We then set out to confirm in a more direct and targeted way the physical interaction between Rnh201 and Rif2. We performed a Co-Immunoprecipitation (Co-IP) assay with endogenously expressed 9myc-tagged Rif2 and HA-tagged Rnh201, which was transiently overexpressed from a plasmid under the control of a galactose-inducible promoter. When HA-Rnh201 was immunoprecipitated with anti-HA antibodies, we were
able to recover Rif2-9myc in the eluate as seen by western blot (Figure 6A). When the HA-immunoprecipitation was performed in cells containing RIF2-9MYC and an empty vector (EV), or when cells expressed HA-Rnh201 but untagged Rif2, we could not detect Rif2-9myc in the eluate (Figure 6A). Thus, we could confirm that Rif2 and Rnh201 can physically interact with each other in the conditions described.
Rnh201 is the catalytic component of the RNase H2 trimeric complex, which additionally contains the proteins Rnh202 and Rnh203. We therefore asked whether the interaction between Rif2 and Rnh201 is mediated by the other components of the RNase H2 complex. To test this, we performed an immunoprecipitation against transiently overexpressed HA-tagged Rnh201 in cells lacking either RNH202, RNH203 (Figure 6B) or both (Figure 6C) and bearing the RIF2-9MYC allele. In the tested settings we were always able to recover Rif2-9myc in the eluate, suggesting that the interaction between Rif2 and Rnh201 is independent of the presence of the other two components of the RNase H2 complex. As a control, HA-immunoprecipitations performed in cells containing an empty vector (EV) did not lead to Rif2-9myc recovery in the eluate (Figures 6B and C). Moreover, each immunoprecipitation reaction presented in this work (Figures 6, 7 and 8) was treated with DNase I to exclude DNA-mediated interactions, and the Co-IP of Rif2- 9myc and Rnh201-HA in Figure 6B was additionally treated with RNase A to exclude RNA- mediated interactions.
Figure 6. Rif2 interacts with RNase H2.
(A-C) Rif2-9myc co-immunoprecipitates with RNase H2. Cell extracts derived from exponentially growing cells of the indicated genotypes containing either an empty vector (EV) or transiently overexpressed HA-Rnh201 (A) or Rnh201- HA (B-C) were immunoprecipitated with anti-HA antibody coupled magnetic beads. Samples were then analyzed by western blot and probed with anti-HA and anti-myc antibodies. 2.5% of input protein was loaded as control. (A-C) All samples were treated with DNase I during immunoprecipitation and sample Rif2-9myc Rnh201-HA (B) was treated additionally with RNase A.
We were also able to co-immunoprecipitate Rif2-9myc with overexpressed HA-tagged Rnh202 and Rnh203 (data not shown), indicating that the entire RNase H2 complex is able to interact with Rif2.
We next wondered whether the interaction between Rif2 and Rnh201 is regulated throughout the cell cycle. To assess this, we arrested Rif2-9myc tagged cells overexpressing HA-RNH201 in G1 with α-factor, and subsequently released them into media containing either 250 mM or 75 mM hydroxyurea (HU), thereby halting the cultures in early and late S phase, respectively (Figure 7A)(Graf et al., 2017). Interestingly, even though HA-Rnh201 was expressed from a GAL-inducible promoter, we observed a drop of its levels in G1 and an increase towards the end of S phase (Figure 7B); nonetheless, we were able to immunoprecipitate similar amounts of the protein in all cell cycle phases using antibodies against HA. We observed interaction between Rif2 and Rnh201 through all the tested phases, but while interaction was clearly lowest in G1 and early S, the highest amount of Rif2-9myc was recovered when the Co-IP was performed in late S phase-arrested cells (Figure 7B).
Figure 7. Rif2-Rnh201 interaction is strongest in late S phase.
(A) Efficient synchronization of cultures in G1 and early/late S phase. Exponentially growing cultures of the indicated strains were arrested in G1 with α-factor and subsequently released at 30˚C in media containing either 250 mM or 75 mM HU for 2 h 30 min, when samples for FACS and Co-IP were collected. (B) Rif2-Rnh201 interaction varies in the cell cycle. Cell extracts deriving from cells of the indicated genotypes in different cell cycle stages (A) and containing either an empty vector (EV) or transiently overexpressing HA-Rnh201 were immunoprecipitated with anti-HA antibody coupled magnetic beads. Samples were then analyzed by western blot and probed with anti-HA and anti-myc antibodies. 2.5% of input protein was loaded as control. (A-B) All samples were treated with DNase I during immunoprecipitation.
This result is consistent with the finding that Rnh201 binding to telomeres is highest in late S phase (Graf et al., 2017), and suggests that the increased interaction between Rif2 and Rnh201 in late S phase is reflected in increased recruitment of RNase H2 to telomeres. We then decided to test whether Rif2 also interacts with RNase H1, and furthermore if Rif1 is capable of interacting with RNase H enzymes as well. To do this, we transiently overexpressed either RNH1-HA or HA-RNH201 from plasmids in cells in which Rif1 or Rif2 were 9myc-tagged (Figure 8A). We observed that in all tested conditions, HA- immunoprecipitation led to recovery of Rif1 and Rif2 in the eluate, indicating that both Rif2 and Rif1 are able to interact with RNase H1 and RNase H2. As a control, we tested for presence of Rap1, an interactor of both Rif1 and Rif2 in the immunoprecipitate. We were not able to detect Rap1 in the eluate following Rnh1-HA and HA-Rnh201 immunoprecipitations (Figure 8B), indicating specificity of the interactions of RNases H with Rif1 and Rif2. Although both RNase H enzymes can interact physically with both Rif1 and Rif2 (Figure 8A), it seems that only RNase H2 can be found at telomeres by ChIP (Figures 5A and B). Perhaps there is a competition between the two enzymes that we were unable to detect in these conditions (see below for more on this).
Figure 8. Rif1 and Rif2 interact with RNase H1 and H2.
(A-B) Rif1 and Rif2 co-immunoprecipitate with Rnh1 and Rnh201. Cell extracts deriving from exponentially growing cells of the indicated genotypes containing either an empty vector (EV) or transiently overexpressing HA-RNH201 or
RNH1-HA were immunoprecipitated with anti-HA antibody coupled magnetic beads. Samples were then analyzed by
western blot and probed with anti-HA and anti-myc antibodies. In addition, samples in (B) were also probed with anti- Rap1 antibodies. 2.5% of input protein was loaded as control. (A-B) All samples were treated with DNase I during immunoprecipitation.