The Identification of Nuclear FMRP Isoform Iso6 Partners

Abstract

A deficiency of FMRP, a canonical RNA-binding protein, causes the development of Fragile X Syndrome (FXS), which is characterised by multiple phenotypes, including neurodevelopmental disorders, intellectual disability, and autism. Due to the alternative splicing of the encoding FMR1 gene, multiple FMRP isoforms are produced consisting of full-length predominantly cytoplasmic (i.e., iso1) isoforms involved in translation and truncated nuclear (i.e., iso6) isoforms with orphan functions. However, we recently implicated nuclear FMRP isoforms in DNA damage response, showing that they negatively regulate the accumulation of anaphase DNA genomic instability bridges. This finding provided evidence that the cytoplasmic and nuclear functions of FMRP are uncoupled played by respective cytoplasmic and nuclear isoforms, potentially involving specific interactions. While interaction partners of cytoplasmic FMRP have been reported, the identity of nuclear FMRP isoform partners remains to be established. Using affinity purification coupled with mass spectrometry, we mapped the nuclear interactome of the FMRP isoform iso6 in U2OS. In doing so, we found FMRP nuclear interaction partners to be involved in RNA processing, pre-mRNA splicing, ribosome biogenesis, DNA replication and damage response, chromatin remodeling and chromosome segregation. By comparing interactions between nuclear iso6 and cytoplasmic iso1, we report a set of partners that bind specifically to the nuclear isoforms, mainly proteins involved in DNA-associated processes and proteasomal proteins, which is consistent with our finding that proteasome targets the nuclear FMRP iso6. The specific interactions with the nuclear isoform 6 are regulated by replication stress, while those with the cytoplasmic isoform 1 are largely insensitive to such stress, further supporting a specific role of nuclear isoforms in DNA damage response induced by replicative stress, potentially regulated by the proteasome.

Fragile X Messenger Ribonucleoprotein Protein and Its Multifunctionality: From Cytosol to Nucleolus and Back

“…FMRP is associated with several kinases, including protein kinase RNAactivated (PKR) and phosphatidylinositol 4-kinase alpha (PI4KA), as well as a wide range of stress-related proteins, such as RAS-GTP-activating protein SH3 domain-binding protein (G3BP), ubiquitin-associated protein 2-like (UBAP2L), Ataxin-2-Like Protein (ATXN2L), cell cycle-associated protein 1 (CAPRIN1), FXR1P, FXR2P, DEAD-Box Helicase 3 X-Linked (DDX3X), and Nuclear FMRP Interacting Protein 2 (NUFIP2) (Figure 1 SG) [46][47][48][49]. This suggests that FMRP acts as a stress sensor for different types of stressors, such as viral infection, cellular toxicity, and DNA damage [47,50]. Consequently, other working groups have anticipated the role of FMRP in stress-related pathways, such as cell apoptosis and tissue tumor necrosis [51,52].…”

“…The second study used nuclear FMRP isoform 6, which is approximately 86% homologous to FMRP isoform 1, in osteosarcoma cells. Importantly, FMRP isoform 6 has a different carboxyl-terminal domain and lacks the nuclear export signal (NES) present in FMRP isoform 1 [50]. In the mouse brain, approximately 30% of FMRP is associated with ribosomal proteins [43].…”

“…In mice, ER stress induces FMRP phosphorylation through the activation of inositol-requiring enzyme-1 (IRE1), which, in turn, reduces atherosclerosis [83]. Recently, Phosphatidylinositol 4-kinase III α (PI4KA) has been identified as an interacting partner with the nuclear isoform of FMRP (isoform 6) [50]. PI4K produces phosphatidylinositol 4-phosphate (PI4P), which is then converted to Phosphatidylinositol 4,5-bisphosphate (PIP2).…”

“…It controls, in complex with staufen double-stranded RNA-binding protein 1 (STAU1) and TAR DNA-binding protein 43 (TDP-43), the expression and synthesis of SIRT1, a deacetylase involved in the repair of DNA double-strand breaks (DSBs) [123]. The induction of DNA damage by DNA polymerase inhibitors alters the nuclear FMRP interaction protein network through the recruitment of DNA damage response proteins such as aurora kinase B (AURBK), DNA topoisomerase II alpha (TOP2A), heterochromatin protein 1 binding protein 3 (HP1BP3), kinesin family member 22 (KIF22), and epithelial cell transforming 2 (ECT2) [50]. It has been reported that nuclear isoforms of FMRP regulate or inhibit the formation of DNA bridges through direct binding during mitotic anaphase.…”