Full UPF3B function is critical for neuronal differentiation of neural stem cells

Abstract

BackgroundMutation in the UPF3B gene on chromosome X is implicated in neurodevelopmental disorders including X-linked intellectual disability, autism and schizophrenia. The protein UPF3B is involved in the nonsense-mediated mRNA decay pathway (NMD) that controls mRNA stability and functions in the prevention of the synthesis of truncated proteins.ResultsHere we show that NMD pathway components UPF3B and UPF1 are down-regulated during differentiation of neural stem cells into neurons. Using tethered function assays we found that UPF3B missense mutations described in families with neurodevelopmental disorders reduced the activity of UPF3B protein in NMD. In neural stem cells, UPF3B protein was detected in the cytoplasm and in the nucleus. Similarly in neurons, UPF3B protein was detected in neurites, the somatic cytoplasm and in the nucleus. In both cell types nuclear UPF3B protein was enriched in the nucleolus. Using GFP tagged UPF3B proteins we found that the missense mutations did not affect the cellular localisation. Expression of missense mutant UPF3B disturbed neuronal differentiation and reduced the complexity of the branching of neurites. Neuronal differentiation was similarly affected in the presence of the NMD inhibitor Amlexanox. The expression of mutant UPF3B proteins lead to a subtle increase in mRNA levels of selected NMD targets.ConclusionsTogether our findings indicate that, despite the down-regulation of NMD factors, functional NMD is critical for neuronal differentiation. We propose that the neurodevelopmental phenotype of UPF3B missense mutation is caused by impairment of NMD function altering neuronal differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0122-1) contains supplementary material, which is available to authorized users.

Nonsense‐mediated mRNA decay: novel mechanistic insights and biological impact

“…In these cells, increased expression of endogenous NMD targets involved in neuronal plasticity and branching was observed (e.g., ATF4 and ARHGAP24 isoform 1 ). This work shows that despite the observed downregulation of NMD factors during neuronal differentiation, a fully functional NMD machinery appears to be critical for proper differentiation …”

“…UPF3B knockdown altered the expression of SIX3 (a master regulator of cortical development) in NPCs and NRCAM and ROBO1 (involved in axon guidance and growth) in hippocampal neurons, genes whose expression is also deregulated in human patient cells harboring loss‐of‐function mutations in UPF3B . A recent study suggests that the neurodevelopmental phenotype of UPF3B missense mutations results from altered neuronal differentiation induced by impaired NMD . In rat neural stem cells, the number and complexity of the branching of neurites was significantly reduced in cells expressing UPF3B protein mutants found in patients with neurodevelopmental disorders.…”

Germline Chd8 haploinsufficiency alters brain development in mouse

“…For example, Dhx9 (M.1) decreases across neurodevelopment and has not been functionally characterized in brain but has been reported in autism-risk networks 20 , while Upf3b (M.3) expression increases across development and is a neuron-specific factor required during neuronal differentiation that is implicated in ID 28,29 . We examined whether Chd8 +/del5 mice exhibited aberrant splicing during brain development linked to DE of RNA processing genes.…”

The exon junction complex in neural development and neurodevelopmental disease

“…Moreover, they find that shRNA mediated depletion of Upf3b in mouse primary NSCs promotes progenitor proliferation, at the cost of reduced differentiation. Recent studies of UPF3B in NSCs also suggest NMD is required for neuronal maturation (Alrahbeni et al, 2015). Depletion of another NMD factor, Upf1, however, tells a different story (Lou et al, 2014).…”

“…Conditional Upf2 loss from neurons caused increased accumulation of the axonal guidance receptor Robo3.2 , which the authors establish as a NMD substrate, providing a molecular mechanism to explain the Upf2 and Upf1 phenotypes. Two additional studies have shown the NMD factor Upf3b also influences axon outgrowth and neurite branching (Jolly et al, 2013; Alrahbeni et al, 2015). Together these findings argue that peripheral EJC components are important for establishing proper neuronal connectivity in the developing brain.…”