Mutation of the conserved polyadenosine RNA binding protein, ZC3H14/dNab2, impairs neural function in Drosophila and humans

Abstract

Here we report a human intellectual disability disease locus on chromosome 14q31.3 corresponding to mutation of the ZC3H14 gene that encodes a conserved polyadenosine RNA binding protein. We identify ZC3H14 mRNA transcripts in the human central nervous system, and we find that rodent ZC3H14 protein is expressed in hippocampal neurons and colocalizes with poly(A) RNA in neuronal cell bodies. A Drosophila melanogaster model of this disease created by mutation of the gene encoding the ZC3H14 ortholog dNab2, which also binds polyadenosine RNA, reveals that dNab2 is essential for development and required in neurons for normal locomotion and flight. Biochemical and genetic data indicate that dNab2 restricts bulk poly(A) tail length in vivo, suggesting that this function may underlie its role in development and disease. These studies reveal a conserved requirement for ZC3H14/dNab2 in the metazoan nervous system and identify a poly(A) RNA binding protein associated with a human brain disorder.

Fig. 1.

ZC3H14 is mutated in NS-ARID patients. (A) Pedigree of Family-1. (B) Schematic of four ZC3H14 splice variants indicating exons encoding the N-terminal PWI-like domain and C-terminal Cys₃His zinc-finger (ZnF) RNA binding motif (CCCH) domain. Positions of patient mutations are indicated by red stars. (C) Anti-ZC3H14 immunoblot of two control lymphoblast lines and one derived from a Family-1 R154X patient. The ZC3H14 antibody (5) recognizes ZC3H14 isoforms 1 and 2/3. Anti-PABPN1 is shown as a loading control (20). (D) Immunofluorescent detection of ZC3H14 (green) in control or patient (R154X) fibroblasts using a commercial ZC3H14 antibody (Abcam) directed against the common ZnF domain. DAPI (blue) marks nuclei. (Scale bar: 10 μm.)

Fig. 2.

ZC3H14 is expressed in vertebrate hippocampal neurons. (A) RT-PCR analysis of ZC3H14 splice variants: variants 1–4 (Top), variant 1 (Middle), or variant 4 (Bottom) from indicated tissues. For B–D, ZC3H14 was detected with the N-terminal antibody that recognizes ZC3H14 isoforms 1 and 2/3 (5). (B) Immunofluorescent detection of ZC3H14 protein (red) in a mouse hippocampal section expressing oligodendroglia-GFP (green) (21). Cornu ammonis fields 1 and 3 (CA1 and CA3, respectively) and dentate gyrus granular cells (DGC) regions of the hippocampus are indicated. The white boxes indicate the zoom-in region in the merge. (C) Adolescent mouse brain sections probed with an oligo-dT FISH probe to detect poly(A) RNA (red) and costained for ZC3H14 (green). ZC3H14 appears in poly(A) RNA-positive nuclear speckles in hippocampal pyramidal neurons. DAPI (blue) marks nuclei. (Scale bars: 20 μm.) (D) Poly(A) RNA FISH (red) and indirect immunofluorescence in cultured rat embryonic hippocampal neurons reveal colocalization of ZC3H14 protein (green) with poly(A) RNA speckles in the nucleus (blue). (Scale bar: 5 μm.)

Fig. 3.

dNab2 is a putative D. melanogaster ortholog of ZC3H14. (A) Domain alignment of S. cerevisiae Nab2, Drosophila dNab2, and human ZC3H14. The conserved N-terminal PWI-like fold, Q-rich, RGG/predicted nuclear localization signal (NLS), and C-terminal tandem Cys₃His ZnF RNA binding motif (CCCH) domains are indicated (5, 10). Amino acid alignment of the five Cys₃His tandem ZnFs from fly dNab2 and human ZC3H14 shows conserved spacing and intervening basic and aromatic residues (underlined) that are required for RNA binding in S. cerevisiae Nab2 (10). (B) RNA binding properties of dNab2 analyzed by RNA electrophoretic mobility shift assay. GST-dNab2 ZnF (ZnFs 1–5) but not GST binds to polyadenosine 25-mer (pA₂₅) RNA. The top arrow indicates a shift. Unlabeled pA₂₅ but not randomized polyN 25-mer (pN₂₅) RNA competitor oligonucleotide competes efficiently for binding to dNab2 ZnF. (C) Schematic of the dNab2 locus indicating the location of the EP3716 and EY08422 elements (inverted triangles) and the five imprecise excision alleles (ex1–ex5). (D) Quantitative real-time RT-PCR analysis of dNab2 transcript levels in adults flies. All genotypes were analyzed in triplicate and normalized to dNab2 transcript levels in w¹¹¹⁸ control animals (set to 1.0). β-tub is an internal control. Error bars = SD. (E–O) Light microscopic images of adult flies of indicated genotypes. (E and F) The majority of dNab2^ex3 and dNab2^ex3/Df [Df(3R)Exel8178] animals die at pharate adult stage, often as partially emerged adults. (G and H) The remainder emerge with a wings held-out phenotype that is (I) absent in controls (p-ex). (J–O) Front and side views of the thorax showing thoracic bristles. dNab2^ex3 (J and K) and dNab2^ex3/Df (L and M) mutants show bent major thoracic bristles (arrowheads in J and L are enlarged in J Inset and L Inset, respectively) and disorganized minor thoracic bristles (arrows in J and L) compared with p-ex controls (N and O).

