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. 2019 Apr 5;14(4):e0206336.
doi: 10.1371/journal.pone.0206336. eCollection 2019.

The mRNA export adaptor Yra1 contributes to DNA double-strand break repair through its C-box domain

Valentina Infantino  1 , Evelina Tutucci  2 , Noël Yeh Martin  3 , Audrey Zihlmann  1 , Varinia Garcia-Molinero  1 , Géraldine Silvano  1 , Benoit Palancade  4 , Françoise Stutz  1
Affiliations

The mRNA export adaptor Yra1 contributes to DNA double-strand break repair through its C-box domain

Valentina Infantino et al. PLoS One. .

Abstract

Yra1 is an mRNA export adaptor involved in mRNA biogenesis and export in S. cerevisiae. Yra1 overexpression was recently shown to promote accumulation of DNA:RNA hybrids favoring DNA double strand breaks (DSB), cell senescence and telomere shortening, via an unknown mechanism. Yra1 was also identified at an HO-induced DSB and Yra1 depletion causes defects in DSB repair. Previous work from our laboratory showed that Yra1 ubiquitination by Tom1 is important for mRNA export. Here, we found that Yra1 is also ubiquitinated by the SUMO-targeted ubiquitin ligases Slx5-Slx8 implicated in the interaction of irreparable DSB with nuclear pores. We further show that Yra1 binds an HO-induced irreparable DSB in a process dependent on resection. Importantly, a Yra1 mutant lacking the evolutionarily conserved C-box is not recruited to an HO-induced irreparable DSB and becomes lethal under DSB induction in a HO-cut reparable system. Together, the data provide evidence that Yra1 plays a crucial role in DSB repair via homologous recombination. While Yra1 sumoylation and/or ubiquitination are dispensable, the Yra1 C-box region is essential in this process.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Yra1 is a sumoylated protein targeted for ubiquitination by the SUMO-dependent ubiquitin ligase Slx5-8.
(A) Yra1 ubiquitination depends on the STUbL Slx5-Slx8 and Tom1. Ubiquitination assay of shuffled HA-YRA1 in wild-type and in Δtom1, Δslx8, Δslx5, Δslx8Δtom1, Δslx5Δtom1 and Δslx8Δslx5 mutant backgrounds. His-Ubiquitinated proteins were affinity-purified and the Ubiquitinated forms of Yra1 detected by Western Blot with αHA antibodies. One representative experiment of 3 is shown. (B) Yra1 is sumoylated. Sumoylation assay in wild-type and HA-YRA1 backgrounds. His-sumoylated proteins were affinity-purified and the sumoylated forms of Yra1 detected by Western Blot with αHA antibodies. One representative experiment of 3 is shown. (C) Yra1 is sumoylated by Siz1/Siz2. Yra1 sumoylation assay in wild-type as well as Δsiz1, Δsiz1/siz2, Δsiz2 and mms21-11 mutant backgrounds was performed as described above. His-sumoylated proteins were affinity-purified and the sumoylated forms of Yra1 detected by Western Blot with αYra1 antibodies. One representative experiment of 2 is shown.
Fig 2
Fig 2. The Yra1 C-box, but not its ubiquitination and sumoylation, is important for genome stability.
(A) Scheme of Yra1 mutants used in this study with corresponding ubiquitination and sumoylation assays. Left: Ubiquitination assay of shuffled HA-YRA1 WT, HA-yra1(1–210) and HA-yra1allKR mutants performed as described in Materials and Methods. Right: Sumoylation assay of shuffled HA-YRA1 WT, HA-yra1allKR and HA-yra1(1–210) mutants performed as described in Materials and Methods. One representative experiment of at least three is shown. Note that Yra1 is a very basic and charged protein and its non-modified forms tend to be retained on the Ni-NTA agarose beads. This is particularly striking with the HA-yra1allKR protein whose expression is increased in these strains. (B) Western Blot analysis of HA-Yra1 levels in integrated HA-YRA1 WT, HA-yra1(1–210), HA-yra1allKR, was performed using an αHA antibody; an αPgk1 antibody was used as loading control. One representative Western blot is shown. Below: Western blot quantification showing the mean of the HA-Yra1/Pgk1 ratio of three experiments. (C) Spot test analysis of confluent cells at 25°C, 30°C, 34°C, 37°C of YRA1 WT (No Tag), integrated HA-YRA1 WT, HA-yra1(1–210), and HA-yra1allKR strains on YEPD 2% Glucose. (D). Spot test analysis on YEPD 2% Glu, Zeocin 25 μg/ml, Zeocin 50 μg/ml, Zeocin 100 μg/ml at 25°C of confluent cells of integrated HA-YRA1 WT and HA-yra1 mutants as well as Δrad52 strains.
