# Sen1: The Varied Virtues of a Multifaceted Helicase

Umberto Aiello, Odil Porrua, Domenico Libri

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Umberto Aiello, Odil Porrua, Domenico Libri. Sen1: The Varied Virtues of a Multifaceted Helicase. Journal of Molecular Biology, 2024, 437 (1), pp.168808. 10.1016/j.jmb.2024.168808. hal-04799533

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# Sen1: The Varied Virtues of a Multifaceted Helicase

Umberto Aiello¹,*, Odil Porrua² and Domenico Libri²,∗

1 - Stanford University School of Medicine, Department of Genetics, Stanford, CA, USA

2 - Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Montpellier, France

Correspondence to Umberto Aiello and Domenico Libri: *Corresponding author uaiello@stanford.edu (U. Aiello), domenico.libri@igmm.cnrs.fr (D. Libri)

https://doi.org/10.1016/j.jmb.2024.168808

Edited by Stephen Buratowski

## Abstract

Several machineries concurrently work on the DNA, but among them RNA Polymerases (RNAPs) are the most widespread and active users. The homeostasis of such a busy genomic environment relies on the existence of mechanisms that allow limiting transcription to a functional level, both in terms of extent and rate. Sen1 is a central player in this sense: using its translocase activity this protein has evolved the specific function of dislodging RNAPs from the DNA template, thus ending the transcription cycle. Over the years, studies have shown that Sen1 uses this same mechanism in a multitude of situations, allowing termination of all three eukaryotic RNAPs in different contexts. In virtue of its helicase activity, Sen1 has also been proposed to have a prominent function in the resolution of co-transcriptional genotoxic R-loops, which can cause the stalling of replication forks. In this review, we provide a synopsis of past and recent findings on the functions of Sen1 in yeast and of its human homologue Senataxin (SETX).

© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Highly trafficked genomes

The multiplicity of protein complexes that interact with the DNA generates complex and crowded structures that have to be dynamically remodeled to allow the proper expression, maintenance and transmission of the genetic information. The accurate and timely orchestration of these activities is required for avoiding interferences and is operated by many factors that use energy to remodel protein-nucleic acids, protein-protein and nucleic acid complexes.

RNA transcription is possibly the quintessential genome occupancy process. Not long ago believed to be exclusively confined inside genes coding for mRNAs or functional non-coding RNA (e.g. snoRNAs), RNA polymerase II (RNAPII) has been shown to transcribe virtually the entirety of the genome, including intergenic regions and

antisense to coding genes. The widespread nature of transcription in yeast has been unveiled by many genome-wide studies, revealing the existence of thousands of cryptic units scattered all along the genome.¹⁻³ Importantly, analyses of other genomes have shown no exception so far to the evolutionary conservation of the phenomenon.⁴

In the context of the crowded nature and the 3D architecture of genomes, widespread transcription is a potentially disturbing element. Transcription over regulatory regions has been shown to be inhibitory via a mechanism referred to as transcriptional interference,⁵ and can be generally used for regulatory purposes.⁶⁻¹⁰ Potential conflicts are of course not a mere transcription family business. Elongating transcription complexes can obstruct the function of other DNA-associated machines, including the replisome. It is now well established that collisions between DNA replicative
forks and RNAPs can jeopardize genome stability, and consequently they must be prevented or resolved.¹¹

Transcription can also favor the generation and stabilization of non-canonical nucleic acid structures and alter the topology of the DNA, which can both impact other DNA-associated processes. The progression of an active machinery that unwinds a DNA duplex introduces downstream positive supercoils and upstream negative supercoils. While excessive positive supercoils can hinder the usage of DNA by impeding its accessibility, the accumulation of upstream negative torsional stress lowers the energetic barrier to form a variety of non-B-DNA structures. Among those, R-loops are three-stranded structures generally formed during transcription when the nascent RNA hybridizes to the DNA template in the wake of RNAPII and outside of the transcription bubble, thus extruding a single strand DNA loop. Because they are prone to DNA damage, R-loops have been shown to impact many aspects of genome stability but also to have important physiological roles.¹²

Mechanisms are in place for coordinating transcription and R-loop formation with other DNA-associated processes. These mechanisms contribute to safeguard the stability and accurate expression of the genome. Transcription initiation is generally directed to positions that favor the expression of functional molecules by the architecture of promoters and the structure of chromatin. However, maybe the most important mechanism that operates to coordinate transcription with other processes is transcription termination. RNA polymerases are released at defined positions at the end of transcription units by several mechanisms, not only to properly partition functional sequences, but also to limit inappropriate extragenic transcription. Termination can also occur within genes for regulatory purposes and for transcription initiated in extragenic regions for limiting interferences with other processes such as replication.

This review will focus on Sen1, an enzyme that has a pivotal role in transcription termination genome-wide and that is also functionally associated with the metabolism of R-loops. Although we will mainly focus on work performed in the yeast system, we will also discuss the known functions of the human homologue, Senataxin, and its implications in human diseases.

