Complex interplay between FMRP and DHX9 during DNA replication stress

Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP. Overall design: HEK293T cells containing FMR1 KO and expressing CMV-EGFP-FMRP (wild type or I304N mutant) were used for ChIP-seq for FMRP or DHX9. Before chromatin immunoprecipitation,, cells were treated with 0.6 or 1 micromolar aphidicolin (APH), with DMSO or no treatment as controls. Input DNA (whole genomic DNA) for each sample was used as control for library construction.

脆性X信使核糖核蛋白（fragile X messenger ribonucleoprotein，FMRP）的突变或缺失，是导致脆性X综合征（Fragile X syndrome，FXS）的原因，而FXS是遗传性智力障碍最常见的诱因。FMRP是一种核质分布蛋白，最初被鉴定为主要的翻译抑制因子，但其核内功能仍不甚明确。我们此前的研究表明，缺乏FMRP的脆性X综合征患者细胞相较于正常细胞，会积累更高水平的DNA双链断裂（DNA double-strand breaks，DSBs），且断裂位点多集中于易形成R环（R-loops）的序列；该表型在DNA复制应激条件下会进一步加重。此外，唯有野生型FMRP的表达，而非已知会引发FXS的FMRPI304N突变体，能够降低与R环相关的DNA双链断裂水平。后续我们的研究证实，重组FMRP可直接结合R环，结合主要依赖于其羧基端的内在无序区域（carboxyl terminal intrinsically disordered region）。

本研究显示，FMRP可与RNA解旋酶DHX9直接相互作用。该相互作用由FMRP的氨基端结构化结构域介导，且FMRPI304N突变会削弱这一相互作用。我们同时证实，FMRP可抑制DHX9对RNA:DNA杂交链的解旋酶活性，且该抑制作用同样依赖于FMRP的氨基末端区域。进一步研究发现，FMRPI304N突变会导致FMRP与DHX9在复制应激条件下持续滞留于染色质上。

上述结果提示，FMRP与DHX9在染色质上存在拮抗关系：二者的正常相互作用可促使两者均从完全解离的R环上解离下来。我们据此提出，FMRP的缺失或功能丧失，会分别造成DHX9持续滞留于未被解离的R环上，或两种蛋白共同附着于该区域，最终引发DNA双链断裂。本研究为我们理解FMRP的基因组功能提供了全新视角。

整体实验设计：我们使用携带FMR1基因敲除（FMR1 KO）并表达CMV-EGFP-FMRP（野生型或I304N突变体）的HEK293T细胞，针对FMRP或DHX9开展染色质免疫沉淀测序（chromatin immunoprecipitation sequencing，ChIP-seq）。在进行染色质免疫沉淀前，细胞分别用0.6或1微摩尔的阿非迪霉素（aphidicolin，APH）处理，以DMSO处理组或未处理组作为对照。每个样本的输入DNA（全基因组DNA）均被用作文库构建的对照。