The completion of DNA synthesis is crucial for maintaining genome stability and survival but a variety of obstacles to DNA replication have the ability to stall replication forks (Magdalou et al., 2014). Stalled forks are usually stabilised by the Intra-S phase checkpoint such that they can ordinarily resume DNA synthesis once the obstacle has been removed or resolved (Lambert and Carr, 2013). However, if replication cannot be resumed and/or the replication fork becomes non-functional, this is known as replication fork collapse. In the majority of cases collapsed replication forks are rescued by a convergent fork, allowing the completion of DNA synthesis. Nevertheless, in regions of the genome with a paucity of origins, or when two convergent forks collapse without an intervening origin, collapsed replication forks must be actively restarted in order to complete replication.

Homologous recombination underpins several mechanisms that have evolved to restart collapsed replication forks. Recombination-dependent replication (RDR) mechanisms include the restart of replication forks following fork reversal plus DNA end processing (Ait Saada et al., 2018) and break-induced replication (BIR; Malkova and Ira, 2013), where replication is initiated from one end of a DNA double strand break (DSB). Yeast model systems have been instrumental in identifying and characterising RDR mechanisms: BIR has been extensively characterised in Saccharomyces cerevisiae and has been shown to occur in G2 phase and to involve initial DSB processing followed by Rad51-dependent strand invasion that results in conservative DNA synthesis via a migrating D-loop. In contrast, DSB-independent RDR (Mizuno et al., 2009) has been characterised mainly in Schizosaccharomyces pombe and has been shown to involve fork reversal, processing of the resulting DNA double strand end and Rad51-dependent strand invasion that results in semi-conservative replication (Teixeira-Silva et al., 2017; Miyabe et al., 2015).

A key tool used in S. pombe to investigate the mechanisms involved in replication fork arrest, collapse and restart is a site-specific replication fork barrier (RFB) that was initially identified close to the mating type locus (Dalgaard and Klar, 2000). This RFB, known as RTS1 (Replication Termination Sequence 1), acts to ensure that replication across the mating type locus is unidirectional. It achieves this by acting as a polar barrier – i.e. it only arrests replication forks travelling from a single ‘restrictive’ direction (Dalgaard and Klar, 2001). Forks travelling in the opposite ‘permissive’ direction are unaffected by the barrier. The RTS1 barrier was first defined as an 859 bp EcoRI fragment that was further refined into two regions, A and B, by deletion analysis. Region B contains four repeat sequences that bind to a Myb-domain protein, known as Rtf1 (Replication Termination Factor 1). Fork arrest at RTS1 is entirely dependent on Rft1 binding (Eydmann et al., 2008) and, in the absence of Rtf1, replication of the RTS1 sequence is entirely normal. In contrast, region A is defined as being required for enhancing barrier activity. Loss of region A has been reported to reduce barrier activity by approximately three quarters and this function was described as being dependent on a second protein, Rtf2 (Codlin and Dalgaard, 2003).

Interestingly, while Rtf1 appears to be S. pombe specific and is not evolutionarily conserved beyond the Myb-like DNA binding domain (Eydmann et al., 2008), Rtf2 is conserved in many eukaryotes including humans (Codlin and Dalgaard, 2003). Rtf2 belongs to a family of proteins that are characterised by a C2HC2 Ring Finger motif predicted to fold up into a RING-finger structure with the ability to bind one Zn²⁺ ion (Inagawa et al., 2009) and is likely to mediate protein-protein interactions. Studies in human cells have shown that HsRTF2 acts to reduce the levels of replication fork restart and thus its removal from arrested replication forks via proteasomal shuttle proteins (DDI1 and DDI2) is important to allow replication fork restart to occur (Kottemann et al., 2018). Additionally, the nuclear receptor interacting protein 3 (NIRP3) has been shown to upregulate DDI1 and increase polyubiquitylation of HsRTF2, promoting HsRTF2 removal and replication fork restart upon replication stress (Suo et al., 2020). Within disease models, HsRTF2 has also been identified as a causal factor for Alzheimer’s Disease, although the exact mechanism remains unclear (Ou et al., 2021; Wingo et al., 2021). To further elucidate the conserved function of Rtf2 at stalled replication forks, we investigated this protein further using a previously described RTS1-RFB system in S. pombe (Naiman et al., 2021).

In agreement with previous studies on Rtf2 at the RTS1 RFB, we observe reduced barrier activity upon rtf2 deletion as assayed both by polymerase-usage sequencing (Pu-seq) and a replication slippage assay. However, in our system, the mechanism of action of Rtf2 at RTS1 does not occur via region A of RTS1, as had been previously reported. Using a proximity-based mass spectrometry method, we identified Rtf2 to be physically associated with mRNA processing and splicing factors. cDNA-Seq of mature polyA-mRNA revealed a modest global increase in the levels of intron retention in rtf2Δ cells. Intron retention was not uniform and specifically affected a subset of introns, with one of the most affected introns residing within the rtf1 transcript. Using an intronless rtf1 (rtf1Δint), that does not require splicing to encode a functional Rtf1 protein, we find that, in the absence of Rtf2, there was no reduction in RFB activity in comparison to when Rtf2 is present. These data demonstrate that the presence of Rtf2 is essential for the correct splicing of the rtf1 transcript in order to allow optimal barrier activity at RTS1.

The finding that the removal of human RTF2 from stalled replication forks is important to allow replication restart and maintain genome stability (Kottemann et al., 2018) led us to investigate the function of S. pombe Rtf2 in more detail. The Rtf2 protein was originally identified as having the role of enhancing the blocking capacity of the RTS1 RFB in S. pombe (Codlin and Dalgaard, 2003). 2D gel analysis of replicating plasmids containing RTS1 revealed a reduced pausing signal when cells were deleted for rtf2. A further study identified a visible increase in large Y-intermediates that were dependent on the Srs2 helicase (Inagawa et al., 2009). This was interpreted as Rtf2 acting downstream of Rtf1 to convert replication barrier activity at the RTS1 locus into a replication termination site.

Here, we confirm that the loss of Rtf2 indeed results in a decrease in replication fork arrest and restart by RDR at the RTS1 RFB. However, we clearly identify Rtf2 as interacting with splicing factors and demonstrate increased intron retention in a subset of transcripts when rtf2 is deleted. Importantly, we found that intron 2 of Rft1, a Myb-domain binding protein required for RTS1 barrier activity (Eydmann et al., 2008), is not spliced in rtf2Δ cells and this results in an Rtf1 protein that contains a 15 amino acid insertion between residues 202–203. Rtf1 contains 5 domains that resemble Myb/SANT domains. A DALI search of an AlphaFold2 model of Rtf1 indicates a high degree of similarity with the crystal structure of S. pombe Reb1, which promotes replication pausing within the rDNA spacer region (Figure 4—figure supplement 2). The 15 amino acid insertion is unlikely to disrupt DNA binding directly, but appears to extend a helix that, based on our observations, renders the resulting Rtf1 protein dysfunctional (Figure 5). Replacing the genomic rtf1 gene with an intronless copy fully rescued the rft2Δ-dependent defect in fork arrest at RTS1, clearly demonstrating that Rft2 acts upstream of Rtf1 to allow the correct splicing of the rft1 mRNA and thus the production of fully functional Rtf1 protein. We also confirmed that expression of an intron-less rtf1 gene that encodes the same additional 15 amino acids between residues 202–203 (Rtf1 intron2NE) does not provoke increased replication slippage downstream of RTS1 (Figure 4—figure supplement 3), indicating that this protein is indeed dysfunctional.

Figure 5

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Using 2D gel analysis of plasmids containing various deletion constructs of RTS1, previous work suggested Rtf2-dependent enhanced blocking capacity at RTS1 functions via an interaction with Region A of the RTS1 sequence (Codlin and Dalgaard, 2003). In our genomic RTS1 system, we showed that deletion of Region A (RTS1_AΔ), does not affect the levels of non-canonical (δ/δ) replication forks or the levels of replication fork slippage downstream of active RTS1 (Figure 2). Therefore, these results contrast with the previous finding and demonstrate that region A of RTS1 is dispensable for the efficiency of barrier activity and not to be the site of Rtf2 interaction.

RTF2 in human cells has been shown to be enriched at nascent chromatin (Dungrawala et al., 2015; Kottemann et al., 2018). However, we see no evidence to suggest that S. pombe Rtf2 is associated with the replication fork from our biotin proximity labelling experiments: Mass spectrometry analysis of proteins within close proximity to Rtf2 did not identify any replication associated factors (Figure 3—figure supplement 1). The studies in human cells specifically enriched for nascent chromatin and it is therefore possible that this allowed higher sensitivity for Rtf2 enrichment when compared to our TurboID experiments. Of note, a previous study Inagawa et al., 2009 found Rtf2 to co-precipitate with S. pombe Pcn1 (PCNA). We did not find any evidence that Pcn1 was enriched in our TurboID MS data. It is possible that a Pcn1:Rtf2 interaction does occur, but is very transient and only visualised in the previous study due to the use of overexpression plasmids.

