Project 4. Understanding RNase H1 regulation at telomeres and beyond.

Telomere repeat-containing RNA (TERRA) R-loops (telR-loops) are formed at telomeres once per cell cycle and are removed prior to telomere replication. telR-loops likely have the function of preventing excessive DNA resection, which slows the rate of telomere shortening and prevents the early onset of replicative senescence in the absence of telomerase. Importantly, when telomeres become critically short, they get extended through homology-directed repair (HDR) and this process also requires the presence of stable R-loops. Paradoxically, HDR requires extensive resection to generate ssDNA for strand invasion. This begs the question of how R-loops can prevent resection in one instance (in pre-senescent cells) but are required for a process that requires resection in another (at critically short telomeres). We hypothesize that in pre-senescent cells (longer telomeres) TERRA R-loops are removed in a cell cycle-dependent manner by RNase H2. When critically short telomeres arise, RNase H2 is no longer localized to telomeres and R-loops become stabilized, which induces DNA replication stress. We hypothesize that for resection and HDR to occur at critically short telomeres, R-loops must also eventually become metabolized. Our preliminary genetic data suggests that RNase H1 gets activated at short telomeres, where R-loops are stable, and removes the stable R-loop to promote HDR. Indeed, we have genetic evidence demonstrating that RNase H1 is only acting at R-loops when they become hyper-stabilized.

Figure 1. Hypothetical model of RNase H1 regulation in response to elevation of R-loop levels.

To test these hypotheses we will:

  1. Probe whether replication stress is indeed occurring at telomeres in an R-loop-dependent manner.
  2. Test whether RNase H1 is recruited to very short telomeres to remove stable hybrids and allow repair.
  3. In addition, we will test whether telR-loops can be formed in trans and evaluate if this affects telomere dynamics.

In the second part of this project, we will try to understand how the RNase H1 is regulated to respond to R-loops. We have observed that in the absence of Sen1, where R-loops accumulate, RNase H1 levels increase. We hypothesize that there may be a feedback loop from stable R-loops to the upregulation of RNase H1. We will address the following questions:

  1. Is RNase H1 upregulated on the transcriptional or post-transcriptional level?
  2. Is the presence of stable R-loops responsible for the increased levels of RNase H1?
  3. Is a specific pool of RNase H up-regulated (chromatin- or non-chromatin -bound)?
  4. What signaling pathways triggers RNase H1 upregulation?

Together, these studies will contribute to understanding how RNase H regulates R-loops and also give insights into how R-loops regulate RNase H. Using replicative senescence as a physiological readout we will be able to show how these regulatory events impinge on telomere regulation.