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Deciphering the cellular roles and dissecting functional regions of UPF3A and UPF3B in human NMD

ABSTRACT: The paralogous proteins UPF3A and UPF3B are involved in recognizing defective mRNAs that are degraded by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, UPF3A was reported to act either an NMD activator or an NMD inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using overexpression, knockdowns, knockouts (KO) and rescue experiments. Overexpression or knockout of UPF3A did not result in detectable changes in global NMD activity or other specific transcriptome alterations . NMD activity was also virtually unchanged in UPF3B KO cells. In contrast, the co-depletion or co-knockout of UPF3A and UPF3B resulted in a marked NMD inhibition and a global upregulation of PTC-containing transcripts. In rescue experiments UPF3B was fully functional when either UPF2- or EJC binding was impaired . However, the deletion of both interaction sites or one interaction site and a central region of UPF3B impaired its NMD function. Altogether, our work identifies critical functional domains of UPF3B and establishes redundant roles of UPF3A and UPF3B during NMD.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Prerana Wagle

LAB HEAD: Niels H. Gehring

PROVIDER: PXD027120 | Pride | 2022-05-04

REPOSITORIES: Pride

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			Action	DRS
	2070_msms.txt	Txt
	2070_proteinGroups.txt	Txt
	2532_msms.txt	Txt
	2532_proteinGroups.txt	Txt
	LFQ_2532_01.raw	Raw

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Project description:UPF3A and UPF3B are paralogous genes in human cells that are involved in the nonsense-mediated decay (NMD) pathway. NMD is a cellular quality control mechanism that monitors mRNAs during translation. Aberrant translation due to features such as the presence of a premature stop codon downstream on an exon-exon junction activates NMD and leads to the degradation of the mRNA. To investigate the role of UPF3B in NMD, we have generated FLAG-TurboID-UPF3B wild type or mutant expressing human Flp-In T-Rex 293 UPF3B knockout cells using the PiggyBac transposon system. We generated proteomic data from the proximity labeled interactome of these UPF3B variants.

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