Molecular consequences of PQBP1 deficiency, involved in the X-linked Renpenning syndrome

Mutations in the PQBP1 gene (polyglutamine-binding protein 1) are responsible for a syndromic X-linked form of intellectual disability (XLID), the Renpenning syndrome. PQBP1 encodes a protein that plays a role in the regulation of gene expression, splicing and mRNA translation. To investigate the consequences of variants in PQBP1, we performed transcriptomic studies in 1) patients’ lymphoblastoid cell lines (LCL) carrying pathogenic variants in PQBP1 and 2) in human neural stem cells (hNSC) knocked-down (KD) for PQBP1. This led to the identification of a hundred dysregulated genes. In particular, we identified an increase in the expression of a non-canonical isoform of another XLID gene, UPF3B. UPF3B plays a crucial role during neurodevelopment by coding for an important actor of the nonsense mRNA mediated decay (NMD) system involved in regulation of protein translation, however, the exact function of the non-canonical isoform,UPF3B_S, is currently unknown. In order to investigate the role of UPF3B_S isoform, we compared the protein interactome of UPF3B_S to the canonical isoform (UPF3B_L). We confirmed that, on the contrary to UPF3B_L, UPF3B_S does not interact with the UPF2/UPF1 complex while it still interacts with exon junction complexes (EJC). However, no notable decrease of NMD pathways was observed in patient’s LCL or in hNSC KD for PQBP1. We identified several additional protein interactors specific to UPF3B_S. Moreover, we used the increase of UPF3B_S mRNA as a molecular marker to test the pathogenicity of variants of unknown clinical significance identified in individuals with ID in PQPB1. We analyzed patients’ LCL mRNA as well as blood mRNA samples and performed complementation studies in HeLa cells by overexpressing Wild-type and mutant PQBP1 cDNA. We showed that all these three approaches were efficient to test the effect of variants, at least for variants affecting the CTD domain of the protein. In conclusion, our study provides information on how PQBP1 deficiency may affect the expression of genes and isoforms, such as UPF3B. This informs about the pathological mechanisms involved in Renpenning syndrome but also allows to propose a functional test for variants of unknown significance identified in PQBP1.

