Biallelic mutations in the 3’ exonuclease TOE1 cause Pontocerebellar Hypoplasia Type 7 and result in snRNA processing defects

Processing of RNA polymerase II-transcribed spliceosomal small nuclear RNAs (snRNAs) initiates with cleavage by the Integrator complex, but the factors that subsequently remove 3’ end extensions from metazoan snRNAs have remained unknown. We studied human families with a unique recessive syndromic constellation of pontocerebellar atrophy with ambiguous genitalia, uncovering biallelic inactivating mutations in TOE1, which encodes a conserved unconventional deadenylase. TOE1 demonstrated tight association with snRNA-protein (snRNP) complexes with specificity for incompletely processed pre-snRNAs containing 3’ end extensions that often included post-transcriptionally added tails. Human cells deficient for TOE1 catalytic activity showed accumulation of 3’-end extended U1, U2, U4 and U5 pre-snRNAs, and TOE1 immuno-isolated from human cells was capable of processing 3’-end extended snRNPs in vitro. The missense mutations identified in patients impaired TOE1 stability and snRNA processing in patient cells, which was associated with defects in splicing. Our findings reveal the cause of a unique brain malformation and uncover a long-sought 3’ exonuclease required for snRNA processing. We performed 154 experiments to profile snRNA tails for U1, U2, U4, and U5 snRNAs. Samples profiled include HEK 293, primary patient fibroblasts, and NPCs derived from primary patient fibroblasts, which were subjected to various treatments to study TOE1/cap1Z function (including siRNA of TOE1 compared to control siRNA, and over-expression of various TOE1 mutants compared to wild-type TOE1 and a FLAG-only control). For some indicated samples total RNA was used; for others in which FLAG-tagged TOE1 was over-expressed, profiling was performed on RNA isolated by RIP (RNA immunoprecipitation) using an anti-FLAG antibody.