Telomere dysfunction is a significant source of genomic instability and contributes to the development of cancer. The multi-protein complex shelterin binds telomeric DNA to mitigate telomere dysfunction and ensure overall telomere stability. In addition to shelterin, the telomeric cap includes the telomeric repeat-containing RNA, TERRA, which associates with telomeric proteins and the telomeric DNA itself, often forming RNA:DNA hybrids or R-loops. TERRA is most abundant in cancer cells that utilize the alternative lengthening of telomeres (ALT) pathway, where it has been suggested that TERRA R-loops act as a source of replication stress at telomeric DNA that ultimately contributes to the activation of the ALT mechanism. In an effort to evaluate the effect TERRA may have on the emergence of the ALT phenotype, we sought to identify the enzyme(s) that regulate TERRA degradation in mammalian cells. Here, we leveraged an auxin-inducible degron (AID) system to identify the 5’-3’ exoribonuclease XRN2 as a direct modulator of TERRA stability in mammalian cells. Following XRN2 depletion, we demonstrate a significant increase in TERRA on chromatin in both non-ALT and ALT-positive cell lines. While the stabilization of TERRA on chromatin alone was insufficient to drive replication stress and activation of ALT in telomerase cells, depletion of XRN2 in the ALT-positive context led to a significant increase in R-loops and DNA damage signaling at telomeric DNA. Thus, increased TERRA stability alone is unlikely to activate ALT but may instead exacerbate ALT activity. Taken together, we demonstrate that XRN2 regulates TERRA stability, that defects in TERRA metabolism can alter telomere stability, and dysfunction in both factors drive telomere dysfunction in cells that rely on the ALT pathway. / 2024-08-12T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/48079 |
| Date | 12 February 2024 |
| Creators | Reiss, Matthew Evan |
| Contributors | Flynn, Rachel Litman |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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