The missense mutation Y65C in PQBP1 causes microcephaly and cognitive deficits through a combination of partial loss-of-function and gain-of-function effects

Domain: neuroscience
Published: 2026-01-08
Editorial outcome: minor_revision

Abstract
The missense mutation Y65C in polyglutamine-binding protein 1 (PQBP1) is associated with Renpenning syndrome, characterized by X-linked intellectual disability and microcephaly. However, the pathogenic mechanism underlying the microcephaly induced by the Y65C mutation remains unclear. In this study, we generated Pqbp1Y65C/Y knock-in male mice and discovered that the Y65C mutation impairs the proliferation of apical progenitors and their subsequent transition to basal progenitors, resulting in microcephaly and cognitive deficits like those observed in Renpenning syndrome patients. This Y65C substitution induces PQBP1 misfolding, which reduces PQBP1 protein levels and consequently impedes apical progenitor proliferation. Unexpectedly, the Y65C mutation also induces a gain-of-function that interferes with the transition from apical to basal progenitors by enhancing interactions with the core components of the mRNA 3’ end processing machinery, thereby preserving proliferative alternative polyadenylation (APA) profiles. Our study demonstrates that a combination of loss-of-function and gain-of-function contributes to the microcephaly caused by the Y65C mutation.

Introduction
Mutations in the polyglutamine-binding protein 1 gene (Pqbp1), located on chromosome Xp11.23, are linked to Renpenning syndrome. This syndrome is characterized by microcephaly, intellectual deficiency, short stature, small testes, and distinct facial dysmorphism1–4. To date, 22 mutations in the Pqbp1 gene have been documented: 6 small deletions1,4–14, 10 missense mutations6,15–22, 3 small insertions1,5,23, and 3 gross deletions2. Moreover, microduplications at Xp11.23p11.22 have been reported in patients with syndromic MR24–27. Notably, the Renpenning syndrome associated with the Y65C missense mutation of Pqbp1 exhibits more severe clinical features than other mutations15,28. The reason for the severity caused by a single amino acid mutation remains elusive.

PQBP1 is predominantly expressed in neural stem progenitor cells and striatal progenitors during mouse embryonic development29–31. Several conditional knockout (cKO) mouse models of the Pqbp1 gene have been created, and studies using these Pqbp1-cKO mice have highlighted the crucial roles of PQBP1 in neural development and cognition30–34. The missense Y65C mutation, identified in Renpenning syndrome, is distinct among the reported Pqbp1 mutations. This mutation alters the conserved tyrosine residue at position 65 within the functional WW domain to a cysteine (Y65C)15. In vitro biochemical studies have shown that this Y65C mutation interferes with the interactions between PQBP1 and its corresponding partners in the splicing complex35,36. Several studies have shown normal levels of mutant PQBP1 protein and dysregulated pre-mRNA splicing in the patient’s lymphoblasts36,37, suggesting that abnormal splicing might be a primary feature of Renpenning syndrome with the Y65C mutation. However, no animal models are currently available for Renpenning syndrome with the Y65C mutation, and the exact pathological mechanism by which this mutation causes Renpenning syndrome remains unclear.

In this study, we developed a Pqbp1Y65C/Y-knock-in mouse model to explore Renpenning syndrome further. Our findings reveal that Pqbp1Y65C/Y mice display microcephaly and cognitive deficits. We demonstrate that the Y65C mutation significantly reduces the PQBP1 protein level, with the partial loss of PQBP1 function impairing apical progenitor proliferation. Unexpectedly, we discovered that the Y65C mutation also induced a gain-of-function that disrupts the transition from apical to basal progenitors by enhancing interactions with the core components of the mRNA 3’ end processing machinery, there by preserving proliferative alternative polyadenylation (APA) profiles. Our results elucidate two previously unrecognized post-transcriptional regulatory mechanisms by which the Y65C mutation causes Renpenning syndrome, offering new insights into the pathogenesis of this disease.

Results
Pqbp1Y65C/Y mice exhibit microcephaly and cognitive deficits
To generate Pqbp1Y65C-knock-in mice, we microinjected Cas9 mRNA, Pqbp1 sgRNA, and a donor oligonucleotide of the Pqbp1-Y65C DNA into zygotes. This procedure resulted in two founder mice with substitutions of tyrosine 65 to cysteine in PQBP1 (Supplementary Fig. 1a, b). The newborn Pqbp1Y65C/Y mice exhibited reduced body weight, brain weight, and decreased cortical area compared to their littermate controls (Fig. 1a–e and Supplementary Fig. 1c–f). Immunostaining analyses revealed a notably thinner neocortex in the newborn PqbpY65C/Y mice compared to their littermate controls (Fig. 1f–h). We further conducted immunostaining using cortical layer-specific markers Tbr1 (Layer VI), Ctip2 (Layer V), and Brn2 (Layer II–IV) to assess the thickness of the cerebral cortex. Although the cortices of newborn Pqbp1Y65C/Y mice showed a normal lamination pattern (Fig. 1f), the thickness of each layer was uniformly reduced in the mutant mice (Fig. 1g–i). Moreover, the proportion of neurons in each layer of the cortex of the mutant mice remained unchanged (Fig. 1j), suggesting no significant abnormalities in the migration and differentiation processes of neurons. The Pqbp1Y65C/Y mice displayed short stature and microcephaly, similar to the major clinical symptoms observed in Renpenning patients.

Next, we performed a behavioral test to evaluate the cognition of Pqbp1Y65C/Y mice at 8–13 weeks. The distances traveled by Pqbp1Y65C/Y mice and the littermate WT controls in the open-field test were comparable (Supplementary Fig. 2a, b), indicating normal locomotion of Pqbp1Y65C/Y mice. However, Pqbp1Y65C/Y mice entered the center less frequently and spent less time in the center than WT controls (Supplementary Fig. 2c). Moreover, the Pqbp1Y65C/Y mice spent significantly less time in the open arms at the elevated plus maze test (Supplementary Fig. 2d–f), suggesting that Pqbp1Y65C/Y mice display increased anxiety. We also performed a novel object-recognition test to evaluate recognition memory (Supplementary Fig. 2g). In the training session, Pqbp1Y65C/Y mice and littermate WT mice explored two identical objects for similar durations (Supplementary Fig. 2h). In the testing session, WT mice spent more time on the novel object, while Pqbp1Y65C/Y mice displayed no preference for either the familiar or novel object (Supplementary Fig. 2i), suggesting impaired recognition memory in Pqbp1Y65C/Y mice. Moreover, we conducted a three-chamber test to evaluate social behavior. We found that the Pqbp1Y65C/Y mice displayed normal social ability, but deficits in social novelty (Supplementary Fig. 2j–n). These social deficits may be associated with underlying cognitive impairments. Our findings suggest that Pqbp1Y65C/Y mice exhibit impaired recognition memory.

Pqbp1Y65C/Y mice exhibit a reduction in both NSPC proliferation and AP-to-BP transition
The proliferation and differentiation of Neural Stem/Progenitor Cells (NSPCs) drive normal corticogenesis. NSPCs comprise Pax6+ apical progenitors (APs) in the ventricular zone (VZ) and Tbr2+ basal progenitors (BPs) in the subventricular zone (SVZ). To investigate the cellular mechanism underlying microcephaly in Pqbp1Y65C/Y mice, we first examined and quantified APs. Our findings revealed comparable AP numbers during early (E12.5), middle (E15.5), and late (E17.5) neurogenesis phases (Fig. 2a, b). Neural proliferation analysis using 5-bromo-2’-deoxyuridine (BrdU) demonstrated a significant reduction in BrdU+ APs (Pax6+ BrdU+) in the VZ of Pqbp1Y65C/Y cortices compared to the controls (Fig. 2c, d). For a more precise understanding of AP proliferation, we employed a dual labeling strategy with BrdU and EdU to accurately measure the cell cycle parameters of AP progenitors (Fig. 2e–g). We injected BrdU first (2h before sampling) to label all cells that were in S-phase, and subsequently injected EdU (0.5h before sampling) to label the cells that entered S-phase later. By analyzing the ratio of labeled cell populations, we can track the progression of a cohort of cells through the cell cycle over time. This analysis revealed an extended cell cycle length for APs in Pqbp1Y65C/Y cortices (Fig. 2e–g). Furthermore, by measuring the proportion of mitotic cells, we found that there is an increase of APs in the M phase in Pqbp1Y65C/Y VZ cortices (Fig. 2h, i), suggesting that the Y65C mutation prolongs the mitotic M phase of APs.

During corticogenesis, APs emerge first and subsequently produce BPs, which differentiate into projection neurons across all cortical layers. In this study, we observed that Pqbp1Y65C/Y mice exhibited a significant reduction in BP numbers (Fig. 3a, b). Further examination following BrdU injection revealed that the proportions of BrdU+ BPs (Tbr2+; BrdU+) in the SVZ were considerably lower in Pqbp1Y65C/Y cortices compared to the controls (Supplementary Fig. 3a, b). Despite the normal number of APs in Pqbp1Y65C/Y mice, APs exhibit lower proliferation, and consequently, BPs are notably reduced. This prompted us to investigate the differentiation potential of APs towards BPs. Our findings indicate that the Y65C mutation decreases the proportion of double-positive Pax6+ Tbr2+ cells within the APs (Fig. 3a, c), indicating the compromised ability of APs to transition into BPs. APs possess a unique polarity, characterized by differential protein distribution at their apical and basal poles relative to the epithelial surface. Any deviation in the cell division plane might result in asymmetric cell division and consequently affect cellular lineage. Upon measuring the mitotic angles of APs, we discovered that those from Pqbp1Y65C/Y mice displayed a stronger inclination towards symmetric division (Fig. 3d–f). Using Cadherin to delineate the plasma membrane, we further confirmed the increased tendency for symmetric division in Pqbp1Y65C/Y mice (Fig. 3g, h). Moreover, apoptosis infrequently exists in both groups, with no significant disparities observed (Supplementary Fig. 3c, d). Taken together, these results suggest that the compromised proliferation of NSPCs and the disrupted transition from APs to BPs contribute to a limited BP pool and compromised corticogenesis in Pqbp1Y65C/Y cortices.

The Y65C mutation significantly reduces PQBP1 protein levels in Pqbp1Y65C/Y cortices
To investigate the pathological mechanism underlying the neural progenitor defects caused by the Y65C mutation, we first assessed its impact on PQBP1 protein levels. Immunofluorescence staining and western blot analyses revealed a significant reduction in PQBP1 protein levels in Pqbp1Y65C/Y cortices (Fig. 4a, b). This was evident using both anti-full-length-PQBP1 polyclonal antibodies (anti-PQBP1FL) and polyclonal antibodies specific to the C-terminus of PQBP1 (anti-PQBP1CTD) (Supplementary Fig. 4a–c). A prior study noted a slight reduction in PQBP1 protein levels in lymphoblasts from Renpenning syndrome patients with the Y65C mutation37. Indeed, PQBP1 protein levels were slightly decreased in Pqbp1Y65C/Y mouse lymphocytes (Supplementary Fig. 4d). However, PQBP1 protein levels were markedly reduced in the Pqbp1Y65C/Y heart, lung, and liver (Supplementary Fig. 4e–g), indicating that the decline in PQBP1 levels due to the Y65C mutation is not brain-specific. We further examined PQBP1 protein levels across different tissues to show that PQBP1 protein was highly abundant in the cortex and lung, moderate in the heart and liver, and very low in lymphocytes (Supplementary Fig. 4h). These findings indicate that the decline in PQBP1 levels caused by the Y65C mutation exhibits tissue-specific differences, and the very low basal expression of PQBP1 in lymphocytes likely mask the observation of any further reduction.

