Renilla Luciferase mRNA

Mutations in the UPF3B gene on the X chromosome are associated with neurodevelopmental disorders, including X-linked intellectual disability, autism, and schizophrenia. The UPF3B protein participates in the nonsense-mediated mRNA decay pathway (NMD), which controls mRNA stability and is involved in preventing the synthesis of truncated proteins. This study shows that UPF3B and UPF1, components of the NMD pathway, are downregulated during the differentiation of neural stem cells into neurons. Through association function analysis, the researchers found that the UPF3B missense mutations found in families with neurodevelopmental disorders reduced the activity of the UPF3B protein in NMD. In neural stem cells, the UPF3B protein was detected in both the cytoplasm and the nucleus. Similarly, in neurons, the UPF3B protein was detected in neurites, the somatic cytoplasm, and the nucleus. In both cell types, the UPF3B protein was enriched in the nucleolus. Using GFP-tagged UPF3B protein, they found that the missense mutation did not affect cellular localization. Expression of missense mutant UPF3B interfered with neuronal differentiation and reduced the complexity of neurite branching. Similar effects on neuronal differentiation were observed in the presence of the NMD inhibitor amlexanox. Expression of mutant UPF3B protein resulted in slightly increased mRNA levels of specific NMD targets.

Here, the researchers wanted to determine whether differentiating neural stem cells retain the ability to mediate UPF3B-promoted neural microenvironment (NMD) and whether this ability is sensitive to UPF3B mutations. First, they determined that the expression of these proteins is similar in neural stem cells (Figure 1a). Then, tethering experiments were performed with wild-type UPF3B, UPF3B-Ala423, and patient UPF3B mutants by transfecting neural stem cells with the appropriate plasmid combination and then inducing differentiation for two days (Figure 1b). Reverse transcription followed by qPCR analysis showed that the test Renilla luciferase mRNA level was reduced by about 40-fold when wild-type UPF3B was tethered compared to UPF3B-Ala423 tethering. This phenomenon was specific for UPF3B tethering, as the average mRNA level changed by only 7% in the reaction with control Renilla luciferase RNA. This suggests that differentiating neural stem cells retain the ability to undergo UPF3B-promoted neuroblastoma (NMD). Binding of mutant UPF3B resulted in 10- to 20-fold higher levels of Renilla luciferase mRNA than wild-type UPF3B. Together, these experiments demonstrate that differentiating neural stem cells are capable of UPF3B-promoted NMD and that NMD in these cells is sensitive to UPF3B variants found in patients with neurodevelopmental disorders.

Figure 1. Mutations in UPF3B impair UPF3B protein NMD activity in neural stem cells. (Alrahbeni T, et al., 2015)