PQBP1 encodes a protein involved in different cellular processes such as regulation of transcription, splicing, translation or even response to retroviral infection. PQBP1 was initially described as a protein interacting with polyglutamine tracts of huntingtin or ataxin1, through its polar amino-acid-rich domain (PRD) (7). It also contains on its N-terminal side a WW domain which has a transcriptional activity and interacts with the splicing factor SIPP1/WP11. The C-terminal part of PQBP1 includes a domain (CTD) interacting with other splicing factors such as U5-15kDa/TXNL4A (8). Moreover, a nuclear localization signal (NLS) allows its addressing to the nucleus through the Kapβ2 receptor (9,10). In the nucleus, PQBP1 is involved in transcription regulation through its interaction with activated RNA polymerase II (7) and various transcription factors such as POU3F2/Brn2 (11). PQBP1 has been found to be located in nuclear speckles, suggesting a role in splicing regulation, consistent with its interactions with the different splicing factors cited above and with the general splicing alterations observed after PQBP1 knock-down in a model of murine primary neurons (12). PQBP1 is a nuclear-cytoplasmic shuttling protein playing also various roles in the cytoplasm. Indeed, it can be localized in stress granules (13) and was shown to play a role in translation of messenger RNAs (14). PQBP1 binds to the elongation factor eEF2 via its WW domain and suppresses its phosphorylation-mediated inactivation. Loss of PQBP1 leads to an increase of the phosphorylation of eEF2 stopping translational elongation and resulting in a global decrease of protein synthesis, which affect protein synthesis-dependent synaptic plasticity in the hippocampus (14). PQBP1 is involved in the regulation of neuronal ciliogenesis via its interaction with Dynamin2 (15). Finally PQBP1 can also play a role in response to retroviral infection, interacting with reverse-transcribed HIV-1 DNA, probably through its CTD domain, and to the viral DNA sensor cGAS probably through its WW domain, and therefore contributing to the innate immune response (16).
Constitutive knock-out of Pqbp1 in mice appears to be lethal (www.mousephenotype.org/), mice models where Pqbp1 was knocked-down (~50%) display abnormal anxiety-related behavior, and a decrease in anxiety-related cognition (17). Nestin-Cre conditional knock-out (Pqbp1 cKO) display abnormal anxiety-related behavior, as well as abnormal fear conditioning and motor dysfunction at the rotarod test (18). Recently, the same group demonstrated that cKO mice showed a short stature and a reduction in bone mass, and display impairment in bone formation and chondrocyte deficiency with reduced osteoblast and chondrocyte-related gene expression (19). The Pqbp1 cKO showed microcephaly, probably resulting from an elongated cell cycle of neural progenitors (18).
In humans, most of the variants described are small indels, located in the AG hexamer of exon 4 or downstream, resulting in truncated proteins lacking their C-terminal domain, but few additional truncating variants were also reported (6,(20)(21)(22). Only few missense variants have been reported in PQBP1 and for years, only a unique missense variant (p.Tyr65Cys) was considered as pathogenic (4). This missense variant affects a very conserved amino acid position located in the WW domain and disrupts the interaction between PQBP1 and the splicing factor WBP11 leading to a decreased pre-mRNA splicing efficiency (23). Two other missense variations (p.Arg243Trp and p.Pro244Leu), located in the CTD domain were identified more recently in patients with ID by our group and others (24,25). The Pro244Leu change was shown to disrupt PQBP1 binding to the splicing factor U5-15kDa/TXNL4A (10).
We describe in this study the changes in gene expression induced by a loss of function of PQBP1 in human cells, using both lymphoblastoid cell lines (LCL) of patients carrying pathogenic variants in PQBP1 and human neural stem cells (hNSC) where PQBP1 was knocked-down (KD) by siRNA. We report in particular the increase of expression of a noncanonical isoform of the UPF3B gene, another gene involved in XLID playing a role in nonsense-mediated mRNA decay (NMD) and regulation of translation. Finally, we showed that we can use this increase as a biomarker for Renpenning syndrome to test the pathogenicity of variants of unknown significance in PQBP1 located in the C terminal part of the protein.

RT-qPCR
Total RNAs were extracted from cells using the RNeasy extraction kit and treated with RNase free DNAse set during 20 min (Qiagen, Valencia, CA, USA) or from blood using PaxGene blood RNA kit extraction (Preanalytix, Hombrechtikon, Switzerland). RNA levels and quality were quantified using a Nanodrop spectrophotometer and then with a 2100 Bioanalyzer All qPCR reactions were performed in triplicate. Primer sets are listed in Table S2.

Western Blot
Cells were lysed in RIPA buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 0.25% sodium deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail. 5 to 50μg of protein lysate were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride membrane. Membranes were blocked in 5% nonfat dry milk diluted in tris buffered saline with tween 20 (50mM Tris, 150mM NaCl, 0.05% Tween 20) and probed using the antibodies overnight at 4°C. GAPDH was used as loading control.
Incubation with appropriate secondary HRP-labelled antibody (less than 1h) was followed by detection with Immobilon western chemiluminescent HRP substrate (Merck Millipore, Darmstadt, Germany).

Immunoprecipitation and Mass Spectrometry analyses
Protein were extracted from HEK293 cells overexpressing UPF3B_L (ENST00000276201) or UPF3B_S (ENST00000636792) and subjected to immunoprecipitation with Pierce Anti-HA Magnetic Beads (Thermofisher 88836) according to manufacturer protocol.

Identification of genes differentially expressed in individuals with Renpenning syndrome
In order to identify the effect of PQBP1 deficiency on the regulation of gene expression and alternative splicing, we performed sequencing of mRNA extracted from lymphoblastoid cell lines (LCL) obtained from unrelated control males (n=3) and males carrying pathogenic or likely pathogenic variants in PQBP1 (n=5) (Figure 1, Figure S1-S2-S3, Table S1)

Consequences of PQBP1 knock-down in human neural stem cells (hNSC)
In order to identify additional DEG or DEE after PQBP1-KD in hNSC, we performed RNAseq of mRNA extracted from the first hNSC line (hNSC-1, in duplicates) ( Figure S2).