Next, we examined Pqbp1 mRNA levels in the cortex of Pqbp1Y65C/Y mice. Quantitative RT-PCR revealed that Pqbp1 mRNA levels in control and Pqbp1Y65C/Y mice were comparable (Fig. 4c and Supplementary Fig. 4d–g), indicating that the mutation does not influence Pqbp1 mRNA transcription. To understand how the Y65C mutation reduces PQBP1 protein levels, we transiently expressed flag-tagged full-length PQBP1 (Flag-PQBP1) or its Y65C mutant (Flag-PQBP1Y65C) in SK-N-BE(2) cells and monitored their protein levels. The expression levels of Flag-PQBP1Y65C were notably lower than those of Flag-PQBP1 at every time point (Fig. 4d). Polysome profiling demonstrated that the Y65C mutation did not alter the distribution of Pqbp1 mRNA in ribosomes (Supplementary Fig. 5a, b), implying no significant abnormalities in PQBP1 protein synthesis. Cycloheximide (CHX), a protein synthesis inhibitor, binds to the E-site on the ribosome, inhibiting nascent protein synthesis. Upon monitoring expressed PQBP1 protein levels at various time points post-CHX addition, we observed that the levels of Flag-PQBP1Y65C protein declined swiftly after CHX treatment (Fig. 4e). Moreover, the addition of the proteasome inhibitor, MG132, significantly decelerated the degradation rate of the PQBP1Y65C mutant protein (Fig. 4f). These findings indicate that the Y65C mutated protein undergoes rapid degradation via the ubiquitin-proteasome pathway. We further modulated the expression levels of PQBP1-Y65C in 293T cells and monitored the corresponding degradation rate of the mutant protein. We did not detect significant degradation of the Y65C protein at very low expression levels, which is consistent with our in vivo observation in lymphocytes, suggesting that low abundance of the Y65C protein may not adequately activate the protein degradation machinery (Supplementary Fig. 5c, d).

Considering that PQBP1 contains a single-cysteine residue at position 60, it is logical to deduce that the Y65C mutation forms anomalous disulfide bonds, subsequently interfering with protein folding and leading to premature degradation. To corroborate this hypothesis, we replaced the tyrosine at position 65 with Phe (Y65F), Ser (Y65S), or Trp (Y65W). These replacements notably elevated the expression levels of PQBP1 (Fig. 4g). Converting the cysteine to Ser at position 60 in the mutant protein (PQBP1C60S,Y65C) also substantially restored PQBP1 expression (Fig. 4g). Conversely, substituting codon TGC with codon TGT, which still encodes the cysteine residue at position 65, did not amplify PQBP1 expression (Fig. 4g). Furthermore, the two mutants, Flag-PQBP1C60S,Y65C and Flag-PQBP1Y65S, which impede disulfide bond formation, exhibited stability comparable to that of Flag-PQBP1 (Supplementary Fig. 5e, f). These findings demonstrate that the Y65C mutation substantially destabilizes PQBP1.

Nestin-cKO mice display microcephaly without deficits in the AP-to-BP transition. Given that the level of PQBP1 protein was significantly decreased in Pqbp1Y65C mice, we asked whether the impaired NSPC proliferation and defective AP-to-BP transition were due to the loss of PQBP1 function. To answer this question, we specifically knocked out PQBP1 in NSPCs by crossing floxed Pqbp1 mice with Nestin-Cre mice38. Through real-time quantitative PCR and immunofluorescence analysis, we confirmed that PQBP1 is significantly decreased at E12.5 and nearly completely knocked out at E14.5 in cKO mice (Supplementary Fig. 6a–c). As predicted, newborn cKO mice (Nestin-Cre; Pqbp1fl/Y) displayed an approximately 6.5% reduction in brain weight compared to their littermate controls (Pqbp1fl/Y) (Fig. 5a). In contrast, Pqbp1Y65C/Y mice exhibited a more severe reduction of approximately 15%. (Fig. 1c, d). Histological analysis demonstrated a significant reduction in the thickness of each layer (Cux1+ for the layer II–IV, Ctip2+ for the layer V, and Tbr1+ for the layer VI) in newborn cKO mice (Fig. 5b–d). Further immunohistochemical analysis revealed a substantial decrease in the number of APs and BPs in cKO mice (Fig. 5e–g). A previous study has shown that the depletion of PQBP1 in NSPCs leads to impaired NSPC proliferation and microcephaly32. These findings suggest that the loss of PQBP1 function impacts NSPC proliferation. However, consistent with the previous observations32, cKO mice show normal AP-to-BP transition (Fig. 5e, h, and Supplementary Fig. 6d, e), indicating that the defective AP-to-BP transition in Pqbp1Y65C/Y mice may be attributed to the Y65C mutation-induced gain of function.

The Y65C mutation induces a gain-of-function to affect the AP-to-BP transition. We next investigated whether the Y65C mutation confers a gain-of-function effect to perturb the AP-to-BP transition in Pqbp1Y65C/WT heterozygous female mice. Although the heterozygous mice expressed normal levels of PQBP1 protein (Supplementary Fig. 7a, b), they showed defective AP-to-BP transition, but not as severe as that in Pqbp1Y65C/Y mice (Fig. 6a, b). We also performed an in utero electroporation experiment by introducing the expression plasmids into the cortex of WT mice at E14.5, which corresponds to the peak period of the AP-to-BP transition (Fig. 6c). Immunofluorescence staining showed that expressing wild-type PQBP1 did not affect the AP-to-BP transition, while expression of PQBP1Y65C mutant proteins caused a significant deficit in the AP-to-BP transition in WT mice (Fig. 6d, e). These results indicate that the Y65C mutation confers a gain-of-function to affect the AP-to-BP transition.

The Y65C mutation preserves the proliferative APA profiles to influence the AP to-BP transition. To elucidate the mechanism behind the gain-of-function exhibited by the Y65C mutant protein, we isolated proteins associated with PQBP1 from the embryonic cortices of both wild-type and Pqbp1Y65C/Y mice using co-immunoprecipitation experiments. Subsequently, these proteins were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Across two independent replicates, 37 proteins displayed increased interactions (Normalized iBAQ Y65C / iBAQ wild-type > 1.5) and 77 proteins displayed decreased interactions (Normalized iBAQ Y65C / iBAQ wild-type < 0.67) with the Y65C protein (Fig. 7a and Supplementary Data 1). Notably, the strengthened interactome was significantly enriched in mRNA processing factors, including known splicing factors such as PRPF19, HNRNPA3, and core components of the mRNA 3’ end processing machinery, such as FIP1L1 (Fig. 7b, c), and the weakened binding proteins related to chromatin remodeling and RNA splicing (Fig. 7b). These results suggest that the gain-of-function effects observed with the Y65C mutant protein may be linked to mRNA processing.

To assess the influence of the Y65C mutation on mRNA processing and pinpoint the targets affecting the AP-to-BP transition, we isolated the cortices at E12.5 and performed high-depth RNA-seq. We initially employed rMATS to analyze alternative splicing and identify target genes impacted by the Y65C mutation. We observed a total of 125 splicing events with significant differences (88 in skipped exons [SE], 14 in retained introns [RI], 9 in alternative 5’ splice sites [A5SS], 6 in alternative 3’ splice sites [A3SS], and 8 in mutually exclusive exons [MXE]) (Supplementary Fig. 8a and Supplementary Data 2). Although these events were statistically significant, we did not identify the critical target genes directly linked to NSPC differentiation among them. Subsequently, we utilized the APAtrap39, a bioinformatic tool that detects the differential polyadenylation site (PAS) usage across experimental conditions, to evaluate the impact of the Y65C mutation on the APA profile of NSPCs. Our analysis revealed 578 genes with significant APA changes (false discovery rate [FDR] < 0.05) in Pqbp1Y65C/Y NSPCs compared to the littermate controls. Among these genes, 441 genes (76.3%) exhibited shifts from distal to proximal PAS in Pqbp1Y65C/Y NSPCs (Fig. 7d; Supplementary Data 3), indicating that the Y65C mutation favors proximal PAS selection in NSPCs, generating the shorter isoforms. It has been extensively documented that the proximal PAS-biased APA pattern is correlated with cell proliferation30,40. To further determine whether these PAS shifts are the cause or the consequence of the prolonged M phase observed in Y65C APs, we analyzed APA profiles of E12.5 cortices from Pqbp1-cKO mice that also exhibit prolonged M phase. In contrast to the constitutive Pqbp1Y65C/Y knock-in model, the Pqbp1-cKO model at E12.5 still retains about 25% residual wild-type protein expression (Supplementary Fig. 6b). Only 179 genes showed significant APA changes in Pqbp1-cKO mice, with 56.4% (101) shifting toward proximal and 43.6% (78) toward distal PAS — an almost balanced pattern (Fig. 7e; Supplementary Data 4). Moreover, merely 39 of the proximal-shifted genes overlapped with the Y65C set (Fig. 7f). We further performed a direct comparative analysis of the APA profiles between the Pqbp1Y65C/Y and Pqbp1-cKO models. This analysis identified 1060 genes with significant APA differences. Among these, 649 genes (61.2%) exhibited a significant preference for the proximal PASs in the Pqbp1Y65C/Y model compared to the cKO, while 411 genes (38.8%) favored distal sites (Supplementary Fig. 8b; Supplementary Data 5). These results underscore substantial differences between the two models and demonstrate that the pronounced proximal-APA bias is a specific molecular signature of the Y65C gain-of-function and is not a consequence of M-phase prolongation. Furthermore, the genes showing distal-to-proximal shifts were significantly enriched in functional categories related to cerebral cortex development and neuronal differentiation (Supplementary Fig. 8c; Supplementary Data 3). Notably, 18 targets were previously identified in genome-wide RNAi screens as potential ESC self-renewal factors41,42 (Fig. 7g, Supplementary Data 6). We further confirmed the APA switches through quantitative real-time PCR (Fig. 7h, i). These findings indicate that the Y65C mutation fosters the selection of proximal PAS, thereby preserving the proliferative APA profiles and influencing the AP-to-BP transition.

To further investigate the molecular mechanism underlying the Y65C mutation-promoted selection of the proximal PAS, we conducted a co-immunoprecipitation experiment. This confirmed the strengthened association between the Y65C mutant protein and FIP1L1, which is essential for activating mRNA 3’ end processing at the cleavage step and facilitating the utilization of the proximal PAS to maintain ESC self renewal43 (Fig. 7j). To assess whether this strengthened association influences the PAS cleavage efficiency, we used the dual luciferase reporter system pPASPORT. In this system, the canonical PAS is inserted between Renilla (Rluc) and Firefly (Fluc) luciferases. An increase in the Rluc/Fluc ratio indicates the cleavage at the inserted PAS. Convincingly, the expression of the Y65C mutant protein significantly increased the Rluc/Fluc ratio, facilitating the PAS cleavage (Fig. 7k, l). Taken together, these findings demonstrate that the Y65C mutation promotes the selection of the proximal PAS to preserve the proliferative APA profiles, thereby influencing the AP-to-BP transition.

