The work in this thesis aimed at understanding for which function, distinct from mRNA export, Yra1 may be regulated by Slx5-Slx8. We showed that Yra1 regulation by Slx5-Slx8 does not lead to proteasomal degradation and that Yra1 is characterized by a long half-life. However, we cannot exclude that Yra1 undergoes proteasomal degradation in specific conditions (for example under irradiation or in Δdia2), although we could not strictly correlate Yra1 stability with Yra1 ubiquitination levels. For technical reasons we could not conclude whether the regulation by Slx5-Slx8 has a role in Yra1 splicing auto-regulation. Nevertheless, our data point to the importance of the highly conserved 16 amino acids Yra1 C-terminal box, which forms a alpha helix containing 4 lysines, in different processes.
The Yra1 C-terminal box contributes to splicing auto-inhibition.
The mechanism through which Yra1 mediates its splicing auto-inhibition is still not elucidated. A possibility is that the splicing auto-regulation defect observed in the yra1(1-210) mutant is due to a compromised interaction with the splicing factor Sub2 (Johnson et al., 2011a; Strasser and Hurt, 2001). To better investigate the interplay between Yra1 and Sub2 in YRA1 splicing auto-regulation, it would be interesting to define whether Sub2 mutants that increase Yra1 occupancy on genes (Johnson et al., 2009a) would rescue the YRA1 splicing auto-regulation defects observed in the yra1(1-210) mutant. This will underline the importance of Yra1 recruitment at the YRA1 intron for its splicing auto-regulation. It will be interesting to identify the key interactors of the C-box region that are involved in the YRA1 splicing auto-regulation mechanism.
The Yra1 C-box mediates the detrimental effects of Yra1 overexpression in promoting the DDR.
The negative effect of the Yra1 C-terminal box is suggested by the fact that the overexpression of the yra1Δintron, which contains the C-box, has a stronger phenotype compared to the yra1(1-210) mutant, which lacks these terminal 16 amino acids, although this truncated protein is expressed to the same level than the yra1Δintron. The detrimental effect of the C-box was observed in different contexts.
First, Rad52 foci, which visualize the centers of homologous recombination, accumulate in the yra1Δintron but are almost abolished in the yra1(1-210) mutant.
This observation suggests that overexpression of the C-box region may increase DNA lesions or impair the DNA repair by homologous recombination. In this regard, a recent study has proposed that Yra1 overexpression induces DNA damage by increasing the levels of RNA::DNA hybrids, resulting in hyper-recombination (Gavalda et al., 2016). However the region of Yra1 responsible for this phenotype has not been defined. Our observations strongly support that the Yra1 C-terminal box could be mediating these effects. In agreement with this possibility, we performed transcription-dependent hyper-recombination assays and confirmed that Yra1 overexpression induces transcription-dependent hyper-recombination, as shown by the Aguilera laboratory (Gavalda et al., 2016). Interestingly, our experiments also show that this phenotype is abolished in the yra1(1-210) mutant (Supplementary Figure 4), suggesting that the C-box domain, when overexpressed, may favor RNA::DNA hybrids formation.
This view is potentially consistent with another recent study indicating that the C-terminal domain of Yra1 inhibits the Dbp2-dependent duplex unwinding activity and prevents aberrant Dbp2 accumulation on mRNPs (Ma et al., 2013; Ma et al., 2016).
Besides Rad52 foci accumulation and hyper-recombination phenotypes, the sensitivity of yra1 OE mutants to HU and Zeocin, the synthetic sickness with Δrad52 as well as the additive effect of Yra1 overexpression on the sensitivity of DDR mutants to DNA damage agents may suggest an involvement of Yra1 in the response to DSB lesions. In this context, however, it is less clear to what extent overexpression of the Yra1 C-box contributes to these phenotypes.
We cannot exclude that the observed phenotypes linked to the DNA damage response pathway are partially due to mRNA export defects of specific transcripts important for the DDR. In this regard, we observed partial rescue of the DNA damage sensitivity of yra1 OE mutants when Nab2 or Mex67, two factors essential for mRNA export, were overexpressed (Figure 3.9 and Table 3). It would be interesting to test by FISH whether the export of specific mRNAs involved in DNA repair by HR is affected in the yra1 OE mutants.
Importance of the Yra1 C-box in DNA locus relocation to the nuclear periphery in G1 cell cycle phase (Figure 4.1).
Our artificial tethering experiments show that Yra1 is able to move a DNA locus to the nuclear periphery and this relocalization depends on the Yra1 C-terminal box specifically in G1/S phase. Yra1 was shown to interact with the NPC-associated Mlp2 protein but the interaction domains are unknown (Vinciguerra et al., 2005). It will be interesting to define whether the relocalization of the DNA locus depends on the interaction of the Yra1 C-terminal box with Mlp2.
Interestingly, irreparable DSBs relocate to the nuclear pore in G1/S (Horigome et al., 2016). We showed that wild-type Yra1 is recruited to DSBs and that the recruitment of the yra1(1-210) mutant is reduced, which could potentially be due to a compromised interaction between yra1(1-210) and Mlp2.
