IGF2BPs directly regulate the noncanonical translation of toxic proteins from mutant FMR1 mRNA containing expanded CGG repeats

Domain: neuroscience
Published: 2025-12-10
Editorial outcome: major_revision

Abstract
Mutant mRNA of the fragile X messenger ribonucleoprotein 1 gene (FMR1) containing expanded CGG repeats in its 5’ UTR is a primary cause of fragile X premutation associated conditions. It serves as a template for the biosynthesis of the major open reading frame encoding canonical protein and the downstream open reading frame containing expanded CGG repeats encoding toxic FMRpolyG protein that comprise a long polyglycine stretch, produced via repeat-associated non-AUG initiated translation. Here, we show that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) binds directly to the 5’ UTR of FMR1 RNA, and the sequence in the vicinity of near-cognate start codons of non-AUG translation is pivotal for IGF2BP3 binding. Upon IGF2BP3’s knockdown, FMRpolyG biosynthesis and cell toxicity evoked by FMRpolyG, significantly decreased in cells expressing mutant FMR1 with expanded CGG repeats. Disruption of IGF2BP ortholog in novel fragile X premutation associated conditions C. elegans model rescues the disease phenotype induced by expression of a human FMR1 RNA fragment containing expanded CGG repeats. Our results suggest that IGF2BP3 positively regulates the noncanonical translation of expanded CGG repeats and may be a promising target for clinical applications.

Introduction
Short tandem repeats occur frequently within exons and noncoding regions of the human genome. They are polymorphic in length and, when long enough, prone to expansion. Expansions over a certain threshold within different regions of protein-coding genes have been linked to over 50 human-inherited repeat expansion disorders, mainly neurodegenerative or neuromuscular. One such genetic loci is the fragile X messenger ribonucleoprotein 1 gene (FMR1), in which CGG repeats occur in the 5’ UTR. In healthy individuals, this locus contains between 5 and 54 CGGs. However, the expansion of CGG repeats (CGGexp) to intermediate size, called premutations (PMs, 55–200 CGG repeats), can cause different fragile X-PM-associated conditions (FXPACs)1, including fragile X-associated tremor/ataxia syndrome (FXTAS), fragile X-associated primary ovarian insufficiency (FXPOI), and fragile X-associated neuropsychiatric disorders (FXANDs). Longer expansions, called full mutations (above 200 CGG repeats) lead to the development of early-onset neurodevelopmental fragile X syndrome (FXS). At the molecular level, a full mutation causes hypermethylation and silencing of the FMR1 promoter, which results in the loss of the protein product, fragile X messenger ribonucleoprotein 1 (FMRP)2.

FXTAS is a late-onset, incurable neurodegenerative disorder characterized by tremors, ataxia, cognitive decline, and Parkinsonian features3. PMs occur at a frequency of 1 in 150–300 females and 1 in 400–850 males in the general population, but due to the location of the FMR1 gene on the X chromosome, male carriers are primarily affected. The prevalence of FXTAS is 40–75% of males with PM, and 16–20% of female carriers4. The pathological hallmark of FXTAS is the presence of large, ubiquitin-positive inclusions in the nuclei of neurons and astrocytes4. FXPOI symptoms include reduced fertility, premature menopause, and ovarian dysfunction5, with a prevalence of 20–30% in females with PM6. FXAND manifests as anxiety and depression in adults7. The pathogenesis of FXPAC remains unclear, and several nonexclusive pathogenic mechanisms have been proposed. One involves the cotranscriptional formation of aberrant R-loops in the CGGexp locus triggering a DNA-damage response8–10. Another suggests a toxic RNA gain-of-function leading to the sequestration of proteins on RNA-containing CGGexp (rCGGexp) within nuclear foci11–15. This results in the functional depletion of these proteins and impairment of their physiological functions. The third mechanism underlying rCGGexp toxicity in FXPAC is the repeat-associated non-AUG initiated (RAN) translation of rCGGexp into aberrant proteins that contain stretches of repeated amino acids despite CGG repeats being located in the 5’ UTR16,17. These three mechanisms can cause downstream effects, such as mitochondrial dysfunction, oxidative stress, impaired miRNA biogenesis, alternative splicing, or the presence of intranuclear inclusions, which contribute to the pathogenesis of FXPAC.

When FMR1 mRNA containing short or long CGG repeats is exported to the cytoplasm, its translation is initiated at the AUG start codon downstream of the CGGs, resulting in the production of the main protein product, FMRP. However, rCGGexp embedded within the FMR1 5’ UTR can also be translated via RAN translation, initiating at non-canonical, near-cognate start codons, resulting in the biosynthesis of toxic RAN proteins16,17. These proteins contain repeated glycine, alanine, or arginine tracts (FMRpolyG, FMRpolyA, and FMRpolyR, respectively), depending on the reading frame, or a combination of them as a result of translational frameshifting18. The FMRpolyG translation is the most efficient and initiates at the near-cognate ACG or GUG codons located upstream of the rCGGexp16,17, while translation initiation of FMRpolyA probably occurs within the repeats (GCG codon)16,19. FMRpolyG is neurotoxic and tends to aggregate in cytoplasmic or ubiquitin-positive nuclear inclusions20,21. Its important role in FXTAS pathogenesis is underlined by the fact that FMRpolyG-positive aggregates have been identified in various tissues, including the frontal cortex, hippocampus, heart, kidney, adrenal gland, and thyroid of FXTAS patients17,22,23, and the ovarian stromal cells of FXPOI patients24. Although FMR1 expression is not limited to neurons, the toxicity of intranuclear inclusions appears to be most prominent in the central nervous system.

A number of studies have contributed to the understanding of the CGG RAN translation mechanism, but knowledge about this process remains limited. Recent reports have shown that the RAN translation of rCGGexp requires a 5’ cap and the translation initiation factors eIF4A and eIF4E for initiation25. Additionally, a hairpin formed by rCGGexp or a nearby 5’ UTR sequence causes steric blockage for the scanning of the 43S preinitiation complex (PIC)25,26. This leads to an increased dwell time of 43S PIC, lowered codon fidelity, and initiation at near-cognate codons (ACG or GUG)27. RNA molecules containing expanded repeats and RAN proteins were shown to activate protein kinase R, which phosphorylates eIF2\alpha and causes the activation of the integrated stress response28. This upregulates the RAN translation of rCGGexp and leads to a global translation shutdown. Genetic and proteomic studies have revealed that the efficiency of RAN translation may be regulated by various proteins, including the translation initiation factors: eIF1 and eIF5; helicases: DEAD box helicase eIF4A25, DDX3X29, and DHX3630; small ribosomal subunit proteins: e25 and e26 (RPS25 and RPS26, respectively); and some of the ribosomal quality control factors: nuclear export mediated facilitator, E3 ligase listerin, ankyrin repeat, and zinc finger peptidyl tRNA hydrolase 131.

From a therapeutic perspective, identifying modifiers of RAN translation is likely to be crucial for the development of FXTAS treatment strategies. In this study, we employed a pull-down approach targeting rCGGexp and combined it with a proteomic analysis to identify proteins binding to the 5’ UTR of FMR1 mRNA in vitro. Selected candidates were further stratified to assess their contribution to RAN translation efficiency in FXTAS cell models. Among the selected proteins, we focused on insulin growth factor 2 mRNA binding protein 3 (IGF2BP3, also called IMP3), a nucleocytoplasmic protein composed of six RNA-binding domains: two N-terminal RNA-recognition motifs and four hnRNP K-homology domains connected by short linkers32. Studies performed using the Xenopus laevis model suggest that this protein plays a role in neuronal development33. Interestingly, IGF2BP3 has also been detected in human placentas and testes, indicating its potential role in reproductive organs. Depending on the target mRNA, IGF2BP3 can act as a modulator of mRNA cellular localization, RNA stability, miRNA biogenesis, or as an m6A reader34. Importantly, binding of IGF2BP3 to the 5’ UTR of insulin growth factor 2 (IGF2) leader-3 mRNA activates its translation35. In this study, we show that IGF2BP3 directly binds to the 5’ UTR of mutant FMR1 mRNA and specifically promotes the RAN translation of toxic FMRpolyG. We also found that two other paralogs of the IGF2BP family, IGF2BP1 and IGF2BP2, can regulate FMRpolyG levels similarly to IGF2BP3, and their action is restricted to RAN-translated proteins. We propose that the inhibition of IGF2BP paralogs is a promising therapeutic approach to decrease the level of toxic RAN-translated FMRpolyG proteins in FXPAC. Finally, we show that IGF2BP3 knockdown mitigates necrosis of FXTAS iPSC-derived neurons, and functional knock out of imph-1, an IGF2BP ortholog, improves the disease phenotype observed in the FXPAC C. elegans model.

Results
RNA-protein pull-down identifies sets of putative interactors of the 5’ UTR of FMR1 mRNA
To identify proteins that may bind to the 5’ UTR of mutant FMR1 mRNA, we performed a pull-down assay using a set of biotinylated RNAs, followed by a proteomic analysis. The bait RNAs included the 5’ UTR of FMR1 containing ca. 100 CGG repeats (FMR1-99CGG), the 5’ UTR of FMR1 devoid of CGG repeats (FMR1-delCGGs), control RNA of similar length and GC content (GC-rich), and RNA containing 23 CGG repeats, without FMR1 flanking sequence (23CGG) (Fig. 1A). First, we incubated in vitro transcribed, biotinylated RNAs with the protein extract of human neuroblastoma SH-SY5Y cells to assemble RNA-protein complexes, which were further purified on streptavidin-coated beads. Different elution profiles were observed on SDS-PAGE gels for different RNA baits (Supplementary Fig. 1A). Using mass spectrometry (MS), we identified ca. 25 proteins significantly enriched on FMR1-99CGG RNA compared to control GC-rich RNA (Fig. 1B, Supplementary Data 1). These proteins included Pur Beta, nucleophosmin, and heterogeneous nuclear ribonucleoprotein M (hnRNP M), which were previously described as binding to rCGGexp11,13. We also identified RPS26, which was previously shown to regulate the translation of FMRpolyG from the 5’ UTR of mutant FMR1 containing ca. 100 CGG repeats36. A gene ontology (GO) analysis of proteins binding to FMR1-99CGG RNA revealed significant enrichment of the following GO terms: structural constituent of the ribosome, translation, and mRNA-5’ UTR binding (Supplementary Fig. 1B).

We then compared proteins binding to either FMR1-99CGG RNA or FMR1-delCGGs devoid of CGG repeats. A label-free analysis revealed that more than half of the identified proteins were eluted with both RNAs (Supplementary Fig. 1C), including IGF2BP3. Twelve proteins were enriched more than two-fold on FMR1-delCGGs RNA, including multiple ribosomal proteins, and the most enriched protein being ubiquilin 2, which was previously detected in nuclear inclusions of FXTAS neurons20. Fifteen proteins preferentially bound to FMR1-99CGG, including nucleophosmin and nucleolin. Finally, we analyzed proteins binding to FMR1-99CGG RNA in comparison to 23CGG RNA probe missing FMR1 flanking sequences (Supplementary Fig. 1D). In this dataset, we quantified 135 proteins binding to both RNAs, of those 50 proteins were significantly enriched on FMR1-99CGG RNA, including Pur Alpha, Pur Beta, HNRNPM, and IGF2BP3.

Among the identified proteins, IGF2BP3 protein, involved in RNA transport, translation, stability, and reading m6A-modified RNAs34, drew our particular attention.

To validate the direct in vitro interaction of IGF2BP3 with FMR1-99CGG RNA and to decipher whether this interaction depends on CGG repeats, we used several orthogonal approaches. First, an RNA-protein pull-down followed by Western blot (WB) indicated that IGF2BP3 binds to both FMR1-99CGG RNA and FMR1-delCGGs mRNA, and, to a lesser extent, the control GC-rich RNA (Fig. 1C). Using filter binding and electrophoretic mobility-shift assays (Fig. 1D and Supplementary Fig. 1F), we confirmed that recombinant IGF2BP3 binds with high affinity to 32P-labeled FMR1-5’ UTR RNA containing 16 CGG repeats, with a low nanomolar dissociation constant (Kd = 3.15 ± 0.79 nM). We did not observe an effective interaction between IGF2BP3 and control (UAA) RNA, even in the highest protein concentration range (Kd » 100 nM) (Supplementary Fig. 1F). Finally, we analyzed publicly available enhanced UV-cross-linking and immunoprecipitation (eCLIP) data37 and identified IGF2BP3 reads along FMR1 5’ UTR (Supplementary Fig. 1G) in HepG2 cells containing normal size of CGG repeats, confirming our in vitro data. Together, our experiments confirmed that, under in vitro conditions, IGF2BP3 directly interacts with the FMR1 5’ UTR in a CGG-independent manner.

