNA:RNA Hybrid Accumulation at DNA Double-Strand Breaks to Control End Resection and Repair Fidelity.

An important but still enigmatic function of DNA:RNA hybrids is their role in DNA double-strand break (DSB) repair. Here, we show that Sen1, the budding yeast ortholog of the human helicase Senataxin, is recruited at an HO endonuclease-induced DSB and limits the local accumulation of DNA:RNA hybrids. In the absence of Sen1, hybrid accumulation proximal to the DSB promotes increased binding of the Ku70-80 (KU) complex at the break site, mutagenic non-homologous end joining (NHEJ), micro-homology-mediated end joining (MMEJ), and chromosome translocations. We also show that homology-directed recombination (HDR) by gene conversion is mostly proficient in sen1 mutants after single DSB. However, in the absence of Sen1, DNA:RNA hybrids, Mre11, and Dna2 initiate resection through a non-canonical mechanism. We propose that this resection mechanism through local DNA:RNA hybrids acts as a backup to prime HDR when canonical pathways are altered, but at the expense of genome integrity.

Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD interaction domain and a CTD-mimicking region in Sen1.

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1.

Sen1 Is Recruited to Replication Forks via Ctf4 and Mrc1 and Promotes Genome Stability.

DNA replication and RNA transcription compete for the same substrate during S phase. Cells have evolved several mechanisms to minimize such conflicts. Here, we identify the mechanism by which the transcription termination helicase Sen1 associates with replisomes. We show that the N terminus of Sen1 is both sufficient and necessary for replisome association and that it binds to the replisome via the components Ctf4 and Mrc1. We generated a separation of function mutant, sen1-3, which abolishes replisome binding without affecting transcription termination. We observe that the sen1-3 mutants show increased genome instability and recombination levels. Moreover, sen1-3 is synthetically defective with mutations in genes involved in RNA metabolism and the S phase checkpoint. RNH1 overexpression suppresses defects in the former, but not the latter. These findings illustrate how Sen1 plays a key function at replication forks during DNA replication to promote fork progression and chromosome stability.

RNA Polymerase II CTD Tyrosine 1 Is Required for Efficient Termination by the Nrd1-Nab3-Sen1 Pathway.

In Saccharomyces cerevisiae, transcription termination at protein-coding genes is coupled to the cleavage of the nascent transcript, whereas most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3, and Sen1 (NNS). Termination involves RNA polymerase II CTD phosphorylation, but a systematic analysis of the contribution of individual residues would improve our understanding of the role of the CTD in this process. Here we investigated the effect of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this genes class. Instead, mutating Tyr1 led to widespread termination defects at non-coding genes terminating via NNS. Finally, we showed that Tyr1 is important for pausing in the 5' end of genes and that slowing down transcription suppresses termination defects. Our work highlights the importance of Tyr1-mediated pausing in NNS-dependent termination.

Senataxin, A Novel Helicase at the Interface of RNA Transcriptome Regulation and Neurobiology: From Normal Function to Pathological Roles in Motor Neuron Disease and Cerebellar Degeneration.

Senataxin (SETX) is a DNA-RNA helicase whose C-terminal region shows homology to the helicase domain of the yeast protein Sen1p. Genetic discoveries have established the importance of SETX for neural function, as recessive mutations in the SETX gene cause Ataxia with Oculomotor Apraxia type 2 (AOA2) (OMIM: 606002), which is the third most common form of recessive ataxia, after Friedreich's ataxia and Ataxia-Telangiectasia. In addition, rare, dominant SETX mutations cause a juvenile-onset form of Amyotrophic Lateral Sclerosis (ALS), known as ALS4. SETX performs a number of RNA regulatory functions, including maintaining RNA transcriptome homeostasis. Over the last decade, altered RNA regulation and aberrant RNA-binding protein function have emerged as a central theme in motor neuron disease pathogenesis, with evidence suggesting that sporadic ALS disease pathology may overlap with the molecular pathology uncovered in familial ALS. Like other RNA processing proteins linked to ALS, the basis for SETX gain-of-function motor neuron toxicity remains ill-defined. Studies of yeast Sen1p and mammalian SETX protein have revealed a range of important RNA regulatory functions, including resolution of R-loops to permit transcription termination, and RNA splicing. Growing evidence suggests that SETX may represent an important genetic modifier locus for sporadic ALS. In cycling cells, SETX is found at nuclear foci during the S/G2 cell-cycle transition phase, and may function at sites of collision between components of the replisome and transcription machinery. While we do not yet know which SETX activities are most critical to neurodegeneration, our evolving understanding of SETX function will undoubtedly be crucial for not only understanding the role of SETX in ALS and ataxia disease pathogenesis, but also for delineating the mechanistic biology of fundamentally important molecular processes in the cell.

RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination.

Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast Saccharomyces cerevisiae, Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance. In particular, it is unclear how much crosstalk occurs between CPF-CF and NNS complexes and how Pol II attenuation is modulated during stress adaptation. In this study, we have identified an attenuator in the DEF1 DNA repair gene, which includes a portion of the 5'-untranslated region (UTR) and upstream open reading frame (ORF). Using a plasmid-based reporter gene system, we conducted a genetic screen of 14 termination mutants and their ability to confer Pol II read-through defects. The DEF1 attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 cis-acting point mutations that clustered into four regions, including a polyadenylation site efficiency element that genetically interacts with its cognate binding-protein Hrp1. Outside of the reporter gene context, a DEF1 attenuator mutant increased mRNA and protein expression, exacerbating the toxicity of a constitutively active Def1 protein. Overall, our data support a biologically significant role for transcription attenuation in regulating DEF1 expression, which can be modulated during the DNA damage response.

Cell-Cycle Modulation of Transcription Termination Factor Sen1.

Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other termination factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant termination. We suggest that ncRNA levels and coding gene transcription termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.

Helicases as transcription termination factors: Different solutions for a common problem.

Helicases are enzymes that remodel nucleic acids or protein-nucleic acid complexes in an ATP-dependent manner. They are ubiquitous and can play many diverse functions related to the metabolism of nucleic acids. A few helicases from both the prokaryotic and the eukaryotic worlds have the ability to induce transcription termination. Here we discuss how the same biological function is achieved by different helicases with quite divergent structures and mechanisms of action.

A global function for transcription factors in assisting RNA polymerase II termination.

The role of transcription factors (TFs) on nucleosome positioning, RNA polymerase recruitment, and transcription initiation has been extensively characterized. Here, we propose that a subset of TFs such as Reb1, Abf1, Rap1, and TFIIIB also serve a major function in partitioning transcription units by assisting the Nrd1p-Nab3p-Sen1p Pol II termination pathway.

Senataxin: Genome Guardian at the Interface of Transcription and Neurodegeneration.

R-loops comprise an RNA/DNA hybrid and a displaced single-stranded DNA. They play crucial biological functions and are implicated in neurological diseases, including ataxias, amyotrophic lateral sclerosis, nucleotide expansion disorders (Friedreich ataxia and fragile X syndrome), and cancer. Currently, it is unclear which mechanisms cause R-loop structures to become pathogenic. The RNA/DNA helicase senataxin (SETX) is one of the best characterised R-loop-binding factors in vivo. Mutations in SETX are linked to two neurodegenerative disorders: ataxia with oculomotor apraxia type 2 (AOA2) and amyotrophic lateral sclerosis type 4 (ALS4). SETX is known to play a role in transcription, neurogenesis, and antiviral response. Here, we review the causes of R-loop dysregulation in neurodegenerative diseases and how these structures contribute to pathomechanisms. We will discuss the importance of SETX as a genome guardian in suppressing aberrant R-loop formation and analyse how SETX mutations can lead to neurodegeneration in AOA2/ALS4. Finally, we will discuss the implications for other R-loop-associated neurodegenerative diseases and point to future therapeutic approaches to treat these disorders.

Flocculation in Saccharomyces cerevisiae is regulated by RNA/DNA helicase Sen1p.

The Nrd1-Nab3-Sen1 (NNS) complex terminates transcription of non-coding RNA genes and mediates degradation of the produced transcript by the nuclear exosome. The NNS complex also represses some stress response genes, by stimulating premature termination. A well-characterized stress response in yeast is flocculation, where cells aggregate to form flocs under expression of lectin-encoding genes designated as FLOs. In this study, we demonstrated the role of the NNS complex and Rrp6p in the expression of flocculation genes: FLO1, FLO5, FLO9, and FLO10. Furthermore, a deletion mutant of the RNA processing machinery (RNT1), and SEN1 mutants that are unable to interact with Rnt1p, exhibit a flocculation phenotype. In summary, we have identified a cooperative role of Rnt1p, Rrp6p and the NNS complex in the repression of FLO genes.

Termination of Transcription of Short Noncoding RNAs by RNA Polymerase II.

The RNA polymerase II transcription cycle is often divided into three major stages: initiation, elongation, and termination. Research over the last decade has blurred these divisions and emphasized the tightly regulated transitions that occur as RNA polymerase II synthesizes a transcript from start to finish. Transcription termination, the process that marks the end of transcription elongation, is regulated by proteins that interact with the polymerase, nascent transcript, and/or chromatin template. The failure to terminate transcription can cause accumulation of aberrant transcripts and interfere with transcription at downstream genes. Here, we review the mechanism, regulation, and physiological impact of a termination pathway that targets small noncoding transcripts produced by RNA polymerase II. We emphasize the Nrd1-Nab3-Sen1 pathway in yeast, in which the process has been extensively studied. The importance of understanding small RNA termination pathways is underscored by the need to control noncoding transcription in eukaryotic genomes.