Methods
Animals
Mice in the C57BL/6 background were used in this study and bred in the SPF core animal facility of Southeast University at 25 °C with a relative humidity of 40%–60% and a 12 h light/12 h dark cycle. Mice had free access to food and water. Mouse breeding and all animal experiments were approved by the Southeast University Institutional Animal Care and Use Committee (#20211101003, Nanjing, China) and were performed by institutional and national guidelines. Given that PQBP1 is located on the X chromosome and that all reported human cases are male, all experimental groups and control groups in this study consist of male mice, except for the heterozygous female Pqbp1Y65C/WT mice. WT represents Pqbp1WT/Y, Y65C represents Pqbp1Y65C/Y, and Y65C/WT represents Pqbp1Y65C/WT.

Generation of Pqbp1Y65C mice
Pqbp1Y65C knock-in mice were generated using homologous recombination in zygotes and implanted into pseudopregnant mice51,52. The sgRNA sequences (5S8: 5’-CATGAGTTTTGACCTATA- and 3S1: 5’-CGGACTCCCTTACTAT) were cloned into the CRISPR/Cas9 system and verified by DNA sequencing. The donor vector included the DNA fragments containing exons 3-4; the mutation of Tyr 65 to Cys (codon TAT to TGT) was also performed. After plasmid verification, the donor vector and CRISPR/Cas9 system were injected into zygotes. Then, the zygotes were injected into 0.5 d.p.c. pseudopregnant females for generating chimeric mice (F0). F0 mice were crossed with wild-type (WT) C57BL/6 mice to establish germline-transmitted founders. Genotyping of Pqbp1Y65C mice was performed by amplifying and sequencing a 0.41-kb product from purified mouse tail DNA (forward: 5’-CCATTCTTCCAGAGCCAGAG and reverse: 5’-TTCTTGGCGGATTTGGTAAC).

Generation of Nestin-cre; Pqbp1flox/Y mice
To generate neural progenitor cell-specific Pqbp1 conditional knockout (cKO) mice, Pqbp1flox/flox mice, which were generated by the Model Animal Research Center of Nanjing University53, were crossed with Nestin-Cre mice38 (Stock# 003771, Jackson Laboratory, Bar Harbor, ME, USA) to produce Pqbp1flox/Y (control) and Nestin-cre; Pqbp1flox/Y (Pqbp1-cKO) mice.

Animal mating strategies
1) Mating Strategy for Pqbp1Y65C knock-in mice
Upon obtaining F0-generation mice, we crossed them with wild-type (WT) C57BL/6 mice to generate F1-generation Pqbp1Y65C/WT (Y65C/WT) heterozygous female mice. These heterozygous females were subsequently backcrossed with WT mice to produce experimental Pqbp1Y65C/Y (Y65C) male mice and littermate control WT male mice for phenotypic analysis.
2) Mating Strategy for Pqbp1 Nestin-cKO mice.
We crossed Nestin-Cre heterozygous males with Pqbp1flox/flox females to generate Nestin-Cre; Pqbp1flox/Y males (Pqbp1-cKO) mice and control Pqbp1flox/Y males (control) mice for experimental analysis.

Nissl staining
Mice brains were perfused with cold phosphate-buffered saline (PBS, pH 7.4), fixed in 4% paraformaldehyde (PFA) overnight at 4°C, then processed for paraffin embedding and serially sectioned (10 μm) by a cryostat (Leica, CM 3050S, Germany). Sagittal sections were deparaffinized, ethanol hydrated, and treated with 0.5% cresyl-violet for 10 min, then rinsed with distilled water three times. Slides were ethanol dehydrated and treated with toluene, and finally coverslipped using neutral resin. Images were collected by LAS X 3.3 (Leica, Germany).

Plasmid construction
The full-length mPQBP1 and mPQBP1Y65C cDNAs, obtained from WT and Y65C mutant mice respectively, were cloned between the XhoI and EcoRI sites of the pCAG-EGFP vector to generate the expression plasmids. The Flag-PQBP1-FL plasmid was were obtained from Zhang et al.44. The pPASPORT plasmids for Luciferase reporter asssys were obtained from Liu et al.30. Other constructs were generated by gene-directed mutation PCR (12351010, Thermo Scientific). A list of primers that were used in all experiments is shown in the Supplementary Data 7.

Cell culture and transfections
293T (#GNHu17) and SK-N-BE(2) cell (# TCHu200) lines were purchased from Cell bank of Chinese Academy of Sciences and cultured under standard conditions (37°C, 5% CO2) in their respective media: 293T cells in DMEM and SK-N-BE(2) cells in DMEM/F12, both supplemented with 10% fetal bovine serum (FBS). For transfection experiments, cells were seeded in 6-well plates at a density of 1×10⁵ cells per well. Upon reaching 70-80% confluence, plasmid DNA transfection was performed using cell line-specific reagents: Lipofectamine 3000 (L3000075, Thermo Fisher Scientific) for 293T cells and X-tremeGENE HP DNA Transfection Reagent (06366236001, Roche Diagnostics, Basel, Switzerland) for SK-N-BE(2) cells.

Antibody generation
FL-PQBP1 antibody was raised in rabbits against the full length of the GST-PQBP1 protein. For the production of polyclonal anti-PQBP1-CTD antibody, a specific peptide N-KKATSRKDEELDPMDPSSYSDAPRGTWSTGLPKRNEAKTGADTTAAGPLFQQRPYPSPGAVLRANAEASRTKQQD-C was synthesized. The peptide was injected into rabbits (YUROGEN, Wuhan, China). After three boosts at 1- to 2-week intervals, high-titer sera were collected and then purified using Sulfo Link Coupling Gel (Pierce, Rockford, IL). For Western blots, FL-PQBP1 and CTD-PQBP1 antibodies were used at a 1:500 and 1:100 dilution, respectively.

Western blots
Mice cortices and cell lines were lysed in cold radio-immunoprecipitation assay (RIPA) lysis buffer [consisting of the following: 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS] containing protease and phosphatase inhibitors (04693132001, Thermo Fisher, USA; 04906837001, Thermo Fisher, USA). Lysates were cleared by centrifugation (12,000×g for 20 min). Normalized volumes of samples (10 μg total protein) were resolved in 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Massachusetts, US). After blocking with 0.5% (w/v) nonfat dry milk in Tris-buffered saline (TBS: 25 mM Tris-HCl, pH 7.4, 150 mM NaCl), membranes were probed with primary antibodies overnight at 4°C. Primary antibodies used were PQBP1 (16264-1-AP, Proteintech, 1:1000 for use); RPS3 (ab128995, abcam, 1:1000 for use); RPL13 (ab134961, abcam, 1:1000 for use); FIP1L1 (83863-2-RR, Proteintech, 1:1000 for use); anti-α-tubulin (T9026, Sigma-Aldrich, 1:5000 for use); anti-actin (T0022, Affinity, 1:5000 for use). The following secondary antibodies were used for western blot, all diluted at 1:1000: Goat anti-rabbit IgG (H+L)-HRP conjugate (401315, Millipore); Goat anti-mouse IgG (H+L)-HRP conjugate (401215, Millipore); Goat anti-Guinea Pig IgG (H+L)-HRP conjugate (AS025, Abclonal). Western blot images were acquired either using an OPTIMAX 2010 automatic film processor for X-ray film development or a Tanon 5200 imaging system for chemiluminescence image capture. To avoid signal saturation, a standardized protocol was strictly followed: an initial short pre-exposure (e.g., 1–2 sec) was performed to estimate signal intensity, followed by a series of graded exposures (e.g., 5 s, 10 s, 30 s, 60 s). Images without overexposed signals were selected for subsequent quantitative analysis. The captured 8-bit TIFF images were analyzed using ImageJ 1.53k software (Fiji). The integrated densities of rectangular regions of interest (ROIs) for target bands and internal control bands were measured, followed by background subtraction. Finally, the normalized ratios of the target proteins to the internal control proteins β-Actin were calculated.

Lymphocyte isolation
Mice were euthanized, and the blood was collected through the orbital vein and collected in an anticoagulant tube. Then, it was diluted with PBS buffer and transferred to lymphocyte separation buffer (7211011, DAKEWE, Shenzhen, China). The lymphocytes were isolated after centrifugation and washed twice with PBS buffer. Finally, lymphocytes are used to extract proteins and RNA.

Polyribosome profiling by sucrose gradient ultracentrifugation
For the polyribosome profiling assay, 1-month-old WT and Pqbp1Y65C/Y mouse brains were quickly removed, and whole brain slices (360 μm) were prepared (Leica, VT1000S) in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 120 NaCl, 3.0 KCl, 1.2 MgSO4, 1.0 NaH2PO4, 26 NaHCO3, 2.0 CaCl2, 11 D-glucose saturated with 95% O2/5% CO2. The slices were recovered at 28°C in ACSF saturated with 95% O2/5% CO2 for at least 2 h before a single slice was transferred to a submersion chamber perfused with 95% O2/5% CO2 saturated ACSF. The brain slices were incubated in ACSF containing 100 mg/ml cycloheximide for 1 h, and then were homogenized in ice-cold PMS buffer containing 20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.3% (v/v) NP-40, 100 U/ml of SUPERase-In TM RNase inhibitor (Invitrogen), 1% w/v protease inhibitor, 1 mM PMSF, and 0.5 mM DTT. The lysates were subjected to centrifugation at 10,000×g for 10 min at 4°C, and the resulting supernatants were retained as PMS. PMS was then layered onto 11 mL of 10%-50% (w/v) sucrose gradients and centrifuged at 22,000×g for 2.5 h at 4°C using a P40ST rotor (HITACHI). The fractions were monitored using an OD254 nm ultraviolet spectrometer (Thermo Scientific, USA). Samples from the fractions were collected for western blot analysis and real-time RT-PCR analysis, respectively.

Quantitative polymerase chain reaction (qPCR)
RNA was isolated using Trizol reagent (9109, TaKaRa, Japan), and 1 µg of the total RNA was reverse transcribed using the Revert Aid First Strand cDNA Synthesis Kit (RR037Q, Takara, Japan). Real-time PCR was performed with 1 μL of the cDNA template added to 10 μL of 2× TB Green Premix Ex Taq (RR820Q, Takara, Japan) and specific primers (10 pM each). Real-time PCR (Applied Biosystems, USA) was carried out for 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 30 s. GAPDH was used as an internal control gene. Amplicons were calculated using the 2−ΔΔCt method, and data are represented as the mean of three independent experiments ± SD. The primer sequences used for qRT-PCR are shown in the Supplementary Data 7.