## Sen1, the many facets of a multitask helicase

Sen1 (Splicing Endonuclease 1) is a 252 kDa RNA and DNA helicase belonging to the Super Family 1B (SF1B), Upf1-like group of helicases. It consists of a large N-terminal domain, a central conserved helicase domain and a C-terminal

region predicted to be intrinsically disordered. As other helicases of the Upf1-like group, Sen1 is endowed with an ATPase-dependent nucleic acid unwinding activity, able to dissociate both homo-(DNA-DNA), and hetero-duplexes (DNA-RNA) with a 5'-3' directionality.¹³,¹⁴ Although it was originally proposed to be involved in the processing of tRNAs, it has later become clear that the tRNA endonuclease phenotype observed in mutants is an indirect effect of its implication in transcription termination. Indeed, the very unique function of Sen1 resides in the ability to evict RNA polymerases (RNAPs) from the DNA template, a property that is not shared even by the closely related helicase Upf1.¹⁵ It has been shown that Sen1 can evict the three yeast RNA polymerases (RNAPI, RNAPII and RNAPIII) from the DNA *in vitro*,¹⁵,¹⁶ and many lines of evidence demonstrate a direct role in termination *in vivo*, although with different implications.²,¹⁶⁻¹⁸

## Sen1 and transcription termination of non-coding RNA genes within the NNS complex

The best studied activity of Sen1 relates to termination of a large set of transcription events that produce unstable non-coding RNAs, namely Cryptic Unstable Transcripts (CUTs)¹⁹,²⁰ and Small Nucleolar RNAs (snoRNAs).²¹,²² While the function of snoRNAs is clearly established in the modification and maturation of rRNAs, there is no clear, individual function for CUTs, which likely have important roles in the evolution of new genes.²³ Termination of CUTs and SnoRNAs occurs by a different pathway than the one responsible for terminating transcription of mRNA-coding genes and other classes of non-coding transcripts such as XUTs (Xrn1-sensitive Unannotated Transcripts) and SUTs (Stable Unannotated Transcripts).²⁴ This latter pathway depends on the activity of the Cleavage and Polyadenylation Factor (CPF) and the Cleavage Factor (CF) complexes (hereafter denoted as the CPF pathway), which generates stable RNAs that are polyadenylated by Pap1 and rapidly exported to the cytoplasm, contrary to CUTs and SnoRNAs that remain nuclear and are polyadenylated by other factors that favor 3'-5' degradation activities (see below).

Sen1 exerts its termination functions at CUTs and snoRNAs within the NNS (Nrd1, Nab3, Sen1) complex (Figure 1). Nrd1 and Nab3 are two sequence-specific RNA-binding proteins whose mutation generates transcription readthrough phenotypes at the same target genes as does mutation of Sen1. All three subunits of the NNS complex are essential in budding yeast,²⁵⁻²⁷ but Sen1 is the only one to be evolutionarily conserved and with a catalytic activity. Nrd1 is a 64 kDa protein with some homology to the mammalian SCAF8 and SCAF4 anti-terminator factors.²⁸ The three proteins hold an N-terminal CTD-Interacting Domain (CID), a single RNA recognition motif (RRM) and a
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Model of transcription termination by the NNS pathway. Black dashed arrows indicate interactions. Blue arrows indicate movement or release. More details in the text.</figcaption></figure>

segment enriched in alternating charged residues, containing several arginine-glutamate and arginine-serine (RE/RS) dipeptides.²⁶ Nab3 is a 90 kDa acidic protein containing an RRM motif, flanked by two low-complexity domains. The N-terminal domain is an aspartic/glutamic acid rich region (D/E rich) whose function is unknown.²⁹,³⁰ The C-terminal domain is a glutamine/proline rich region (Q/P rich) and, unlike the N-terminal domain, is essential and might play important roles in Nab3 self-association.²⁹,³⁰

Mutation or depletion of any subunit of the NNS complex has no general impact on transcription termination of CPF targets,²,¹⁷,³¹ although a few mRNA-coding genes can be prematurely terminated by the NNS complex, leading to the production of truncated, non-functional transcripts, thus effectively downregulating gene expression.²¹,³²,³³ The *NRD1* gene itself is the stereotypical case of such regulation.³²

Several studies have allowed to define a relatively detailed picture of NNS-dependent termination (Figure 1).

In the most widely accepted model, exposure of the RNA outside of the transcribing polymerase allows the recruitment of the Nrd1-Nab3 heterodimer close to the 5'-end of the transcription unit. Nrd1 and Nab3 have been shown by biochemical approaches to form a tight heterodimer,³⁴,³⁵ which allows the cooperative binding of the two proteins to their target sequences (GUAA/G and UCUUG, for Nrd1 and Nab3, respectively).³⁵⁻³⁹ These motifs are often enriched at the 5'-end of CUTs and their mutation prevents efficient NNS-dependent transcription termination¹⁹,²⁰ indicating that the binding of Nrd1, Nab3 or both proteins to the nascent RNA is essential to trigger termination. Nrd1 interacts via its CID domain with the Ser5-phosphorylated CTD of RNAPII, typically prominent at the beginning of the transcription cycle,⁴⁰,⁴¹ which has been suggested to favor the

recruitment of Nrd1 (and possibly Nab3) to the nascent RNA.⁴²,⁴³

Sen1 is the enzyme that, alone, can dissociate a transcribing or arrested RNAPII from a synthetic transcription scaffold with purified components (Sen1 and RNAPII) *in vitro*. However it has low abundance (between 300 and 1000 molecules per cell),⁴⁴⁻⁴⁶ suggesting the requirement for a recruitment step to its many targets. The more abundant Nrd1-Nab3 heterodimer, which is recruited to the nascent non-coding RNA and interacts directly with Sen1⁴⁷,⁴⁸ represents an obvious candidate for mediating its recruitment (Figure 1). Interaction with the RNA-bound Nrd1-Nab3 dimer would then favor the access of Sen1 to the nascent RNA and the RNA polymerase for the actual termination step (see below). Consistent with this notion, Sen1 contains a Nrd1-Interaction-Motif (NIM) in its C-terminal end that is responsible for the association with Nrd1 and Nab3.⁴²,⁴⁹ Deletion of this motif decreases, albeit does not abolish, the interaction of Sen1 with its target non-coding RNAs. Surprisingly, however, it does not impair NNS-dependent termination⁴² strongly suggesting that alternative and redundant mechanisms for Sen1 recruitment exist and that the role of Nrd1 and Nab3 is likely more complex than simply mediating the recruitment of Sen1.

