title: Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways.
abstract: Nonsense-mediated mRNA decay (NMD) is a eukaryotic post-transcriptional gene regulation mechanism that eliminates mRNAs with the termination codon (TC) located in an unfavorable environment for efficient translation termination. The best-studied NMD-targeted mRNAs contain premature termination codons (PTCs); however, NMD regulates even many physiological mRNAs. An exon-junction complex (EJC) located downstream from a TC acts as an NMD-enhancing signal, but is not generally required for NMD. Here, we compared these "EJC-enhanced" and "EJC-independent" modes of NMD with regard to their requirement for seven known NMD factors in human cells using two well-characterized NMD reporter genes (immunoglobulin μ and β-Globin) with or without an intron downstream from the PTC. We show that both NMD modes depend on UPF1 and SMG1, but detected transcript-specific differences with respect to the requirement for UPF2 and UPF3b, consistent with previously reported UPF2- and UPF3-independent branches of NMD. In addition and contrary to expectation, a higher sensitivity of EJC-independent NMD to reduced UPF2 and UPF3b concentrations was observed. Our data further revealed a redundancy of the endo- and exonucleolytic mRNA degradation pathways in both modes of NMD. Moreover, the relative contributions of both decay pathways differed between the reporters, with PTC-containing immunoglobulin μ transcripts being preferentially subjected to SMG6-mediated endonucleolytic cleavage, whereas β-Globin transcripts were predominantly degraded by the SMG5/SMG7-dependent pathway. Overall, the surprising heterogeneity observed with only two NMD reporter pairs suggests the existence of several mechanistically distinct branches of NMD in human cells.
authors: Stefanie
authors: S
authors: Metze
authors: Veronika A
authors: VA
authors: Herzog
authors: Marc-David
authors: MD
authors: Ruepp
authors: Oliver
authors: O
authors: Mühlemann
mesh_terms: D015153:Blotting, Western; D018389:Codon, Nonsense / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D059365:Nonsense Mediated mRNA Decay / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D020365:RNA Helicases; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D018389:Codon, Nonsense; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D020365:RNA Helicases; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C468286:UPF2 protein, human; C435487:UPF3A protein, human; C514704:SMG1 protein, human; C101828:UPF1 protein, human
keywords: endo- and exonucleolytic mRNA degradation; UPF1; UPF2; UPF3b; SMG1; SMG5; SMG6; SMG7; exon-junction complex; mRNA surveillance; mRNA turnover; nonsense-mediated mRNA decay; post-transcriptional gene regulation
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