Transcriptomic analysis revealed 59 significant DEG (12 up-regulated and 47 downregulated) in PQBP1-KD cells compared to cells treated with transfection agent only
(INTERFERin), while no gene was found significantly DE in the scramble siRNA condition (Table S5, Figure 3D). Unsurprisingly, the most significant DE gene was PQBP1 (log2FC=-0.82, adjusted p-value =3.7E-8). To confirm these changes in gene expression, RT-qPCR was performed for the best candidate genes on a third series of hNSC-1 (n=3 samples per condition) as well as on three series of hNSC-2 (n=9 samples per condition in total) ( Figure   3E). Among others, we identified that PQBP1-KD leads to a decrease in expression of genes encoding proteins essential for brain development, including transcription factors (FOXJ1, A similar tendency was obtained for hNSC-2 (p=0.059). Despite the described role of PQBP1 in splicing regulation, difference in exon coverage were observed in a few genes only using DEXSeq (n=3) ( Table S6), but could not be confirmed by RT-PCR.

The increase of UPF3B_S expression induced by PQBP1 deficiency does not affect NMD
The UPF3B_L isoform encodes a protein involved in mRNA nonsense mediated decay (NMD), but the role of UPF3B_S is currently not known. This isoform is particularly expressed in testis and in EBV-immortalized lymphocytes but is also detected at lower level in other tissues such as cerebellum (GTex database). Therefore, to test if this increase in UPF3B_S expression caused by PQBP1 deficiency affects NMD, we first searched if known NMD target genes (35) were DE in LCL from individuals with Renpenning syndrome or in PQBP1-KD hNSC but found no significant overlap. Finally, we proceeded to PQBP1-KD in fibroblasts carrying a truncating variant in the DYRK1A gene (c.1232dup, p.Arg413fs) known to be degraded by the NMD system (36). We observed an increase of UPF3B_S expression (2,4 and 6 days after inactivation) but with no effect on the DYRK1A mutant transcript, confirming that PQBP1 inactivation has no obvious effect on NMD (data not shown). To study the role of UPF3B_S, we generated plasmids containing human UPF3B_L (ENST00000276201) and UPF3B_S cDNA sequences (ENST00000636792) and performed immunoprecipitation-coupled mass spectrometry on proteins extracted from HEK293 cells transfected with each of the constructs. We identified a total of 101 proteins: 13 proteins interacting with both isoforms, 74 proteins interacting preferentially or exclusively with UPF3B_L and only 14 preferentially or exclusively with UPF3B_S (Table S7). We retrieved eight of the known UPF3B interactors annotated in the STRING and BioGRID databases ( Figure 4). As expected, we found that UPF1, UPF2 and ERF3A/GSPT1 interact only with UPF3B_L and not UPF3B_S, which lacks the interaction domain (Figure 4), while the EJC binding protein RBM8A (alias Y14) interacts with both isoforms. Among the proteins interacting preferentially with UPF3B_S, we identified proteins involved in antibacterial humoral response (LTF, SEMG1, IGHA1) and in redox homeostasis (PRDX4, SDHA).

As the increase of UPF3B_S expression was observed in both cells from individuals with
Renpenning syndrome and in all types of cells KD for PQBP1 (Figure 3C), we intent to use it as a biomarker of PQPB1 deficiency in order to test the functional consequences of variants of uncertain significance in this gene. We tested UPF3B_S expression in LCLs from individuals with PQBP1 variants. We observed a significant increase of UPF3B_S in LCL obtained from two brothers carrying a distal frameshift variant, p.Phe240fs, leading to a protein 10 amino acids longer than the wild-type and previously reported by Hu et al.  Figure 5C). Interestingly, the UPF3B_S increase was even more pronounced for these mutants than for p.Arg153fs* and an increase was observed even without PQBP1-KD, suggesting that these mutants have a stronger effect than just a loss of PQBP1. The increase was not significant for the p.Tyr65Cys and p.Arg10Pro variants, confirming what we previously observed in LCL ( Figure 2B) and suggesting that expression, suggesting a likely pathogenic effect ( Figure 5C).