IGF2BP3 positively regulates the level of RAN-translated FMRpolyG in cells with expanded CGG repeats
To check whether IGF2BP3 regulates the RAN translation of FMRpolyG we first knocked down (KD) IGF2BP3 levels using short interfering RNA (siRNA) in human embryonic kidney cells (HEK-293T), human cervical carcinoma (HeLa), and human neuroblastoma cells (SH-SY5Y) with a transient expression of RAN-translated FMRpolyG containing tract of either 16 or 99 glycine residues tagged with green fluorescent protein, GFP (FMR16xG and FMR99xG, respectively). We also silenced IGF2BP3 in previously generated HEK293 FlipIn T-Rex cell models with a stable expression of FMRpolyG containing either 16 or 95 glycine residues (S-FMR16xG and S-FMR95xG lines, respectively). In all of the tested cell models, including transient and stable expression systems, we observed a significant, approximately two-fold, reduction of FMR99xG protein levels upon IGF2BP3 KD (Fig. 2A, Supplementary Fig. 2A-C). Importantly, we also observed (although to a lower extent (20–25%)), a decrease in the shorter protein, FMR16xG, upon IGF2BP3 KD (Fig. 2A and Supplementary Fig. 2A), suggesting that IGF2BP3 regulates RAN-translated FMRpolyG synthetized from both normal and PM CGG length thresholds. Additionally, in tested cell models, we did not observe an effect of IGF2BP3 KD on endogenous FMRP levels, which is produced from the same mRNA as FMRpolyG, but different open reading frame (Fig. 2A). On the other hand, IGF2BP3 KD led to an increase of endogenous FMR1 mRNA level (Fig. 2A), suggesting that FMR1 is a target of IGF2BP3, which is in line with eCLIP data (Supplementary Fig. 1G).

Since IGF2BP3 plays a role in various aspects of RNA metabolism, we also investigated the effects of IGF2BP3 KD on FMR1 transcripts containing either 16 or 99 CGG repeats. The quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed that, in a stable expression system, the mRNA levels of FMR16xG and FMR95xG increased approximately 2-fold (Fig. 2A). To our surprise, in the transient expression system, the relative FMR99xG mRNA levels increased 2.5-fold in HEK293T cells, while the FMR16xG mRNA with a short repeat tract remained unchanged (Supplementary Fig. 2A).

We also performed nuclear-cytoplasmic fractionation to verify whether the transport of FMR99xG mRNA is impaired upon IGF2BP3 KD. In both nuclear and cytoplasmic fractions isolated from the FMR99xG cell model, we observed a significant increase of FMR99xG mRNA, with a similar trend as in FMR99xG measured for the total RNA (Fig. 2B). Together, these data suggest that the decrease in FMRpolyG level is not a result of a reduced RNA level, impaired nuclear retention, or defective nucleocytoplasmic transport.

Importantly, we found that overexpression of IGF2BP3 induced opposite effects on FMRpolyG levels than KDs (Fig. 2C). Co-transfection of HEK293T cells with an IGF2BP3-overexpressing construct and FMR16xG- or FMR99xG-constructs led to a 3-fold and 1.7-fold increase of FMR16xG and FMR99xG protein levels, respectively. IGF2BP3 overexpression did not affect FMR16xG mRNA levels, however, it slightly increased the FMR99xG mRNA level by 1.3-fold.

Toxic FMRpolyG proteins can be present in cells in soluble, insoluble, and aggregated form, therefore, we investigated whether both protein states would be affected by IGF2BP3 depletion. We monitored the number of GFP-positive FMRpolyG aggregates in HeLa cells and found that their number was significantly decreased upon IGF2BP3 KD (Fig. 2D). This finding was further confirmed using a flow cytometry assay, where the average fluorescence signal of FMRpolyG-GFP, emitted by both soluble and aggregated forms of FMR99xG, was significantly decreased in HEK293T cells with lowered IGF2BP3 expression (Fig. 2E). Moreover, we found that apoptosis evoked by toxic FMR99xG was also significantly diminished in the same cell model (Fig. 2F).

IGF2BP3-dependent regulation is specific to the RAN translation of FMRpolyG and dependent on the FMR1 RNA sequence context
Next, we investigated whether IGF2BP3-dependent regulation is specific to noncanonical RAN biosynthesis, characterized by initiation at non-AUG codons. Therefore, we tested the effect of IGF2BP3 KDs on the FMRpolyG produced from mRNA in which the near-cognate ACG codon was replaced by the AUG codon (ATG-FMR99xG). As expected, this substitution significantly increased the basal FMRpolyG level. However, the protein level of AUG-FMR99xG derived from the AUG codon, and its mRNA level, remained unchanged upon IGF2BP3 KD (Fig. 3A).

We then tested whether supplementation of an in vitro translation reaction with recombinant IGF2BP3 affects the biosynthesis of FMRpolyG. We used cell-free rabbit reticulocyte lysate to translate in vitro RNA encoding the 5’ UTR sequence of the FMR1 gene with 16 CGG repeats. In accordance with previous studies25, a WB analysis confirmed the efficient production of FMRpolyG. Importantly, the addition of recombinant IGF2BP3 to in vitro transcribed FMR16xG RNA at a 2:1 molar ratio significantly enhanced FMR16xG synthesis (Fig. 3B). This finding confirms the direct effect of IGF2BP3 on RAN translation.

Recently, an integrative study revealed that the IGF2BP3 protein recognizes RNA containing CA-motifs and GGC core elements (either GGCA or CGGC) separated by appropriate spacing38. Since multiple CA motifs are located upstream of CGG repeats in the 5’ UTR of FMR1, we investigated whether one of these CA dinucleotides is critical for IGF2BP3 binding. First, we overexpressed the construct encoding the 5’ UTR region of FMR1 containing 16 CGG repeats fused with nanoluciferase (FMR16xG-nLuc) in HEK293T cells, and found that depletion of IGF2BP3 in this model resulted in decreased FMR16xG protein levels (Fig. 3D). Of note, the FMR16xG-nLuc transcript level was upregulated upon IGF2BP3 silencing, similar to the FMR16xG transcript produced from the construct with the GFP tag. This experiment confirmed that the type of tag used in transgene generation does not affect the sensitivity of tested RNA and proteins to the amount of IGF2BP3. Then, using mutagenesis, we replaced CA with the TG dinucleotide motif located 20 nucleotides upstream of the CGG repeats and 14 nucleotides downstream of the ACG near cognate start codon (mutFMR16xG-nLuc, Fig. 3C). We found that this mutation did not impede production of FMR16xG, but the mutant FMR16xG and its mRNA level were not sensitive to IGF2BP3 silencing (Fig. 3D). This indicates that the CA dinucleotide is crucial for IGF2BP3 binding, and mutation of this site abolishes the regulatory role of this protein on the RAN translation of FMR1 mRNA.

The results suggest that the IGF2BP3 protein controls FMRpolyG protein levels, independent of CGG repeat length, and that the regulation is specific to RAN but not canonical translation, as no impact on AUG-translated FMRpolyG nor endogenous FMRP proteins was observed upon IGF2BP3 silencing. IGF2BP3 modulates the RAN translation of CGG repeats via direct interaction with the 5’ UTR of normal and mutant FMR1 mRNA.

Other IGF2BP paralogs regulate the biosynthesis of RAN-translated FMRpolyG
The IGF2BP family is composed of three members: IGF2BP1, IGF2BP2, and IGF2BP3, which are characterized by high sequence identity (Supplementary Fig. 4E) and similar composition of RNA binding domains32,39,40. IGF2BP paralogs govern a different pool of cellular RNAs by multiple mechanisms, including sequestration in mRNP granules and the protection or enhancement of RNA degradation (reviewed in ref. 41), and the binding specificity is determined by differences in amino acid sequence in variable loops of KH domains40. Importantly, some of the target RNA overlap, such as CD44, CRC, MYC and TNBC (reviewed in Duan et al. 42). Additionally, IGF2BP1 was previously reported, by us and others, as protein binding to the FMR1 5’ UTR containing expanded CGG repeats in cellulo36,43, or CGG repeats without FMR1 sequence context44. Thus, we aimed to check whether IGF2BP1 and IGF2BP2 paralogs can regulate levels of FMR99xG similarly to IGF2BP3. We found that silencing IGF2BP1 and IGF2BP2 resulted in a 2-fold decrease of transiently expressed FMR99xG protein levels, without impairing its mRNA level (Fig. 4A), and co-expression of FMR99xG with IGF2BP1 or IGF2BP2 resulted in 9- and 6-fold increase in FMR99xG protein levels, respectively (Fig. 4B). Additionally, we found that KDs of IGF2BP1 or IGF2BP2 paralogs did not influence the level of FMRpolyG translated from a canonical AUG codon, similar to that of the IGF2BP3 paralog (Fig. 4C). We then asked whether IGF2BP1 and IGF2BP2 can compensate for IGF2BP3 level and found that only IGF2BP1 mRNA level increased upon IGF2BP3 KD (Fig. 4D). However, combined silencing of three paralogs revealed that IGF2BP2 and IGF2BP3 contribute most significantly to the regulation of FMRpolyG level in HEK293T cells (Fig. 4E). Together, this data suggests that all IGF2BP paralogs specifically affect the level of RAN translated FMRpolyG containing long polyG tracts.

Small molecule inhibitors targeting IGF2BP3 modulate the level of FMRpolyG
Several preclinical studies have demonstrated successful downregulation of IGF2BP3 expression using different small compounds, including isocorydine derivative45 and bromo- and extra-terminal domain (BET) inhibitors such as JQ146–48 and I-BET15149,50 (Fig. 5A). Here, we aimed to verify whether these small molecules can modulate the level of FMR99xG through the inhibition of IGF2BP3. We administered these compounds to HEK293T cells with transient expressions of FMR16xG or FMR99xG and found that, although all three molecules induced an efficient decrease of IGF2BP3 protein levels in line with previous reports, only JQ1 and I-BET151 decreased the IGF2BP3 mRNA level (Fig. 5B). This suggests that isocorydine inhibits the IGF2BP3 level via a different mechanism than BET inhibitors. Importantly, cells treated with all of the tested inhibitors significantly reduced FMR99xG but not FMR16xG protein levels (Fig. 5C, D). Moreover, both JQ1 and I-BET151 induced the upregulation of mRNA levels of both FMR16xG and FMR99xG, similar to the knockdown of IGF2BP3 (Fig. 5C, D). The actions of JQ1 and I-BET151 appear to be specific to RAN-translated FMR99xG, as administration of these molecules to HEK293T cells with transient expressions of ATG-FMR99xG did not affect the level of FMRpolyG produced from the canonical start codon (Fig. 5E). Additionally, only JQ1 significantly decreased FMRP protein level, while both JQ1 and I-BET151 decreased FMR1 mRNA level (Supplementary Fig. 5B, D). We have also found that administration of JQ1 decreased the relative mRNA level of IGF2BP1 and IGF2BP2 (Supplementary Fig. 5D), which is in line with previous findings51.

In sum, we conclude that the tested small compounds, which have well-described anticancer activities, significantly reduced the biosynthesis of toxic FMR99xG similar to IGF2BP3 KDs, which suggests a therapeutic potential for these and similar molecules in FXPAC.

