Senataxin: A key actor in RNA metabolism, genome integrity and neurodegeneration.

The RNA/DNA helicase senataxin (SETX) has been involved in multiple crucial processes related to genome expression and integrity such us transcription termination, the regulation of transcription-replication conflicts and the resolution of R-loops. SETX has been the focus of numerous studies since the discovery that mutations in its coding gene are the root cause of two different neurodegenerative diseases: Ataxia with Oculomotor Apraxia type 2 (AOA2) and a juvenile form of Amyotrophic Lateral Sclerosis (ALS4). A plethora of cellular phenotypes have been described as the result of SETX deficiency, yet the precise molecular function of SETX as well as the molecular pathways leading from SETX mutations to AOA2 and ALS4 pathologies have remained unclear. However, recent data have shed light onto the biochemical activities and biological roles of SETX, thus providing new clues to understand the molecular consequences of SETX mutation. In this review we summarize near two decades of scientific effort to elucidate SETX function, we discuss strengths and limitations of the approaches and models used thus far to investigate SETX-associated diseases and suggest new possible research avenues for the study of AOA2 and ALS4 pathogenesis.

Human senataxin is a bona fide R-loop resolving enzyme and transcription termination factor.

Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.

Mechanisms of eukaryotic transcription termination at a glance.

Transcription termination is the final step of a transcription cycle, which induces the release of the transcript at the termination site and allows the recycling of the polymerase for the next round of transcription. Timely transcription termination is critical for avoiding interferences between neighbouring transcription units as well as conflicts between transcribing RNA polymerases (RNAPs) and other DNA-associated processes, such as replication or DNA repair. Understanding the mechanisms by which the very stable transcription elongation complex is dismantled is essential for appreciating how physiological gene expression is maintained and also how concurrent processes that occur synchronously on the DNA are coordinated. Although the strategies employed by the different classes of eukaryotic RNAPs are traditionally considered to be different, novel findings point to interesting commonalities. In this Cell Science at a Glance and the accompanying poster, we review the current understanding about the mechanisms of transcription termination by the three eukaryotic RNAPs.

Pervasive transcription: a controlled risk.

Transcriptome-wide interrogation of eukaryotic genomes has unveiled the pervasive nature of RNA polymerase II transcription. Virtually, any DNA region with an accessible chromatin structure can be transcribed, resulting in a mass production of noncoding RNAs (ncRNAs) with the potential of interfering with gene expression programs. Budding yeast has proved to be a powerful model organism to understand the mechanisms at play to control pervasive transcription and overcome the risks of hazardous disruption of cellular functions. In this review, we focus on the actors and strategies yeasts employ to govern ncRNA production, and we discuss recent findings highlighting the dangers of losing control over pervasive transcription.

Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals.

RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on polythymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryoelectron microscopy (cryo-EM) structures of the yeast Pol III PTC and complementary functional states at resolutions of 2.7-3.9 Å. Pol III recognizes the poly(dT) termination signal with subunit C128 that forms a hydrogen-bond network with the NT strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo, confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly(dT) signals.

An integrated model for termination of RNA polymerase III transcription.

RNA polymerase III (RNAPIII) synthesizes essential and abundant noncoding RNAs such as transfer RNAs. Controlling RNAPIII span of activity by accurate and efficient termination is a challenging necessity to ensure robust gene expression and to prevent conflicts with other DNA-associated machineries. The mechanism of RNAPIII termination is believed to be simpler than that of other eukaryotic RNA polymerases, solely relying on the recognition of a T-tract in the nontemplate strand. Here, we combine high-resolution genome-wide analyses and in vitro transcription termination assays to revisit the mechanism of RNAPIII transcription termination in budding yeast. We show that T-tracts are necessary but not always sufficient for termination and that secondary structures of the nascent RNAs are important auxiliary cis-acting elements. Moreover, we show that the helicase Sen1 plays a key role in a fail-safe termination pathway. Our results provide a comprehensive model illustrating how multiple mechanisms cooperate to ensure efficient RNAPIII transcription termination.

Modulated termination of non-coding transcription partakes in the regulation of gene expression.

Pervasive transcription is a universal phenomenon leading to the production of a plethora of non-coding RNAs. If left uncontrolled, pervasive transcription can be harmful for genome expression and stability. However, non-coding transcription can also play important regulatory roles, for instance by promoting the repression of specific genes by a mechanism of transcriptional interference. The efficiency of transcription termination can strongly influence the regulatory capacity of non-coding transcription events, yet very little is known about the mechanisms modulating the termination of non-coding transcription in response to environmental cues. Here, we address this question by investigating the mechanisms that regulate the activity of the main actor in termination of non-coding transcription in budding yeast, the helicase Sen1. We identify a phosphorylation at a conserved threonine of the catalytic domain of Sen1 and we provide evidence that phosphorylation at this site reduces the efficiency of Sen1-mediated termination. Interestingly, we find that this phosphorylation impairs termination at an unannotated non-coding gene, thus repressing the expression of a downstream gene encoding the master regulator of Zn homeostasis, Zap1. Consequently, many additional genes exhibit an expression pattern mimicking conditions of Zn excess, where ZAP1 is naturally repressed. Our findings provide a novel paradigm of gene regulatory mechanism relying on the direct modulation of non-coding transcription termination.

