Poster abstracts
Poster number 43 submitted by Zhongxia Yi
NMD activation by UPF3 paralogs is dictated by their mid-domain but not their EJC binding.
Zhongxia Yi (Center for RNA Biology, the Ohio State University), Rene M. Arvola, Sean Myers, Corinne N. Dilsavor, Rabab Abu Alhasan, Bayley N. Carter, Robert D. Patton, Ralf Bundschuh, Guramrit Singh
Abstract:
Nonsense-mediated mRNA decay (NMD) degrades aberrant transcripts with premature termination codons (PTC) and also regulates ~10% of mammalian transcriptome. While three core NMD factors, Upf1p, Upf2p and Upf3p, are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, with its paralog UPF3A suggested to only weakly activate or even suppress NMD. The mechanism of UPF3B-independent NMD and the mRNA substrates of this distinct NMD branch remains unknown. NMD is enhanced by the presence of an exon junction complex (EJC) downstream of a stop codon. EJC is a multi-protein complex of heterogenous composition that marks exon-exon junctions. In current models of NMD, UPF3B activates NMD by bridging the UPF complex (UPF1-UPF2-UPF3B) at the terminating ribosome to the downstream EJC. This model, however, fails to explain how UPF3 can activate NMD without EJC in various organisms from yeast to human. To characterize the UPF3B-dependent and -independent human NMD pathway, we have used CRISPR-Cas9 to knockout UPF3B in the human colorectal carcinoma HCT116 cell line. RNA-seq from the knockout and wildtype (WT) cells show that, in HCT116 cells, a vast majority of NMD targets can undergo UPF3B-independent NMD. UPF3B loss preferentially affects some, but not all, EJC-mediated NMD substrates. We find that while UPF3A is almost completely dispensable for NMD in WT HCT116 cells, it strongly activates NMD after UPF3B loss. Together, UPF3A and UPF3B regulate a significant portion of EJC-mediated NMD. Further, UPF3-dependent NMD can be regulated by a specific composition of EJC that contains the CACS3 protein. Surprisingly, complementation of human UPF3 double knockout cells with human and mouse UPF3A paralogs shows that the EJC-binding domain is not essential for UPF3 function in NMD but the conserved mid domain, previously shown to engage with translation release factors, is required for a full UPF3 NMD activity. Taken together, we show that while UPF3-dependent NMD can be regulated by EJC, UPF3 plays a more active role in triggering NMD than simply bridging the EJC to the UPF complex.
Keywords: Nonsense-mediated mRNA decay, UPF3, Exon junction complex