IGF2BP3 regulates the level of FMR1 mRNA in FXTAS patient derived cells
To validate our findings in an endogenous context, we silenced IGF2BP3 in control and FXTAS patient-derived fibroblasts and found that the relative FMR1 mRNA level increases upon IGF2BP3 KD (Fig. 6A, Supplementary Fig. 6A), similarly as in cell lines with stable expression of FMRpolyG (Fig. 2A). Furthermore, we differentiated two control and two FXTAS-patient derived induced pluripotent stem cells (iPSCs), into neural progenitor cells (NPCs, Supplementary Fig. 6E). We found that silencing of IGF2BP3 in this model resulted in increased relative FMR1 mRNA levels in FXTAS NPC (Supplementary Fig. 6B), similarly as observed in fibroblasts (Fig. 6A). The NPCs were then differentiated into neurons, and after two weeks of culture, cells were transfected with either a non-targeting control siRNA or IGF2BP3 siRNA. In this model, we also observed the increase of relative FMR1 mRNA level upon IGF2BP3 KD (Fig. 6B, Supplementary Fig. 6C), confirming our observations in overexpression models (Fig. 2A, Fig. 6A). Expression of CGG RAN translation products was previously shown to decrease cell viability52 and induce neuronal cell death17,18. In line with these findings, we measured the impact of IGF2BP3 silencing on necrosis progression in control and FXTAS iPSC-derived neurons, starting at forty-eight hours post siRNA treatment. We found that lower IGF2BP3 expression rescued the necrosis phenotype in FXTAS iPSC-derived neurons (Fig. 6C), but not in control cells (Supplementary Fig. 6F). After three weeks of neuronal differentiation, using immunofluorescence, we detected FMRpolyG in 5 to 10% of FXTAS iPSC-derived neurons (Fig. 6D, Supplementary Fig. 6G), but not in controls (Supplementary Fig. 6H), in agreement with previous reports17. We also found that IGF2BP3 KD in these cells resulted in a modest, but not statistically significant reduction in the number of FMRpolyG aggregates (Fig. 6D). We suppose that this can be due to lower IGF2BP3 KD efficiency (Supplementary Fig. 6D), which was not sufficient to inhibit FMRpolyG production and aggregate formation. Finally, we administered BET inhibitors: JQ1 and I-BET151, to control and FXTAS iPSC-derived neurons and measured apoptosis rate after 6 days of treatment. We observed that both JQ1 and I-BET151 decreased the apoptosis rate in FXTAS iPSC-derived neurons, but not in controls (Supplementary Fig. 6I).

imph-1/IGF2BP disruption rescues 99xCGG phenotype in Caenorhabditis elegans
To determine whether the effects of IGF2BP knockdown on the FXPAC phenotype could be replicated in an animal model, we generated transgenic C. elegans expressing RNA of human FMR1 5’ UTR with 99 CGG repeats, fused to GFP at the 3’ end (polyG(agaEx7[99xCGG])), here in referred to as 99xCGG model (Fig. 7A). Microscopy analysis revealed that these worms expressed low level of FMRpolyG-GFP in the head (Fig. 7B, Supplementary Fig. 7A), which was further confirmed by fluorescence lifetime imaging (FLIM), showing a GFP lifetime of 2.49 ns (Supplementary Fig. 7A). We then sought to assess the role of C. elegans IGF2BP ortholog, imph-1, which shares almost 30% amino acid sequence with human IGF2BP paralogs (Supplementary Fig. 7B), in the 99xCGG C. elegans model. To do so, we crossed the 99xCGG transgenic strain with the imph-1(sy1993) mutant animals, which carries a stop-in cassette insertion disrupting imph-1 expression. In this 99xCGG; imph-1\Delta genetic background, we observed a decrease of FMRpolyG-GFP fluorescence (Fig. 7B, C), and increase in FMR1-99CGG mRNA levels compared to the 99xCGG strain alone (Fig. 7D), consistent with the upregulation seen in FXTAS fibroblast and FXTAS iPSC-derived neurons (Fig. 6A, B).

Furthermore, we observed that 99xCGG animals exhibited severe phenotypic defects, including reduced brood size, impaired thrashing behavior and decreased lifespan, relative to wild-type animals (Fig. 7E–G, Supplementary Movies 1–4). Notably, these phenotypes were significantly rescued in the 99xCGG; imph-1\Delta background, suggesting that the product of imph-1 positively regulates FMRpolyG expression and that its loss may mitigate the detrimental effects of CGG repeat expansion in model organism.

Methods
Genetic constructs
The genetic construct FMR1-99CGG (Addgene plasmid #63091), was a kind gift from N. Charlet-Berguerand. This construct contains 5’ UTR sequence of FMR1 gene with ~99 CGG repeats and located downstream GFP sequence17 and was used to produce FMRpolyG. The FMR1-16CGG construct, was obtained by spontaneous contraction of CGG repeats from FMR1-99CGG template during bacterial growth. The FMR1-delCGGs construct, lacking CGG repeats, was generated from FMR1-99CGG template via inverse polymerase chain reaction (PCR), using InFusion Cloning technology (Takara), with primers F1/R1, followed by DNA fragments assembly with NEBuilder (New England Biolabs). The already described ATG-99CGG construct78, contains canonical ATG start codon instead of near-cognate ACG upstream of the repeats. Note that the part of FMR1 5’ UTR upstream the start codon is lacking in this plasmid. Construct ACG-16CGG-NLuc, encoding 5’ UTR sequence of FMR1 gene with 16 CGG repeats in frame with nanoluciferase and FLAG, was obtained via amplification of FMR1 5’ UTR sequence from ACG-16xCGGCompleteSTOP plasmid78 by primers F2/R2. The FLAG tag was added downstream nanoluciferase in pNL1.1 CMV construct (Promega) via inverse PCR with primers F3/R3. To ensure the transcription start site at the beginning of FMR1 5’ UTR, the sequence of 109 nt between the CMV promoter and the FMR1 sequence was removed. The generated pNL1.1 CMV-FLAG backbone was linearized by primers F4/R4, mixed with FMR1 5’ UTR amplicon and cloned via NEBuilder system. Construct ACG-16CGG-mutCA-NLuc, with mutation of CA nucleotides to TG in region predicted for binding IGF2BP3 was obtained by inverse PCR based on InFusion cloning, using primers F5/R5. GC-rich PCR product, corresponding to TMEM170 mRNA and serving as a control for RNA pull-down, was amplified by PCR from HEK293T cDNA, using primers F6/R6 and Phusion High-Fidelity DNA Polymerase (ThermoFisher). PCR product was used as a template in second PCR reaction with primers F7/R6, in order to add the T7 promoter sequence, recognized by T7 RNA polymerase. Sequences of all constructs were confirmed by Sanger sequencing. All plasmids containing CGG repeats were transformed and isolated from NEB stable competent E. coli (New England Biolabs). pDESTmycIGF2BP3 construct79 used for the overexpression of IGF2BP3 was a gift from Thomas Tuschl (Addgene plasmid #19879). pMSCVPIGIMP-1-short construct80, used for overexpression of IGF2BP1, was a gift from David Bartel (Addgene plasmid #21659). pcDNA3-GFP-IMP2-2, used for overexpression of IGF2BP281, was a gift from Alexandra Kiemer (Addgene plasmid #42175). The list of all primers used in the study is available in a Supplementary Table 1.

In vitro transcription
Biotinylated RNA probes were prepared by in vitro transcription on the FMR1-99CGG and FMR1-delCGGs constructs containing T7 polymerase promoter upstream to 5’ UTR FMR1 sequence, and linearized by AvrII enzyme (New England Biolabs). Template for control GC rich RNA was obtained by PCR amplification of TMEM107 gene fragment from HEK293T cDNA and adding T7 polymerase promoter by PCR. One µg of template DNA was used per 10 µL of transcription reaction containing 1x transcription buffer (Promega), 4U of T7 polymerase (Promega), 10 mM DTT, 10 U of RNAsin (Promega), rNTPs (0.5 mM each), rCTP was mixed with Biotin-14-CTP analog (Invitrogen) in 10:1 ratio, to incorporate biotinylated cytidines in a random manner. Reaction was performed at 37°C for 2h. Transcribed RNA was precipitated using sodium acetate and resuspended in DNAse and RNase free water. All in vitro transcription reaction products were analyzed on 1% agarose gels with ethidium bromide (0.5 µg/ml) run in 1x Tris-Borate-EDTA buffer at 70V for 2h.

For electromobility shift assay, RNA fragments were prepared in transcription reaction in 50 µL containing DNA template (~1 µg), 4 mM of each ribonucleotide (Promega), 1x transcription buffer (Promega), 10 U of T7 polymerase (Promega), 10 mM DTT, 10 U of RNAsin (Promega). Reaction was performed at 37°C for 2h. Transcripts were submitted to DNAse treatment (Invitrogen), incubated with 1U of Alkaline Phosphatase at 37°C for 10 min, purified and concentrated on spin columns (A&A Biotechnology).

Template RNA for in vitro translation was obtained using mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen). FMR1-16CGG construct was linearized using PmlI enzyme (New England Biolabs). The reaction was assembled at room temperature according to the manufacturer’s instructions and performed at 37°C for 1.5h. The transcribed RNA was treated with Turbo DNase (Invitrogen), purified using Clean-Up RNA Concentrator (A&A Biotechnology) and denatured (65°C for 3 minutes) immediately before in vitro translation.

Biotinylated RNA-protein pull down
Cellular extracts were prepared by lysing 2.5×106 SH-SY5Y or HEK293T cells in 200 µL of lysis buffer containing 50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% Na-deoxycholate supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific) for 30 min on ice. Lysates were cleared by centrifugation and 200 µL of supernatant was incubated for 20 min at 21°C with 5 µg of RNA in 200 µL of 2x TENT buffer (100 mM Tris pH 7.8, 2 mM EDTA, 500 mM NaCl, 0.1% Tween) supplemented with RNAsin (Promega). RNA-protein complexes were then incubated with MyOne Streptavidin T1 DynaBeads (Thermo Fisher Scientific) for 20 min, followed by washing steps in 1x TENT buffer. For SDS-PAGE and Western blot analysis, bound proteins were released by heat denaturation in Bolt LDS Sample Buffer (Thermo Fisher Scientific) at 95°C for 10 min. For MS analysis, bound proteins were released by incubation in digestion buffer containing 100 mM Tris HCl, 6M Urea, 2M Thiourea, 2% ASB-14, at 37°C for 10 min.

SDS-PAGE and Western blot (WB)
Harvested 0.24×106 HEK293T or HeLa cells or 0.5×106 SH-SY5Y cells were lysed in lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% Na-deoxycholate) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific) for 30 min on ice, followed by sonication and centrifugation at 14,000g for 10 min at 4°C. Extracted proteins were denatured in Bolt LDS Sample Buffer mixed with Bolt Reducing agent (Thermo Fisher Scientific) at 95°C for 5 min. Proteins were separated in Mini Gel Tank (Invitrogen) using Bolt™ 4–12% Bis-Tris Plus gels (Thermo Fisher Scientific) in Bolt™ MES SDS Running Buffer (Thermo Fisher Scientific). For silver staining analysis, gels were stained with Silver Stain for Mass Spectrometry kit (Pierce) according to the manufacturer’s instructions.

For WB, proteins were electroblotted to PVDF membrane (0.2 µm, GE Healthcare) for 1h at 100V in ice-cold Bolt™ Transfer Buffer (Thermo Fisher Scientific). Membranes were blocked in room temperature for 1h in 5% skim milk (Sigma) in Tris-buffered saline with 0.1% Tween-20 (TBS-T). Incubation with antibodies was performed in following conditions: rabbit anti-FMRP antibody (ab17722, Abcam) 1:1000, rabbit anti-IGF2BP3 antibody (ab177477, Abcam) 1:1000, anti-FMRpolyG antibody (MABN1788, Sigma) 1:1000, anti-Vinculin (sc-73614 HRP, Santa Cruz Biotechnology) 1:4000, anti-GAPDH antibody (sc-47724 HRP, Santa Cruz Biotechnology) 1:2000, diluted in 5% skim milk in TBS-T for 16h at 4°C. Membranes were washed in TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies, anti-rabbit (AS09 602, Agrisera) 1:4000 in TBS-T or anti-mouse (A9044, Sigma) 1:10,000 in TBS-T for 1h at RT. Membranes were washed in TBS and covered with Immobilon Forte Western HRP Substrate (Sigma) according to the manufacturer’s instructions. Images were captured with the use of G:Box Chemi-XR5 (Syngene) and ChemiDoc Imaging System (BioRad) and quantified using GeneTools 4.02 (Syngene) or Image Lab (BioRad), respectively. Relative protein level was normalized to Vinculin.

Proteomic digestion
Proteins that bound to biotinylated RNA probes (FMR1-99CGG RNA, FMR1-delCGG RNA and GC-rich RNA as a control) were eluted in digestion buffer as described above. Each dataset included three independent pull-downs (N=3). Then, samples were reduced by the addition of 3 µL of 5M dithioerythritol (Sigma) for 1h at RT, and alkylated with 6 µL of 5M iodiacetamide (Sigma) for 30 min at RT in dark. After this step, 1 µg of Trypsin/Lys-C (Promega) solution was added, followed by incubation for 3h at 37°C. Then, sterile MilliQ water was added to the samples and digestion was continued at 37°C for 16h. After digestion, samples were desalted using solid phase extraction (SPE). 180 µL of 0.2% trifluoroacetic acid (Sigma) was added into every sample prior to the SPE. Samples were purified using C18 Isolute columns (Biotage) according to the manufacturer’s instructions. Collected peptides were dried in speed-vac and submitted to proteomic analysis.

Mass spectrometry (MS) analysis
LC-MS analysis was performed in Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a Street, 02-106 Warsaw, Poland. The samples were analyzed using a nanoAcquity UPLC system (Waters) connected to a Q Exactive mass spectrometer (Thermo Scientific). Peptides were trapped on a C18 precolumn (180 µm x 20 mm, Waters) with 0.1% formic acid (FA) in water as the mobile phase. Then, transferred to a nanoAcquity BEH C18 column (75 µm x 250 mm, 1.7 µm, Waters) using an acetonitrile (ACN) gradient (0–35% ACN over 160 min) with 0.1% FA at a flow rate of 250 nL/min. Data was acquired in data-dependent mode, selecting the top 12 precursors for MS2 within an isolation window of 3 m/z. Full MS scans were conducted for 300–2000 m/z at resolution of 70,000, with a maximum injection time of 60 ms and an AGC target value of 1e6. MS2 scans were performed at a resolution of 17500, with a maximum injection time of 300 ms and an AGC target of 2e5. Dynamic exclusion was applied for 30 s.

