Interactome analysis reveals ZNF804A, a schizophrenia risk gene, as a novel component of protein translational machinery critical for embryonic neurodevelopment

Abstract

Recent genome-wide association studies identified over 100 genetic loci that significantly associate with schizophrenia (SZ). A top candidate gene, ZNF804A, was robustly replicated in different populations. However, its neural functions are largely unknown. Here we show in mouse that ZFP804A, the homolog of ZNF804A, is required for normal progenitor proliferation and neuronal migration. Using a yeast two-hybrid genome-wide screen, we identified novel interacting proteins of ZNF804A. Rather than transcriptional factors, genes involved in mRNA translation are highly represented in our interactome result. ZNF804A co-fractionates with translational machinery and modulates the translational efficiency as well as the mTOR pathway. The ribosomal protein RPSA interacts with ZNF804A and rescues the migration and translational defects caused by ZNF804A knockdown. RNA immunoprecipitation-RNAseq (RIP-Seq) identified transcripts bound to ZFP804A. Consistently, ZFP804A associates with many short transcripts involved in translational and mitochondrial regulation. Moreover, among the transcripts associated with ZFP804A, a SZ risk gene, neurogranin (NRGN), is one of ZFP804A targets. Interestingly, downregulation of ZFP804A decreases NRGN expression and overexpression of NRGN can ameliorate ZFP804A-mediated migration defect. To verify the downstream targets of ZNF804A, a Duolink in situ interaction assay confirmed genes from our RIP-Seq data as the ZNF804A targets. Thus, our work uncovered a novel mechanistic link of a SZ risk gene to neurodevelopment and translational control. The interactome-driven approach here is an effective way for translating genome-wide association findings into novel biological insights of human diseases.

Figure 1

ZFP804A expression during brain development. (a) qRT-PCR shows Zfp804a mRNA is increased during brain development. n=2, mean ±s.e.m. (b) Endogenous ZFP804A expression increases from E10 to E16 and peaks at E13, at the whole brain level. n=2, mean ±s.e.m. (c) Cortical staining of ZFP804A in E14 brain with TUJ1, a new-born neuron marker. Green, TUJ1; red, ZFP804A. Scale bar, 20 μm. (d) Cortical staining of ZFP804A at E14 brain with SOX2, an NPC marker. Green, SOX2; red, ZFP804A. Scale bar, 20 μm. (e) ZFP804A is highly expressed in the cortical and hippocampal regions of the mouse brain. (f–i) ZFP804A expression in the CA1 (f), the CA3 (g), the DG (h) and the cortex (i). Scale bar, 50 μm. DG, dental gyrus; NPC, neuronal progenitor cell; qRT-PCR, quantitative PCR with reverse transcription.

Figure 2

ZNF804A regulates neuronal migration and NPC proliferation. (a) ZNF804A overexpression enhances neural migration in E18 neocortex. Vector or ZNF804A plasmids were electroporated into E15 embryonic mouse brains, and mice were killed at E18. Percentage of GFP cells in each region. n=3 for each group, *P<0.05, n.s., mean ±s.e.m., t-tests. Scale bar, 100 μm. (b) Zfp804a knockdown induces migration defects of neural stem cells in E17.5 neocortex, which are rescued by ZNF804A overexpression. Control or Zfp804a shRNA plasmids were electroporated into E14.5 embryonic mouse brains alone or with ZNF804A, and mice were killed at E17.5. Percentage of GFP cells in each region. n=6 for vector+control and n=3 for other groups, *P<0.05, **P<0.01, ***P<0.001, n.s., not significant, mean ±s.e.m., one-way ANOVA with Tukey's multiple comparison test. Scale bar, 100 μm. (c) Brain slices of embryonic brains electroporated with control and shRNA constructs are stained with pH3. The SVZ/VZ area is shown. Percentages of pH3 and GFP double-positive cells in total GFP-positive cells are measured. n=3 for each group, ***P<0.001, mean ±s.e.m., t-tests. Scale bar, 20 μm. White arrows indicate GFP and pH3 double-positive cells. (d) Embryonic brains were electroporated with vector and ZNF804A OE constructs at E15 and harvested at E18. Coronal sections stained with intermediate neural progenitor marker, TBR2 (red). Percentages of TBR2 and GFP double-positive cells in total GFP-positive cells are shown. n=3, *P<0.05, mean ±s.e.m., t-tests. Scale bar, 20 μm. (e) Embryonic brains were electroporated with control and Zfp804a shRNAs constructs at E14.5 and harvested at E17.5. Brain sections stained with TBR2 (red). Percentages of TBR2 and GFP double-positive cells in total GFP-positive cells are shown. n=3 for each group, **P<0.01, mean±s.e.m., t-tests. scale bar, 20 μm. ANOVA, analysis of variance; CP, cortical plate; GFP, green fluorescent protein; n.s., not significant; IZ, the intermediate zone; SVZ/VZ, the subventricular zone/ventricular zone.