BrdU and Edu injection
Since both EdU and BrdU are thymidine analogues that incorporate into DNA during the S-phase, administering them sequentially allows us to track the progression of a cohort of cells through the cell cycle over time. For proliferation analysis, pregnant mice were intraperitoneally injected with BrdU (100 mg/kg, B5002, Sigma) one hour before embryo collection. For cell cycle calculation, we injected BrdU first (2 h before sampling) to label all cells that were in S-phase at that time. This population is referred to as BrdU⁺ cells. Subsequently, we injected EdU (50 mg/kg, A10044, Invitrogen) 0.5 h before sampling to label a later, distinct population of cells that entered S-phase just before collection (Edu⁺ cells). The embryos were then perfused with PBS, fixed in 4% PFA for approximately 8 h, cryoprotected, and sectioned into 14 μm thick slices for immunohistochemistry54.

Immunofluorescence
Paraffin brain sections were deparaffinized and then boiled in 0.01 M sodium citrate buffer solution, pH 6.0. Frozen sections were air-dried and treated with 0.5% Triton in PBS. After blocking in 3% BSA for 1 h, they were immunostained with antibodies raised against Tbr1 (ab183032, Abcam, 1:200 for use); Ctip2 (ab18465, Abcam, 1:500 for use); Brn2 (sc-6029, Santa Cruz, 1:100 for use); Cux1 (sc-13024, Santa Cruz, 1:100 for use); Pax6 (901302, biolegend, 1:500 for use); Tbr2 (ab23345, Abcam, 1:500 for use; 14-4875-82, Invitrogen, 1:200 for use); BrdU (ab6326; Abcam, 1:500 for use), Ki67 (ab16667, Abcam, 1:500 for use); pH3 (9701, CST, 1:500 for use); γ-tubulin (ab11316, Abcam, 1:500 for use); Cadherin (ab51034, Abcam, 1:500 for use); cleaved caspase 3 (9661, CST, 1:500 for use) and EGFP (A10262, Invitrogen, 1:500 for use). The following secondary antibodies diluted at 1:200 were used for immunofluorescence: Alexa Fluor 555 goat anti-rabbit IgG (ab150078, Abcam); Alexa Fluor 488 goat anti-rabbit IgG (ab150081, Abcam); Alexa Fluor 488 goat anti-mouse IgG (ab150117, Abcam); Alexa Fluor 555 goat anti-mouse IgG (ab150118, Abcam); Alexa Fluor 647 goat anti-rabbit IgG (ab150079, Abcam); Alexa Fluor 647 goat anti-mouse IgG (ab150115, Abcam); Alexa Fluor 488 goat anti-Chicken IgG (ab150169, Abcam); Alexa Fluor 555 goat anti-Rat IgG (ab150158, Abcam); Alexa Fluor 488 goat anti-Rat IgG (ab150157, Abcam). DAPI (A606584, Sangon Biotech, China, 1:5000 for use) was used to stain nuclei. Images were obtained using a Zeiss LSM700 and LSM900 confocal microscope (Oberkochen, Germany).

Morphometry
For quantifying cell area and number, images were analyzed and processed using ZEN 2.3 (Zeiss, Germany), LAS X 3.3 (Leica, Wetzlar, Germany), and ImageJ 1.53k (Fiji) software (National Institutes of Health, Bethesda, MD, USA). Three sections from each cortex, spaced 200 μm apart, were used for cell counting.

Behavioral assessments
Age-matched WT littermates of 8–13-week-old healthy male mice were used for all behavioral tests. All behavioral assays in this study were performed during the light cycle and were blinded to genotype.
Open field test
A mouse was positioned adjacent to the wall within a 50×50 cm open field arena, and its movement was captured by a camera for a duration of 5 min. The recorded video footage was subsequently analyzed using EthoVision XT 7.0 software (Noldus). Quantitative measures, including the number of entries into and the total time spent in the central area of the arena (15×15 cm), as well as the total moving distance, were recorded. Between each trial, the open field arena was thoroughly cleaned with 70% ethanol and dried using paper towels.
Elevated plus maze test
The test mice were placed on the central platform of an elevated plus maze (45 cm above the floor), facing the open arm of the maze, and allowed to explore for 5 min. Their entries into the open and closed arms and the times spent in each location were recorded and analyzed using EthoVision XT 7.0 software (Noldus).
Novel-object recognition test
A test mouse was initially habituated to an open field arena (50×50 cm) for a period of 5 min. Following the habituation phase, the mouse was temporarily removed from the arena, and two identical objects, each measuring 6×6×4 cm and of the same color, were strategically placed on the same side of the arena, positioned 10 cm from the side walls. The mouse was then reintroduced to the center of the arena and allowed to explore the environment, including the two novel objects, for a duration of 5 min. After a 24-hour interval, one of the original objects was replaced with a novel object of similar size but distinct in shape and color. The same test mouse was once again placed in the arena to investigate the arena and the two objects. The mouse’s movements were recorded using a camera for a total of 5 min and subsequently analyzed with EthoVision XT 7.0 software (Noldus).
Three-chamber test
The three-chambered apparatus is designed with three equally sized rectangular compartments, each measuring 20 cm in width, 40 cm in length, and 22 cm in height. The two adjacent compartments are separated by transparent plexiglass walls, each featuring a rectangular door (5 cm wide×8 cm high) to permit the subject mice to access each compartment. To isolate the stranger mice from the subject mice, wire pencil cups (7.5 cm wide×7.5 cm long×10 cm high) are used, with a heavy cup placed on top to prevent the subject mice from climbing. These wire pencil cups are positioned on the outer sides of the apparatus. Before testing, stranger mice are acclimated to the wire pencil cups for 10 min. Between tests, the apparatus, wire pencil cups, and heavy cups are thoroughly cleaned with 75% ethanol and dried with paper towels. Each testing session is composed of three 10-minute stages. During the first stage, subject mice are allowed to freely explore the compartments for 10 min. In the second stage, a stranger mouse (stranger 1) is introduced into one of the wire pencil cups, and the subject mice are given another 10 min to investigate the compartments. Following the sociability test, the subject mice are separated from stranger 1 for 10 min. In the third stage, a novel stranger mouse (stranger 2) is placed into another wire pencil cup, and the subject mice are permitted to explore the three-chambered box. The duration of time that mice spent in each chamber was measured and analyzed for comparison. All stranger mice are male, age-matched, and unfamiliar to the subject mice, with a C57BL/6J background. The tests are recorded using an overhead camera and Ethovision XT 7.0 software (Noldus). Additionally, a trained experimenter records the sniffing time with a stopwatch to measure sociability and social recognition memory. A “social memory index” is calculated to quantify social recognition memory, using the formula: Sociability index = The time spent with S1/Total time x 100%. Social novelty index = The time spent with S2/Total time x 100%.

Analysis of cleavage plane angle
The mitotic orientation and cleavage plane of neuroepithelial cells in the dorsal telencephalon were analyzed after double staining for γ-tubulin (ab11316, Abcam, 1:500 for use), with DAPI (A606584, Sangon Biotech, China, 1:5000 for use) co-stain. The cleavage plane was defined as the perpendicular axis to the mitotic orientation; its angle relative to the apical surface was calculated for each genotype55.

In utero electroporation (IUE)
Pregnant WT mice at E14.5 were anesthetized with 0.8% pentobarbital sodium (80 mg/kg). After anesthesia, a small incision of about 2 cm was made in the lower abdominal region. Embryos were carefully extracted using forceps, and a glass electrode was utilized to inject 1 μg of plasmid (pCAG-EGFP; pCAG-PQBP1; pCAG-PQBP1Y65C) into the lateral ventricle of the embryos, mixed with 0.05% Fast Green (F7252, Sigma). Five electric pulses (36 V, 50 ms duration at 1 s intervals) were generated by using an electroporator (ECM830, BTX) and delivered to the head of embryos using 5 mm forceps-like electrodes (BTX). Following the procedure, the uterus was carefully returned to the abdominal cavity, and the peritoneum and abdominal skin were meticulously sutured. The mice were then placed on a 47°C heating pad to facilitate recovery. After 24 h (at E15.5), the male embryos were collected for further analysis56.

CO-IP and mass spectrum
Brain tissues from E15.5 embryonic male mice were harvested for cryopreservation, and then lysed in cold HNTG buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 1 mM DTT, 1% w/v protein inhibitor, 1% w/v PhosStop) for 30 min on ice. The lysates were subjected to centrifugation at 12,000 g for 10 min at a temperature of 4°C. Following this, the protein lysates were divided into two equal portions. For each aliquot, either pre-immune serum or the guinea pig polyclonal anti-mPQBP1 antibodies was added accordingly. The mixtures were gently rotated and incubated at 4°C for 2 h to allow for optimal binding. Following this incubation, an appropriate volume of Protein A agarose beads (10002D, Invitrogen) was introduced to the lysate and allowed to incubate overnight at 4°C. The next day, the supernatant was carefully removed, and the beads were washed with HNTG buffer to eliminate any unbound material. Following elution with SDS buffer, the samples were denatured in a boiling water bath for 10 min to prepare for protein interaction analysis via Western blot. Before MS characterization, all immunoprecipitation (IP) products underwent Western blot validation. For MS processing, IP products from two biological replicates were co-separated on 10% SDS-PAGE. Specific gel lanes corresponding to each IP sample were excised and submitted to the Proteomics Core Facility at Nanjing Medical University (Nanjing, China) for nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) analysis. Mass spectrum data was collected by Orbitrap Fusion Lumos mass spectrometer (Thermofisher, USA), and the data-dependent acquisition (DDA) mode was applied. The raw files were searched using the MaxQuant (version: 1.5.2.8) software against the mouse UniProt sequence database with default settings.

Luciferase reporter assay
The pPASPORT plasmids inserted with L3 polyA site were co-transfected with pFLAG-CMV2, pFLAG-hPQBP1-WT, and pFLAG-hPQBP1-Y65C, respectively. After 24 h, the transfected cells were lysed with 100 μL of the Passive Lysis Buffer from the Dual-Luciferase Reporter Assay (E1910, Promega), and 30 μL of the lysates were added to 75 μL of Luciferase Assay Reagent. The firefly signals were then quantified by the SpectraMax iD3 multi-function reader (MD, USA). 75 μL of Stop & Glo Buffer was added to each sample before the Renilla signals were quantified. The Rluc/Fluc ratio was then normalized to the control cells.

