Biallelic mutations in the 3' exonuclease TOE1 cause pontocerebellar hypoplasia and uncover a role in snRNA processing

Abstract

Deadenylases are best known for degrading the poly(A) tail during mRNA decay. The deadenylase family has expanded throughout evolution and, in mammals, consists of 12 Mg²⁺-dependent 3'-end RNases with substrate specificity that is mostly unknown. Pontocerebellar hypoplasia type 7 (PCH7) is a unique recessive syndrome characterized by neurodegeneration and ambiguous genitalia. We studied 12 human families with PCH7, uncovering biallelic, loss-of-function mutations in TOE1, which encodes an unconventional deadenylase. toe1-morphant zebrafish displayed midbrain and hindbrain degeneration, modeling PCH-like structural defects in vivo. Surprisingly, we found that TOE1 associated with small nuclear RNAs (snRNAs) incompletely processed spliceosomal. These pre-snRNAs contained 3' genome-encoded tails often followed by post-transcriptionally added adenosines. Human cells with reduced levels of TOE1 accumulated 3'-end-extended pre-snRNAs, and the immunoisolated TOE1 complex was sufficient for 3'-end maturation of snRNAs. Our findings identify the cause of a neurodegenerative syndrome linked to snRNA maturation and uncover a key factor involved in the processing of snRNA 3' ends.

Figure 1

TOE1 mutations lead to pontocerebellar hypoplasia with abnormal genitalia (PCH type 7). (a) Pedigrees of affected families showing recessive inheritance. Double bar, consanguineous marriage; open circle, unaffected female; open square, male; filled circle, affected female, filled square, affected male; triangle, spontaneous abortion; open diamond, unaffected unknown sex; diagonal line, deceased; arrow, proband. (b) Magnetic resonance midline sagittal (top) and axial (bottom) images showing reduced cerebellar volume (yellow arrows).

Figure 2

TOE1 mutations occur in conserved domains and amino acid residues and reduce protein levels. (a) TOE1 is a 510 amino acid protein containing a DEDD deadenylase domain (red), C₃H-type Zinc finger (orange), and a nuclear localization signal (NLS, blue). Identified homozygous mutations (bold) above, compound heterozygous mutations below, with corresponding family number. (b) TOE1 missense mutations showing vertebrate conservation. (c) TOE1 mutations (blue) modeled onto solved structures of paralog CNOT7 and Zn Finger C₃H domain of TOE1 (both teal). Residues affecting deadenylase activity (D64, E66; red) occur in the RNA cleft (grey arrow), while the patient mutations are located on the protein surface. (d) Western blots (cropped) showing reduced expression of TOE1 in affected (A) compared with related unaffected (U) fibroblasts. Control: ATCC fibroblast cell line. GAPDH loading control. See Supplementary Fig. 3b for full length blots (e) Impaired accumulation of TOE1 protein in affected (A) compared with related unaffected (U) patient-derived neural progenitor cell (NPC) lines. GAPDH loading control.

Figure 3

Toe1 depletion in zebrafish results in structural brain defects, mimicking human PCH7 pathology. (a) Comparison of zebrafish at 48 hours post fertilization (hpf) injected with 6 ng Control, toe1 ATG morpholino (MO), toe1 Splice-blocking MO, or toe1 Splice-blocking MO together with 0.1 pg zebrafish (z) or human (h) TOE1 mRNAs. MO injected fish show abnormal head shape and thin, curved tails, which is rescued by addition of zebrafish or human WT, but not DE mutant TOE1 mRNA. Scale bar = 500 µm. Quantification at bottom right. Normal: no observable phenotype; Moderate: small eyes, slight reduction in head size; Severe: small eyes, small head, thin curved tail. n=100/condition. (b) Maximum confocal projection of whole mount pan-neuronal HuC immunofluorescence at 48 hpf. Diagram of zebrafish anatomy (right). Midbrain, cerebellum, and hindbrain regions of toe1 MO injected zebrafish have reduced HuC protein levels, which were restored with WT zebrafish and human TOE1, but not human DE mutant mRNA. Scale bar = 100 µm. n = 6.

Figure 4

TOE1 targets 3’ extended pre-snRNAs. (a) Northern (top) and Western (middle and bottom - cropped) blots for cells treated with control (siCtrl) or TOE1 siRNA (siTOE1), then induced to express either WT or DE FLAG-TOE1 at near endogenous levels. Input samples (left). Samples from RNA immunoprecipitation with anti-FLAG (right). Arrows mark slower migrating snRNA species associated with TOE1 DE suggesting impaired processing. (b) Left: Cumulative plots of sequence reads for snRNA 3’ ends from the average of 3 independent experiments. Position ‘0’ refers to mature 3’ end of snRNAs with shaded areas indicating 3’ end positions within the mature snRNAs. Reads terminating at position -4 or after are represented (see Supplementary Figure 8b for all reads). Dotted curves represent 3’ end positions of genome templated snRNA sequences. Solid curves mark 3’ end positions of snRNA sequences, including untemplated tails. Right: Bar graphs showing the average percent of snRNA reads with 3’ tails from 3 independent experiments. Independent experiments represented by dots. Error bars: standard deviation (S.D.) from three independent experiments and p-values (Student’s two-tailed paired t-test) *: p<0.05, **: p<0.01. Cumulative plots and bar graphs for U1 and U5 snRNAs represent reads from all snRNA variants, whereas for U2 snRNA, reads are from RNU2-1 genes only (see Supplementary Figure 8b for RNU2-2P genes) and the 3’ adenosine added to mature RNU2-1 snRNAs was left out of the analysis to allow visualization of exonucleolytic processing. (c) Schematic of U1 snRNA processing intermediate with 3’ end templated tails (encoded nucleotides, pink) and untemplated tails (unencoded nucleotides, purple).

Figure 5

TOE1 enzymatic activity processes snRNA 3’ ends. (a) Northern blot for RNA from TOE1 activity assays. Left: input samples from RNA of cells treated with siCtrl or siTOE1, then induced to express either FLAG-TOE1-WT or -DE at near endogenous levels. Right: samples from RIP with anti-FLAG were divided and treated either with 2mM Mg²⁺ or 2mM EDTA on-bead, post-IP; asterisks mark TOE1-processed snRNA. (b) Cumulative plots of sequence reads for U1 and U5 snRNA 3’ end positions after incubation with TOE1-WT or -DE. Dark shaded area indicates reads terminating within the mature U5 snRNA and before the alternative 3’ end (Alt 3’ end). (c) Primary Pol-II transcribed snRNAs (pri-snRNAs) are co-transcriptionally cleaved by the Integrator complex. Remaining 3’ tails are subsequently processed to mature length by TOE1, in a process that might involve a 3’ terminal nucleotidyltransferase (3’ TnT).

Figure 6

Patient-derived fibroblasts and neuronal progenitor cells (NPCs) show defects in snRNA 3’ ends. (a) Cumulative U1 and U2 snRNA 3’ end sequence reads from affected (A) and unaffected (U) patient fibroblasts, showing TOE1 mutations result in snRNA tails (top). Bar graph showing percent reads with 3’ tails (bottom). (b) Cumulative U1 and U2 snRNA 3’ end sequence reads from affected and unaffected patient-derived neuronal progenitor (NPC) lines showing TOE1 mutations result in snRNA tails for NPCs (top). Percent reads with 3’ end tails (bottom).