Bypass forzado: celdas sin fallo

RNase H enzymes can catalyze the nucleolytic degradation of RNA molecules that are hybridized to DNA (RNA-DNA hybrids or R-loops; reviewed in (Cerritelli and Crouch, 2009)). Several studies in recent years have identified the presence of R-loops at telomeres in diverse organisms, including budding yeast (Arora et al., 2014; Balk et al., 2013; Pfeiffer et al., 2013; Wahba et al., 2016; Yu et al., 2014). Given the interaction between Rif2 and RNase H2, as well as the Rif2-mediated association of RNase H2 to telomeres, we speculated that the Rif2-RNase H2 axis might impact R-loop levels at telomeres.

To assess this, we utilized the S9.6 antibody, which selectively binds RNA-DNA hybrids of at least 6-8 bp (Phillips et al., 2013), to perform a DNA-RNA Immunoprecipitation (DRIP) experiment followed by qPCR. When we performed the DRIP in wild type and Rif2 depleted cells, we could observe that rif2 mutants displayed an approximate 1.5-2 fold increase of R-loops at telomeres compared to wild type cells (Figure 9A), consistent with the loss of Rnh201 from telomeres in this mutant (Figure 5B). Furthermore, constitutive in vivo overexpression of RNH1 from a plasmid reduced the telomeric R-loop signal in rif2 mutants to wild type levels, while not significantly affecting the wild type telomeric R-loop levels. As a positive control, we measured R-loops at the 18S rDNA locus, which is highly prone to forming R-loops (El Hage et al., 2010). RIF2 deletion had no effect on R-loops at rDNA, suggesting that Rif2 regulates RNA-DNA hybrids specifically at telomeres. Altogether, these results suggest that the Rif2-mediated Rnh201 localization to telomeres is functional in restricting R-loops, and that the R-loops that accumulate at telomeres in rif2 mutants are sensitive to RNH1 overexpression. The latter result also further confirms that the telomeric DRIP signal that accumulates in rif2 mutants stems from RNA-DNA hybrids, and furthermore indicates that Rnh1 can act at telomeres even though we could not detect it by ChIP (Figure 5A).

rif2 mutants, as well as rif1 mutants, display elongated telomeres (Wotton and Shore, 1997). To control for possible effects of telomere length on the DRIP results, we made use of auxin-inducible degron (AID) alleles of these proteins. This system allows rapid degradation of the targeted proteins upon addition of auxin (indole-3-acetic acid, IAA) to

the growth media (Morawska and Ulrich, 2013)(Figure 9B). We performed DRIP experiments in RIF1AID_{, RIF2}AID _{and RIF2}AID _RIF1AID _{in presence or absence of IAA}

treatment, in order to analyze the effect of the depletion of these proteins on R-loops without affecting telomere length. We could confirm that, also in these conditions, depletion of Rif2 leads to the accumulation of R-loops at telomeres, but not at the rDNA locus (Figure 9C); furthermore, depletion of Rif1 had no effect on telomeric R-loop levels and did not contribute to the R-loop accumulation in RIF2AID _RIF1AID _{cells. This result}

confirms previous findings and suggests that Rif1 is not a regulator of R-loops at wild type telomeres.

RNase H2 has a genome-wide role in promoting genome stability (Lazzaro et al., 2012; O'Connell et al., 2015), so we decided to investigate whether the Rif2 interaction with Rnh201 has implications on the genome-wide role of RNase H2. To test this, we again took advantage of the high sensitivity of the rnh1 rnh201 mutant to MMS, as a readout. We performed a spotting assay to test sensitivity to MMS of several mutants. As seen previously (Figure 5D), rnh1 rnh201 mutants are highly sensitive to MMS, while the double mutant rif2 rnh1 grew at wild type levels (Figure 9D), indicating that Rif2 is not required for RNase H2 function in response to genome-wide MMS-induced genotoxic stress.

Figure 9. Rif2 restricts telomeric R-loops.

(A) rif2 mutants accumulate telomeric R-loops. Exponentially growing cultures of the indicated genotypes containing either an empty vector (-) or overexpressed RNH1 (+) were crosslinked and subjected to DRIP using the S9.6 antibody. R-loop associated chromatin was further analyzed by qRT-PCR. Values are presented as % input of DNA recovered relative to wild type, which is set to 1 for each primer set. Data are shown as mean + SEM; n=3. *: p<0.05; **: p<0.01 (Student’s t-test). (B) IAA-inducible degradation of Rif1 and Rif2. Cells of the indicated genotypes were collected before and after 1 h treatment with 500 µM IAA. Protein extracts were analyzed by western blot. Rif1 and Rif2 were detected with anti-myc antibodies, and Pgk1 serves as a loading control. (C) RIF2AID _{mutants accumulate R-loops in the presence}

of IAA. Cultures of the indicated genotypes were grown to exponential phase and half of each culture was treated with 500 µM IAA for 3 h, after which cultures were crosslinked and subjected to DRIP using the S9.6 antibody. R-loop associated chromatin was analyzed by qRT-PCR. Values are plotted as % input of DNA recovered relative to the correspondent uninduced control, which is set to 1 for each primer set. Data are shown as mean + SEM; n=4. *: p<0.05; **: p<0.01 (Student’s t-test). (D) Rif2 does not contribute to Rnh201 genome-wide function. Cells of the indicated genotypes were spotted in serial dilutions onto YPD and MMS-containing YPD plates. The YPD plate was imaged after 48 h of incubation at 30˚C and the MMS plate was further incubated 48 h at room temperature before being imaged. Experiments presented in (B-C) were performed by Diego Bonetti.