Rtf2 is an abundant protein that is predicted to contain a RING motif similar to that found in the E3 SUMO ligases Pli1 and Nse2 in S. pombe (Watts et al., 2007) and potentially related to RING domains of ubiquitin E3 ligases. Furthermore, deletion of pmt3 (S. pombe SUMO) has been reported to result in a similar decrease in replication fork stalling at RTS1 as rtf2Δ (Inagawa et al., 2009), leading to speculation that Rtf2 acts at the RTS1 stall site to SUMO modify replication factors. However, it has also been shown that ubiquitylation and sumoylation of splicing factors are important to maintain efficient splicing within a cell (Pozzi et al., 2017) and therefore it is also conceivable that the efficient splicing of rtf1 intron 2, as well as other introns, is dependent on either sumoylation and/or ubiquitylation (Pozzi et al., 2018) of splicing factors by Rtf2. The balance of evidence suggests that Rtf2 has evolved a new function(s) in human cells to modulate replication fork restart: we are unable to identify any evidence that Rtf2 associates with replication proteins, rtf2 deletion does not directly affect RTS1-dependent fork arrest or subsequent restart by RDR; rtf2 deletion cells are not sensitive to the replication inhibitor HU. Furthermore, in human cells RTF2 is actively degraded via the DDI1/2 proteasomal shuttle system to regulate its activity at stalled forks. In S. pombe, we saw no evidence that the DDI1/2 homolog, Mud1, is influencing Rtf2 stability or the response to HU treatment.

Rtf2 has also been identified in Arabidopsis thaliana as an essential protein that contains an additional non-conserved N-terminal extension (Sasaki et al., 2015). Mass spectrometry to identify AtRtf2 interacting proteins revealed proteins involved in mRNA splicing, RNA binding and metabolism, as well as DNA binding and ribosomal proteins. Mass spectrometry for proteins interacting with AtRtf2 truncated for the non-conserved N-terminal domain identified many of the same proteins, indicating the interactions and their specificity to the conserved core of RTF2 are likely to be conserved between S. pombe and humans. Furthermore, the cDNA-Seq we conducted identified a subset of introns that were inefficiently spliced in rtf2Δ cells (Figure 3—figure supplement 2). This is consistent with the study in A. thaliana, which also detected intron retention defects when AtRTF2 was deleted (Sasaki et al., 2015). Interestingly, a genome wide screen in S. pombe for factors affecting mRNA splicing similarly identified deletion of rtf2 to result in intron retention defects (Larochelle et al., 2019), supporting our cDNA-Seq results (Figure 3). Whether Rtf2 acts directly as a splicing factor or indirectly effects spliceosome function remains to be elucidated.

Rtf2 is a conserved protein that affects splicing in fission yeast and plants and is likely to affect splicing in humans. In S. pombe, the major phenotype that has been observed relates to replication fork arrest at the RTS1 barrier, but other biological processes (for example a cellular response to MMS in fission yeast - Figure 2—figure supplement 1) are likely to be affected by inefficient spicing of other proteins. It is also likely that other pathways are directly influenced by Rft2 function, as appears to be the case in human cells (Kottemann et al., 2018; Suo et al., 2020). Recently, Rtf2 has been implicated in a number of additional physiological functions and pathologies including restriction of viral infection (Chia et al., 2020) and a possible causal factor for Alzheimer’s disease in humans (Wingo et al., 2021; Ou et al., 2021). Alzheimer’s has previously been associated with differential splicing patterns (Raj et al., 2018) raising the possibility that the mis-splicing observed in Alzheimer’s patients could be connected to changes in RTF2 protein function in these patients. This needs to be investigated further but could provide a better understanding of the disease’s progression.

In summary, we identify S. pombe Rtf2 as a key factor for the efficient splicing of a subset of introns and demonstrate that this explains the effects of rtf2Δ at the site-specific replication fork barrier RTS1 (Figure 5).

Sequence data is available under GEO dataset GSE192344.

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Author details

1. Alice M Budden

   Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

2. Murat Eravci

   Department of Biochemistry and Biomedicine, University of Sussex, Brighton, United Kingdom

   Present address

   Medicines and Healthcare Products Regulatory Agency (MHRA), Hertfordshire, United Kingdom

   Contribution

   Data curation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

3. Adam T Watson

   Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Eduard Campillo-Funollet

   Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom

   Present address

   Department of Mathematics and Statistics, Lancaster University, Lancaster, United Kingdom

   Contribution

   Data curation, Validation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

5. Antony W Oliver

   Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom

   Contribution

   Formal analysis, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2912-8273

6. Karel Naiman

   Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom

   Present address

   Cancer Research Center of Marseille (CRCM), INSERM U1068, CNRS, Aix Marseille Université, Institut Paoli-Calmettes, Marseille, France

   Contribution

   Conceptualization, Data curation, Supervision, Investigation, Writing – review and editing

   For correspondence

   karel.naiman@inserm.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3594-1516

7. Antony M Carr

   Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Project administration, Writing – review and editing

   For correspondence

   A.M.Carr@sussex.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2028-2389

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