RNA sequencing and data processing
Total RNA was extracted from the cortices of wild-type and Pqbp1Y65C mice at E12.5. RNA quality was examined using gel electrophoresis and a Nanodrop spectrophotometer (NanoDrop 2000, Thermo Fisher), and sequencing was carried out using the Illumina Novaseq 6000 instrument.
RNA seq data quality was assessed using FastQC v0.11.2, and clean reads were aligned to the mouse reference genome via Hisat v2.2.1. Differential alternative splicing from replicate RNASeq data was detected by rMATs v4.1.2. APA site usage was analyzed via the APAtrap v1.0 tool39, a bioinformatic tool designed to identify novel 3’ untranslated regions (UTRs) and quantify differential alternative polyadenylation (APA) usage between samples. The annotated 3’-UTRs were refined, and the new 3’-UTRs or 3’-UTR extensions were identified using the identifydistal3’UTR module. All potential APA sites were inferred, and their corresponding usages were estimated using the predictAPA module. Significant differences in APA site usage under different conditions were identified using the R package deAPA. A stringent multiple testing correction was applied, and an mRNA was considered to exhibit significantly different 3’-UTR isoforms if the adjusted P value (Padj.) was less than 0.05. The PD index represents the percentage difference in APA site usage between two compared groups, and the Pearson product-moment correlation coefficient (r) quantifies the significant changes in APA site usage within a gene across two samples. A positive r value (r > 0) indicates that the Y65C group preferentially uses distal poly(A) sites (longer 3′-UTRs) compared to the control, whereas a negative r value (r < 0) indicates preferential usage of proximal poly(A) sites (shorter 3’-UTRs) in the Y65C group relative to the control. Gene ontology enrichment analysis was performed with the web-based DAVID v6.8 software (https://david.ncifcrf.gov/). Functional enrichment was determined using a one-sided hypergeometric test with Benjamini and Hochberg correction.