MS data analysis
Raw data obtained from the LC-MS/MS runs were analyzed using MaxQuant v2.6.4.0 using the label-free quantification (LFQ) with default parameters. UniProtKB database for reviewed human canonical and isoform proteins of May 2023 was used. The false discovery rate (FDR) at the peptide spectrum matches and protein level was set to 0.01; variable peptide modifications: oxidation (M) and acetyl (N-term), fixed modification: carbamidomethyl (C), two missed cleavages were allowed. Statistical analyses were performed using Perseus software v2.1.2.0 after filtering for “reverse”, “contaminant” and “only identified by site” proteins. The LFQ intensity was logarithmized (log2[x]), and imputation of missing values was performed with a normal distribution (width=0.3; shift=1.8). Proteomes were compared using t-test statistics with a permutation-based FDR of 5% and P-values < 0.05 were considered to be statistically significant.

Gene Ontology (GO) analysis
Lists of proteins that were identified by MS as binding to FMR1-99CGG, FMR1-delCGGs, and GC-rich RNAs were submitted to gene ontology (GO) analysis using Panther82 software, released 2024-01-17. Analysis type was Statistical Overrepresentation test, with annotation sets: GO biological process complete, GO molecular function complete, and GO cellular component complete. Used test type was Fisher’s exact with the use of Bonferroni correction for multiple testing.

Electrophoretic Mobility Shift Assay (EMSA)
RNA fragments were prepared using in vitro transcription, as described above. For 5’-end radiolabeling, 2 pmol of RNA was incubated with 2 pmol of [\gamma-P32]ATP, 1U of RNasin (Promega), and 10U of T4 polynucleotide kinase (Thermo Scientific) in 1x reaction buffer in a total volume of 10 µL and at 37°C for 30 min. The excess of [\gamma-P32]ATP was removed on spin columns (A&A Biotechnology). EMSA was carried out by incubating 5’ radiolabelled RNA with recombinant IGF2BP3 protein (Origene) of indicated concentrations (ranging from 0 to 100 nM) in a volume of 10 µL, in buffer containing 10 mM Tris-Cl pH 7.5, 50 mM KCl, 1 mM EDTA, 0.05% Triton X, 5% Glycerol and 1 mM DTT, and incubated at 37°C for 20 min. The samples were run on a native 6% polyacrylamide gel in 0.5x TBE at 100V for 1h. The gel was subsequently dried, and the signal was detected overnight, visualized on phosphoimager Amersham Typhoon IP (GE Healthcare), and quantified using Multi Gauge 3.0 software (FujiFilm).

Filter Binding Assay (FBA)
FBA was carried out by incubating 5’-end radiolabelled RNA with recombinant IGF2BP3 protein (Origene) of indicated concentrations (ranging from 0 to 100 nM) in a volume of 10 µL, in buffer A containing 10 mM Tris-Cl pH 7.5, 50 mM KCl, 1 mM EDTA, 0.05% Triton X, 5% Glycerol and 1 mM DTT, and incubated at 37°C for 20 min. Next, samples were loaded under vacuum-induced pressure onto two membranes: nitrocellulose (Protran BA 85, Whatman) and nylon (Hybond™ N+, Amersham) placed in dot blotter apparatus. Following wash with buffer A, RNA- protein complexes remained on the top nitrocellulose membrane, while free RNA was retained on a nylon membrane. The signal from membranes was detected O/N, visualized on phosphoimager Amersham Typhoon IP (GE Healthcare) and quantified using Multi Gauge software (FujiFilm). The dissociation constant (Kd) of the RNA/IGF2BP3 interaction was calculated in the GraphPad program using the following equation: one site specific binding curve (Y=Bmax*X/(Kd+X)).

Cell culture and transfection
The human HEK293T, HeLa and SH-SY5Y cells were grown in a high glucose DMEM medium with L-Glutamine (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1x antibiotic/antimycotic solution (Sigma). S-95xCGG and S-16xCGG cells were grown in DMEM medium containing certified tetracycline-free FBS (Biowest). All cells were grown at 37°C in a humidified incubator containing 5% CO2. Cell lines were routinely tested for mycoplasma using mycostrip test (Invivogen) and found negative.

siRNA were delivered by reverse transfection protocol using jetPRIME® reagent (Polyplus) according to the manufacturer’s instructions. Plasmids were delivered 24h post siRNA silencing using jetPRIME® reagent (Polyplus) and manufacturer’s transfection protocol and harvested 48h post siRNA silencing and 24h post transient plasmids expression. The list of all siRNAs and appropriate concentrations used in the study is available in a Supplementary Table 2.

RNA isolation and quantification of mRNA level
Cells were harvested in TRIReagent (Thermo Fisher Scientific), and total RNA was isolated with Total RNA Zol-Out D (A&A Biotechnology) kit according to the manufacturer’s protocol. 500ng of RNA was reversely transcribed using High Capacity Reverse Transcription Kit with RNAse inhibitors and random primers (Invitrogen). Quantitative real-time RT-PCRs were performed in a QuantStudio 6 Flex System (Thermo Fisher Scientific) using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific) with 5ng of cDNA in each reaction. Transgene FMR1-99CGG mRNAs were amplified with primers F8/R8 with a note that reverse primer was anchored in GFP sequence in order to distinguish endogenously expressed FMR1 transcripts (amplified with primer F9/R9 pair). Reactions were run at 58°C annealing temperature and Ct values were normalized to GAPDH mRNA level (amplified with primer F10/R10 pair). Fold differences in expression level were calculated according to the 2−\Delta\DeltaCt method.

Flow cytometry
Cells were harvested 72h post siRNA delivery and 48h post transient plasmids expression by trypsinization followed by centrifugation. Cell pellets were suspended in 200 µL of PBS. Dead cells were stained with propidium iodide (PI) at final concentration 1 µg/mL and excluded from analysis. Cells were analyzed with Sony MA900 cell sorter. GFP was excited by 488 nm laser and collected after a 525/50 nm band pass (BP) filter. For each sample, 10.000 GFP-positive cells were collected. Threshold for GFP-positive cells was set based on signal from non-transfected cells. Forward scatter high (FSC-H) and forward scatter width (FSC-W) were plotted to permit doublet discrimination.

Microscopic analysis of FMRpolyG-GFP aggregates
FMRpolyG aggregates were detected by fluorescence microscopy as described previously78. Briefly, HeLa cells were grown on 48-well plates, siRNA and plasmid transfections were delivered as described in Cell culture and transfection section. Fourty-eighth hours post siRNA delivery and 24h post FMR1-99CGG delivery, HeLa cells were incubated in standard growth medium with final concentration of 5 µg/mL of Hoechst 33342 (Thermo Fisher Scientific) for 10 min. Images were taken with Axio Observer.Z1 inverted microscope equipped with A-Plan 10×/0.25 Ph1 or LD Plan-Neofluar 20×/0.4 Ph2 objective (Zeiss), Zeiss Colibri 7 excitation band 385/30 nm, emission filter 425/30 nm (Hoechst) and Zeiss Colibri 7 excitation band 469/38 nm, emission filter 514/30 nm (GFP), Zeiss AxioCam 506 camera and ZEN 2.6 pro software. Presented values were quantified from 10 images, number of cells and aggregates were calculated using ImageJ and AggreCount plugin.

Cytoplasm/nucleus fractionation
For fractionation of the nuclei and cytoplasm, HEK293T cells were seeded on T25 flasks, and transfected with siRNA and FMR1-99CGG plasmid as described above. Cells were harvested 24h post plasmid delivery and fractionated using PARIS™ Kit (Invitrogen), according to manufacturer’s instructions. Total, cytoplasmic and nuclear RNA was isolated using Filter Cartridges supplied in the PARIS™ Kit. DNA contamination was removed using TURBO DNA-free™ kit (Invitrogen) according to manufacturer’s instructions. cDNA synthesis and qPCR were performed as described in “RNA isolation and Quantification of mRNA” section.

Apoptosis assay
HeLa cells were grown on 96-well plate and transfected with siIGF2BP3 or siControl at 50 nM concentration using JetPrime reagent (Polypus). 24h post-siRNA transfection, plasmid encoding FMR1-99CGG was delivered to the cells using JetPrime reagent (Polypus). 46h post siRNA and 22h post plasmid delivery, apoptosis was tested with RealTime-Glo™ Annexin V Apoptosis Assay (Promega) according to the manufacturer’s instructions. Luminescence signal corresponding to apoptosis was measured 4h after addition of the reaction mix (50h post siRNA and 26 post-plasmid delivery, respectively), using SPARK microplate reader (TECAN).

In vitro translation
Flexi Rabbit Reticulocyte Lysate System (Promega) was used for in vitro translation. In vitro translation reactions contained 10 nM mRNA, 70% rabbit reticulocyte lysate, 10 µM amino acid mix minus leucine, 10 mM amino acid mix minus methionine, 70 mM KCl, 0.5 mM MgOAc, 0.8 U/µL RNasin (Promega) and recombinant IGF2BP3 protein (Origene) at various concentrations (0, 10, 50 nM). Reactions were performed at 30°C for 60 min and terminated by incubation on ice. Reactions were then sonicated, diluted 1:10 in concentrated Bolt LDS Sample Buffer mixed with Bolt Reducing agent (Thermo Fisher Scientific) and incubated at 70°C for 15 min to denature the proteins. The prepared samples were then analyzed by SDS-PAGE and WB.

iPSC culture
Two human FXTAS iPSC lines, expressing 72 and 83 CGG repeats in the FMR1 5’ UTR, were a kind gift from Nicolas Charlet-Berguerand, and were described earlier17. Legal ethical approval and informed consent from the patients were obtained to generate and reuse these iPSC cells for scientific and research purpose. Two human control iPSC lines, harboring 28 and 29 CGG repeats in the 5’ UTR of FMR1 were reprogrammed in our laboratory using Episomal iPSC Reprogramming Vectors (Thermo Fisher Scientific). iPSCs were cultured in StemFlex (Thermo Fisher Scientific) medium on Geltrex-coated (Thermo Fisher Scientific) plates at 37°C humidified incubator containing 5% CO2. Cells were passaged every 3–4 days using PBS-EDTA. Cell lines were routinely tested for mycoplasma using mycostrip test (Invivogen) and found negative.

iPSC neuronal differentiation
iPSCs were differentiated into neurons as described previously17 with modifications. Briefly, 2 million iPSCs were plated on one Geltrex-coated well (Thermo Fisher Scientific) of 6-well plate in Essential 8 medium (Gibco) with 10 µM Y-27632 dihydrochloride (MedChemExpress). The next day, medium was changed to NFS medium (N2B27 supplemented with 20 ng/mL FGF2 (Peprotech), 0.25 µM LDN-193189 (Sigma) and 10 µM SB431542 (MedChemExpress)), and cells were grown for 10 days with daily medium change. After the appearance of neural rosette, cells were passaged onto poly-L-ornithine (Merck) and laminin (Merck)-coated 48-well plates, and NFS medium was replaced with NSC medium (N2B27 supplemented with 20 ng/mL hBDNF (Peprotech), 1 µM LY-411575 (MedChemExpress) and 0.2 µM Ascorbic Acid (Sigma)). Media change was performed every two days and cells were differentiated for 21 days in NSC medium. For measuring the impact of IGF2BP3 KD on FMR1 level, control siRNA and siRNA targeting IGF2BP3 were delivered by lipofection using RNAiMAX (Thermo Fisher Scientific) after 14 days of differentiation and cells were harvested 60h post siRNA treatment. For measuring the number of FMRpolyG inclusions experiment, control siRNA and siRNA targeting IGF2BP3 were delivered by lipofection using RNAiMAX (Thermo Fisher Scientific) every 7 days, starting from day 7 after beginning of differentiation.