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.

Purification and In Vitro Analysis of the Exosome Cofactors Nrd1-Nab3 and Trf4-Air2.

In many eukaryotic organisms from yeast to human, the exosome plays an important role in the control of pervasive transcription and in non-coding RNA (ncRNA) processing and quality control by trimming precursor RNAs and degrading aberrant transcripts. In Saccharomyces cerevisiae this function is enabled by the interaction of the exosome with several cofactors: the Nrd1-Nab3 heterodimer and the Trf4-Air2-Mtr4 (TRAMP4) complex. Nrd1 and Nab3 are RNA binding proteins that recognize specific motifs enriched in the target ncRNAs, whereas TRAMP4 adds polyA tails at the 3' end of transcripts and stimulates RNA degradation by the exosome. This chapter provides protocols for the purification of recombinant forms of these exosome cofactors and for the in vitro assessment of their activity.

High-resolution transcription maps reveal the widespread impact of roadblock termination in yeast.

Transcription termination delimits transcription units but also plays important roles in limiting pervasive transcription. We have previously shown that transcription termination occurs when elongating RNA polymerase II (RNAPII) collides with the DNA-bound general transcription factor Reb1. We demonstrate here that many different DNA-binding proteins can induce termination by a similar roadblock (RB) mechanism. We generated high-resolution transcription maps by the direct detection of RNAPII upon nuclear depletion of two essential RB factors or when the canonical termination pathways for coding and non-coding RNAs are defective. We show that RB termination occurs genomewide and functions independently of (and redundantly with) the main transcription termination pathways. We provide evidence that transcriptional readthrough at canonical terminators is a significant source of pervasive transcription, which is controlled to a large extent by RB termination. Finally, we demonstrate the occurrence of RB termination around centromeres and tRNA genes, which we suggest shields these regions from RNAPII to preserve their functional integrity.

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.

Sen1 has unique structural features grafted on the architecture of the Upf1-like helicase family.

The superfamily 1B (SF1B) helicase Sen1 is an essential protein that plays a key role in the termination of non-coding transcription in yeast. Here, we identified the ~90 kDa helicase core of Saccharomyces cerevisiae Sen1 as sufficient for transcription termination in vitro and determined the corresponding structure at 1.8 Å resolution. In addition to the catalytic and auxiliary subdomains characteristic of the SF1B family, Sen1 has a distinct and evolutionarily conserved structural feature that "braces" the helicase core. Comparative structural analyses indicate that the "brace" is essential in shaping a favorable conformation for RNA binding and unwinding. We also show that subdomain 1C (the "prong") is an essential element for 5'-3' unwinding and for Sen1-mediated transcription termination in vitro Finally, yeast Sen1 mutant proteins mimicking the disease forms of the human orthologue, senataxin, show lower capacity of RNA unwinding and impairment of transcription termination in vitro The combined biochemical and structural data thus provide a molecular model for the specificity of Sen1 in transcription termination and more generally for the unwinding mechanism of 5'-3' helicases.

Biochemical characterization of the helicase Sen1 provides new insights into the mechanisms of non-coding transcription termination.

Pervasive transcription is widespread and needs to be controlled in order to avoid interference with gene expression. In Saccharomyces cerevisiae, the highly conserved helicase Sen1 plays a key role in restricting pervasive transcription by eliciting early termination of non-coding transcription. However, many aspects of the mechanism of termination remain unclear. In this study we characterize the biochemical activities of Sen1 and their role in termination. First, we demonstrate that the helicase domain (HD) is sufficient to dissociate the elongation complex (EC) in vitro. Both full-length Sen1 and its HD can translocate along single-stranded RNA and DNA in the 5΄ to 3΄ direction. Surprisingly, however, we show that Sen1 is a relatively poorly processive enzyme, implying that it must be recruited in close proximity to the RNA polymerase II (RNAPII) for efficient termination. We present evidence that Sen1 can promote forward translocation of stalled polymerases by acting on the nascent transcript. In addition, we find that dissociation of the EC by Sen1 is favoured by the reannealing of the DNA upstream of RNAPII. Taken together, our results provide new clues to understand the mechanism of Sen1-dependent transcription termination and a rationale for the kinetic competition between elongation and termination.

Transcription Termination: Variations on Common Themes.

Transcription initiates pervasively in all organisms, which challenges the notion that the information to be expressed is selected mainly based on mechanisms defining where and when transcription is started. Together with post-transcriptional events, termination of transcription is essential for sorting out the functional RNAs from a plethora of transcriptional products that seemingly have no use in the cell. But terminating transcription is not that easy, given the high robustness of the elongation process. We review here many of the strategies that prokaryotic and eukaryotic cells have adopted to dismantle the elongation complex in a timely and efficient manner. We highlight similarities and diversity, underlying the existence of common principles in a diverse set of functionally convergent solutions.