Statistics
Data were analyzed using GraphPad Prism 8.3.0 and are presented as the means ± SD. Statistical significance was determined using the two-tailed, unpaired Student’s t test, the two-tailed Mann–Whitney rank-sum test and two-tailed one-way ANOVA with Tukey post hoc test. P values for each comparison were set as *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001. No significance (ns) was set as P > 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this work, the authors generated the first knock-in mouse model of Renpenning syndrome carrying the pathogenic Y65C mutation in PQBP1, with the aim of investigating the pathological mechanisms underlying the disease-associated microcephaly. Through a well-thought-out and elegant series of experiments—including diverse methodological approaches and comparison with a conditional KO mouse—they demonstrate that the mutation is responsible for two distinct molecular phenotypes, corresponding to a loss-of-function and a gain-of-function, respectively.\nSpecifically, the introduction of a new cysteine in the mutant likely results in altered folding and subsequent increased degradation of PQBP1 Y65C via the proteasome. These reduced levels of PQBP1 are shown to underlie the impaired proliferation of NSPCs in the embryonic cortex of KI animals, which accounts for the microcephaly that mirrors the human condition. This phenotype, resulting from the marked reduction in protein levels, as well as the microcephaly itself, is also observed in the conditional knockout model.\nIn contrast, only PQBP1 Y65C animals exhibit a defective apical-to-basal progenitor transition. The authors propose—and support with evidence—that this defect is the result of a mutation-induced gain-of-function. Through protein–protein interaction studies and deep RNA sequencing, they demonstrate that the mutant form has increased association with several proteins, many of which are involved in transcript maturation—particularly splicing—and a preferential usage of proximal polyadenylation sites. Many of the affected genes are key regulators of cortical development and neuronal differentiation, offering a plausible explanation for the observed brain phenotype.\nI believe the results are relevant not only for the specific disease context but also for researchers in the broader field of neurodevelopment. In my opinion, the study is thorough, well-designed, and the conclusions are well supported by experimental evidence.\nMinor criticisms:\n• I am not entirely convinced that the reduction in PQBP1 Y65C levels correlates with the endogenous protein levels in each tissue. In particular, this doesn’t seem to hold true for the cortex, where the reduction appears less marked compared to other tissues that normally express higher protein levels. If the degradation is caused by misfolding, why would a tissue with more protein experience a greater drop? Could tissue-specific differences in degradation mechanisms explain this?\nOn Materials and Methods:\n• Animal mating strategies are not explained.\n• The age of the animals used in experiments is not always reported.\n• I assume male mice were used in most experiments, but this should be explicitly stated. Indeed, it is never mentioned that the genotype of Pqbp1 Y65C mice is Pqbp1Y65C/y.\n• For Western blots, there is no description of image acquisition procedures, so it is difficult to assess whether the use of Fiji is appropriate for analysis—it is important to ensure no band is saturated.\nIn the discussion, it might be worth mentioning any behavioral differences observed between Pqbp1 Y65C mice and conditional knockouts.\nMinor issues:\n1. Line 347 – “The mutation-induced reduction in PQBP1 levels does not appear to be due to the dominant effects of the mutation, as Pqbp1Y65C/+ heterozygous female mouse cortices expressed normal PQBP1 protein levels; endogenous PQBP1 protein levels in transfected SK-N-BE(2) cells were also unaffected.”\nThis sentence is unclear. Since the gene is X-linked, heterozygous cells would never co-express both isoforms, so a dominant effect wouldn't be expected. Also, I could not find the experiment with SK-N-BE(2) cells in the manuscript.\n2. Line 131 – Consider replacing “cortices” with “VZ”.\n3. Please briefly explain the concept of dual labeling with EdU and BrdU.\n4. I would include the angle measurement for Figure 6e.\n5. Line 152 – “exist” should be corrected to “exists”.\n6. Line 173 – Replace “brain” with “cortex”.\n7. The abbreviation NSPC (Neural Stem/Progenitor Cell) is never defined. Also missing are the expression pattern of Nestin-Cre and a reference; it might help to briefly introduce what APAtrap is.\n8. Lines 227–228 – This passage is hard to follow; please clarify.\n9. Line 342 – Remove the extra “on”.\n10. Line 658 – A closing parenthesis is missing.", "reviewer_2": "The manuscript by Han et al is an impressive body of work where authors model a pathogenic variant of PQBP1 (Y65C) in mice to understand the cellular and molecular mechanisms underlying microcephaly.\nThey show a gain of function effect in the form of a reduction of basal progenitors in the subventricular zone not seen in the KO mice and show associated alteration of mitotic cleavage planes in the ventricular zone. Supporting a gain of function effect is the fact that they see basal progenitor depletion in het females and when they introduce the variant into developing cortex via electroporation. The authors identify a molecular mechanism in the form of favored proximal alternative polyadenylation sites that would favor self-renewal of apical progenitors over generation of basal progenitors. A dual luciferase system is used to demonstrate that expression of the mutant protein promotes use of proximal polyA sites in contrast to the known activity of the WT protein. This is in addition to the reduced stability of the mutated protein and resulting lower protein levels that lead to loss of function effects that mirror previously reported findings in KO models including impaired progenitor proliferation. The combined gain and loss of function effects presumably account for the more severe clinical phenotype observed in individuals affected by the Y65C variant.\nOverall, I find the work to be thorough and that it expands the knowledge in this field. My only comment is a rather banal one regarding differences in cortical development vis a vis differences in cortical progenitor populations and the need to follow this work up in a different model system to better understand how these findings apply to the human disease.", "reviewer_3": "Context: In 2006, Lubs et al. reported a family with X-linked mental retardation (XLMR), microcephaly, postnatal growth deficiency, and other anomalies—including atrial septal defect—previously described by Golabi, Ito, and Hall in 1984. Given recent findings implicating PQBP1 in four XLMR disorders, they identified a rare missense variant (p.Y65C) affecting a conserved tyrosine in the WW domain.\nCompared to other individuals with Renpenning syndrome and harbouring pathogenic hemizygous PQBP1 premature stop variants, affected males in this family had more severe growth and head circumference restriction.\nIn 2009, Musante et al. showed that PQBP1 frameshift variants affecting the AG hexamer in exon 4 variably reduced PQBP1 mRNA levels, partly rescued by inhibiting nonsense-mediated decay. These variants also led to exon 4 skipping, likely due to nonsense-associated altered splicing. Truncated proteins were observed in patient-derived lymphoblastoid cells.\nPQBP1 encodes an RNA-binding protein involved in alternative splicing, and its WW domain functions as a transcriptional activator in yeast two-hybrid assays (Komuro et al., 1999). Tapia et al. (2010) showed that Y65C disrupts interactions with splicing factors, impairing pre-mRNA processing. Wang et al. (2013) studied two pathogenic variants: a ΔAG deletion and the Y65C missense mutation. Both disrupted splicing-related functions, as proteomic profiling revealed altered or lost interactions with splicing factors, indicating these associations are critical for PQBP1 function.\nUsing high-resolution NMR and molecular dynamics, Pucheta-Martinez et al. (2016) found that Y65C destabilizes the residual fold of the WW domain and predisposes the protein to form a disulfide bond, potentially explaining the loss of functon(PMID: 27456546).\nLiu et al. (2020) showed that while frameshift mutants (Δ461–462, Δ575–576) lost binding to TXNL4A, the Y65C variant did not. Similarly, the Y65C mutation preserved binding to Kapβ2, unlike the Δ461–462 frameshift mutant, which lacked PRD, NLS, and CTD.\nThese differences may underlie the distinct clinical phenotypes seen with truncating versus Y65C variants.\nIto et al. (2014) generated a conditional Pqbp1 knockout mouse with microcephaly and observed a prolonged cell cycle, particularly in the M phase, in neural stem/progenitor cells (NSPCs).\nIn this study: Yuan et al. analyzed a Pqbp1-Y65C-knock-in mouse model to investigate the behavioral and molecular consequences of the Y65C variant in vivo and to further elucidate potential mechanisms underlying genotype–phenotype correlations in Renpenning syndrome. The authors interpret their findings as evidence of a dual effect of the Y65C mutation: a loss-of-function component—consistent with observations in the conditional Pqbp1 knockout mouse and mirroring the clinical features of Renpenning syndrome—and a gain-of-function effect, characterized by enhanced association with the core mRNA 3′ end processing machinery. This gain-of-function leads to increased utilization of proximal polyadenylation sites (PAS) over distal PAS in neural stem progenitor cells (NSPCs), disrupting the transition from apical to basal progenitors.\nThe article presents a compelling dataset, particularly regarding the loss-of-function effect, which aligns with findings from previously cited studies. However, I have some reservations about the interpretation of the proposed gain-of-function mechanism, which, if better substantiated, could make their results even more compelling.\nThe authors analyze neural stem/progenitor cells (NSPCs), including Pax6⁺ apical progenitors (APs) in the ventricular zone (VZ) and Tbr2⁺ basal progenitors (BPs) in the subventricular zone (SVZ). Their analysis reveals comparable numbers of APs at early (E12.5), middle (E15.5), and late (E17.5) neurogenesis stages, a reduced number of Tbr2⁺ BPs at E12.5, and a decreased proportion of Pax6⁺Tbr2⁺ double-positive cells among APs. These findings suggest a compromised transition from APs to BPs, likely due to a prolonged M phase in APs.\nIn the Nestin-Cre conditional Pqbp1 knockout model, the authors report reduced numbers of both Pax6⁺ and Tbr2⁺ cells and a comparable proportion of Pax6⁺Tbr2⁺ double-positive cells in the Pqbp1 conditional knockout compared to controls at E15.5. Based on this, they conclude a gain-of-function effect of the Y65C mutation.\nHowever, this interpretation is problematic, as the models used are not directly comparable—not only because the analyses were conducted at different embryonic stages, but also due to limitations in the conditional knockout model. Notably, Liang et al. (2012; PMID: 23259054) demonstrated that the Nestin-Cre transgenic line from The Jackson Laboratory, employed in this study, does not drive efficient recombination in early embryonic neural stem and progenitor cells (NSPCs). At E12.5 and E15.5, Nestin-Cre activity is largely confined to the intermediate zone and cortical plate—regions composed primarily of post-mitotic neurons—and is largely absent from the ventricular and subventricular zones (VZ and SVZ), where proliferating NSPCs reside. Efficient recombination in NSCs and NPCs occurs predominantly during late embryonic and early postnatal stages.\nTherefore, the conclusion of a gain-of-function effect associated with the Y65C mutation, based on this model, should be interpreted with caution. The recombination pattern in the Nestin-Cre line does not adequately overlap with the early progenitor populations affected in the knock-in model. Can the authors provide evidence that, despite the characterization by Liang et al., the Nestin-Cre line used here achieves sufficient recombination in NSPCs at the stages analyzed?\nAdditionally, with regard to genotype–phenotype correlation, it remains unclear whether the microcephaly observed in the Pqbp1-Y65C-knock-in model is more severe than that seen in the conditional knockout mice, thereby better reflecting the phenotype seen in patients. The manuscript does not provide a direct comparative analysis between the two models in this respect. Clarification and quantitative comparison of the severity of microcephaly between the knock-in and knockout models would strengthen the argument for a mutation-specific effect and better reflect the phenotype observed in patients carrying the Y65C variant.\nTo investigate the proposed gain-of-function mechanism, the authors conducted co-immunoprecipitation experiments to compare the protein interactome of wild-type and Pqbp1Y65C proteins in embryonic mouse cortices. However, it is not entirely clear why the analysis focused exclusively on proteins exhibiting increased association with the mutant form. Was this choice made to support the gain-of-function hypothesis? A broader analysis, including both increased and decreased interactions, might provide a more comprehensive understanding of the mutation's impact on protein networks.\nThe authors report enhanced interactions between Pqbp1-Y65C and various splicing factors, as well as with core components of the mRNA 3′ end processing machinery. These changes were accompanied by splicing alterations, as revealed by high-depth RNA-seq, and a global shift from distal to proximal polyadenylation site usage, as detected by PAS-seq, in E12.5 cortices. Given that proximal polyadenylation sites are typically utilized in proliferating or undifferentiated cells—resulting in mRNAs with shorter 3′ UTRs—whereas distal sites are preferentially used in differentiated cells, it remains unclear whether these molecular changes are the cause or the consequence of the prolonged M phase observed in apical progenitors. Whether these molecular alterations are causative or merely secondary to splicing defects that perturb cell cycle regulation remains unclear. Although this is an interesting result—showing an effect opposite to that observed in the previously described conditional knockout model—the authors may wish to elaborate further on this point to clarify the directionality and mechanistic significance of these findings.", "reviewer_4": "Although I do believe the study is interesting, I currently find it hard to interpret the results as the n is missing for many of the experiments."}, "author_response": "Point-by-point responses\nReviewer #1: In this work, the authors generated the first knock-in mouse model of Renpenning syndrome carrying the pathogenic Y65C mutation in PQBP1, with the aim of investigating the pathological mechanisms underlying the disease-associated microcephaly. Through a well-thought-out and elegant series of experiments—including diverse methodological approaches and comparison with a conditional KO mouse—they demonstrate that the mutation is responsible for two distinct molecular phenotypes, corresponding to a loss-of-function and a gain-of-function, respectively.\nSpecifically, the introduction of a new cysteine in the mutant likely results in altered folding and subsequent increased degradation of PQBP1 Y65C via the proteasome. These reduced levels of PQBP1 are shown to underlie the impaired proliferation of NSPCs in the embryonic cortex of KI animals, which accounts for the microcephaly that mirrors the human condition. This phenotype, resulting from the marked reduction in protein levels, as well as the microcephaly itself, is also observed in the conditional knockout model. In contrast, only PQBP1 Y65C animals exhibit a defective apical-to-basal progenitor transition. The authors propose—and support with evidence—that this defect is the result of a mutation-induced gain-of-function. Through protein–protein interaction studies and deep RNA sequencing, they demonstrate that the mutant form has increased association with several proteins, many of which are involved in transcript maturation—particularly splicing—and a preferential usage of proximal polyadenylation sites. Many of the affected genes are key regulators of cortical development and neuronal differentiation, offering a plausible explanation for the observed brain phenotype.