Immunofluorescence
For immunofluorescence, iPSC-derived neurons were grown on poly-L-ornithine (Merck) and laminin (Merck) cell culture chamber slides (Ibidi). After 21 days of differentiation, cells were fixed in 4% paraformaldehyde/PBS for 15 min at room temperature, washed with PBS, and permeabilized in 0.5% Triton X-100/PBS and blocked by incubation for 45 min with 5% normal donkey serum (Jackson ImmunoResearch) 0.1% tween-20 in PBS. Samples were incubated overnight at 4°C with primary antibody against FMRpolyG (Millipore 8FM, 1:100) and MAP2 (ab5392, Abcam, 1:5000). The next day, slides were washed with 0.1% Tween-20 in PBS before incubation with secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 647 for 1h at room temperature, and the excess of secondary antibody was removed by two washes in 0.1% Tween-20 in PBS. The samples were then incubated with Hoechst 33342 (Thermo Fisher Scientific) as a counterstain. Slides were examined using a confocal microscope Leica SP8 equipped with white laser, HyDS detectors, and HC PL APO CS2 40x/1.10 WI objective. Cells were imagined as z-stacks with voxel size 0.57×0.57×0.42 µm. Alexa 488 (FMRpolyG), DAPI, and Alexa 647 (neuronal marker MAP) were imagined as separate channels with fluorochrome-specific excitation and emission settings. All images utilized for aggregates counting were done with the same microscopy settings. FMRpolyG aggregates as high fluorescence intensity objects were identified in Fiji ImageJ by Auto-Threshold (method: Internodes) on Maximum Z-Projection images with Alexa 488 signal. For avoiding non-specific aggregates, only objects identified in 10 µm distance from DAPI-stained nucleus were counted. At least 350 cells per line were imagined for counting aggregates.

Necrosis and apoptosis assay of FXTAS iPSC-derived neurons
Control and FXTAS iPSC-derived neurons were grown on 96-well plate covered with laminin for 10 days and transfected with siIGF2BP3 or siControl at 50 nM concentration using RNAiMAX (Thermo Fisher Scientific). 48h post-siRNA transfection, apoptosis and necrosis were tested with RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay (Promega) according to the manufacturer’s instructions. Luminescence or fluorescence signal corresponding to apoptosis or necrosis, respectively, was measured at different time points starting from 1h after addition of the reaction mix, using SPARK microplate reader (TECAN).

C. elegans maintenance
Experiments conducted using Caenorhabditis elegans are not subjected to institutional ethical approval for animal research. Work was carried out in accordance with established guidelines for handling C. elegans. C. elegans strains were maintained according to standard methods83. Animals were grown on nematode growth medium (NGM) agar plates seeded with Escherichia coli OP50 as a food source. Healthy populations were maintained by transferring worms to fresh OP50-seeded plates every 3–4 days to prevent starvation and overcrowding. To synchronize the worms, we extracted the eggs from gravid adults with bleaching solution (30% (v/v) Sodium hypochlorite solution 5% pure Warchem Catalog number: 7681-52-9), 0.75M KOH). The eggs were left in M9 buffer (42 mM Na2HPO4, 22 mM KH2PO4, 86 mM NaCl, 1 mM MgSO4) without any food for 12–16h, thus generating synchronised, arrested L1 stage worms, which were then plated on plates with bacterial food as described above.

Transgenic animal generation
Transgenic C. elegans animals were generated by microinjection of DNA into the gonads of young adult Bristol N2 worms, following standard procedures84. Injection mixtures contained 100 ng/μL of the plasmid containing FMRpolyG ORF and 20 ng/μL of a co-injection marker (pCFJ104 Pmyo-3::mCherry). Injections were performed using a Narishige micromanipulator and an Eppendorf InjectMan 4 microinjection system.

Injected worms (P0s) were singled onto NGM plates seeded with E. coli OP50 and incubated at 20°C. F1 progeny were screened for the presence of the co-injection marker under a fluorescent dissecting microscope (Nikon SMZ800N). Animals expressing the marker were picked to establish stable transgenic lines carrying extrachromosomal arrays.

Brood size counting
Brood size counting was done according to the protocol described in Gudipati et al.85. C. elegans hermaphrodites were singled onto 55 mm plates seeded with E. coli OP50 at the fourth larval stage and incubated at 25°C. These animals were referred to as P0s. Once egg-laying commenced, P0s were transferred to fresh plates, and this process was repeated until egg-laying had completely stopped. The number of F1 viable progeny was counted. The data was represented as a violin with each dot referring to the total number of viable progenies from a single P0.

Thrashing assay
Locomotor activity of C. elegans was evaluated using a thrashing assay. L4 stage larvae were transferred onto fresh NGM plates seeded with E. coli OP50 and maintained at 25°C for three days, to obtain synchronised 3-day-old adult animals. For thrashing analysis, individual worms were placed into 1 mL of M9 buffer on an empty 55 mm plate. After a 1 min acclimatization period, worms were observed under a dissecting microscope. A single thrash was defined as a complete bending movement in which the head, mid-body, and tail of the worm were oriented in opposite directions, forming a “C” shape. Thrashing events were manually counted for 1 min using a dissecting microscope (Leica S9E). At least 15–20 animals were scored per condition for each biological replicate. Data was represented as a violin where each dot corresponds to the thrashes per minute of a single 3-day adult animal.

Longevity assay
Age-synchronized L4 animals were transferred to fresh NGM plates seeded with OP50. These plates were labelled as Day 0 plates (the beginning of the experiment) and maintained at 25°C. About 220–235 animals were scored for each genotype. The animals were transferred to fresh plates every other day until the end of their reproductive phase to prevent mixing up with the progeny. Animals were scored daily for survival by gently poking their heads with a platinum worm pick. If the animals did not respond to multiple poking, they were scored as dead and were removed from the plate. Animals missing from the plate or showing bagging and bursting phenotypes were scored as censored. Survival data were compiled and analysed in GraphPad Prism version 9 using the Kaplan–Meier method, and statistical significance was assessed with the log-rank test.

C. elegans image acquisition
Animals were mounted on a 3% w/v agarose pad on a clean glass slide containing 10 μL of 10 mM levamisole solution. Images were acquired on a Nikon ECLIPSE NI-E 931349 using NIS Elements software. The Fluorescent and Differential Interference Contrast (DIC) images were obtained with a 40x oil immersion objective. Selected representative images were processed using the Fiji software.

Fluorescence lifetime images of FMRpolyG-GFP expressed in the head of 99xCGG model were obtained with Leica SP8 confocal microscope equipped with FAst Lifetime CONtrast (FALCON) module. HC PL APO CS2 40x/1.10 WI objective was used. GFP was excited with 488 nm and detected with HyD S detector in 494-525 nm range. Lifetimes from pharynx were marked with ROI and calculated in LasX FLIM module by fitting with single exponential in reconvolution fit model. For FMRpolyG fluorescence measurements, 99xCGG and 99xCGG; imph-1\Delta animals were imaged as z-stacks. The same settings of imaging were applied for all C. elegans genotypes. The EGFP intensity (Raw Int Den; ImageJ) was measured in pharynx area of 26 animals for each strain, from Sum Intensity Projection images.

