Human UPF3A and UPF3B enable fault‐tolerant activation of nonsense‐mediated mRNA decay

Abstract

The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). UPF3B has been demonstrated to support NMD, presumably by bridging an exon junction complex (EJC) to the NMD factor UPF2. The role of UPF3A has been described either as a weak NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not substantially change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. In rescue experiments, UPF2 or EJC binding-deficient UPF3B largely retained NMD activity. However, combinations of different mutants, including deletion of the middle domain, showed additive or synergistic effects and therefore failed to maintain NMD. Collectively, UPF3A and UPF3B emerge as fault-tolerant, functionally redundant NMD activators in human cells.

Mammalian UPF3A and UPF3B can activate nonsense‐mediated mRNA decay independently of their exon junction complex binding

“…Thus, UPF3A/3B can activate NMD without EJC binding, thereby challenging the decades‐old bridging model for function of UPF3 proteins in the pathway. Our conclusion is further bolstered by the findings of Wallmeroth et al where a complete deletion of UPF3B C‐terminal domain can still support functional NMD (Wallmeroth et al , 2022 ).…”

“…A parallel study by Wallmeroth et al also shows that UPF3A functions as a NMD activator in HEK293 cells that lack UPF3B. The redundancy between UPF3A and UPF3B is more pronounced in these cells as NMD inhibition is observed only upon depletion of both proteins (Wallmeroth et al , 2022 ). Thus, in human patients with UPF3B inactivating mutations, UPF3A can likely fill in for UPF3B during NMD, which is critical for several physiological processes (e.g., hematopoiesis (Weischenfeldt et al , 2008 )).…”

“…Therefore, additional factors such as the complement of proteins bound to an mRNA in a particular cell type can influence UPF3A/3B dependence. In addition to the complete dispensability of UPF3 proteins, the redundant nature of individual UPF3B activities like UPF2 or EJC binding (Wallmeroth et al , 2022 ), also raises questions about contexts where such redundant activities of UPF3 proteins become important for NMD activation.…”

“…More work in the future will be necessary to resolve if the differences in NMD activating versus repressive abilities of UPF3A observed in various studies stem from the use of transformed cell lines (our work and (Kunz et al, 2006;Chan et al, 2009;Wallmeroth et al, 2022)) versus primary cells (Shum et al, 2016). It is important to note that UPF3A function as an NMD repressor in various primary cells (neural stem cells, embryonic fibroblasts, spermatocytes, olfactory sensory precursor cells and neurons) was described based on only a handful of NMD targets (Shum et al, 2016).…”

No-nonsense: insights into the functional interplay of nonsense-mediated mRNA decay factors

“…UPF3B associates with the EJC in the nucleus and is exported with the spliced EJC-bound mRNA, while UPF2 is recruited by UPF3B to the EJC at the nuclear envelope after export [ 49 ]. Recent work suggests a role of CASC3 in promoting the association of UPF3B with the EJC and in enhancing UPF3B-dependent NMD [ 50 , 51 ]. This agrees with reports of a link between CASC3-containing EJCs and UPF3B [ 39 , 47 ].…”