Investigating the role of RNase H enzymes in the regulation of telomeric R-loops during replicative senescence

Telomeres are nucleoprotein structures that protect and maintain the ends of eukaryotic linear chromosomes. Telomeres shorten at each round of DNA replication due to the end replication problem. The enzyme telomerase, by adding telomeric repeats to chromosome ends, can counteract this process. In the absence of telomerase, telomeres progressively shorten until they reach a critical length that activates the DNA damage response, thereby halting the cell cycle in a condition referred to as replicative senescence. Telomeres are transcribed into a long, non-coding RNA dubbed TERRA, which can hybridize with its template strand, thereby forming R-loops at S. cerevisiae and human telomeres. Recent data implicate telomeric R-loops in the promotion of homologous recombination at telomeres, leading to telomere lengthening events which can partially compensate for telomere shortening in the absence of telomerase. Telomeric R-loops are regulated by RNase H1 and H2 enzymes, which can degrade the RNA moiety of RNA-DNA hybrids. While the accumulation of telomeric R-loops in cells lacking both enzymes delays senescence onset by promoting homologous recombination at telomeres, the depletion of telomeric R-loops by overexpressing RNase H1 leads to premature senescence onset. This PhD thesis aims to better understand how telomeric R-loops are regulated especially during replicative senescence in S. cerevisiae. We found that RNase H2 localizes to long telomeres and physically interacts with the telomere-associated protein Rif2, which is required for RNase H2 recruitment to telomeres. Accordingly, in the absence of Rif2 telomeric R-loops accumulate, indicating that Rif2 and RNase H2 play a pivotal role in restricting R-loops at long telomeres. Importantly, the interaction between RNase H2 and Rif2 is strongest in late S phase, which is reflected in the degradation of telomeric R-loops in this time frame. We propose that this cell cycle regulated telomeric R-loop degradation is required to avoid collisions of the replication machinery, which replicates long telomeres in late S phase, with R-loops, an event that could have detrimental effects on telomere stability. It was previously shown that, as telomeres shorten, Rif2 localization to telomeres is diminished. We show that decreased Rif2 association to short telomeres leads to impaired recruitment of RNase H2, which is functionally reflected in the accumulation of R-loops at short telomeres. Moreover, while RNase H1 could not be detected at long telomeres, we observed its localization to short telomeres, thereby indicating a distinct requirement for the RNase H enzymes. By analyzing the effect of single RNase H enzymes deletion on the kinetics of senescence onset in telomerase negative cells, we revealed an opposing effect of the two enzymes, suggesting that, differently from what was proposed, RNase H enzymes do not have redundant functions at telomeres. In conclusion, we propose that, while at long telomeres R-loops are timely regulated by Rif2-RNase H2 to avoid collisions with the replication machinery, at short telomeres R-loops are allowed to accumulate, thereby promoting homologous recombination-mediated telomere extension

The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage

<div><p>RNA-DNA hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1’s helicase activity plays a crucial role in responding to persistent RNA-DNA hybrids.</p></div

Mph1 forms foci at telomeres during senescence, when RNA-DNA hybrids accumulate.

<p><b>A.</b> Both wild type and <i>tlc1</i> cells were derived from the <i>TLC1/tlc1</i> heterozygous diploid yAL95 and grown for approximately 60 population doublings (PD) before RNA was extracted from exponentially growing cells. Following reverse transcription with a telomeric sequence, TERRA levels were analysed at the indicated telomeres via qPCR with subtelomeric specific primer pairs (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007136#sec009" target="_blank">methods</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007136#pgen.1007136.s006" target="_blank">S1 Table</a> for details). Three biological replicates were used for each genotype. Error bars indicate 95% confidence intervals. * represents significance relative to wild type determined by Student’s t-test (P<0.05). <b>B.</b> Both wild type and <i>tlc1</i> cells were grown for approximately 60 population doublings. ChIP with the S9.6 antibody that specifically recognizes RNA-DNA hybrids followed by qPCR. Error bars represent 95% confidence intervals, *, significance relative to wild type as determined by a Student’s t-test (P<0.05). <b>C.</b> Representative images for senescing cells showing co-localization between Mph1-YFP, Rad52-yEmRFP and Cdc13-CFP. Scale bar, 3 μm. Quantification of Mph1 (<b>D</b>.) and Cdc13-foci (<b>E</b>.). RNase H1 was expressed from a plasmid (pBB39).</p

The SMC5/6 complex regulates Mph1 at RNA-DNA hybrids.

<p><b>A.</b> Yeast haploid cells with the indicated genotypes were generated following tetrad dissection of ySLG419. Cells were grown overnight in liquid YPD at 23°C and spotted in 10-fold serial dilutions at the indicated temperatures on YPD agar. Digital images were acquired following 2 days of incubation. <b>B.</b> Haploids were derived from dissection of ySLG419 and ten-fold serial dilutions were spotted. Images were taken after 2 days. <b>C.</b> <i>MPH1</i>-dependent accumulation of Rad52 foci in <i>rnh201 smc6-9</i> mutants. Spontaneous Rad52-mCherry foci were quantified in wild type (yBL1052), <i>mph1</i> (YBL1051), <i>smc6-9</i> (yBL1047), <i>rnh201</i> (yBL1053), <i>mph1 smc6-9</i> (yBL1050), <i>rnh201 smc6-9</i> (yBL1048), and <i>mph1 smc6-9 rnh201</i> (yBL1049) after shifting an exponentially growing culture (OD₆₀₀ = 0.3) from 25°C to 30°C for 3 hours in SC medium supplemented with 100 μg/ml adenine. Upper panel: Representative images of Rad52 foci. Arrowheads indicate foci. Scale bar, 3 μm. Lower panel: Quantification of Rad52 foci. Two replicates of 200–600 cells were examined. Error bars indicate 95% confidence intervals. *, significance relative to the wild type determined by Fisher’s exact test (P < 0.05). <b>D.</b> The heterozygous diploid strain yBL1022 was micro-dissected, and the haploid offsprings with the indicated genotype were spotted in 10-fold serial dilutions onto YPD-agar and incubated for 2 days at 30°C. <b>E.</b> <i>smc6-9 rnh201 mph1</i> was transformed with either empty vector or <i>MPH1</i> expression plasmids (<i>WT</i> allele or helicase-dead mutants). The transformants were spotted on selective media and grown for three days at 25°C or for two days at 30°C. <b>F.</b> The indicated genotypes were derived via tetrad dissection of ySLG115. Cells were diluted daily (every 24 hours) to OD₆₀₀ 0.01 before the density was re-determined and cells were re-diluted. 6 biological replicates were used for every genotype indicated. Error bars represent SEM.</p

Mph1’s fork reversal activity might allow dissolution of RNA-DNA hybrids.

<p>When replication forks are paused at an RNA-DNA hybrid (either an R-loop as depicted, or consecutively incorporated ribonucleotides), Mph1 gets recruited to the stalled fork, potentially through its C-terminal interaction domain with RPA. Mph1 may directly remove the RNA-DNA hybrid (in the case of an R-loop) through its helicase activity, or promote the resolution by other factors (e.g. the RNase H enzymes as depicted). The Smc5/6 complex negatively regulates Mph1’s pro-recombinogenic activity at RNA-DNA hybrids to prevent toxic recombination intermediates from accumulating (see text for detailed explanation). Persistent R-loops at shortened telomeres may represent a natural scenario where such a regulation occurs.</p