Statistics and reproducibility
All data obtained in this study were processed and analyzed using Microsoft Excel and GraphPad Prism. Statistical analysis of all experiments performed on cell lines was done using unpaired two-tailed Student’s t-test. Tests that result in P<0.05 have been reported to be statistically significant. The symbols; *,**,***,**** represent values of P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Error bars represent standard deviation (SD). All cellular and in vitro experiments presented in this work were repeated at least two times with similar results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Fragile X Tremor and Ataxia Syndrome (FXTAS) is a rare neurodegenerative disease caused by an expansion of CGG repeats located within the 5’UTR of the FMR1 gene. These CGG repeats are translated into a novel protein, FMRpolyG, which is prone to form cellular inclusions, and which expression is toxic in cell and animal models. Importantly, translation of these repeats depends on initiation at near-cognate start codons (GUG, ACG) located upstream of the repeats. Fidelity of translation initiation at near cognate codons vs canonical AUG start sites is closely regulated by several proteins (eIF1, eIF 2 and eIF5, among others), and thus of potential clinical interest in FXTAS to modulate expression of the toxic FMRpolyG protein.\nIn that aspect, Baud and collaborators found that the IGF2BP RNA binding proteins binds to the FMR1 5’UTR sequence and regulate FMRpolyG expression. This is an important finding as it open routes toward identifying a therapeutic approach for this devastating syndrome. Overall, presented data are solid and clear, experiments are technically well controlled, and the results are novel and of general interest for people working on FXTAS and/or on regulation of translation initiation. However, interest for this work is tempered by several points:\nMain comments:\n- All data have been generated in vitro or using transformed cell lines (HeLa, HEK or SH-SY-5Y), thus this work, especially the use of drugs inhibiting IGF2BP as a potential therapeutic approach, suffers from a lack of validation in physiological iPS FXTAS cell or animal models.\n- IGF2BP is a family of 3 proteins, hence effects of overexpressing or siRNA-mediated depletion of IGF2BP3 in Figure 2 could be fused to the analysis of IGF2BP1 & 2 in Figure 4. Importantly, siRNA-depletion needs to consider compensation between IGF2BP proteins, and a depletion of 2 or all 3 members by siRNA should be investigated. This is especially important as according to database, RNA expression of these proteins change according to cell type: only IGF2BP3 mRNA is expressed in SH-SY-5Y, but both IGF2BP1 and 3 transcripts are expressed in HeLa and all three are predicted to be expressed in HEK293, etc.). Thus, levels of these 3 proteins in the cell lines analyzed by the authors (with and without their corresponding siRNA treatment) should be presented. Moreover, out of the 3 IGFBP proteins, only IGF2BP2 mRNA is predicted to be expressed in human brain, and only weakly (~10 nPTM in human cerebellum), thus this could be discussed for a future potential therapeutic strategy. Alternatively, as mRNA and protein levels not always correlate, a western on various human or mouse tissue, including the ones affected in FXTAS, showing expression of these protein would strengthen a proposed strategy based on decreasing IGF2BP proteins levels.\n- Minor comments: the RNA structure presented in figure 3C would gain in clarity with symbols or colors indicating FMRpolyG and its near-cognate codons different from the color or symbol of other proteins/RAN product and their start sites. Same comment for the CA element required for IGF2BP binding, etc.\n- Finally, various recent reports (including by the same Sobczak’s group) show modulation of CGG repeat translation at the initiation, elongation or degradation level, notably by PSMB5 (Kong et al., 2022), RPS25/26 (Tutak et al., 2024), ANKZF1 (Tseng et al., 2024), etc. Thus, it could be of interest to show extend of the IGF2BP effect compared to other reported modulators.", "reviewer_2": "The manuscript by Sobczak and colleagues investigates the role of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in the regulation of noncanonical translation of toxic proteins associated with CGG repeat expansions in the FMR1 gene, a mechanism implicated in fragile X-associated tremor/ataxia syndrome (FXTAS). The study demonstrates that IGF2BP3 binds to the 5’ untranslated region (UTR) of FMR1 mRNA, specifically near expanded CGG repeats, and enhances repeat-associated non-AUG (RAN) translation, which produces neurotoxic polyglycine proteins (FMRpolyG).\nBelow are some of my specific comments:\n1. In Vivo Validation of IGFBP3 Binding to CGG Repeats: The authors present comprehensive in vitro data showing IGFBP3 binding to CGG repeats; however, without in vivo confirmation of this interaction, the physiological relevance remains uncertain. Demonstrating IGFBP3 binding to CGG repeats within the context of living cells or in a relevant animal model would significantly strengthen their claims. Specifically, assays such as RNA immunoprecipitation (RIP) coupled with qPCR or cross-linking and immunoprecipitation (CLIP) would validate IGFBP3’s interaction with endogenous FMR1 mRNA. This data is essential to rule out potential artifacts from in vitro binding conditions, which may not fully mimic the cellular environment. The absence of in vivo validation leaves a critical gap, questioning the translatability of these findings to biological settings.\n2. Limitations of Overexpression Systems in CGG Translation Assays: The reliance on overexpression systems to study CGG repeat translation limits the applicability of the findings. Overexpression can alter cellular dynamics, potentially leading to non-physiological binding events or artifacts that would not occur at endogenous levels. Thus, it would be critical for the authors to assess RAN translation using native FMR1 transcript levels. Including in vivo or endogenously expressed FMR1 models would validate the observed regulatory effects and reinforce the physiological relevance of IGFBP3’s role in CGG translation. Without this, it is challenging to determine if the reported effects are an artifact of overexpression.\n3. Specificity of Small Molecule Inhibitors on IGF2BP Paralogs: While the authors indicate that small molecule inhibitors reduce IGFBP3 expression, it remains unclear whether these inhibitors selectively target IGFBP3 or also affect IGF2BP1 and IGF2BP2. Given the close structural similarity among IGF2BP family members, cross-reactivity is likely, and off-target effects could complicate the interpretation of results. Additional experiments are needed to dissect whether these inhibitors selectively target IGFBP3 without interfering with other paralogs. For example, parallel knockdown or CRISPR knockout experiments of IGF2BP1 and IGF2BP2 would clarify if these paralogs contribute similarly to CGG repeat translation. This analysis needs to be more robust to the therapeutic implications of their findings, as off-target effects may reduce the efficacy or safety of IGFBP3 inhibitors in clinical applications.\n4. Mechanistic Insight into IGFBP3’s Role in RAN Translation: The manuscript presents IGFBP3 as a regulator of RAN translation, yet it does not sufficiently explore the mechanistic basis underlying this regulation. Specifically, further investigation into whether IGFBP3 impacts translation initiation factors, ribosomal recruitment, or RNA secondary structures would provide greater mechanistic clarity. This would also address whether IGFBP3’s influence is generalizable across other noncanonical translation events or specific to CGG repeats. Such insights are crucial for understanding the broader implications of IGFBP3 function in RNA biology and neurodegenerative disorders.\n5. Experimental Controls and Replicability: There needs to be more discussion regarding experimental controls and biological replicates, particularly for complex assays like in vitro translation and protein-RNA interaction studies. Including appropriate controls—such as non-targeted RNA for pull-down assays and non-binding RNA mutants—would strengthen the reliability of the data. Additionally, clear reporting of the number of biological replicates would enhance reproducibility and lend robustness to the findings.", "reviewer_3": "The study explores the role of IGF2BP3, in aiding the translation of FMR1 mRNA with expanded CGG repeats. Using RNA-pull down followed by mass spectrometry and western blots, the authors identified IGF2BP3 binds to FMR1 mRNA probs either with or without CGG repeats. IGF2BP3 knockdown decreased the protein level of FMRpolyG but increased mRNA level, while OE of IGF2BP3 increased the protein level of FMR plyG but no changes on mRNA level. Then, by replacing the near-cognate ACG codon with AUG in FMRplyG mRNA as a mutant FMR16xG, OE or KD of IGF2BP2 does not affect either protein or mRNA level of FMR16xG. Next, the authors showed that other IGF2BP family proteins including IGF2BP2 and IGF2BP1 also have similar effects in aiding FMRpolyG biosynthesis. Targeting IGF2BP3 by some BET inhibitors showed decreased protein levels of IGF2BP3 as well as FMR99xG. The whole part of the work stands overall on less solid ground, with unclear rationale for looking at sometimes FMR16xG but sometimes FMR99xG expression levels and with insufficient controls to determine that this is indeed the functional ability of IGF2BP3 to modulate FMRpolyG expression. In general, no experiment in the study is up to current technological standards, and no robust conclusion can be drawn in the absence of unbiased readouts, including at the very least measuring the levels of endogenous FMR1, FMR16xG, FMR96xG simultaneously upon KD or OE of IGF2BP2 in the key experiments, as well as the rescue experiments to confirm FMRpolyG is the essential target of IGF2BP2. Additional comments below, in no particular order of importance.\nIn figure 1, the authors concluded that IGF2BP3 binds to the FMR1 5’UTR in a CGG-independent manner. To be convinced, biotinylated RNA probes designed with expanded CGG repeats but without FMR1 5’ UTR region should be used as a negative control instead of GC-rich RNA in figure 1A, as well as for all the protein binding assay experiments in figure 1B-D.\nFigure1D, the authors turned to use FMR1-5’utr RNA containing 16CGG repeats in filter binding assay. Why not use FMR1-99CGG and FMR1-delCGG in this binding assay and do a comparison? What is the rational to use FMR1-16CGG as research objective suddenly? In addition, only showing one Kd value means nothing. Please show the Kd value with either one negative or positive control.\nFigure 2A, if as authors concluded that IGF2BP3 binds to FMRP in a CGG-independent manner, why is there no influence on normal FMRP protein level? how was the mRNA level of endogenous FMRP? The authors need to provide additional data to clarify this point.\nFigure 2D-F, the study should also perform with FMR16xG. Additionally, the apoptosis induced by FMR99xG upon KD of IGF2BP3 is not convinced unless rescue experiments are performed.\nRIP-seq should be performed to find the exact binding sites in FMRpolyG mRNA. Otherwise, can authors check the RIP-seq public data to find the motif?\nAUG-FMR16xG, normal FMRP should also be tested in figure 3A as controls.\nIn fig 3B, more IGF2BP3 concentrations should be added to confirm a dose-dependent manner.\nIn figure 3C, no protein or mRNA level of mut16xG protein changed upon IGF2BP3 knockdown, which is not direct evidence to show mut16xG does not bind to IGF2BP2. RNA pull down or RIP-qPCR need to be performed.\nFigure 4 shows IGF2BP paralogs regulate the protein level of FMRpolyG. What is the rationale to look for other IGF2BP paralogs??? Does mass spectrometry data also show the binding of FMRpolyG with IGF2BP family? Or, if no direct binding? What is the mechanism?\nIn figure5, all the inhibitors that the authors used are not directly targeting IGF2BP3, which made this part meaningless.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response do the Reviewer Comments\nWe thank Reviewers for the critical comments and experimental suggestions, which were instrumental in enhancing the quality of our research. We considered most of the advices in the revised version of the manuscript, which allowed us to significantly improve our work and further support our statement that IGF2BP proteins regulate the noncanonical translation of toxic polyglycine protein (FMRpolyG) from mutant FMR1 mRNA.\nWe have addressed all specific concerns below and made changes to the new version of the manuscript (indicated in red).\nReviewers' comments:\nReviewer #1 (Remarks to the Author):\nFragile X Tremor and Ataxia Syndrome (FXTAS) is a rare neurodegenerative disease caused by an expansion of CGG repeats located within the 5’UTR of the FMR1 gene. These CGG repeats are translated into a novel protein, FMRpolyG, which is prone to form cellular inclusions, and which expression is toxic in cell and animal models. Importantly, translation of these repeats depends on initiation at near-cognate start codons (GUG, ACG) located upstream of the repeats. Fidelity of translation initiation at near cognate codons vs canonical AUG start sites is closely regulated by several proteins (eIF1, eIF 2 and eIF5, among others), and thus of potential clinical interest in FXTAS to modulate expression of the toxic FMRpolyG protein.\nIn that aspect, Baud and collaborators found that the IGF2BP RNA binding proteins binds to the FMR1 5’UTR sequence and regulate FMRpolyG expression. This is an important finding as it open routes toward identifying a therapeutic approach for this devastating syndrome. Overall, presented data are solid and clear, experiments are technically well controlled, and the results are novel and of general interest for people working on FXTAS and/or on regulation of translation initiation. However, interest for this work is tempered by several points:\nMain comments:\n1. All data have been generated in vitro or using transformed cell lines (HeLa, HEK or SH-SY-5Y), thus this work, especially the use of drugs inhibiting IGF2BP as a potential therapeutic approach, suffers from a lack of validation in physiological iPS FXTAS cell or animal models.\nWe thank Reviewer for this crucial remark. In the current version of the manuscript, we have validated our findings in FXTAS patient-derived cells (fibroblasts, iPSC-derived neuronal progenitor cells and iPSC-derived neurons)- a new paragraph IGF2BP3 regulates the level of FMR1 mRNA in FXTAS patient derived cells; Figure 6. In these models, we observed that silencing of IGF2BP3 resulted in increased relative FMR1 mRNA levels, similarly as in cell lines with stable or transient expression of FMR99xG, what we showed in previous version of the manuscript. Importantly, we observed that IGF2BP3 KD rescued the necrosis phenotype in FXTAS iPSC-derived neurons. Additionally, we tested whether silencing of IGF2BP3 will decrease the number of FMRpolyG inclusions in FXTAS iPSC-derived neurons, and observed small but non-significant decrease (Fig. 6D). This can be explained by the fact that in this experimental set-up, siRNA targeting IGF2BP3 was added in cyclic manner, every 7 days, starting from day 7 after beginning of differentiation, thus the low level of IGF2BP3 was not constant during the experiment and in average was decreased by around 30% when comparing to control (Fig. S6D). On the other hand, IGF2BP3 level was decreased by around 70% comparing to control (Fig. S6B) in iPSC-drived neurons which were harvested 60h post siRNA treatment,. We have also observed that BET inhibitors decreased the toxicity of CGGexp in FXTAS iPSC-derived neurons, but not in controls (Fig. S6G).\nAdditionally, we have generated a novel FXPAC animal model- C. elegans expressing RNA of human FMR1 5'UTR with 99 CGG repeats, fused to GFP at the 3’ end, which exhibited phenotypic defects, described in a new paragraph imph-1/IGF2BP disruption rescues 99xCGG phenotype in Caenorhabditis elegans; Figure 7. We observed that these phenotypes were rescued, when we crossed the 99xCGG strain with strain imph-1Δ, characterized by disrupted expression of IGF2BP ortholog. What is more, in the 99xCGG; imph-1Δ genetic background, we observed an increase in FMR1-99CGG RNA levels compared to the 99xCGG strain alone (Fig. 7C), consistent with the upregulation seen in FXTAS fibroblast and iPSC-derived neurons (Fig. 6A & 6B).