For instance, it has been shown that Sen1 also interacts with the RNAPII itself (Figure 1), by binding to both the Ser2- and Ser5-phosphorylated (S2P and S5P) isoforms of the CTD.⁴²,⁵⁰ The significance of the S2P interaction is unclear: although Sen1 was shown to crosslink to the 3'-region of transcripts (where S2P would be predominant),¹⁷,⁴² inactivation of Sen1 does not generally affect termination of mRNA-coding genes.¹⁷,² S2P interaction is unlikely relevant for NNS-dependent termination, as this CTD modification is virtually absent in polymerases transcribing NNS-dependent genes.⁵¹ S5P is instead typically abundant at the beginning of the transcription cycle,
although its role in promoting NNS-dependent termination is still under debate.⁴¹,⁵²,⁵³

A distinctive feature of the NNS termination pathway is the handing over of the RNAs released after termination to the RNA exosome, a multisubunit exonuclease that degrades the RNA from the 3'-end (Figure 1). The transcripts are released in association with the Nrd1-Nab3 heterodimer that interacts directly with a poly(A) polymerase, Trf4, part of the TRAMP4 complex (Trf4, Air1/2, Mtr4).⁴³,⁵⁴ Polyadenylation by Trf4, and also the direct interaction of TRAMP with the exosome, favors 3'-end trimming of snoRNAs and full degradation of CUTs, which are poorly detectable in wild type cells.³,⁵⁵,⁵⁶

## SEN1 and termination of RNAPIII and RNAPI

While RNAPII requires extrinsic factors, termination of RNAPIII is mainly thought to be intrinsically dependent on the core enzyme.⁵⁷ The end of RNAPIII-transcription units, such as tRNA genes, is pinpointed by a stretch of thymidines of variable lengths (T-tracts) in the non-template DNA strand that act as the primary termination signal. RNAPIII recognizes this sequence via its subunit C128,⁵⁸ which induces pausing and subsequently termination (Figure 2). There is evidence that the dissociation of RNAPIII is also favored by the folding of secondary, hairpin-like structures in the nascent RNA, with a mechanism that resembles intrinsic termination by bacterial RNAP.⁵⁹,¹⁶ Supposedly, these hairpins function by invading the RNA exit channel and disrupt the stability of the RNAPIII elongation complex.

More recently, however, new studies have shed light on how Sen1 also contributes to RNAPIII termination in yeast (Figure 2). In fission yeast there are two non-essential homologs of Sen1, Sen1 and Dlb8, which, contrary to budding yeast, are not involved in RNAPII termination.⁶⁰,⁶¹

It was shown⁶² that deletion of *S. pombe* Sen1 results in the accumulation of RNAPIII downstream of tRNA genes and the accumulation of extended, unstable RNAs. It was proposed that in *S. pombe* Sen1 functions to facilitate release of the polymerase at the main T-stretch terminator. In a second study¹⁶ it was shown that *S. cerevisiae* Sen1 interacts with RNAPIII via its N-terminal domain. This interaction is mediated by the same residues responsible for the binding of Sen1 to the replisome (see below) although binding to the replisome or to RNAPIII is mutually exclusive. The use of a mutant, *sen1-3*, harboring substitutions in the N-terminal domain that abolish these interactions but do not affect RNAPII termination allowed to study in more detail the role of *S. cerevisiae* Sen1 in RNAPIII termination, revealing a more complex scenario than previously anticipated.

While T-tracts remain the main determinant for RNAPIII termination, short T stretches (less than 5 Ts) require secondary structures to efficiently

induce polymerase release, but Sen1 does not appear to be required at this step.¹⁶ Rather, polymerases that escape termination at the canonical site require Sen1 for efficient release downstream, particularly at sites containing suboptimal T-tracts and lacking auxiliary secondary structures. Sen1 is therefore responsible for a failsafe termination pathway (Figure 2), preventing RNAPIII from running uncontrolled and thus interfering with other DNA-associated processes.

Sen1 has also been implicated in termination of RNAPI, the polymerase responsible for the production of ribosomal RNA (Figure 3).

In this case termination is triggered by a DNA-bound factor (Nsi1 in yeast) that roadblocks the polymerase at the end of the *RDN37* genes.⁶³,⁶⁴ Cleavage of the nascent rRNA by the Rnt1 endonuclease creates a 5' entry site for Rat1 5'-3' exonuclease that is also involved in termination of RNAPII genes. Degradation by Rat1 of the 3' RNA fragment still associated with the polymerase leads to RNAPI termination via a mechanism that is unknown but that might resemble the one described for RNAPII. It was shown that Sen1 depletion exacerbates the termination defects of Rat1-deficient cells, suggesting that it also contributes to the removal of RNAPI.¹⁸ Because depleting only Sen1 does not lead to substantial RNAPI termination defects it is likely that the protein only functions in the absence of Rat1 or that it only functions downstream as a fail-safe mechanism as shown for RNAPIII. In further support of this role it was shown that Sen1 can terminate RNAPI transcription *in vitro*.¹⁶

## The mechanism of RNA polymerase release by Sen1

The mechanism by which Sen1 releases the RNA Polymerase is relatively well understood, thanks to a combination of *in vitro* assays, single-molecule and structural studies.¹³,¹⁵,⁴²,⁶⁵,⁶⁶ Nrd1 and Nab3 are dispensable for termination from synthetic substrates *in vitro*,¹⁵ suggesting that they might not be involved in the step of elongation complex dismantling. Consistent with this notion, it has been recently shown that Sen1 handles many termination events independently of the heterodimer.¹⁷

To remove the elongation complex, Sen1 translocates on the nascent RNA in the 5' to 3' direction, which requires ATP hydrolysis, until it makes contacts with the RNA polymerase (Figure 1). Neither binding to RNA, nor translocation have apparent sequence specificity,⁶⁷,¹⁵ but a single-stranded region of at least 15 nt immediately upstream of RNAPII is required¹⁴ because termination *in vitro* is impaired by the presence of RNA secondary structures¹⁶ or DNA:RNA hybrids,¹⁵ presumably because double stranded regions hamper Sen1 binding and/or efficient translocation. Although Sen1 can unwind RNA-DNA or DNA-DNA duplexes, its helicase activity has been shown to be poorly proces-
<figure><img src="image_4.png" /></figure>

**Figure 2.** Scheme of the implication of Sen1 in fail-safe termination of RNAPIII transcription units (TU) in *S. cerevisiae*. Arrows as in Figure 1. Sen1 is proposed to function downstream of the primary terminator, where the T-tract and RNA secondary structures concur to RNAPIII removal. In *S. pombe* the Sen1 homologue might also function at the primary terminator.

