Complex interplay between FMRP and DHX9 during DNA replication stress

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.