\n2. IGF2BP is a family of 3 proteins, hence effects of overexpressing or siRNA-mediated depletion of IGF2BP3 in Figure 2 could be fused to the analysis of IGF2BP1 & 2 in Figure 4. Importantly, siRNA-depletion needs to consider compensation between IGF2BP proteins, and a depletion of 2 or all 3 members by siRNA should be investigated. This is especially important as according to database, RNA expression of these proteins change according to cell type: only IGF2BP3 mRNA is expressed in SH-SY-5Y, but both IGF2BP1 and 3 transcripts are expressed in HeLa and all three are predicted to be expressed in HEK293, etc.). Thus, levels of these 3 proteins in the cell lines analyzed by the authors (with and without their corresponding siRNA treatment) should be presented. Moreover, out of the 3 IGFBP proteins, only IGF2BP2 mRNA is predicted to be expressed in human brain, and only weakly (~10 nPTM in human cerebellum), thus this could be discussed for a future potential therapeutic strategy. Alternatively, as mRNA and protein levels not always correlate, a western on various human or mouse tissue, including the ones affected in FXTAS, showing expression of these protein would strengthen a proposed strategy based on decreasing IGF2BP proteins levels.\nWe thank Reviewer for this comment. In our manuscript we focus mostly on the role of IGF2BP3 paralog, therefore we decided to keep Figures 2 and 4 as separate parts, which, in our opinion, facilitates understanding of our work.\nIn the current version of the manuscript we investigated whether depletion of IGF2BP3 could be compensated by the increase in expression of another IGF2BP paralog and found, that only IGF2BP1 expression increases upon IGF2BP3 KD (Figure 4D). We also included the analysis of combined depletion of two or all three IGF2BP paralogs (Figure 4E), suggesting that IGF2BP2 and IGF2BP3 contribute most significantly to the regulation of FMRpolyG levels. Additionally, we now included the analysis of impact of BET inhibitors on the levels of IGF2BP1 and IGF2BP2 (Figure S5D). These changes, suggested by Reviewer, add a broader insight into the role of IGF2BP paralogs on regulation of FMRpolyG level.\n3. Minor comments: the RNA structure presented in figure 3C would gain in clarity with symbols or colors indicating FMRpolyG and its near-cognate codons different from the color or symbol of other proteins/RAN product and their start sites. Same comment for the CA element required for IGF2BP binding, etc.\nWe have included changes suggested by the Reviewer in the current version of the manuscript.\n4. Finally, various recent reports (including by the same Sobczak’s group) show modulation of CGG repeat translation at the initiation, elongation or degradation level, notably by PSMB5 (Kong et al., 2022), RPS25/26 (Tutak et al., 2024), ANKZF1 (Tseng et al., 2024), etc. Thus, it could be of interest to show extend of the IGF2BP effect compared to other reported modulators.\nAs suggested, in the current version of the manuscript, we discuss different mechanisms of RAN translation modulation more extensively.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Sobczak and colleagues investigates the role of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in the regulation of noncanonical translation of toxic proteins associated with CGG repeat expansions in the FMR1 gene, a mechanism implicated in fragile X-associated tremor/ataxia syndrome (FXTAS). The study demonstrates that IGF2BP3 binds to the 5’ untranslated region (UTR) of FMR1 mRNA, specifically near expanded CGG repeats, and enhances repeat-associated non-AUG (RAN) translation, which produces neurotoxic polyglycine proteins (FMRpolyG).\nBelow are some of my specific comments:\n1. In Vivo Validation of IGFBP3 Binding to CGG Repeats: The authors present comprehensive in vitro data showing IGFBP3 binding to CGG repeats; however, without in vivo confirmation of this interaction, the physiological relevance remains uncertain. Demonstrating IGFBP3 binding to CGG repeats within the context of living cells or in a relevant animal model would significantly strengthen their claims. Specifically, assays such as RNA immunoprecipitation (RIP) coupled with qPCR or cross-linking and immunoprecipitation (CLIP) would validate IGFBP3’s interaction with endogenous FMR1 mRNA. This data is essential to rule out potential artifacts from in vitro binding conditions, which may not fully mimic the cellular environment. The absence of in vivo validation leaves a critical gap, questioning the translatability of these findings to biological settings.\nWe thank Reviewer for this valuable suggestion. In the current version of the manuscript, we included eCLIP analysis of IGF2BP3 binding to 5’UTR of FMR1 transcript in HepG2 cells, deposited in the ENCODE eCLIP resource (Nostrand et al., Nature Methods 2016). In this data, IGF2BP3 enrichment can be observed at the FMR1 5’UTR (Figure S1G), which validates our in vitro data.\n2. Limitations of Overexpression Systems in CGG Translation Assays: The reliance on overexpression systems to study CGG repeat translation limits the applicability of the findings. Overexpression can alter cellular dynamics, potentially leading to non-physiological binding events or artifacts that would not occur at endogenous levels. Thus, it would be critical for the authors to assess RAN translation using native FMR1 transcript levels. Including in vivo or endogenously expressed FMR1 models would validate the observed regulatory effects and reinforce the physiological relevance of IGFBP3’s role in CGG translation. Without this, it is challenging to determine if the reported effects are an artifact of overexpression.\nWe thank Reviewer for this crucial remark. In the current version of the manuscript, we have validated our findings in FXTAS patient-derived cells (fibroblasts, iPSC-derived neuronal progenitor cells and iPSC-derived neurons)- a new paragraph IGF2BP3 regulates the level of FMR1 mRNA in FXTAS patient derived cells; Figure 6. In these models, we observed that silencing of IGF2BP3 resulted in increased relative FMR1 mRNA levels, similarly as in cell lines with stable or transient expression of FMR99xG, what we showed in previous version of the manuscript. Importantly, we observed that IGF2BP3 KD rescued the necrosis phenotype in FXTAS iPSC-derived neurons. Additionally, we tested whether silencing of IGF2BP3 will decrease the number of FMRpolyG inclusions in FXTAS iPSC-derived neurons, and observed small but non-significant decrease (Fig. 6D). This can be explained by the fact that in this experimental set-up, siRNA targeting IGF2BP3 was added in cyclic manner, every 7 days, starting from day 7 after beginning of differentiation, thus the low level of IGF2BP3 was not constant during the experiment and in average was decreased by around 30% when comparing to control (Fig. S6D). On the other hand, IGF2BP3 level was decreased by around 70% comparing to control (Fig. S6B) in iPSC-drived neurons which were harvested 60h post siRNA treatment,. We have also observed that BET inhibitors decreased the toxicity of CGGexp in FXTAS iPSC-derived neurons, but not in controls (Fig. S6G).\nAdditionally, we have generated a novel FXPAC animal model- C. elegans expressing RNA of human FMR1 5'UTR with 99 CGG repeats, fused to GFP at the 3’ end, which exhibited phenotypic defects, described in a new paragraph imph-1/IGF2BP disruption rescues 99xCGG phenotype in Caenorhabditis elegans; Figure 7. We observed that these phenotypes were rescued, when we crossed the 99xCGG strain with strain imph-1Δ, characterized by disrupted expression of IGF2BP ortholog. What is more, in the 99xCGG; imph-1Δ genetic background, we observed an increase in FMR1-99CGG RNA levels compared to the 99xCGG strain alone (Fig. 7C), consistent with the upregulation seen in FXTAS fibroblast and iPSC-derived neurons (Fig. 6A & 6B).\n3. Specificity of Small Molecule Inhibitors on IGF2BP Paralogs: While the authors indicate that small molecule inhibitors reduce IGFBP3 expression, it remains unclear whether these inhibitors selectively target IGFBP3 or also affect IGF2BP1 and IGF2BP2. Given the close structural similarity among IGF2BP family members, cross-reactivity is likely, and off-target effects could complicate the interpretation of results. Additional experiments are needed to dissect whether these inhibitors selectively target IGFBP3 without interfering with other paralogs. For example, parallel knockdown or CRISPR knockout experiments of IGF2BP1 and IGF2BP2 would clarify if these paralogs contribute similarly to CGG repeat translation. This analysis needs to be more robust to the therapeutic implications of their findings, as off-target effects may reduce the efficacy or safety of IGFBP3 inhibitors in clinical applications.\nIn the current version of the manuscript, we have included an analysis of the effect of small molecule inhibitors on the expression of all three IGF2BP paralogs (Figure S5D). As supposed by the Reviewer, mRNA level of all paralogs was decreased by administration of BET inhibitors. However, as shown now in Figure 4E, simultaneous KD of IGF2BP1, IGF2BP2 and IGF2BP3 has the greatest impact of FMR99xG expression, which may suggest that even if the level of all paralogs is decreased upon BET inhibitors administration, these small molecules may still have favourable effects in FXTAS cell models. What is more, our new data suggest the beneficial effect of JQ1 and IBET-151 on the apoptosis phenotype in FXTAS iPSC-derived neurons, strengthening our proposition that BET inhibitors could be new therapeutic option, in addition to mostly studied antisense oligonucleotide-based therapy. Nevertheless, off-target effects of BET inhibitors should be taken into account as written in the Discussion.\n4. Mechanistic Insight into IGFBP3’s Role in RAN Translation: The manuscript presents IGFBP3 as a regulator of RAN translation, yet it does not sufficiently explore the mechanistic basis underlying this regulation. Specifically, further investigation into whether IGFBP3 impacts translation initiation factors, ribosomal recruitment, or RNA secondary structures would provide greater mechanistic clarity. This would also address whether IGFBP3’s influence is generalizable across other noncanonical translation events or specific to CGG repeats. Such insights are crucial for understanding the broader implications of IGFBP3 function in RNA biology and neurodegenerative disorders.\nWe thank Reviewer for this comment. We believe that this investigation, although very interesting, is beyond the scope of the manuscript.\n5. Experimental Controls and Replicability: There needs to be more discussion regarding experimental controls and biological replicates, particularly for complex assays like in vitro translation and protein-RNA interaction studies. Including appropriate controls—such as non-targeted RNA for pull-down assays and non-binding RNA mutants—would strengthen the reliability of the data. Additionally, clear reporting of the number of biological replicates would enhance reproducibility and lend robustness to the findings.\nConclusions included in the manuscript are based on at least three independent biological replicates, as stated below each figure. Non-targeted RNA (called “GC rich”) control was included in pull-down assays (shown in Figure 1A-C), while non-targeted UAA control was included for in vitro binding assays: EMSA and FBA (shown in Figure S1F). In the current version of the manuscript, we have also included a novel proteomic data showing that IGF2BP3 was detected in pull-down of FMR1-99CGG RNA bait, but not synthetic 23CGG RNA bait (Figure S1D), which supports our finding that IGF2BP3 requires the appropriate flanking sequence, possibly with CA dinucleotide motif upstream of CGG repeats for binding (Figure 3D).\nReviewer #3 (Remarks to the Author):\nThe study explores the role of IGF2BP3, in aiding the translation of FMR1 mRNA with expanded CGG repeats. Using RNA-pull down followed by mass spectrometry and western blots, the authors identified IGF2BP3 binds to FMR1 mRNA probs either with or without CGG repeats. IGF2BP3 knockdown decreased the protein level of FMRpolyG but increased mRNA level, while OE of IGF2BP3 increased the protein level of FMR plyG but no changes on mRNA level. Then, by replacing the near-cognate ACG codon with AUG in FMRplyG mRNA as a mutant FMR16xG, OE or KD of IGF2BP2 does not affect either protein or mRNA level of FMR16xG. Next, the authors showed that other IGF2BP family proteins including IGF2BP2 and IGF2BP1 also have similar effects in aiding FMRpolyG biosynthesis. Targeting IGF2BP3 by some BET inhibitors showed decreased protein levels of IGF2BP3 as well as FMR99xG.\nThe whole part of the work stands overall on less solid ground, with unclear rationale for looking at sometimes FMR16xG but sometimes FMR99xG expression levels and with insufficient controls to determine that this is indeed the functional ability of IGF2BP3 to modulate FMRpolyG expression. In general, no experiment in the study is up to current technological standards, and no robust conclusion can be drawn in the absence of unbiased readouts, including at the very least measuring the levels of endogenous FMR1, FMR16xG, FMR96xG simultaneously upon KD or OE of IGF2BP2 in the key experiments, as well as the rescue experiments to confirm FMRpolyG is the essential target of IGF2BP2.\nAdditional comments below, in no particular order of importance.\nWe thank Reviewer for this assessment. However, we hope that Reviewer will appreciate the fact that our conclusions are based on experiments using well established techniques, such as pull downs combined with high resolution mass spectrometry, biochemical assays (FBA, EMSA), gene silencing and overexpression in multiple cell models, including now also FXTAS patient-derived cells, that were analysed by western blots, flow cytometry, apoptosis/necrosis assays, confocal microscopy and quantitative real-time PCR. In our experimental design we paid attention to use orthogonal techniques in order to decrease the possibility of biased read-outs and artifacts.\nFinally, as suggested by the Reviewer we performed simultaneous expression of FMR16xG and FMR99xG in cells with KD of IGF2BP3, but we were not able to draw any conclusions from this experiment since we observed overlap of the bands corresponding to both short and long polyglycine repeats (shown below).\n1. In figure 1, the authors concluded that IGF2BP3 binds to the FMR1 5’UTR in a CGG-independent manner. To be convinced, biotinylated RNA probes designed with expanded CGG repeats but without FMR1 5’ UTR region should be used as a negative control instead of GC-rich RNA in figure 1A, as well as for all the protein binding assay experiments in figure 1B-D.\nAs requested by the Reviewer, in the current version of the manuscript, we included a novel proteomic data showing that IGF2BP3 was detected in pull-down of FMR1-99CGG RNA bait, but not synthetic 23CGG RNA bait (Figure S1D), which supports our finding that IGF2BP3 binds to the FMR1 5’UTR independently of the CGG content.\nOur statement that IGF2BP3 can bind to 5’UTR of FMR1 containing short CGG repeats is also supported by eCLIP analysis of IGF2BP3 binding to 5’UTR of FMR1 transcript in HepG2 cells, containing normal size of CGG repeats, deposited in the ENCODE eCLIP resource (Nostrand et al., Nature Methods 2016). In this data, IGF2BP3 enrichment can be observed at the FMR1 5’UTR (Figure S1G).\n2. Figure1D, the authors turned to use FMR1-5’utr RNA containing 16CGG repeats in filter binding assay. Why not use FMR1-99CGG and FMR1-delCGG in this binding assay and do a comparison? What is the rational to use FMR1-16CGG as research objective suddenly? In addition, only showing one Kd value means nothing. Please show the Kd value with either one negative or positive control.