<figure><img src="image_5.png" /></figure>

**Figure 3.** Scheme of the proposed function of Sen1 in termination of rDNA transcription. Details in the text.

sive.¹⁵,⁶⁶ Indeed, *in vitro* studies have shown that Sen1 dissociates after unwinding 20–30 nt of duplex nucleic acids¹³,⁶⁸ possibly explaining why hybrids located upstream of the polymerase hamper its release. On the contrary, Sen1 has been shown to translocate in a processive manner on single-stranded DNA.⁶⁸ It remains to be established whether Sen1 behaves the same way on RNA, which is, so far, its only known functional target molecule.

Pausing of the polymerase is required for efficient termination, both *in vivo* and *in vitro*, possibly because some form of competition takes place between transcription elongation and efficient translocation by Sen1,¹³,¹⁵,⁶⁹ or because termination cannot occur during transcription elongation. *In vitro* Sen1 can release the polymerase only when this latter is stalled in the presence of a DNA-bound roadblock protein or when one of the required nucleotides is absent.¹³,¹⁵ *In vivo*, it was shown that RNAPII mutants with a faster elongation rate exacerbate read-through at Sen1-dependent terminators, whereas, conversely, the termination defect of a hypomorphic *sen1* mutant is alleviated by the combination with a slower RNAPII elongation rate.⁶⁹ These findings are compatible with the notion that Sen1 is chasing the polymerase during termination but also with the alternative possibility that Sen1 is recruited close to the polymerase and

that a slow transcription elongation rate increases the temporal window of opportunity for a productive release step. Interestingly, it has been shown that *in vitro* Sen1 can “push” a paused RNA polymerase,¹³,¹⁶ which was confirmed by independent evidence from a recent single-molecule study.⁷⁰

Although the mechanism underlying RNAPII “pushing” remains unclear, these studies suggest that the collision of translocating Sen1 with the polymerase generates a tension on the nascent RNA that might contribute either to the forward movement of the polymerase or its release. It is enticing to suggest that only a transiently stalled elongation complex can be resolved by Sen1 “pushing” to resume elongation as opposed to termination for a persistent RNAPII stall.

Structural studies have shed light on the structure of the protein and allowed to propose a model for polymerase eviction.⁶⁵,⁷¹ All known enzymatic activities of Sen1 can be fully recapitulated *in vitro* by its isolated helicase domain,¹³ which is extremely well-conserved.⁶⁵ Similar to other helicases, Sen1 contains two RecA domains (Figure 4A). From the first RecA module emerge the ‘stalk’, the ‘barrel’ and the ‘prong’ auxiliary domains.⁶⁵ The prong subdomain is particularly relevant for Sen1 helicase activity as it has been proposed to play a role in unwinding by inserting into the nucleic acid duplex. The recent structure of the full length *Chae-*
tomium thermophilum Sen1 (CtSen1) in complex with single-stranded RNA<sup>71</sup> has allowed to model the position of a double-stranded RNA-DNA hybrid close to the prong, further supporting a role of the latter in unwinding (Figure 4B). Despite strong structural similarities to the architecture of Upf1, Sen1 exhibits distinctive structural features that underlie its unique function. For instance, Sen1 possesses an exclusive subdomain composed of about 50 residues known as the ‘brace’ that mediates extensive intramolecular contacts that impose a peculiar conformation to the auxiliary domains. Also, the ‘prong’ domain has a more flexible nature compared to the same domain contained in other Upf1-like helicases. This subdomain is essential for viability and for transcription termination and it has been proposed that its insertion in the RNA exit channel of the polymerase provokes conformational changes in the elongation complex (EC) and its destabilization.<sup>65</sup> A conformational change in the EC induced by the interaction with Sen1 was also suggested by single molecule studies, which revealed the existence of an intermediate state of the Sen1-RNAPII complex, prior to the release of RNAPII.<sup>66</sup>

Deletion of the N-terminal domain of Sen1 causes growth impairment,<sup>42</sup> suggesting that the mere catalytic activity is not sufficient for the normal functions of Sen1 *in vivo*. The N-terminal domain likely plays important roles in regulating the function of the protein or its recruitment, for example by binding to other factors. In fact, the N-terminal domain lodges interaction sites for RNAPII, RNAPIII, Rnt1, Rad52 and the replisome subunits Ctf4 and Mrc1.<sup>16,72-74</sup> The region of the protein C-terminal to the helicase domains is not required for viability and mediates the interaction with Glc7 and Nab3/Nrd1.<sup>42,47,49</sup> In a recent study, it was shown that the phosphorylation at a conserved threonine in the catalytic domain of Sen1 reduces the efficiency of transcription termination.<sup>6</sup> Interestingly, several other post-translational modifications have been detected all along Sen1,<sup>6</sup> some of which map to the N- and C-terminal domains, thus opening the possibility that these domains might also directly modulate the activity of the protein. The structure of CtSen1<sup>71</sup> has unveiled an important role for the Sen1 N-terminal domain. It was shown that the N-terminal domain of the protein interacts with the C-terminal via three, well-conserved regions. The main element of this interaction is an insertion in the RecA1 domain extending the prong, the Leucine-Zipper-Zipper (LZZ) motif (Figure 4C). Interaction of the N- and C-terminal domains of Sen1 leads to inhibition of its helicase and ATPase activities, which is consistent with the earlier observation that the presence of the N-terminal domain decreases the efficiency of termination.<sup>13</sup>

Inhibition was also shown to occur *in trans* and to be relieved by deletion of the LZZ motif. The presence of the N-terminal domain stabilizes a

nucleotide-dependent conformation that is incompatible with binding of single-stranded RNA and, interestingly, overlaps with the modelled docking of the RNA-DNA hybrid, suggesting a direct competition for the double-stranded substrate binding.<sup>71</sup> The N-terminal domain might therefore constitute a functional platform for controlling Sen1 autoregulation by partners binding and post-translational modifications.