Figure 3

ZNF804A interacts with genes involved in translation and cell adhesion. (a) Biological processes of ZNF804A-interacting proteins. Black lines indicate new interactions identified here. Green lines indicate interactions reported by NCBI. Purple cluster represents genes in translational control. Blue cluster includes genes involved in cell adhesion and cytoskeleton. (b) Gene Ontology analysis shows enrichment of genes involved in translational regulation. (c–h) Immunoprecipitation studies confirm that ZNF804A associates with APEX1, FEZ1, LGALS1, PDHB, PDZD4 and RPSA. Each experiment has been repeated twice to confirm the results. (i) ZFP804A (red) is co-localized with GM130 (green) in CAD cells. Scale bar, 10 μm. (j) ZFP804A (red) is co-localized with endogenous RPSA (green) in CAD cells. Scale bar,10 μm.

Figure 4

ZNF804A controls protein translation efficiency. (a) ZNF804A sedimented in two fractions of polyribosomes prepared from HEK293 cells. EDTA treatment disassociated ZNF804A from the high-to-low molecular mass fractions. (b) AHA-labeled nascent proteins decrease with Zfp804a shRNAs in mouse CAD cells. n=3 for each group, *P<0.05, mean±s.e.m., t-tests. (c) AHA-labeled nascent proteins increase with ZNF804A overexpression in mouse CAD cells. n=3 for each group, *P<0.05, mean±s.e.m., t-tests. (d) ZNF804A overexpression increases phosphorylation of AKT, mTOR and p70 S6K. n=2, mean±s.e.m. (e) Laminin stimulates ZNF804A and RPSA interaction. Duolink assay detects close interaction of ZNF804A and RPSA in response to PDL and laminin. Quantifications show total area of the loci (unit= μm²), total number of loci each cell, and mean intensity (AU). n=99 for PDL coating and n=107 for laminin coating, **P<0.01, mean±s.e.m., Mann–Whitney U-test. Scale bar, 10 μm. (f) Co-electroporation of RPSA with Zfp804a shRNAs in E14.5 embryonic brain rescues the migration deficit caused by ZNF804A knockdown. Percentage of GFP cells in each region. n=6 for vector+control and n=3 for other groups, *P<0.05, **P<0.01, ***P<0.001, mean ±s.e.m., one-way ANOVA with Tukey's multiple comparison test. Scale bar, 100 μm. (g) Deficiency of AHA-labeled nascent protein caused by Zfp804a shRNAs can be rescued by RPSA in mouse CAD cells. n=5, *P<0.05, **P<0.01, mean±s.e.m., one-way ANOVA with Dunnett's multiple comparison test. AHA, azidohomoalanine; ANOVA, analysis of variance; AU, arbitrary unit; GFP, green fluorescent protein; n.s., not siginificant PDL, poly-d-lysine.

Figure 5

RNA-IP sequencing identified a large number of RNAs bound by ZFP804A. (a) Volcano plot of RIP-seq results showing target genes enriched by ZFP804A IP. (b) Gene length distribution of ZFP804A target genes versus background distribution. (c) Major functional categories enriched in ZFP804A target genes. (d) Enrichment of ribosomal genes by ZFP804A IP. (e) Brain cell-type-specific genes among the ZFP804A target genes. (f) Enrichment of neuron-specific genes, including several interneuron marker genes. (g) Western blot confirms that TYROBP and NRGN are downregulated when ZFP804A is knocked down. n=2, mean±s.e.m. (h) CAD cells expressing vector or ZNF804A construct (GFP+) were transiently labeled with puromycin. The translation efficiency was monitored by the PLA signals amplified from antibodies against puromycin and USMG5. Duolink assay showed that USMG5 translation is significantly increased by ZNF804A. n=105 with vector and n=114 with ZNF804A overexpression, *P<0.05, mean±s.e.m., Mann–Whitney U-test. Scale bar, 10 μm. (i) Co-electroporation of NGRN with Zfp804a shRNAs in E14.5 embryonic brain rescues the migration deficit caused by Zfp804a knockdown. Percentage of GFP cells in each region. n=6 for vector+control, n=7 for NRGN+control, n=3 for other groups, *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA with Tukey's multiple comparison test. Scale bar, 100 μm. ANOVA, analysis of variance; IP, immunoprecipitates; GFP, green fluorescent protein; n.s., not significant.