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Complex interplay between FMRP and DHX9 during DNA replication stress.

Arijita Chakraborty1, Arijit Dutta2, Leonardo G Dettori1

  • 1Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York, USA.

The Journal of Biological Chemistry
|December 18, 2023
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Summary

Fragile X messenger ribonucleoprotein (FMRP) deficiency causes DNA double-strand breaks by preventing the resolution of R-loops. FMRP

Keywords:
DHX9DNA double strand breaks (DSB)DNA replication stressFMR1FMRPFragile X SyndromeI304NKH2 domainR-loopRNA helicase

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Fragile X syndrome (FXS) is linked to FMRP deficiency, causing intellectual disability.
  • FMRP's nuclear functions, particularly in DNA repair and R-loop regulation, are not fully understood.
  • FXS cells exhibit increased DNA double-strand breaks (DSBs) at R-loop-prone sites, especially under replication stress.

Purpose of the Study:

  • To investigate the interaction between FMRP and DHX9.
  • To elucidate the role of FMRP in regulating DHX9 activity and R-loop resolution.
  • To understand how FMRP mutations affect protein localization and DNA damage.

Main Methods:

  • Co-immunoprecipitation to assess FMRP-DHX9 interaction.
  • In vitro helicase assays to measure DHX9 activity inhibition by FMRP.
  • Chromatin immunoprecipitation to analyze protein persistence on chromatin.
  • Analysis of R-loop-associated DSBs in cells with and without FMRP.

Main Results:

  • FMRP directly interacts with the RNA helicase DHX9 via its amino-terminal domain.
  • FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids, an effect dependent on the amino terminus.
  • The FXS-associated FMRP I304N mutation reduces FMRP-DHX9 interaction and leads to persistent FMRP and DHX9 on chromatin during replication stress.
  • Absence or dysfunction of FMRP results in unresolved R-loops and increased DSBs.

Conclusions:

  • FMRP and DHX9 have an antagonistic relationship at chromatin, crucial for resolving R-loops.
  • Proper FMRP-DHX9 interaction facilitates their dissociation from resolved R-loops.
  • FMRP deficiency or mutations disrupt this balance, leading to R-loop accumulation and DNA damage, contributing to FXS pathogenesis.