## Sen1 and R-loop metabolism

Soon after exiting the RNAPII, the nascent RNA is typically processed and packaged with RNA-binding proteins. However, in some instances, the RNA can instead pair with its own DNA template, thus forming a DNA-RNA hybrid outside of the transcription bubble and extruding a single-stranded DNA loop, likely behind the transcribing RNAPII (Figure 5A). These structures, known as R-loops, have been found in genomes from all species, and are believed to be present at steady state in 5–10% of the human genome. Single molecule analyses in the human system have allowed determining that the majority of R-loops range in size from 100 to 450 nt, but kilobase length structures have also been detected.<sup>75</sup> These are Janus-faced structures, in that they are believed to both fulfill physiological roles and constitute a danger for the cell. They are required for immunoglobulin class-switch and mitochondrial replication, but their excess generates DNA damage and genome instability.<sup>12</sup> It has been proposed that genome instability is linked to the endonucleolytic cleavage of the ssDNA moiety<sup>12</sup> but also to the fact that the DNA-RNA hybrid might represent an obstacle for replication or that it prevents stalled forks from resuming replication.<sup>76</sup> Because of this ambivalent position in cell homeostasis, the steady state level of R-loops is tightly controlled. RNase H1 and H2 in yeast and other eukaryotes, have crucial roles in the degradation of the RNA moiety of the R-loop (Figure 5B). Helicases are believed to have a more conservative role by unwinding the RNA-DNA hybrid. Moreover, many factors have been proposed to limit R-loop formation, generally by binding to the nascent RNA and hindering the formation of the RNA-DNA hybrid.

It is generally accepted that Sen1 has a major role in R-loops turnover by displacing the engaged RNA strand and thus promoting the reannealing of the two DNA strands (Figure 5C). Evidence for this role was originally provided by the use of genetic reporters whose read-out depends on the occurrence of transcription-associated recombination induced by DNA damage.<sup>77</sup> Sen1 hypomorphic mutants showed increased recombination, linked to an increase in R-loop levels that was suppressed by the overexpression of RNase H.<sup>77</sup> ChIP analyses have supported the notion that Sen1 localizes to sites of replication in the presence of replication stress induced by hydroxyurea (HU)
<figure><img src="image_6.png" /><figcaption>**Figure 4.** A. Structure of the helicase domain of *S. cerevisiae* Sen1. The structural subdomains are illustrated in the scheme below with same color code. B. The structure of full length *Chaetomium thermophilum* Sen1, with same color code of the *S. cerevisiae* helicase domain. The N-terminal domain is in grey. LZZ: Leucine Zipper Zipper motif, interacting with N-terminal domain alpha helices to establish autoinhibition. C. Scheme of the active (left) and autoinhibited (right) conformations.</figcaption></figure>

and it has been shown that R-loops increase in the absence of Sen1 at the *PDC1* locus, one site prone to transcription-replication conflicts (TRCs).⁷⁸

Genome-wide R-loop mapping by DRIP (DNA-RNA Immunoprecipitation), a method relying on the immunoprecipitation of extracted DNA-RNA hybrids with an antibody, have extended these early studies using hypomorphic mutants or acute depletion of Sen1.⁷⁹⁻⁸¹ In one more recent study⁷⁶ it has been proposed that increased R-loops formation in double RNases H mutants in yeast prevents

replication fork restarting under HU-induced stress, and that this can be suppressed by Sen1 overexpression.

These studies converge towards the notion that R-loop levels, as measured by DRIP-related methods, increase in the absence of Sen1 and decrease upon Sen1 overexpression. However, a few caveats should be noted concerning the mechanisms underlying the relationships between Sen1 function and R-loop metabolism. Although failure to resolve R-loops by unwinding is
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Role of Sen1 and RNases H in controlling R-loop levels. A: scheme of R-loop formation behind a paused RNA polymerase. R-loop levels can be controlled by degradation of the RNA moiety by RNases H (RnH, B) or by unwinding (C) by Sen1 or other helicases. In D, two alternative models whereby Sen1 can control R-loops by removing the RNA polymerase at sites of pausing, either after (top) or before (bottom) R-loop formation. In these cases, R-loop removal would be a consequence of RNAPII removal.</figcaption></figure>

generally accounted for their observed increase in Sen1 lack of function (Figure 5C), it cannot be excluded that Sen1 controls R-loop levels through mechanisms other than by denaturing the hybrid. This would be consistent with *in vitro* studies using purified components showing that Sen1 is a poorly processive helicase, unable to efficiently unwind DNA-RNA hybrids longer than 20–30 nt, which can efficiently inhibit Sen1 translocation on the nascent RNA to reach and dismantle the polymerase.¹³ The poor processivity of Sen1 was also more recently confirmed by single molecule studies.⁶⁶ One possibility (Figures 5D) is that R-loop turnover is not due to unwinding. For instance, it can be envisioned that R-loops are suppressed as a consequence of the release of RNAPII from its template by Sen1 at sites of pausing, either because release of the polymerase precedes their formation (Figure 5D, top) or because the DNA-RNA hybrid is destabilized by the absence of the polymerase (Figure 5D, bottom). In this perspective, R-loop control by Sen1 would be an indirect consequence of RNAPII removal. In the same line of thought, it can be envisioned that R-loops associated to paused RNAPII are released when the

paused elongation complex is “pushed” back to elongation by the action of Sen1.