\nIndeed, when we noticed that in pull-down assays IGF2BP3 binds to both FMR99xG and FMR1-delCGG (Figure 1C), and IGF2BP3 KD decreases levels of both FMR16xG and FMR99xG (Figure 2A), we decided to use FMR16xG RNA in in vitro assays. This was due to the fact, that gel purification and gel migration of transcripts containing 99 CGG repeats is very challenging, because of the stable secondary structure which hampers migration of the RNA in polyacrylamide. Similarly, mutagenesis and cloning of plasmids containing 99 CGG repeats is very difficult due to often occurring contractions of the repeats, therefore we performed mutagenesis on the FMR16xG plasmid. On the other hand, in IGF2BP KD assays, we tested the effect on both FMR16xG and FMR99xG to confirm in vitro data.\nWith respect to the Kd value of negative control (UAA) for EMSA and FBA assays (Fig. S1F), we were not able to determine the Kd since we did not observe any binding between IGF2BP3 and UAA RNA even in the highest, 100nM protein concentration. We can just say that Kd>>100nM.\n3. Figure 2A, if as authors concluded that IGF2BP3 binds to FMRP in a CGG-independent manner, why is there no influence on normal FMRP protein level? how was the mRNA level of endogenous FMRP? The authors need to provide additional data to clarify this point.\nWe thank Reviewer for this important remark. Based on our data presented in the manuscript, we indeed concluded that IGF2BP3 binds to FMR1 5’UTR independently on the CGG repeats (Figure 1, Figure S1). This finding is also supported by IGF2BP3 eCLIP reads coverage of 5’UTR of FMR1 transcript (Figure S1G).\nAs requested by the Reviewer, we have measured the effect of IGF2BP3 KD on the mRNA level of endogenous FMR1 in cell models with stable expression of FMR99xG (Figure 2A), control and FXTAS patients-derived fibroblasts (Figure 6A) as well as control and FXTAS iPSC-derived neurons (Figure 6B). In all these models, we observed an increase of relative FMR1 mRNA level upon IGF2BP3 silencing, when comparing to control, suggesting that FMR1 is a target of IGF2BP3. However, as Reviewer noticed, IGF2BP3 KD impacted level of RAN-translated FMRpolyG but not FMRP. One possible explanation can be the fact that FMRP is translated from canonical AUG codon, and the translation efficiency is much higher than the efficiency of RAN translation of FMRpolyG. Therefore, binding of IGF2BP3 to FMR1 5’UTR will not influence canonical translation initiation of FMRP from optimal Kozak context. This explanation was added to the current version of the manuscript.\n4. Figure 2D-F, the study should also perform with FMR16xG. Additionally, the apoptosis induced by FMR99xG upon KD of IGF2BP3 is not convinced unless rescue experiments are performed.\nWe thank Reviewer for the comment. However, we were not able to repeat experiments shown in Figures 2D-F for FMR16xG due to the fact that protein containing short polyglycine stretch (FMR16xG) does not form aggregates, in contrast to FMR99xG. We confirmed this fact in control iPSC-derived neurons, harboring 29CGG repeats in the 5’UTR of FMR1 gene (Figure S6G). On the contrary, we observed FMRpolyG aggregates in FXTAS iPSC-derived neurons (Figure 6D). Additionally, in control iPSC-derived neurons, necrosis was not decreased upon IGF2BP3 KD (Figure S6F), suggesting that lack of FMRpolyG aggregates does not incur necrosis, as opposed to FXTAS iPSC-derived neurons, where FMRpolyG aggregates were detected.\n5. RIP-seq should be performed to find the exact binding sites in FMRpolyG mRNA. Otherwise, can authors check the RIP-seq public data to find the motif?\nIGF2BP3 is an RNA binding protein with multiple RNA targets. Schneider et al. (Nature communications, 2019) found that IGF2BP3 protein recognizes RNA containing CA- motifs and GGC core elements (either GGCA or CGGC) separated by appropriate spacing. In the 5’UTR of FMR1 we identified three possible CA- followed by CGGC- motifs (shown in Fig. 3C). In line with that, we analysed the publicly available eCLIP data (Figure S1G) and found significant peaks of IGF2BP3 binding to the 5’UTR of FMR1 mRNA, notably in the region where CA- and CGGC motifs occurs.\n6. AUG-FMR16xG, normal FMRP should also be tested in figure 3A as controls.\nWe show the effect of IGF2BP3 KD on FMRP level in four different biological models: 1) HEK293 cells with stable expression of 16CGG repeats, 2) HEK293 cells with stable expression of 99CGG repeats- (shown Figure 2A), 3) HEK 293 cells with transient expression of 16CGG repeats, and 4) HEK293 cells with transient expression of 99CGG repeats (shown in Figure S2A). In neither case, we observed changes of endogenous FMRP level. These results, together with lack of effect of IGF2BP3 KD on FMRpolyG level produced from canonical AUG codon (Figure 3A), suggest that IGF2BP3 KD will possibly not influence level of FMR16xG produced from AUG codon based on canonical model of translation initiation.\n7. In fig 3B, more IGF2BP3 concentrations should be added to confirm a dose-dependent manner.\nWe apologise the Reviewer for not fulfilling this request, which we now included in the discussion as a limitation of our study. Given the limited time, we decided to focus on validation of our initial findings, obtained in vitro and in overexpression systems, in FXTAS patient-derived cells and in novel FXTAS C. elegans model.\n8. In figure 3C, no protein or mRNA level of mut16xG protein changed upon IGF2BP3 knockdown, which is not direct evidence to show mut16xG does not bind to IGF2BP2. RNA pull down or RIP-qPCR need to be performed.\nWe agree with the Reviewer that lack of this validation is a limitation of our study, which we now included in the discussion. However, we believe that including eCLIP data (Figure S1G) in the current version of the manuscript strengthens our conclusion that IGF2BP3 binds to 5’UTR of FMR1, also in an endogenous context.\n9. Figure 4 shows IGF2BP paralogs regulate the protein level of FMRpolyG. What is the rationale to look for other IGF2BP paralogs??? Does mass spectrometry data also show the binding of FMRpolyG with IGF2BP family? Or, if no direct binding? What is the mechanism?\nIGF2BP paralogs share overall high sequence identity- in the current version of the manuscript we included the alignment of IGF2BP1, IGF2BP2 and IGF2BP3 (Figure S4E) to better show similarities between paralogs. Additionally, IGF2BP paralogs may bind the same RNA target, i.e. CD44, MYC (reviewed in Ramesh-Kumar et al., Seminar in Cancer Biology, 2022). What is more, IGF2BP1 was also identified by us (Tutak et al., eLife 2025) and others (Malik et al., EMBO Mol. Med, 2021; Rosario et al., FASEB J., 2022) in the pull-down screen of proteins binding to mutant FMR1 5’UTR RNA. Together, this prompted us to verify whether all IGF2BP paralogs can regulate FMRpolyG level.\n10. In figure5, all the inhibitors that the authors used are not directly targeting IGF2BP3, which made this part meaningless.\nWe thank Reviewer for this comment. We would like to stress that in our manuscript we do not claim that the effect of BET inhibitors on IGF2BP3 is direct. Rather, we show our results as new interesting treatment possibility, which of course requires further investigation. Our point of view can be supported by the fact that BET inhibitors were used by different research teams to inhibit IGF2BP3 expression in various type of cancers (Mancarella et al., Clin. Cancer Res., 2018; Coleman et al. Sci. Rep., 2019; Dai et al., Nat. Commun. 2024). It was shown that JQ1 impairs binding of bromodomain-containing protein 4 (BRD4) to chromatin in cancer cells, and ChIP-seq analysis revealed binding of BRD4 to IGF2BP3 promoter region in PC3 cells, which could be possible explanation of the negative effect of JQ1 on IGF2BP3 protein level.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank Reviewer for supporting Early Career Researchers by training in the revision process."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors adequately addressed my comments, notably with addition of stat of the art iPS cell models. This work is now suitable for publication.", "reviewer_2": "In this revised manuscript, the authors made efforts to address several concerns raised in the previous review, including the addition of patient-derived cell models and a C. elegans model. While these new data and clarifications strengthen some aspects of the study, I remain concerned about the following points:\n1. There is still no mechanistic evidence demonstrating how IGF2BP3 influences CGG repeat RNA activity or RAN translation. The authors argue that this is beyond the scope of the manuscript; however, given the central claim, mechanistic insight (e.g., effects on translation initiation, ribosomal recruitment, or RNA structure) is essential. The current finding that IGF2BP3 KD increases FMR1 mRNA is insufficient to explain functional consequences at the protein or phenotypic level.\n2. While the inclusion of FXTAS patient iPSC-derived neurons is an improvement, the manuscript does not specify how many independent fragile X premutation iPSC lines were used. Current publication standards typically require at least three independent lines to ensure reproducibility. Furthermore, the reported necrosis phenotype in FXTAS iPSC-derived neurons has not, to my knowledge, been described previously, and there is insufficient discussion or validation of this novel observation.\n3. The newly introduced C. elegans model expressing human FMR1 5′UTR with CGG repeats is intriguing; however, its characterization is insufficient. The authors do not clarify whether the observed toxicity is mediated by the RNA itself or by the translated polyG protein. A more detailed phenotypic and molecular characterization is needed to establish the relevance of this model to FXTAS.\n4. The significantly enriched proteins in Supplemental Data 1 need to be listed.", "reviewer_3": "I can see authors has tried their best to answer my questions and concerns. I have no further concerns.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to the Reviewer Comments\nWe thank Reviewer for the critical comments and experimental suggestions, which were instrumental in enhancing the quality of our research. We considered most of the advices in the revised version of the manuscript, which allowed us to significantly improve our work and further support our statement that IGF2BP proteins regulate the noncanonical translation of toxic polyglycine protein (FMRpolyG) from mutant FMR1 mRNA.\nWe have addressed all specific concerns below and made changes to the new version of the manuscript (indicated in blue).\nReviewer #2 (Remarks to the Author):\nIn this revised manuscript, the authors made efforts to address several concerns raised in the previous review, including the addition of patient-derived cell models and a C. elegans model. While these new data and clarifications strengthen some aspects of the study, I remain concerned about the following points:\n1. There is still no mechanistic evidence demonstrating how IGF2BP3 influences CGG repeat RNA activity or RAN translation. The authors argue that this is beyond the scope of the manuscript; however, given the central claim, mechanistic insight (e.g., effects on translation initiation, ribosomal recruitment, or RNA structure) is essential. The current finding that IGF2BP3 KD increases FMR1 mRNA is insufficient to explain functional consequences at the protein or phenotypic level.\nWe thank Reviewer for this comment. In agreement with Senior Editor handling the manuscript, we did not seek further explanations of the mechanism of IGF2BP3 action on CGG repeat RNA activity or RAN translation. However, we completed the Discussion section with more explicit description of the limitations of our studies with regard to the mechanism by which IGF2BP3 regulated the noncanonical translation of the mutant FMR1 mRNA. We hope that we will be able to address these questions in our further research.\n2. While the inclusion of FXTAS patient iPSC-derived neurons is an improvement, the manuscript does not specify how many independent fragile X premutation iPSC lines were used. Current publication standards typically require at least three independent lines to ensure reproducibility. Furthermore, the reported necrosis phenotype in FXTAS iPSC-derived neurons has not, to my knowledge, been described previously, and there is insufficient discussion or validation of this novel observation.\nWe thank Reviewer for this comment. Indeed, latest publications present at least two or three independent iPSC lines. In our work, we performed experiments on two independent FXTAS cell lines, expressing 81 CGG (FXTAS #1) or 72 CGG (FXTAS #2) repeats (Fig. 6B, S6B, S6D and Fig. 6D, S6H), while experiments shown in Fig. 6C and S6I were performed on one FXTAS cell line (72CGG). In the current version of the manuscript, we clarified these details in the Materials & Methods section, paragraph IGF2BP3 regulates the level of FMR1 mRNA in FXTAS patient derived cells and Figure 6 legend. However, we would like to point out that results presented in Fig.6 are confirmatory to results shown in Figures 2 and 5, further supporting our claims about the role of IGF2BP3 in regulation of CGG RAN translation.\nWe have also referenced and discussed three publications describing the cell death (Sellier et al., 2017), reduced cell viability (Hoem et al., 2019) and neurotoxicity (Wright et al., 2022) caused by products of CGG translation. We believe that there is strong link between these reports and the necrosis phenotype in FXTAS iPSC neurons, which we observed.\nTo further support our claim, we have performed additional necrosis measurements in FXTAS iPSC-derived neurons, using as a positive control the antisense oligonucleotide (ASO) targeting directly CGGexp (ASO-CCG), which was previously published by us (Derbis et al., Nature Communications 2021). This experiment aligns with our findings described in Figure 6, and shows that ASO targeting expanded CGG repeats decrease necrosis in FXTAS iPSC-derived neurons, but not in control neurons. These experiments are an integral part of the findings of other research, which we submitted to Nucleic Acids Research. We are planning to resubmit in a few weeks.\nFigure 1. Administration of antisense oligonucleotide targeting directly CGGexp lowers necrosis rate in FXTAS (72 CGG) iPSC-derived neurons. Necrosis was measured as fluorescence signals (relative fluorescence units). The graph presents relative mean values from N = 5 biologically independent samples treated with ASO Control (blue) or ASO CCG (red), with SDs.\n3. The newly introduced C. elegans model expressing human FMR1 5′UTR with CGG repeats is intriguing; however, its characterization is insufficient. The authors do not clarify whether the observed toxicity is mediated by the RNA itself or by the translated polyG protein. A more detailed phenotypic and molecular characterization is needed to establish the relevance of this model to FXTAS.\nWe thank Reviewer for this comment. At this stage we cannot rule out the possibility that the observed phenotype is due to RNA toxicity. However, the fact that the FMRpolyG mRNA level increases in 99xCGG; imph-1Δ genotype (Fig. 7D) and yet the phenotype of animals is rescued (Fig. 7 E-G), suggest that FMRpolyG is the major contributor to the observed toxicity. This is further supported by our novel data showing that the fluorescence signal of FMRpolyG-GFP decreases in the head region of 99xCGG; imph-1Δ animals, comparing to 99xCGG strain (new Fig. 7C).\nAs requested by the Reviewer, we have also performed additional characterization of our animal model: quantification of FMRpolyG-GFP fluorescence in the head region of 99xCGG and 99xCGG; imph-1Δ animals (new Fig. 7C) and longevity assay of 99xCGG and 99xCGG; imph-1Δ animals (new Fig. 7G). These results further support our statement that toxic effect of rCGGexp expression is significantly reduced upon IGF2BP3 insufficiency.\n4. The significantly enriched proteins in Supplemental Data 1 need to be listed.\nWe thank Reviewer for this comment. Significantly enriched proteins were listed in the previous version of Supplemental Data 1, however to increase the understanding of Datasheets, we have changed the names of Spreadsheets, added colour code of spreadsheets, and marked significantly enriched proteins in bold and borders."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have addressed all my comments and this manuscript is ready for publication.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter