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.

Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways

“…We therefore conclude that SMG6-and SMG7-mediated degradation routes appear to be two highly redundant branches of the mammalian NMD pathway. This is consistent with previous observations made with reporter genes (Luke et al 2007;Jonas et al 2013;Metze et al 2013) and in line with what was observed in a study focusing on SMG6 endonucleolytic activity (Schmidt et al 2015). In ′ UTR features between NMD targets and a matched control group.…”

The SMG5–SMG7 heterodimer directly recruits the CCR4–NOT deadenylase complex to mRNAs containing nonsense codons via interaction with POP2

“…7G). We observed that the codepletion of both proteins resulted SMG7 recruits the CCR4-NOT complex to NMD targets in a synergistic inhibition of NMD, leading to a 30-fold increase in the b-globin PTC reporter levels, as previously reported (Luke et al 2007;Jonas et al 2013;Metze et al 2013). This effect was prevented by the expression of a shRNA-resistant version of SMG7, which partially restored NMD (Fig.…”

“…Briefly, an shRNA targeting the ORF of the smg5 mRNA was used to deplete endogenous SMG5 in cell lines constitutively expressing a well-characterized NMD reporter based on the b-globin gene (Thermann et al 1998). Because SMG6 can partially compensate for the absence of SMG5 (Jonas et al 2013;Metze et al 2013), we depleted SMG6 in combination with SMG5. SMG5 and SMG6 codepletion resulted in a 12-fold increase in the level of b-globin PTC mRNA (Fig.…”

“…Furthermore, codepletion of SMG5 and SMG7 does not exacerbate the effects of the individual depletions, suggesting an epistatic interaction. In contrast, codepletion of SMG6 with either SMG5 or SMG7 results in a synergistic inhibition of NMD (Luke et al 2007;Jonas et al 2013;Metze et al 2013), suggesting that the SMG5-SMG7 complex acts redundantly with SMG6 to promote the degradation of NMD targets.…”

An atlas of chromatoid body components

“…In addition to the exon-junction complex (EJC) components that provide the recognition platform for NMD, the CB accumulated UPF1/RENT, which is a key mediator of NMD, SMG1 kinase, which activates UPF1 through phosphorylation, and SMG6, which is an endonuclease involved in the NDM pathway (Huntzinger et al 2008;Eberle et al 2009;Kervestin and Jacobson 2012). SMG1-mediated UPF1 phosphorylation (and the subsequent dephosphorylation) was recently shown to be the only critical requirement for both, EJC-enhanced and the alternative, EJC-independent NMDs (Metze et al 2013). It is important to note that according to our immunofluorescence results, the CB-localized UPF1 is at least partially phosphorylated.…”