The RNA m5C modification in R-loops as an off switch of Alt-NHEJ

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• The roles of RNA modifications and R-loops in DNA double-stranded break (DSB) repair are not well understood.
• Understanding these mechanisms is crucial for developing targeted cancer therapies.

Purpose of the Study:

• To investigate the function of RNA methyl-5-cytosine (m5C) modification in R-loops during DNA damage response.
• To determine how m5C affects DSB repair pathway selection and PARP1 activity.

Main Methods:

• Bisulfite sequencing to map m5C modifications in R-loops genome-wide.
• In vitro and cellular assays to assess the impact of m5C on PARP1-mediated poly ADP-ribosylation (PARylation).
• Analysis of DNA repair pathway choice (transcription-coupled homologous recombination vs. alternative non-homologous end joining) in response to m5C levels.

Main Results:

• DNA damage induces m5C modification in R-loops, primarily by the methyltransferase TRDMT1.
• Absence of m5C leads to increased R-loop-associated PARP1 PARylation.
• m5C promotes transcription-coupled HR (TC-HR) while suppressing alternative non-homologous end joining (Alt-NHEJ), favoring TC-HR in transcribed regions.

Conclusions:

• RNA m5C modification in R-loops is a key regulator of DSB repair pathway choice.
• Simultaneous inhibition of TRDMT1 and PARP or Polymerase θ demonstrates synergistic cytotoxicity in cancer cells.
• Targeting R-loop m5C and alternative DSB repair pathways offers a promising strategy for cancer therapy by exploiting genomic instability.