\nI believe the results are relevant not only for the specific disease context but also for researchers in the broader field of neurodevelopment. In my opinion, the study is thorough, well-designed, and the conclusions are well supported by experimental evidence.\nMinor criticisms:\nI am not entirely convinced that the reduction in PQBP1 Y65C levels correlates with the endogenous protein levels in each tissue. In particular, this doesn’t seem to hold true for the cortex, where the reduction appears less marked compared to other tissues that normally express higher protein levels. If the degradation is caused by misfolding, why would a tissue with more protein experience a greater drop? Could tissue-specific differences in degradation mechanisms explain this?\nWe thank the reviewer for raising this insightful question regarding the apparent tissue-specific differences in the reduction of PQBP1-Y65C protein levels. We agree that this is an important point that merits clarification, and we have conducted additional experiments to address it.\nAs the reviewer rightly suggests, tissue-specific variations in degradation mechanisms—such as differential expression of chaperones, ubiquitin ligases, or proteasome activity—could contribute to these observations. However, our data indicate that the endogenous abundance of PQBP1 protein itself plays a critical role in engaging the quality control machinery.\nIn lymphocytes, where basal PQBP1 expression is inherently low (Supplementary Fig.4c-h), the introduction of the Y65C mutant may not generate sufficient misfolded protein to overwhelm the proteostasis network, thus resulting in less pronounced degradation. In contrast, tissues with higher native expression levels produce more mutant protein, which is more likely to saturate folding and degradation capacities, thereby triggering more robust activation of UPS (ubiquitin-proteasome system) and/or ERAD pathways.\nTo directly test this hypothesis, we performed controlled overexpression of PQBP1-Y65C at varying levels in HEK293T cells and monitored its degradation kinetics. We found that at low expression levels, the Y65C protein was relatively stable, whereas high expression strongly induced its degradation. These new data, now included as Supplementary Fig. 5c and 5d in the revised manuscript, support the notion that protein abundance significantly influences the activation of misfolding-triggered degradation.\nThus, while tissue-specific degradation mechanisms may contribute to the differential reduction, our results highlight the role of substrate abundance in engaging quality control systems. We have expanded our discussion of these concepts in the revised manuscript to better contextualize these findings within the broader framework of protein homeostasis. (Page 10, Line 201-206 ).\nOn Materials and Methods:\n• Animal mating strategies are not explained.\nWe have added the mating strategies of Pqbp1Y65C/Y knock-in mice and Pqbp1-cKO (Pqbp1fl/Y; Nestin-Cre) mice in the revised Materials and Methods (Page 22, Line 479-489 ).\n• The age of the animals used in experiments is not always reported.\nWe have added the age information of the mice for each experiment in our revised manuscript. The detection times for the developmental stages of mice have been indicated in the lower left of the images and figure legends.\n• I assume male mice were used in most experiments, but this should be explicitly stated. Indeed, it is never mentioned that the genotype of Pqbp1 Y65C mice is Pqbp1Y65C/y.\nSince the PQBP1 gene is on the X chromosome, all documented patients carrying PQBP1 mutations are male, and no obvious developmental abnormalities were observed in heterozygous females. Thus, we used the male mice (Pqbp1Y65C/Y) in most experiments. To clarify this, we have now explicitly stated in the revised Materials and Methods: “WT represents Pqbp1WT/Y, Y65C represents Pqbp1Y65C/Y , and Y65C/WT represents Pqbp1Y65C/WT” (Page 21, Line 452-454).\n• For Western blots, there is no description of image acquisition procedures, so it is difficult to assess whether the use of Fiji is appropriate for analysis—it is important to ensure no band is saturated.\nWestern blot images were acquired either using an OPTIMAX 2010 automatic film processor for X-ray film development or a Tanon 5200 imaging system for chemiluminescence image capture. To avoid signal saturation, a standardized protocol was strictly followed: an initial short pre-exposure (e.g., 1–2 seconds) was performed to estimate signal intensity, followed by a series of graded exposures (e.g., 5s, 10s, 30s, 60s). Images without overexposed signals were selected for subsequent quantitative analysis. The captured 8-bit TIFF images were analyzed using ImageJ software (Fiji). The integrated densities of rectangular regions of interest (ROIs) for target bands and internal control bands were measured, followed by background subtraction. Finally, the normalized ratios of the target proteins to the internal control proteins (Actin or Gapdh) were calculated.\nAs suggested, we have added the description of image acquisition procedures in the revised Materials and Methods (Page 25, Line 537-547).\n•In the discussion, it might be worth mentioning any behavioral differences observed between Pqbp1 Y65C mice and conditional knockouts.\nWe thank the Reviewer for this insightful suggestion. Indeed, we did observe behavioral differences between Pqbp1Y65C/Y and Pqbp1-cKO (Pqbp1fl/Y; Nestin-Cre) mice. Both Pqbp1Y65C/Y and Pqbp1-cKO mice showed decreased center engagement in the open field test. Notably, Pqbp1Y65C/Y mice spent significantly less time in the open arms in the elevated plus maze test (New Supplementary Fig.2d-f), whereas no significant difference was observed in Pqbp1-cKO mice (Mol Psychiatry. 2014 Jul 29;20(4):459–471 Supplementary Fig.10). Moreover, Pqbp1Y65C/Y mice exhibit significant cognitive impairments in the novel object recognition test and three-chamber test (Supplementary Fig.2g-n), mimicking the cognitive phenotypes observed in clinical patients. These impairments have not been reported in Pqbp1-cKO mice (Mol Psychiatry. 2014 Jul 29;20(4):459–471). Therefore, animal models harboring disease-specific mutations provide a more accurate representation of human disease pathology.\nAs suggested, we have added this information in the revised Discussion (Page 16, Line 346-355).\nMinor issues:\n1. Line 347 – “The mutation-induced reduction in PQBP1 levels does not appear to be due to the dominant effects of the mutation, as Pqbp1Y65C/+ heterozygous female mouse cortices expressed normal PQBP1 protein levels; endogenous PQBP1 protein levels in transfected SK-N-BE(2) cells were also unaffected.”\nThis sentence is unclear. Since the gene is X-linked, heterozygous cells would never co-express both isoforms, so a dominant effect wouldn't be expected.\nWe thank the Reviewer for this insightful suggestion. To clarify, we have removed this sentence in the revised version.\nAlso, I could not find the experiment with SK-N-BE(2) cells in the manuscript.\nWe sincerely apologize for this mistake in the original manuscript. The experiment presented in Fig. 4d was performed in SK-N-BE(2) cells, not in HEK293T cells. We have corrected it in the revised manuscript (Page 9, Line 188-190).\n2. Line 131 – Consider replacing “cortices” with “VZ”.\nWe have revised the text as the reviewer suggested.\n3. Please briefly explain the concept of dual labeling with EdU and BrdU.\nTo perform the dual labeling with BrdU and EdU (Fig.2e-g), we injected BrdU first (2 hours before sampling to label all cells that were in S-phase), and injected EdU 0.5 hours before sampling (to label the cells that entered S-phase later). We analyzed the ratio of differently labeled cell populations to track the progression of a cohort of cells through the cell cycle over time. We have added a brief description of the concept of dual labeling with EdU and BrdU in the revised manuscript. (Page 6, Line 130-133) and a new diagram (new Fig. 2f) to demonstrate the experimental procedure.\n4. I would include the angle measurement for Figure 6e.\nWe thank the Reviewer for this insightful suggestion. While the angle measurement would substantially strengthen our analysis, it is unfeasible in practice due to temporal uncoupling between electroporation and phenotype analysis. The transfected AP cells were only transiently anchored at the apical plane exclusively during M-phase. However, to assess differentiation fates through lineage marker staining, we fixed and collected tissue samples at 24 hours after electroporation, ensuring the majority of transfected cells had already exited M phase and progressed into subsequent stages of the second cell cycle. Therefore, we are unable to capture and quantify mitotic spindle angles in these cells.\n5. Line 152 – “exist” should be corrected to “exists”.\nWe have revised the text as the reviewer suggested.\n6. Line 173 – Replace “brain” with “cortex”.\nWe have revised the text as the reviewer suggested.\n7. The abbreviation NSPC (Neural Stem/Progenitor Cell) is never defined. Also missing are the expression pattern of Nestin-Cre and a reference; it might help to briefly introduce what APAtrap is.\nWe have defined the abbreviation \"NSPC\" (Neural Stem /Progenitor Cells) upon its first appearance in the revised text. (Page 6, Line 119)\nWe have provided the expression pattern of Nestin-Cre at different developmental stages in the new Supplementary Fig.6a-c, and added the reference for the transgenic mouse line in the revised text. (Page 10, Line 225)\nWe have included a brief introduction to APAtrap in the revised text. (Page 13, Line 283-285)\n8. Lines 227–228 – This passage is hard to follow; please clarify.\nWe have revised the text as the reviewer suggested. (Page 10, Line 244-245)\n9. Line 342 – Remove the extra “on”.\nWe have revised the text as the reviewer suggested.\n10. Line 658 – A closing parenthesis is missing.\nWe have revised the text as the reviewer suggested.\nReviewer #2: The manuscript by Han et al is an impressive body of work where the authors model a pathogenic variant of PQBP1 (Y65C) in mice to understand the cellular and molecular mechanisms underlying microcephaly.\nThey show a gain-of-function effect in the form of a reduction of basal progenitors in the subventricular zone, not seen in the KO mice, and show associated alteration of mitotic cleavage planes in the ventricular zone. Supporting a gain-of-function effect is the fact that they see basal progenitor depletion in het females, and when they introduce the variant into the developing cortex via electroporation. The authors identify a molecular mechanism in the form of favored proximal alternative polyadenylation sites that would favor self-renewal of apical progenitors over the generation of basal progenitors. A dual luciferase system is used to demonstrate that expression of the mutant protein promotes use of proximal polyA sites in contrast to the known activity of the WT protein. This is in addition to the reduced stability of the mutated protein and resulting lower protein levels that lead to loss of function effects that mirror previously reported findings in KO models, including impaired progenitor proliferation. The combined gain and loss of function effects presumably account for the more severe clinical phenotype observed in individuals affected by the Y65C variant.\nOverall, I find the work to be thorough and that it expands the knowledge in this field. My only comment is a rather banal one regarding differences in cortical development vis a vis differences in cortical progenitor populations and the need to follow this work up in a different model system to better understand how these findings apply to the human disease.\nWe fully appreciate the Reviewer’s positive evaluation and constructive suggestions for our work. We totally agree that validating our findings in a human-based model is a crucial next step, and we are currently carrying out an ongoing project to generate human cortical organoids with the Y65C mutation.\nReviewer #3: In this study: Yuan et al. analyzed a Pqbp1-Y65C-knock-in mouse model to investigate the behavioral and molecular consequences of the Y65C variant in vivo and to further elucidate potential mechanisms underlying genotype–phenotype correlations in Renpenning syndrome. The authors interpret their findings as evidence of a dual effect of the Y65C mutation: a loss-of-function component—consistent with observations in the conditional Pqbp1 knockout mouse and mirroring the clinical features of Renpenning syndrome—and a gain-of-function effect, characterized by enhanced association with the core mRNA 3′ end processing machinery. This gain-of-function leads to increased utilization of proximal polyadenylation sites (PAS) over distal PAS in neural stem progenitor cells (NSPCs), disrupting the transition from apical to basal progenitors.\nThe article presents a compelling dataset, particularly regarding the loss-of-function effect, which aligns with findings from previously cited studies. However, I have some reservations about the interpretation of the proposed gain-of-function mechanism, which, if better substantiated, could make their results even more compelling.\nThe authors analyze neural stem/progenitor cells (NSPCs), including Pax6⁺ apical progenitors (APs) in the ventricular zone (VZ) and Tbr2⁺ basal progenitors (BPs) in the subventricular zone (SVZ). Their analysis reveals comparable numbers of APs at early (E12.5), middle (E15.5), and late (E17.5) neurogenesis stages, a reduced number of Tbr2⁺ BPs at E12.5, and a decreased proportion of Pax6⁺Tbr2⁺ double-positive cells among APs. These findings suggest a compromised transition from APs to BPs, likely due to a prolonged M phase in APs.\nIn the Nestin-Cre conditional Pqbp1 knockout model, the authors report reduced numbers of both Pax6⁺ and Tbr2⁺ cells and a comparable proportion of Pax6⁺Tbr2⁺ double-positive cells in the Pqbp1 conditional knockout compared to controls at E15.5. Based on this, they conclude a gain-of-function effect of the Y65C mutation.\nHowever, this interpretation is problematic, as the models used are not directly comparable—not only because the analyses were conducted at different embryonic stages, but also due to limitations in the conditional knockout model. Notably, Liang et al. (2012; PMID: 23259054) demonstrated that the Nestin-Cre transgenic line from The Jackson Laboratory, employed in this study, does not drive efficient recombination in early embryonic neural stem and progenitor cells (NSPCs). At E12.5 and E15.5, Nestin-Cre activity is largely confined to the intermediate zone and cortical plate—regions composed primarily of post-mitotic neurons—and is largely absent from the ventricular and subventricular zones (VZ and SVZ), where proliferating NSPCs reside. Efficient recombination in NSCs and NPCs occurs predominantly during late embryonic and early postnatal stages.\nTherefore, the conclusion of a gain-of-function effect associated with the Y65C mutation, based on this model, should be interpreted with caution. The recombination pattern in the Nestin-Cre line does not adequately overlap with the early progenitor populations affected in the knock-in model. Can the authors provide evidence that, despite the characterization by Liang et al., the Nestin-Cre line used here achieves sufficient recombination in NSPCs at the stages analyzed?\nWe sincerely thank the Reviewer for raising this important point regarding the variability in recombination efficiency across different Nestin-Cre driver lines. We were aware of this potential issue early in our study and therefore took great care to rigorously validate our specific Nestin-Cre model (Jackson Laboratory Stock #003771). Genotyping confirmed that our mice matched all characteristic features of this stock as specified by the Jackson Laboratory protocol. According to the provider's documentation, this line exhibits robust Cre recombinase activity in the nervous system beginning at embryonic day 11 (E11). This early activity profile is consistent with multiple independent studies confirming Cre-mediated recombination throughout the nervous system from E10.5 to E15.5 (Nucleic Acids Res. 2005 Mar 22;33(5):e51, Genesis. 2008 Jun;46(6):308-17, J Neurosci. 2004 Mar 31;24(13):3402-12, Development. 2006 May;133(9):1799-809, J Neurosci. 2005 Oct 26;25(43):9940-8).\nIn our work, we directly assessed recombination efficiency in the cortex across developmental stages. Quantitative PCR revealed 70% target gene knockout by E12.5, and immunofluorescence imaging further demonstrated widespread recombination at E12.5—including knockout in neural stem and progenitor cells (NSPCs) residing in both the ventricular zone (VZ) and subventricular zone (SVZ). Near-complete recombination was observed by E14.5 in the Pqbp1-cKO cortical progenitor cells (New Supplementary Fig.6a-c). These data confirm that the Nestin-Cre line used in our study achieves efficient and early target gene deletion in cortical NSPCs by E12.5.