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Maintenance of R-loop structures by phosphorylated hTERT preserves genome integrity.

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|May 28, 2024
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Phosphorylated human telomerase (hTERT) has RNA-dependent RNA polymerase (RdRP) activity that resolves RNA-DNA hybrids (R-loops), preventing genome instability. Targeting hTERT RdRP activity impairs tumor growth and reveals synthetic lethal interactions with Fanconi anaemia/BRCA genes.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cancer Research

Background:

  • Aberrant RNA-DNA hybrids (R-loops) are linked to DNA damage and genome instability.
  • Cellular regulators are crucial for managing R-loop structures.
  • Understanding R-loop regulation is key to preventing genomic instability.

Purpose of the Study:

  • To investigate the role of human telomerase reverse transcriptase (hTERT) in regulating R-loop formation.
  • To identify novel regulators and functions of hTERT beyond telomere maintenance.
  • To explore therapeutic strategies targeting R-loop homeostasis in cancer.

Main Methods:

  • Utilized RNA-dependent RNA polymerase (RdRP) assays to characterize hTERT activity.
  • Employed immunofluorescence and co-immunoprecipitation to study protein localization and interactions.
  • Conducted genome-scale CRISPR loss-of-function screens to identify synthetic lethal interactions.
  • Targeted TERT gene expression in alternative lengthening of telomeres (ALT) cells.

Main Results:

  • Phosphorylated hTERT (p-hTERT) exhibits RdRP activity in nuclear speckles.
  • p-hTERT associates with TERRA RNAs to resolve R-loops, independent of telomerase RNA.
  • TERT gene targeting in ALT cells reduced RdRP activity and impaired tumor growth.
  • Fanconi anaemia/BRCA genes were identified as synthetic lethal partners of hTERT RdRP.
  • Inactivation of hTERT RdRP and Fanconi anaemia/BRCA genes led to R-loop accumulation and DNA damage.

Conclusions:

  • The RdRP activity of p-hTERT plays a critical role in resolving R-loops and maintaining genome stability.
  • hTERT's function extends beyond telomere maintenance to actively regulate R-loop structures.
  • Targeting hTERT RdRP activity presents a potential therapeutic avenue for cancers, particularly in combination with therapies targeting DNA repair pathways like Fanconi anaemia/BRCA.