One alternative is that the increased R-loop levels in the absence of Sen1 are due to their enhanced formation, rather than (or in addition to) their decreased destruction. As pointed out above, the proximity of Sen1 with regions of replication was interpreted as a necessity to resolve R-loops at sites of TRCs. Surprisingly, however, global R-loop levels are not altered when a Sen1 mutant cannot interact with the replisome (and is therefore presumably not active at TRC sites) but retains its transcription termination functions,¹⁷,⁷² possibly suggesting that the transcription termination defect of Sen1 lack-of-function mutants contributes to the increased R-loops phenotype.

Sen1 lack-of-function has major consequences on transcription termination,²,¹⁷ and it can be envisioned that in the absence of Sen1 the increased load of extragenic transcription events leads to increased frequency of conflicts and a consequential enhanced formation of R-loops. It has been shown that transcription termination defects generated independently of Sen1 are not sufficient alone for inducing increased R-loops and genome insta-
bility.<sup>77,78,80</sup> However, Sen1 is also independently required for the removal of RNAPII at sites of conflicts (described in the next section). Thus, it is possible that in the total absence of Sen1 functions, at least two components contribute to the R-loops/ genomic instability observed, one concerning the increased formation of R-loops, due to defective termination of non-coding RNA genes, and another due to failure to resolve transcription-related conflicts, for instance with replication. Indeed, it has been recently shown that combining these two defects generated independently, phenocopies the genome instability phenotype observed in Sen1 lack of function.<sup>17</sup>

## Sen1 and transcription-driven conflicts

In genomes where a multitude of machineries coexist and share the use of the DNA, a proper coordination is essential to prevent catastrophic collisions. Recent results point to a general role of Sen1 in resolving conflicts with the replisome but also between different transcription machineries, independently of its functions in transcription termination of non-coding RNAs within the NNS complex.

An interaction of Sen1 with the replisome components Ctf4 and Mrc1 has been identified and biochemically characterized and a mutant, *sen1-3*, was isolated that is defective for this interaction but retains the association with RNAPII and is proficient for transcription termination within the NNS complex pathway.<sup>72</sup> It has been proposed that this interaction serves the NNS-independent recruitment of Sen1 at the replisome to dismantle colliding RNAPII complexes and allow fork progression.<sup>17</sup> High resolution analysis of transcription by CRAC (Crosslinking Analysis of cDNAs) and replication by DNA copy number has allowed demonstrating that the interaction of Sen1 with the replisome is important to release RNAPII in the 5'-end of genes (Figure 6A). These are likely sites of TRCs, because no Sen1-dependent release occurs in the absence of replication, and the phenomenon is preferentially observed in genes that are undergoing replication. Interestingly, RNAPII release appears to occur regardless of the relative directions of transcription and replication (head-on, HO, or co-directional, CD), implicating that RNAPII release by Sen1 is a prevention mechanism, independent of the impact of the collision, which is believed to be higher for HO.<sup>82</sup> Another region where Sen1 was shown to release RNAPII at sites of TRC is the replication fork barrier (RFB) in the 3'-end of the rDNA repeats (Figure 6B). This genomic structure halts replication forks, preventing progression of replication in the opposite direction of rDNA transcription. RNA polymerases travelling CD to forks stalled at the RFB pause and are released by Sen1 associated to the replisome. Interestingly, R-loops have also been detected in the wake of paused polymerases, which increase

when RNAPII is not released in the *sen1-3* mutant. However, R-loop increase can be fully explained by RNAPII occupancy, i.e. no increase is observed after normalization to RNAPII levels. This finding suggests that Sen1 is not functioning at these sites for unwinding R-loops independently of RNAPII but rather that removal of R-loops and RNAPII occurs in a coordinated manner that could be consequential (removal of one induces removal of the other) or otherwise coordinated.

Coordinated control of R-loops and RNAPII might explain the strong negative synthetic interaction of losing Sen1 at the replisome in a genetic context deprived of RNases H.<sup>17,72</sup> Although this could imply a shared function in resolving R-loops, as often proposed, it is also possible that it reflects a more complex scenario, whereby both enzymes can potentially concur to the release of the polymerase and/or to control R-loops, in a direct (unwinding or degradation) or indirect manner. A role for RNases H in releasing RNA polymerase at sites of conflicts has been indeed proposed<sup>17,83</sup>; these enzymes could cleave the annealed RNA in R-loops and induce termination by providing an entry site for the Rat1/Xrn2 endonuclease to degrade the nascent RNA, or even favor the binding of Sen1, which would otherwise be impeded by the presence of the RNA-DNA hybrid as suggested by *in vitro* work.

Besides orchestrating relationships with the replisome, Sen1 can also modulate the interactions between different RNA polymerases in Transcription-Transcription Conflicts (TTCs).<sup>17</sup> As mentioned above, Sen1 interacts with RNAPIII via the same residues that promote binding to the replisome,<sup>16</sup> and this interaction is important for the already discussed mechanism of failsafe termination of RNAPIII. However, this is not all, because preventing the interaction of Sen1 with RNAPIII in the *sen1-3* mutant leads to the abnormal accumulation of RNAPII around tRNA genes,<sup>17</sup> suggesting that, akin to what was observed for the replisome, the recruitment of Sen1 to RNAPIII promotes termination of conflicting RNAPII around RNAPIII genes thus limiting negative effects on gene expression (Figure 7).

