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

Abstract

Nonsense‐mediated mRNA decay (NMD) is governed by the three conserved factors—UPF1, UPF2, and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, and its paralog UPF3A is suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here, we characterize the UPF3A/B‐dependence of NMD in human cell lines deleted of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, NMD can operate in a UPF3B‐dependent and ‐independent manner. While UPF3A is almost dispensable for NMD in wild‐type cells, it strongly activates NMD in cells lacking UPF3B. Notably, NMD remains partially active in cells lacking both UPF3 paralogs. Complementation studies in these cells show that EJC‐binding domain of UPF3 paralogs is dispensable for NMD. Instead, the conserved “mid” domain of UPF3 paralogs is consequential for their NMD activity. Altogether, our results demonstrate that the mammalian UPF3 proteins play a more active role in NMD than simply bridging the EJC and the UPF complex.

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

“…Thus, it remains an open question how relevant the individual UPF3 paralogs and their potential NMD activating or repressing function are in different vertebrates and/or different cell types. Interestingly, in a parallel manuscript, Yi et al find that not only human but also mouse UPF3A was able to restore NMD activity in their HCT116 UPF3A‐UPF3B dKO cell line (Yi et al , 2022 ). In case of a general NMD inhibitory function of human or mouse UPF3A, no rescue effect should have been observed, which further questions the proposed broadly acting role of UPF3A as an NMD repressor.…”

“…Our own work and the work of Yi et al have re‐examined the functions of the human UPF3 paralogs UPF3A and UPF3B (Yi et al , 2022 ). Together, the studies confirmed some previous findings and contradict others, thereby successfully (re‐)defining the role of UPF3 proteins and their functional domains in human cells.…”

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

“…This agrees with reports of a link between CASC3-containing EJCs and UPF3B [ 39 , 47 ]. Consistently, in CASC3 knockdown cells, UPF3B's association with the EJC was reduced, and vice versa CASC3 overexpression led to enhanced EJC–UPF3B association in HeLa cells [ 51 ]. The structure of EJC–UPF3B indicates that the interactions of CASC3 and UPF3B with the EJC core are limited to small regions of the two proteins ( Figure 1E ) [ 49 , 52 ].…”

“…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 ].…”

“…UPF3A shares the domain architecture of UPF3B but its EBM has lower binding affinity to the EJC ( Figure 2 ) [ 97 ]. In an UPF3B-deficient context, UPF3A has been shown to be an active NMD factor that can compensate for UPF3B [ 50 , 51 , 97 ] while in other contexts, UPF3A can function as a NMD inhibitor, protecting mRNAs from NMD [ 98 ] (see below).…”