Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways

Aug 22, 2013RNA (New York, N.Y.)

Two partly overlapping pathways for breaking down faulty messenger RNA in human cells, with and without EJC involvement

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Abstract

Both EJC-enhanced and EJC-independent modes of nonsense-mediated mRNA decay (NMD) require UPF1 and SMG1.

Transcript-specific differences were observed in the requirement for UPF2 and UPF3b between the two NMD modes.
EJC-independent NMD demonstrated higher sensitivity to reduced concentrations of UPF2 and UPF3b.
A redundancy of endo- and exonucleolytic mRNA degradation pathways was identified in both NMD modes.
The decay pathways contributed differently to the degradation of mRNA transcripts, with immunoglobulin μ being more affected by SMG6-mediated cleavage and β-Globin primarily degraded by the SMG5/SMG7 pathway.
The findings indicate the presence of several distinct branches of NMD in human cells.

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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.

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Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways

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