Distinct effects on mRNA export factor GANP underlie neurological disease phenotypes and alter gene expression depending on intron content

Abstract

Defects in the mRNA export scaffold protein GANP, encoded by the MCM3AP gene, cause autosomal recessive early-onset peripheral neuropathy with or without intellectual disability. We extend here the phenotypic range associated with MCM3AP variants, by describing a severely hypotonic child and a sibling pair with a progressive encephalopathic syndrome. In addition, our analysis of skin fibroblasts from affected individuals from seven unrelated families indicates that disease variants result in depletion of GANP except when they alter critical residues in the Sac3 mRNA binding domain. GANP depletion was associated with more severe phenotypes compared with the Sac3 variants. Patient fibroblasts showed transcriptome alterations that suggested intron content-dependent regulation of gene expression. For example, all differentially expressed intronless genes were downregulated, including ATXN7L3B, which couples mRNA export to transcription activation by association with the TREX-2 and SAGA complexes. Our results provide insight into the molecular basis behind genotype-phenotype correlations in MCM3AP-associated disease and suggest mechanisms by which GANP defects might alter RNA metabolism.

Figure 1

Novel variants in MCM3AP and structural modeling of Sac3 domain variants. (A) Family pedigrees of EST and NL2. (B) qPCR quantification of MCM3AP mRNA levels in EST1, EST2 and NL2 patient and four control fibroblasts normalized to GAPDH. The mean of three technical replicates is shown for each cell line as dots. Bars show mean of control or patient cells. Error bars indicate SD. ** P < 0.0001. (C) Map of GANP variants investigated in this study. Variants in new families are shown in black and previously reported variants in grey. (D) Modelling of mutations in the Sac3 domain. The secondary structure of the GANP, PCID2 and DCCI chains is shown using different colors to identify specific components. The winged-helix regions of GANP and PCID2 are shown as yellow and red, respectively, their TPR (tetratricopeptide repeats) regions in green and magenta, respectively, and DCCI as light blue. All of the disease-associated missense variants (black) are located either in the GANP winged-helix domain (shown as yellow secondary structure) or in the GANP TPR region (shown as green) immediately adjacent to the winged helix domain. The residues mutated are primarily located in the interior of the structure and so are likely to generate local perturbations in the molecular conformation in this region. Although Ser951 is located on the surface, it lies within a helix, and because it is mutated to Pro, a residue that is incompatible with an α-helix, this mutation would also probably generate a local structural perturbation. It is likely that the local structural perturbations introduced by these mutations could influence the binding of RNAs to the GANP:PCID2:DCCI complex, either directly by changing the conformation of the GANP winged helix domain or indirectly by altering the interaction between GANP and PCID2.

Figure 2

Variants outside Sac3 domain cause loss of GANP protein. Immunocytochemistry with an N-terminal GANP antibody in patient fibroblasts. (A) Confocal images of patient group A (AUS, FIN1, FIN2, USA and NL1) and control fibroblasts stained with GANP antibody show normal localization to the nuclear envelope and interior in control and AUS cells and reduction in staining in other affected individuals’ cell lines. NLM refers to cell line of mother of NL1. (B) Confocal images of patient group B (EST1, EST2 and NL2). (C) Confocal images of patient group C (IR). IRM refers to cell line of mother of IR. (D) Example of categorization of cells into GANP +, GANP intermediate or GANP –. (E) Bar graph shows quantification of GANP protein levels relative to GAPDH for A-C. Bar graph quantification of GANP staining in the nucleus and the nuclear envelope as fraction of cells from blindly counted cells for that sample. Cells were classified into GANP + (black), intermediate (−/+) (dark grey) and GANP—(light grey) cells based on staining in the nuclear envelope and interior. Quantification is based on technical triplicate samples of the same experiment n^average = 112 for samples A and technical duplicates n^average = 136 for samples B, n^average = 90 for duplicate samples in experiment C. Dapi stains nuclei. Scale bars: 10 μm. See Supplementary material figure S1A for control samples for experiments B and C.

Figure 3

Ultrastructural assessment of the nuclear pore with electron microscopy. Electron micrographs of patient FIN1/2, AUS and control fibroblasts. Blue arrows indicate nuclear pore complexes and black arrows the nuclear envelope. Scale bars: 2 μm.

Figure 4

Differentially expressed genes in patient fibroblasts. (A) Pearson hierarchical correlation of gene expression of affected individuals’ and control fibroblasts. C indicates control. (B) Volcano plot showing expression differences and significance for genes. Of the TREX-2/SAGA complex associated genes MCM3AP and ATXN7L3B are indicated in green and non-significant USP22, ENY2 and ATXN7L3 in grey. (C) Heatmap of expression differences (to gene average) for the enriched genes in REACTOME category ‘neuronal system’. See full list in Supplementary material Table S2. (D) Gene-set enrichment analysis. See full list in supplementary material Table S3.

Figure 5

Downregulation of intronless genes in patient fibroblasts. (A) Distribution of exons across differentially expressed genes. (B) Volcano plot indicating expression differences for up- and downregulated intronless genes in log2FC in patient versus control samples and significance as –log10 p-value. (C) qPCR validations of intronless genes ATXN7L3B, EID1 and TSPYL4 relative to GAPDH. Each dot represents average of three technical replicates of an affected (n = 8) or control individual (n = 4). ** P < 0.0001. Error bars show mean with SD.

Figure 6

ATXN7L3B RNA in situ hybridization shows reduced signal in patient fibroblasts. (A) Patient and control fibroblasts hybridized with ATXN7L3B target probe (Cy3) and dapi (blue). White square indicates focus area on the other image. Scale bars: 10 μm. (B) Average of punctae number in cells/line is plotted in the bar graph. P < 0.05. Mean with SD plotted.