Fig 3
Fig 3. Yra1 is recruited to an irreparable DSB HO cut site.
(A) Yra1 recruitment at the HO cut site was defined by ChIP with an αYra1 antibody after 0.5h, 1h, 2h and 4h of HO endonuclease induction with galactose using the GA6844 strain described in [22]. The 2h Glucose time point was used as no cut control. ChIP values are shown as percentage of input at 0.6Kb, 1.6Kb, 4.5Kb, 9.6Kb and 23Kb from the HO cut. The average of 6 experiments is shown with corresponding standard error of the mean. Two way ANOVA test was performed with multiple comparisons; P values < 0.05 (*), < 0.01 (**), < 0.001 (***) that refer to Glu 2h (no cut) are shown. (B) Yra1 mutants are differentially recruited to the irreparable HO cut site. ChIP using αHA antibody of HA-Yra1 WT, HA-yra1(1–210), HA-yra1allKR, Yra1 WT (no tag) at 0.6 Kb from the HO cut site after 2h of HO induction with Galactose using the strains with HA-YRA1 WT or mutants integrated in strain GA6844 described in [22]. The 2h Glucose time point was taken as no cut control. ChIP values are shown as percentage of input. The average of 3 independent experiments is shown with corresponding standard error of the mean. (C) Yra1 recruitment is dependent on extensive resection. HA-Yra1 WT recruitment at the HO cut site was defined by ChIP with an αHA antibody after 2h of HO endonuclease induction with galactose in HA-YRA1 WT combined with Δsae2Δexo1 or Δexo1Δsgs1. The 2h Glucose time point was used as no cut control. ChIP values are shown as percentage of input at 0.6Kb from the HO cut. The average of 4 experiments is shown with corresponding standard error of the mean.
Fig 4
Fig 4. Survival under persistant induction of a reparable HO cut.
(A) The Yra1 C-box is important for DSB repair. NA17 strains [25] containing integrated HA-YRA1 WT (WT), HA-yra1(1–210), HA-yra1allKR were transformed with pGAL-HO endonuclease containing plasmid or Empty Vector, as well as No-Tag (NA17) and Δrad52 strains containing endogenous pGAL-HO endonuclease were transformed with an Empty Vector. Diluted cells were plated on SCLGg Gal 2% or Gal 3%-Raf1% to constantly induce HO cut, and on SCLGg Glu 2% to repress HO endonuclease expression. The percentage colony forming units (CFUs) was determined as described in Materials and Methods. The average of 3 independent experiments for each condition SCLGg Gal 2%/ Glu 2% and SCLGg Gal 3%-Raf 1%/ Glu 2% is shown with corresponding standard error of the mean. One way ANOVA test was performed with multiple comparisons and P value < 0.001 (**) is shown on the graph referring to HA-YRA1 WT. (B) Yra1 C-box is important for DSB repair. Spot test analysis on Leu- SCLGg Glu 2%, SCLGg Gal 2%, SCLGg Gal 3%-Raf 1%, at 25°C of exponentially growing HA-YRA1 WT (WT), HA-yra1(1–210), HA-yra1allKR (transformed with pGAL-HO endonuclease containing plasmid or Empty Vector); No-Tag and Δrad52 strains (containing endogenous pGAL-HO endonuclease and transformed with an empty vector) served as controls. One representative experiment out of 3 is shown. (C) yra1 mutant survival in the Δsae2Δexo1 background under persistant induction of an irreparable HO cut. Dilutions of exponentially growing cells of GA6844 strain [22] containing integrated HA-YRA1 WT (WT), HA-yra1(1–210), HA-yra1allKR combined or not with Δsae2Δexo1 were plated on SCLGg Gal 2%-Raf2% to constantly induce the HO cut. Corresponding dilutions of cells were plated on SCLGg Raf2%. The percentage of Colony Forming Units (CFUs) was determined as described in Materials and Methods. The average of 2 independent experiments is shown with corresponding standard error of the mean. One way ANOVA test was performed with multiple comparisons and P value < 0.05 (*) is shown on the graph.
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This work was funded by NCCR ‘Frontiers in Genetics’ (fellowship to VI), the Swiss National Science Foundation (grant no 31003A 153331 to FS), Polish Swiss Research Programme PSRP NoCore (183/2010 to FS), iGE3 and the Canton of Geneva. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.