\nIn addition, our results demonstrate that Pqbp1Y65C/Y mice exhibit defective apical-to-basal progenitor transition as early as E12.5—corresponding to the initial phase of differentiation. In striking contrast, no such abnormality was detected in Pqbp1-cKO mice at this stage (New Supplementary Fig.6d-e). Even at E15.5, by which time knockout efficiency is maximal, the AP–BP transition remains unaffected in Pqbp1-cKO mice. This phenotypic divergence underscores that impaired progenitor transition is specifically attributable to the gain-of-function effects of the Y65C mutation rather than to loss of PQBP1 function. Furthermore, as a response to Reviewer 1, we also observed behavioral differences between Pqbp1Y65C/Y and Pqbp1-cKO mice, supporting the gain-of-function effect associated with the Y65C mutation.\nWe have included these new data and clarifications in the revised manuscript. (Page 11， Line 224-228 and 237-239)\nAdditionally, with regard to genotype–phenotype correlation, it remains unclear whether the microcephaly observed in the Pqbp1-Y65C-knock-in model is more severe than that seen in the conditional knockout mice, thereby better reflecting the phenotype seen in patients. The manuscript does not provide a direct comparative analysis between the two models in this respect. Clarification and quantitative comparison of the severity of microcephaly between the knock-in and knockout models would strengthen the argument for a mutation-specific effect and better reflect the phenotype observed in patients carrying the Y65C variant.\nWe thank the Reviewer for this insightful comment. As shown in Fig. 1c–d, Pqbp1Y65C/Y mice exhibit a reduction in whole-brain weight of approximately 15% compared to the littermate controls at P0. In contrast, Pqbp1-cKO mice at the same developmental stage show a milder reduction of only about 6.5% (Fig. 5a). This comparative analysis suggests that microcephaly is more severe in the Pqbp1Y65C/Y knock-in model than in the Pqbp1-cKO model.\nWe have added the comparison of the severity of microcephaly between the knock-in and knockout models in the revised manuscript (Page 11, Line 226-231).\nTo investigate the proposed gain-of-function mechanism, the authors conducted co-immunoprecipitation experiments to compare the protein interactome of wild-type and Pqbp1Y65C proteins in embryonic mouse cortices. However, it is not entirely clear why the analysis focused exclusively on proteins exhibiting increased association with the mutant form. Was this choice made to support the gain-of-function hypothesis? A broader analysis, including both increased and decreased interactions, might provide a more comprehensive understanding of the mutation's impact on protein networks.\nWe sincerely thank the Reviewer for raising this important point and fully agree that this broader analysis provides a more complete overview of the altered protein interactome resulting from the Y65C mutation. Using the same statistical threshold (fold change < 0.67) and considering the intersection of two independent experimental replicates, we identified 77 proteins with decreased interaction with PQBP1-Y65C, including WBP11, a known interactor with the WW domain of PQBP1 (The Journal of Biological Chemistry 285, 19391-19401).\nWe have added an analysis of the proteins exhibiting decreased association with the Y65C mutant in the new Fig. 7a and 7b. The full list of decreased interactors and the GO term analysis have now been included in the revised manuscript (New Supplementary Table 1).\nThe authors report enhanced interactions between Pqbp1-Y65C and various splicing factors, as well as with core components of the mRNA 3′ end processing machinery. These changes were accompanied by splicing alterations, as revealed by high-depth RNA-seq, and a global shift from distal to proximal polyadenylation site usage, as detected by PAS-seq, in E12.5 cortices. Given that proximal polyadenylation sites are typically utilized in proliferating or undifferentiated cells—resulting in mRNAs with shorter 3′ UTRs—whereas distal sites are preferentially used in differentiated cells, it remains unclear whether these molecular changes are the cause or the consequence of the prolonged M phase observed in apical progenitors.\nWe thank the reviewer for raising this critical point regarding the causal relationship between APA changes and M-phase prolongation—a question that we have carefully considered in our study. Indeed, as noted, proximal polyadenylation sites are often associated with proliferating or undifferentiated states, while distal sites are linked to differentiation; however, our data support that the observed APA alterations are likely a cause rather than a consequence of the cell cycle defects.\nDuring cortical development, NPC cell cycle duration dynamically changes: lengthening of the G1 phase marks the transition from stem-like apical progenitors to fate-committed basal (intermediate) progenitors; interestingly, proliferating apical and basal progenitors exhibit a significantly prolonged S-phase compared to those committed to neuronal production (Nat Commun 2, 154 (2011). https://doi.org/10.1038/ncomms1155). Importantly, a study by Wang et al. (Proc Natl Acad Sci U S A. 2022 Dec 1;119(49):e2113504119) demonstrated at single-cell resolution that cell cycle genes are enriched for dynamic PAS switching and that perturbation of polyA sites directly disrupts cell cycle progression, indicating APA can actively regulate cell cycle.\nIn our models, both Pqbp1Y65C/Y and Pqbp1-cKO exhibit M-phase prolongation, but their APA profiles diverge strikingly. In Pqbp1-cKO mice, APA changes were minimal and balanced (56.4% proximal vs. 43.6% distal shifts among 179 genes) (New Fig.7e; Supplementary Table 4), whereas Pqbp1Y65C/Y mutants showed a strong proximal bias (76.3% of 441 genes shifting proximally). Critically, among 18 self-renewal-associated genes, 17 displayed APA changes exclusively in Pqbp1Y65C/Y—not Pqbp1-cKO—despite comparable M-phase defects (New Fig 7e and f). This dissociation indicates that the pronounced proximal PAS preference in Y65C is not secondary to M-phase prolongation.\nMechanistically, our proteomic analysis revealed that the PQBP1-Y65C protein displays enhanced binding to the core 3′ end processing factors, including FIP1L1, a factor known to enhance cleavage/polyadenylation efficiency and promote the use of the proximal PAS to maintain the self-renewal of ESCs (EMBO J. 2014 Mar 4;33(8):878–889). Consistently, our APAtrap analysis revealed that the Y65C mutation leads to a marked preference for proximal PASs in genes related to cell self-renewal, and luciferase reporter assays showed that overexpression of the Y65C protein significantly increased the efficiency of PAS cleavage. These support that PQBP1-Y65C may directly affect APA process through associating with core 3′ end processing factors, independent of its splicing regulatory role.\nThese findings, coupled with the cKO vs. Y65C phenotypic disparity, support that APA alterations directly contribute to cell cycle regulation rather than merely reflecting its effects.\nWhether these molecular alterations are causative or merely secondary to splicing defects that perturb cell cycle regulation remains unclear. Although this is an interesting result—showing an effect opposite to that observed in the previously described conditional knockout model—the authors may wish to elaborate further on this point to clarify the directionality and mechanistic significance of these findings.\nWe thank the reviewer for this insightful comment regarding the phenotypic divergence between the Pqbp1Y65C/Y and Pqbp1-cKO models, which we agree is a crucial point for mechanistic interpretation. We have carefully analyzed this issue and present a developmentally contextualized explanation below.\nIn the Pqbp1-cKO model, apical progenitor alternative polyadenylation (APA) profiles did not exhibit strong directional bias at E12.5. However, by E14.5, we observed a pronounced proximal-to-distal (P-to-D) APA shift (Cell Reports 43, 114525, August 27, 2024). We attribute this stage-specific effect to fundamental differences in neural progenitor cell (NPC) states: at E12.5, NPCs are primarily engaged in self-renewal, whereas by E14.5, many are committed to neuronal differentiation. This transition is regulated by dynamic changes in key signaling pathways—for instance, Wnt signaling promotes self-renewal at E12.5 but drives neuronal differentiation at E14.5 (Development, 131(12), 2791–2801).\nConsistent with these developmental dynamics, PQBP1 function is also stage-dependent. The minimal APA change in E12.5 cKO suggests that PQBP1 plays a limited role in APA regulation at this stage, potentially functioning primarily in splicing. By E14.5, however, PQBP1 is essential for gatekeeping NPC differentiation—its loss leads to a P-to-D APA shift in synapse-related genes, thereby promoting neuronal commitment.\nStrikingly, the Y65C mutation produces a fundamentally distinct, distal-to-proximal (D-to-P) APA profile, enriched in nucleosome assembly and Wnt pathways, potently impairing NPC self-renewal. This opposite directionality and divergent pathway engagement strongly support a gain-of-function mechanism, rather than loss of function, underlying the Y65C phenotype.\nWe have expanded the discussion of these concepts in the revised manuscript (Page 19, Line 406-443) to clarify the directionality and mechanistic significance of these findings.\nReviewer #4: Although I do believe the study is interesting, I currently find it hard to interpret the results as the n is missing for many of the experiments.\nWe thank the Reviewer for raising the important point regarding sample sizes. We have explicitly added n numbers in the figure legends, and we also appreciate the opportunity to clarify our experimental design and statistical approach.\nFor all animal experiments, the sample size (n) represents the number of individual embryos analyzed. Due to our research focus on embryonic stages, each pregnant mouse yields a limited number of embryos of the desired genotype and specific developmental stage. Despite this inherent challenge, we ensured that every experiment included n ≥ 3 biologically independent embryos per group, a sample size that is well-established and widely accepted in the field of developmental biology. Furthermore, our experimental design strictly adhered to the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal research. Upon observing a clear and consistent phenotype across multiple litters and achieving statistical significance, we refrained from pursuing unnecessary additional replications. For cell-based assays, each experiment was independently repeated at least three times (n ≥ 3 biological replicates), in line with standard practices in the field.\nWe are confident that the sample sizes presented are sufficient to support our conclusions."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have provided a thorough and thoughtful revision, carefully addressing all the points raised. Overall, the manuscript is elegant, well-structured, and scientifically solid.", "reviewer_2": "The work has been further improved by addressing the concerns raised by other reviewers.", "reviewer_3": "I greatly appreciate the revision work performed by the co-authors to address my previous criticisms, as well as the edits added to the manuscript to acknowledge the limitations of the animal models used in this study.\nFor the sake of clarity, it would be important for the authors to mention the 25% residual protein expression observed in the conditional knock-out model at E12.5 when comparing the outcomes of the APA profiles with those of the constitutive knock-in model.\nIn addition, while I generally prefer to avoid suggesting further analyses, I believe that a direct comparison between the Y65C and cKO models in Figure 7 could be a valuable addition. This would not require new experiments, as all the relevant data have already been generated, and could be performed through an additional bioinformatic analysis. The current Y65C/WT vs. cKO/WT comparison is certainly useful to identify commonly deregulated APA events, but a direct Y65C vs. cKO analysis would more clearly highlight the specific differences between the two models. That said, I would consider this suggestion as a nice-to-have refinement rather than a mandatory request.\nFinally, in line with their findings, I would recommend a minor stylistic edit to the article title. Since the loss-of-function effect prevail over the gain-of-function effect, and is consistent with the main pathogenic mechanism described for this disease, this effect should be placed first in the wording.\nOverall, after addressing these minor revisions, I believe the manuscript should be considered for publication."}, "author_response": "Responses:\nWe are deeply grateful to the Reviewer#1 and Reviewer#2 for their positive feedback on our revised manuscript. We also sincerely thank them for their time and insightful comments throughout the review process, which have significantly strengthened our work.\nReviewer #3:\nI greatly appreciate the revision work performed by the co-authors to address my previous criticisms, as well as the edits added to the manuscript to acknowledge the limitations of the animal models used in this study.\n• For the sake of clarity, it would be important for the authors to mention the 25% residual protein expression observed in the conditional knock-out model at E12.5 when comparing the outcomes of the APA profiles with those of the constitutive knock-in model.\nResponse:\nAs suggested, we have now explicitly mentioned the ~25% residual protein expression in the Pqbp1-cKO model at E12.5 when comparing the outcomes of the APA profiles with those of the constitutive knock-in model. (Page 14, lines 296-298 and Page 20, lines 450-451)\n• In addition, while I generally prefer to avoid suggesting further analyses, I believe that a direct comparison between the Y65C and cKO models in Figure 7 could be a valuable addition. This would not require new experiments, as all the relevant data have already been generated, and could be performed through an additional bioinformatic analysis. The current Y65C/WT vs. cKO/WT comparison is certainly useful to identify commonly deregulated APA events, but a direct Y65C vs. cKO analysis would more clearly highlight the specific differences between the two models. That said, I would consider this suggestion as a nice-to-have refinement rather than a mandatory request.\nResponse:\nWe thank the reviewer for this valuable suggestion. Accordingly, we have now performed a direct comparative analysis of the APA profiles between the Pqbp1Y65C and Pqbp1-cKO models. This analysis identified 1060 genes with significant differential APA usage. Among these, 649 genes (61.2%) exhibited a significant shift towards the proximal polyA site usage in the Pqbp1Y65C model compared to the cKO, while 411 genes (38.8%) favored distal sites. This result underscores substantial differences between the two models and reinforces the conclusion that the Y65C mutation drives a stronger and more specific proximal PAS bias. We have added this result in revised Supplementary Figure 8b.\nNevertheless, we have to cautiously note a potential technical confounder under this direct comparison: the RNA-seq libraries for these two models were prepared and sequenced in separate batches. Although standard normalization procedures were applied, we cannot rule out the possibility that batch effects may contribute to a portion of the observed variance. We have noted this limitation in the Discussion section. Therefore, we maintain that the parallel internal control comparisons presented in Figure 7—Pqbp1Y65C vs. its littermate WT control and Pqbp1-cKO vs. its littermate Pqbp1flox/Y control —provide a more rigorous and conservative assessment, as they are unaffected by batch effects.\n• Finally, in line with their findings, I would recommend a minor stylistic edit to the article title. Since the loss-of-function effect prevail over the gain-of-function effect, and is consistent with the main pathogenic mechanism described for this disease, this effect should be placed first in the wording.\nResponse:\nAs suggested, we have reorganized the title to prioritize the loss-of-function effect over the gain-of-function effect, which more accurately reflects the primary pathogenic mechanism."}]

Decision letter