Fig. 4.

A neuronal-specific requirement for dNab2 in normal behavior. (A) Locomotor phenotypes of genomic alleles and tissue-specific RNAi of dNab2. Data are presented as the average percentage of flies that reach the top of a cylinder after 5 s across all trials. Groups of 10 5-d-old flies were tested for at least 10 independent trials per genotype (*P = 8.36 × 10⁻⁹ and **P = 8.38 × 10⁻⁹ in a two-tailed t test). Error bars = SEM. (B and C) Pan-neuronal expression of dNab2 rescues both eclosion and locomotor defects. (B) Table summarizing the percentage of flies eclosed (of expected) for indicated genotypes. (C) For the locomotor assay, data are presented as the average percentage of flies that reaches the top of a cylinder after indicated time points across all trials. Groups of 10 2-d-old flies were tested for at least 10 independent trials per genotype (*P = 5.12 × 10⁻¹³, **P = 6.59 × 10⁻¹³, ***P = 1.97 × 10⁻⁷, and ****P = 0.002 in a two-tailed t test). Error bars = SEM.

Fig. 5.

dNab2 regulates poly(A) tail length. (A) Light microscopic images of adult fly eye (A) and (B) quantification of eye size (pixels) for the following genotypes: (A, i) GMR-Gal4/+ (GMR control), (A, ii) GMR-Gal4/+;dNab2^EP3716/+ (dNab2 o/e), (A, iii) GMR-Gal4/Pabp2⁵⁵;dNab2^EP3716/+ (dNab2 o/e Pabp2^−/+), (A, iv) GMR-Gal4/Pabp2^EP2264;dNab2^EP3716/+ (dNab2 o/e Pabp2 o/e), (A, v) GMR-Gal4/+;dNab2^EP3716/UAS-eGFP (dNab2 o/e eGFP o/e), (A, vi) GMR-Gal4/Pabp2⁵⁵ (Pabp2^−/+ control), and (A, vii) GMR-Gal4/Pabp2^EP2264 (Pabp2 o/e control). o/e, overexpression. *P = 0.0006 and **P = 0.0014 in a two-tailed t test (n = 5 per genotype). Error bars = SEM. (C) Bulk poly(A) tail length measurements in heads of (1) control (p-ex), (2) dNab2^ex3, (3) GMR-Gal4/+;dNab2^EP3716/+, and (4) GMR-Gal4/Pabp2^EP2264;dNab2^EP3716/+. (D) Densitometric quantification of poly(A) tracts (Image J) from C showing poly(A) tail length profiles of the indicated genotypes (highlighted in colored lines). (E) Bulk poly(A) tail length from whole flies as analyzed by densitometric quantification of poly(A) tracts (Image J) of ∼250 nt normalized to poly(A) tracts of ∼100 nt for WT (w¹¹¹⁸) and dNab2^ex3. w¹¹¹⁸ control was set to 1.0. (*P < 0.04; n = 3, two-tailed t test). Error bars = SD. (F) Poly(A) RNA localization was analyzed in wing disc cells subjected to FISH to visualize poly(A) RNA (green) and costained with anti-Lamin D (red) to visualize the nuclear periphery. Upper shows a dNab2^ex3 mosaic larval wing disc; dotted lines denote boundaries between WT (^+/+) and dNab2^ex3 mutant clones (^−/−). Lower shows a wing disc homozygous for the sbr^ts allele (17) shifted to 33 °C. Arrowheads indicate nuclei accumulating poly(A) RNA.