Finally, there is evidence that Sen1 might play a similar function in preventing TTCs between RNAPI and RNAPII.<sup>17</sup> In *sen1-3* cells RNAPII was found to accumulate within the rDNA locus, both in the sense and antisense direction relative to RNAPI direction of transcription, suggesting that it is normally released by Sen1. Why this phenotype is observed in the *sen1-3* mutant is however unclear as this Sen1 variant still interacts – directly or indirectly – with RNAPI. One possibility is that Sen1 piggybacks on RNAPII during transcription of RNAPII genes to enter the nucleolus,<sup>84</sup> a path that would be missing in a *sen1-3* mutant. Importantly, all the effects observed at the borders of RNAPI and RNAPII transcription units have been shown to be repli-
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Role of Sen1 in transcription replication conflicts at genes (A) and at the replication fork barrier (RFB) bound by Fob1 (B). Sen1 interacts with the replisome components Ctf4 and Mrc1, (black dashed arrows) which are proposed to contribute to the recruitment of Sen1 for releasing RNAPII when a conflict with replication occurs. RNase H (RNH) is also involved in this process at the RFB.</figcaption></figure>

cation independent,¹⁷ confirming that they are inde-
pendent of conflicts with replication.

From Sen1 to Senataxin

A human gene encoding a putative DNA-RNA helicase was discovered while mapping a chromosomal region (chromosome 9q34.1) whose mutations are linked to an autosomal recessive ataxia, Ataxia-ocular apraxia 2 (AOA2).⁸⁵ Because of its sequence similarity to yeast Sen1, the gene was named senataxin (SETX). Mutations in SETX are also found in individuals affected by a juvenile form of Amyotrophic Lateral Sclerosis (ALS4).⁸⁶ The SETX gene encodes for a 302 kDa helicase belonging to the Super Family 1B (SF1B) group. Sen1 and SETX share a similar domain organization, with most sequence homology found within the helicase domain, and both have been attributed common biological functions. SETX has been so far described to partake in three main processes: transcription termination, R-loop metabolism and DNA repair. Because a comprehensive description of SETX and of its functions has recently been covered elsewhere,⁸⁷ we will mainly focus on the impli-

cations of SETX in the transcription process, on the
similarities with its yeast counterpart, and on the
most recent findings.

Because budding yeast Sen1 is clearly implicated
in transcription termination, studies on the impact of
SETX on the same process were conducted soon
after its discovery. Similarly to Sen1, it was shown
that SETX interacts with RNAPII.⁸⁸ Early studies
reported a significant increase in read-through tran-
scription upon SETX depletion at individual loci and
reporter systems.⁸⁹ This increase was found to cor-
relate with an accumulation of R-loops, which
peaked downstream of the polyA signal. Surpris-
ingly, both the overexpression of RNase H1 (lead-
ing to R-loop decrease) and the depletion of SETX
(leading to R-loops accumulation) were shown to
induce transcription termination defects. These
observations led the authors to propose a model
by which DNA-RNA hybrids naturally forming
behind the elongating RNAPII would induce paus-
ing, and resolution by SETX would be necessary
to allow the 5'-3' exonuclease XRN2 to access
the nascent RNA for transcription termination.⁸⁹
Additional studies have reported termination
defects upon SETX deficiency at specific genes
<figure><img src="image_9.png" /><figcaption>**Figure 7.** Scheme depicting the role of Sen1 in resolving conflicts between RNAPII and RNAPIII at RNAPIII transcription units (TU). Interactions are indicated by black dashed arrows. Shown here only the head-on conflicts case, but a similar role is fulfilled by Sen1 in co-directional conflicts.</figcaption></figure>

such as genes involved in the circadian clock⁹⁰ and, more recently, genes involved in epidermal differentiation.⁹¹ A recent biochemical characterization has provided support for a direct role of Senataxin in transcription termination, that, akin to Sen1, can evict RNAPII from the DNA.⁹² However, although depletion of SETX or inactivating mutations have been shown to affect transcription termination of several classes of genes, evidence for a general genome-wide impact is currently missing.⁹³ Additional studies will be required for understanding the features in genes that define a specific SETX dependence. Several proteins have been shown to interact with SETX to promote its recruitment to termination sites. For instance, the breast cancer susceptibility gene 1 (BRCA1) protein enhances the binding of SETX to termination regions to favor R-loop resolution and prevent R-loop-associated DNA damage.⁹⁴ The interaction of SETX with Survival of Motor Neuron (SMN), which recognizes symmetrically demethylated R1810 arginine residues of the CTD of RNAPII was also shown to favor SETX recruitment to transcribing RNAPII.⁹⁵ Disruption of this interaction resulted in transcription termination defects and R-loops accumulation,⁹⁵ albeit the link between SETX-SMN and SETX-BRCA1 interactions, if any, remains unclear.

Extending the similarities with its budding yeast homolog, SETX was proposed to regulate gene expression by premature transcription termination. For instance, it was proposed that SETX inhibits expression from an HIV-1 promoter by inducing early transcription termination via a complex mechanism that involves RRP6, DROSHA and XRN2.⁹⁶ Similarly, the expression of genes involved in the antiviral response was proposed to be negatively affected by SETX, presumably via the induction of premature termination.⁹⁷ However, alterations in the efficiency of premature termination typically lead to changes in the distribution of RNA-

PII, with increased occupancy in the gene body compared to promoter-proximal regions. Such a pattern was not observed by global run-on analysis (GRO-seq) suggesting that, if premature termination occurs, RNAPII should be dismantled very early after initiation. One possible alternative is that these antiviral genes are activated as a consequence of impaired SETX function in R-loop control.⁹⁸