DISCUSSION
In this study, we identified genes and exons differentially expressed in individuals with pathogenic variants in PQBP1 or after a transient knock-down of PQBP1 in human neural stem cells (hNSC). If we observed a high variability in gene expression in LCL, which prevented us to confirm several of the deregulations identified by RNA sequencing such as APP increase, the changes in gene expression observed in hNSC were robust and most of them were successfully replicated in a second independent hNSC line. Decrease in the expression of genes involved in brain development and synapse formation or in lipid metabolism were observed, which are consistent with the roles of PQBP1 previously described by others. For instance, a role of PQBP1 in regulation of dendrite length and branching has been described in mouse embryonic primary cortical neurons (12). PQBP1 was also found to regulate lipid metabolism: a decrease of the lipid content was observed in nematode intestinal cells, which could be related to the low body mass index observed in patients (37). Among the genes deregulated after PQBP1-KD, we found genes involved in other neurodevelopmental disorders such as CELF2 (expression decreased after PQBP1-KD), an RNA binding protein mutated in a neurodevelopmental syndrome with ID and epilepsy (38), or UPF3B (expression increased for its non-canonical isoform UPF3B_S), another Xlinked gene involved in ID (39). This increase of UPF3B_S was observed in different cell types after PQBP1-KD (hNSC, HeLa, fibroblasts, etc) and was also detected from patients' material (LCL and blood). Compared to the canonical UPF3B_L transcript, this isoform involves an alternative transcription initiation site as well as an alternative splicing of exon 11 (the last exon of the canonical isoform).
There is no report about the biological function of this short isoform of UPF3B. UPF3B_S mRNA appears to be low expressed, except in testis, according to GTEx database. It is noticeable that genital manifestations, and especially microorchidia, were reported for individuals with Renpenning syndrome. A mild expression in Epstein Barr Virusimmortalized lymphoblastoid cells is also reported in GTEx, and might explain why we particularly detected this increase in LCL. If no expression of UPF3B_S is reported in GTEx in adult brain tissue, we cannot exclude that it could be expressed in a transient way during brain development. UPF3B_S predicted protein sequence contains motifs known to mediate interaction with RBM8A (alias Y14) but not those mediating interaction with UPF2/UPF1.
Using immunoprecipitation coupled to mass spectrometry (IP-MS), we confirmed that UPF3B_S can bind to the EJC-protein RBM8A but fails to interact with NMD effectors UPF1 and UPF2. We could speculate that UPF3B_S could therefore exert a competitive antagonist activity to UPF3B_L on NMD. However, we did not observe NMD dysfunction after PQBP-KD in fibroblasts carrying a premature stop codon (PTC) and we observed no change in expression of natural target of NMD in hNSC or LCL. We found that UPF3B isoforms interact with proteins involved in mRNA binding and regulation of translation (ribosomal proteins and translation initiation or elongation factors). In particular, both isoforms interact with the translation elongation factor eEF2 (14), which is also known to be a partner of PQBP1. Indeed, PQBP1 binds to eEF2 and prevent its phosphorylation, which decreases translational elongation (14). Further experiments will be necessary to test whether UPF3B, known to play a role in the regulation of normal translation termination in addition to its role in NMD (40), could also be involved in regulation of translation elongation.
Among the few proteins detected as interacting specifically with UPF3B_S, some play a role in regulation of cell cycle (PRDX4, GLUL In conclusion, we describe here for the first time the consequences of PQBP1 inactivation in human neural stem cells. We were able to show that cell proliferation is affected, as well as the expression of a about fifty genes, some of them known to be involved in other neurodevelopmental disorders. Despite the known role of PQBP1 in splicing regulation and the fact that the pathogenic variants are known to disrupt interactions with splicing factors (10,23), we failed to identify important changes in alternative splicing events. The only isoform-specific event we could detect was the increase of a non-canonical isoform of UPF3B, which has a still unknown function, but which can be used as a biomarker for PQBP1 deficiency.

DATA AVAILABILITY
Data have been submitted to Gene Expression Omnibus.