Unfortunately we have not been able to test whether Yra1 is required for the relocation of an irreparable DSB to the pores because we were unable to construct the Yra1 wild-‐type and mutant strains required for these microscopy localization experiments. Another possibility is that Yra1 recruitment to irreparable double strand breaks is the consequence of HO cut relocalization to the Nuclear Pore that occurs within 2h after cut induction (Nagai et al., 2008). Yra1 is mainly linked to transcription and mRNA export; however we showed that Yra1 recruitment to irreparable DSB is independent of transcription, in agreement with a study showing that the resection event inhibits transcription at loci surrounding an HO cut (Manfrini et al., 2015).
In light of these considerations, it will be interesting to define whether Yra1 is recruited not only to irreparable DSBs but also to reparable DSBs located within the
nucleus (Dion et al., 2013) and not at the Nuclear Pore. These experiments should also allow defining whether the yra1 OE mutants show different repair efficiency and whether the differences may implicate the Yra1 C-terminal box. Such experiments may explain the phenotypes that we observed in the DDR pathway linked to HR. In particular: the DNA damage sensitivity to DSBs agents of the yra1 OE mutants, their synthetic sickness with Δrad52, and the additive effect of sensitivity to DNA damage agents of the yra1 OE mutants with the DDR components such as Δrad52, Δexo1, Δsae2 and Δmre11.
Overexpression of the Yra1 C-box promotes chromosome segregation defects.
The chromosome segregation defects observed in the yra1Δintron were less systematically present in the yra1(1-210) mutant (Figure 5.1). This leads for the first time to describe YRA1 as a Chromosome instability gene (referred as CIN) (Spencer et al., 1990; Stirling et al., 2012; Yuen et al., 2007). Interestingly, it is possible that the Chromosome segregation phenotype that we observe may depend on the interaction of Yra1 with Mlp2. Indeed, Mpl2 co-purifies with the SPB component Spc42 and contributes to SPB assembly (Niepel et al., 2005; Vinciguerra et al., 2005). In the yra1Δintron mutant, Yra1 overexpression may titrate Mlp2 affecting the interaction of Mlp2 with Spc42 and leading to defects in SPB assembly. In the yra1(1-210) mutant, although the truncated protein is overexpressed, , the chromosome segregation defect is reduced since the yra1(1-210) may not interact with Mlp2, as previously suggested.
The Yra1 C-box may participate in the Spindle Positioning pathway through functional interaction with Kar9.
Among the yra1 OE mutants tested, only the yra1(1-210) showed sensitivity to benomyl suggesting that the C-box region may be important to prevent microtubule destabilization. Based on genetic interactions, we found that Yra1 overexpression specifically perturbs the Kar9 pathway that is important for spindle positioning.
Consistently, we observed that the yra1 OE mutants show defects in spindle positioning and alignment. Interestingly, Kar9 is targeted by Slx5-Slx8 for proteasomal degradation (Schweiggert et al., 2016). Kar9 has also been shown to interact with Yhr127W, a protein of unknown function that interacts with Yra1 and which loss causes chromosome segregation defects (Oeffinger et al., 2007; Schoner et al., 2008).
The interplay between Yra1, Slx5-Slx8, Kar9 and Yhr127W may explain why we observed chromosome segregation defects in yra1 OE mutants.
We showed that the Yra1 C-terminal box plays a crucial role in different aspects of Yra1 function. These may be depend on the interaction of Yra1 C-box region with a variety of factors that we were unfortunately unable to identify in the context of our Mass Spec analyses. For this reason, it still remains crucial to perform proteomics experiments comparing proteins co-purifying with the wild-type Yra1 vs the yra1(1-210) protein. This may help elucidate the molecular mechanisms through which Yra1 may function in the genome stability pathways analyzed.
In summary, thanks to the analysis of the effects of wild-type or mutant Yra1 overexpression, we identify YRA1 as a new CIN gene. The molecular mechanism of CIN related to the DDR may involve Yra1 recruitment to DSBs, an event potentially important for repair mechanisms by HR. On the other hand, we cannot exclude that the yra1 OE phenotype related to DDR reflects increased DNA damage due to accumulation of RNA::DNA hybrids in the yra1 OE mutants, as suggested by a recent study from the Aguilera lab (Gavalda et al., 2016). Another possibility to consider is that yra1 OE mutants have altered gene expression patterns under DNA damage condition, indirectly affecting the DDR.
The molecular mechanism of CIN related to the Spindle Positioning pathway may involve interplay of Yra1 with Kar9, a factor important for Spindle position. A possible mechanism could be that Yra1 influences SPB position at the nuclear envelope. Consistently, my results indicate that Yra1 overexpression perturbs Spindle Position leading to Chromosome segregation defects. Alternatively, the Spindle Positioning and Chromosome segregation defects observed in the yra1 OE mutants may be due to mis-regulated export of specific transcripts.
The importance of Yra1 in these two pivotal pathways may explain why the YRA1 gene is essential in yeast and has to be tightly regulated.