Besides its role in transcription, SETX has been mostly studied in relation to its function in the maintenance of genome stability. As for Sen1, it is generally proposed that SETX resolves R-loops by unwinding RNA:DNA hybrids, which is consistent with recent *in vitro* data.⁹² Although one genome-wide study did not detect any increased R-loop signal upon SETX depletion,⁹⁹ numerous others over the last decade have reported accumulation of R-loops when the function of SETX is compromised, pointing to a major role for this protein in R-loop control.⁸⁹,⁹¹,⁹⁴,⁹⁵,⁹⁸,¹⁰⁰

As mentioned above, R-loop accumulation in the absence of SETX has been linked to increased DNA damage and genome instability. Upon SETX depletion, the transcription-coupled repair endonucleases XPG and XPF cleave the DNA around R-loops producing DNA breaks that lead to the accumulation of deletions and chromosomal aberrations.¹⁰¹,⁹⁸ Interestingly, cleavage also provokes the release of DNA:RNA hybrids that are exported to the cytoplasm, where they are targeted by sensors of the innate immune system.⁹⁸ These sensors promote the activation of genes such as interferon-induced genes that typically function in the antiviral response.

Similar to Sen1, SETX has also been involved in the resolution of TRCs. SETX was shown to localize at foci that are enriched in markers of DNA damage in a replication-dependent manner¹⁰²,¹⁰³ and at sites of double strand breaks in transcriptionally
active regions.¹⁰⁰ The recruitment of SETX to these putative TRC sites suggests a possible role in regulating such conflicts. Consistent with this notion, a subsequent study⁹³ reported the abnormal RNAPII accumulation at promoter-proximal regions in SETX-deficient cells, often in very long genes containing common fragile sites, which are believed to correspond to TRC sites. This pattern parallels the accumulation of RNAPII in the 5'-end of genes undergoing replication observed in the yeast *sen1-3* mutant that cannot interact with the replisome.¹⁷ Finally, a recent pre-print study reports TRC sites in cells using a proximity-ligation-based approach and has shown that SETX depletion leads to an increase in the number of these sites, further supporting a role for SETX in conflict resolution.¹⁰⁴

SETX also appears to play important roles in the rDNA, but the precise mechanism of function and its biological implication might be different than the roles of yeast Sen1 in releasing RNAPII presumably in conflict with RNAPI. A recent study¹⁰⁵ has reported that RNAPII transcription in the rDNA intergenic region generates an “R-loop shield” that is proposed to prevent RNAPI to transcribe intergenic non-coding RNAs that disturb rRNA expression as well as nucleolar organization. SETX was shown to be required for the establishment of this shield, as its depletion resulted in decreased R-loops and RNAPII occupancy. How the role of SETX relates to the other known functions of the protein remains, however, unclear.

Finally, a recent report has shed light on the enzymatic activities of SETX *in vitro*. The side-by-side comparison of the biochemical properties of SETX and Sen1 helicase domains has revealed that the two proteins have very similar activities, but with some interesting species-specific features. For instance, both helicases interact with single-stranded nucleic acids and can unwind duplex nucleic acids. However, while Sen1 is substantially more efficient at unwinding DNA-DNA duplexes than DNA-RNA hybrids, SETX has a strong preference for R-loops over other types of structures. This suggests that SETX might have evolved a more specific and prominent activity than Sen1 in R-loop resolution. Akin to Sen1, Senataxin can evict RNAPII from the DNA; interestingly, however, the two enzymes display species-specific preferences in that they can only function with their cognate RNAPIIs.⁹² Overall, despite some possible differences in the structure, biochemical properties and interacting partners of the two homologues, it is clear that the main biological roles of Sen1 in the maintenance of genome expression and stability are evolutionary conserved.

## Conclusions and perspectives

Sen1 and its human homologue SETX appear to be central to essential processes such as release of

transcribing or stalled RNA polymerases from the DNA template, R-loop homeostasis and genome stability. In spite of the large amount of data produced since their first characterization, many aspects of their function remain to be defined. We would like to point to a few perspective lines of future investigation for Sen1. The first concerns the mechanism and impact of regulation of Sen1 activity, which could also be relevant for the function of SETX. How autoregulation induced by the interaction of the Sen1 N-terminal with the helicase domain is controlled, possibly by post-translational modifications and the binding of partners that might release autoinhibition, is a very important and, so far, open question.

The poor processivity of Sen1 for unwinding DNA-RNA hybrids might challenge the notion that Sen1 functions on R-loop turn-over as opposed to preventing their formation, for instance by promoting RNAPII removal before R-loop formation. A poor processivity of the enzyme on nascent RNAs that might contain double stranded regions (e.g., R-loops) or bound proteins might also challenge the notion that a race between Sen1 and RNAPII occurs for inducing termination. These aspects of Sen1 activities deserve further investigation. Finally, Sen1 has been shown to be able to translocate on single stranded DNA and to unwind double stranded DNA more efficiently than DNA-RNA hybrids. It is unclear if these biochemical properties are relevant for the biological functions of Sen1, but the possibility that Sen1 has functions beyond the ones around the RNA molecule might open very interesting avenues of investigation.

## CRediT authorship contribution statement

**Umberto Aiello:** Writing – review & editing, Writing – original draft. **Odil Porrua:** Writing – review & editing, Writing – original draft. **Domenico Libri:** Writing – review & editing, Writing – original draft.

## DECLARATION OF COMPETING INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

This work was supported by the CNRS, by the Fondation pour la Recherche Medicale (grant MND202003011479 to O.P.), the ANR (ANR-21-CE12-0040 to D.L.), the University of Montpellier (Soutien à la recherche 2024 – programme
d'excellence I-SITE). U.A. is supported by a
Stanford University School of Medicine Dean's
Fellowship and by an EMBO postdoctoral
fellowship (ALTF 889-2022).

Received 24 July 2024;
Accepted 26 September 2024;
Available online 30 September 2024

**Keywords:**
transcription;
termination;
Sen1;
Senataxin;
SETX

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