Transcription termination and the control of the transcriptome: why, where and how to stop.

Transcription termination occurs when the polymerase is released after a transcription event, thus delimitating transcription units; however, the functional importance of termination extends beyond the mere definition of gene borders. By determining the cellular fate of the generated transcripts, transcription termination pathways shape the transcriptome. Recent reports have underscored the crucial role of these pathways in limiting the extent of pervasive transcription, which has attracted interest in post-initiation events in gene expression control. Transcription termination pathways involved in the production of non-coding RNAs - such as the Nrd1-Nab3-Sen1 (NNS) pathway in yeast and the cap-binding complex (CBC)-ARS2 pathway in humans - are key determinants of transcription quality control. Understanding the mechanisms leading to the timely and efficient dismantling of elongation complexes remains a major unmet challenge, but new insights into the molecular basis of termination at mRNA-coding and non-coding RNA gene targets have been gained in eukaryotes.

Characterization of the mechanisms of transcription termination by the helicase Sen1.

In vitro transcription systems have been widely used to study all the steps of transcription from initiation to termination and many transcription-coupled processes. Here we describe an in vitro transcription-termination assay that we have used for the analysis of the mechanism of termination by the yeast helicase Sen1. In this system, we use highly purified proteins to assemble ternary elongation complexes (RNA polymerase, DNA template, and nascent RNA) on biotinylated DNA that is subsequently immobilized on streptavidin beads. After allowing transcription by the addition of nucleotides, the termination events can be detected and quantified by comparing the amounts of polymerases and transcripts released from the DNA templates in reactions performed in the absence or in the presence of purified Sen1. By modifying different parameters of the assay, this technique allows the study of several aspects of the termination reaction.

Roadblock termination by reb1p restricts cryptic and readthrough transcription.

Widely transcribed compact genomes must cope with the major challenge of frequent overlapping or concurrent transcription events. Efficient and timely transcription termination is crucial to control pervasive transcription and prevent transcriptional interference. In yeast, transcription termination of RNA polymerase II (RNAPII) occurs via two possible pathways that both require recognition of termination signals on nascent RNA by specific factors. We describe here an additional mechanism of transcription termination for RNAPII and demonstrate its biological significance. We show that the transcriptional activator Reb1p bound to DNA is a roadblock for RNAPII, which pauses and is ubiquitinated, thus triggering termination. Reb1p-dependent termination generates a class of cryptic transcripts that are degraded in the nucleus by the exosome. We also observed transcriptional interference between neighboring genes in the absence of Reb1p. This work demonstrates the importance of roadblock termination for controlling pervasive transcription and preventing transcription through gene regulatory regions.

Molecular basis for coordinating transcription termination with noncoding RNA degradation.

The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.

An A-tract at the AtzR binding site assists DNA binding, inducer-dependent repositioning and transcriptional activation of the PatzDEF promoter.

The LysR-type regulator AtzR activates the Pseudomonas sp. ADP atzDEF operon in response to nitrogen limitation and cyanuric acid. Activation involves repositioning of the AtzR tetramer on the PatzDEF promoter and relaxation of an AtzR-induced DNA bend. Here we examine the in vivo and in vitro contribution of an A5 -tract present at the PatzDEF promoter region to AtzR binding and transcriptional activation. Substitution of the A-tract for the sequence ACTCA prevented PatzDEF activation and high-affinity AtzR binding, impaired AtzR contacts with the activator binding site and shifted the position of the AtzR-induced DNA bend. Analysis of a collection of mutants bearing different alterations in the A-tract sequence showed that the extent of AtzR-dependent activation does not correlate with the magnitude or orientation of the spontaneous DNA bend generated at this site. Our results support the notion that indirect readout of the A-tract-associated narrow minor groove is essential for the AtzR-DNA complex to achieve a conformation competent for activation of the PatzDEF promoter. Conservation of this motif in several binding sites of LysR-type regulators suggests that this mechanism may be shared by other proteins in this family.

A bacterial-like mechanism for transcription termination by the Sen1p helicase in budding yeast.

Transcription termination is essential to generate functional RNAs and to prevent disruptive polymerase collisions resulting from concurrent transcription. The yeast Sen1p helicase is involved in termination of most noncoding RNAs transcribed by RNA polymerase II (RNAPII). However, the mechanism of termination and the role of this protein have remained enigmatic. Here we address the mechanism of Sen1p-dependent termination by using a highly purified in vitro system. We show that Sen1p is the key enzyme of the termination reaction and reveal features of the termination mechanism. Like the bacterial termination factor Rho, Sen1p recognizes the nascent RNA and hydrolyzes ATP to dissociate the elongation complex. Sen1p-dependent termination is highly specific and, notably, does not require the C-terminal domain of RNAPII. We also show that termination is inhibited by RNA-DNA hybrids. Our results elucidate the role of Sen1p in controlling pervasive transcription.