SMG6 interacts with the exon junction complex via two conserved EJC-binding motifs (EBMs) required for nonsense-mediated mRNA decay

Kashima, Isao; Jonas, Steven; Jayachandran, Uma; Buchwald, Gretel; Conti, Elena; Lupas, Andrei N.; Izaurralde, Elisa

doi:10.1101/gad.604610

Cited by 75 publications

(78 citation statements)

References 38 publications

(79 reference statements)

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“…Consistent with a dependence of NMD on translation termination, Upf1 interacts with the release factors eRF1 and eRF3 in humans and yeast, possibly to function in termination events using its helicase and RNA binding activities (Czaplinski et al 1998;Wang et al 2001;Ghosh et al 2010). In vertebrates, Upf1 and its regulator Smg-1 interact with the release factors forming the SURF (Smg-1-Upf1-Release factors) complex, and this SURF-associated Upf1 appears to be able to interact with Upf2-Upf3 bound to the exon junction complex (Yamashita et al 2009;Kashima et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Yeast Upf1 CH domain interacts with Rps26 of the 40S ribosomal subunit

Min

Roy

Amrani

et al. 2013

RNA

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The central nonsense-mediated mRNA decay (NMD) regulator, Upf1, selectively targets nonsense-containing mRNAs for rapid degradation. In yeast, Upf1 preferentially associates with mRNAs that are NMD substrates, but the mechanism of its selective retention on these mRNAs has yet to be elucidated. Previously, we demonstrated that Upf1 associates with 40S ribosomal subunits. Here, we define more precisely the nature of this association using conventional and affinity-based purification of ribosomal subunits, and a two-hybrid screen to identify Upf1-interacting ribosomal proteins. Upf1 coimmunoprecipitates specifically with epitope-tagged 40S ribosomal subunits, and Upf1 association with high-salt washed or puromycin-released 40S subunits was found to occur without simultaneous eRF1, eRF3, Upf2, or Upf3 association. Two-hybrid analyses and in vitro binding assays identified a specific interaction between Upf1 and Rps26. Using mutations in domains of UPF1 known to be crucial for its function, we found that Upf1:40S association is modulated by ATP, and Upf1:Rps26 interaction is dependent on the N-terminal Upf1 CH domain. The specific association of Upf1 with the 40S subunit is consistent with the notion that this RNA helicase not only triggers rapid decay of nonsense-containing mRNAs, but may also have an important role in dissociation of the premature termination complex.

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Section: Introductionmentioning

confidence: 99%

Yeast Upf1 CH domain interacts with Rps26 of the 40S ribosomal subunit

Min

Roy

Amrani

et al. 2013

RNA

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“…The EJC is a multimeric protein complex deposited by the splicing machinery 24 nt upstream of the spliced exon-exon boundary in metazoans (Le Hir et al 2000;Sauliere et al 2012;Singh et al 2012). The EJC core consists of the four proteins eIF4AIII, Y14, MAGOH, and MLN51 (also known as Barentz) (Andersen et al 2006;Bono et al 2006), and additional factors associate with the EJC core in a dynamic way, including Pinin, SKAR, SRm160, RNPS1, Acinus, SAP18, UAP56, Aly/REF, and the two NMD factors UPF3b and SMG6 (Le Hir et al 2000Kashima et al 2010;Bono and Gehring 2011;Murachelli et al 2012). UPF3b binds the EJC core via its C-terminal low-complexity region (Gehring et al 2003;Buchwald et al 2010), and two conserved EJC-binding motifs (EBMs) were identified in the N-terminal part of SMG6 that interact with essentially the same surface on the EJC core as UPF3b (Kashima et al 2010).…”

Section: Introductionmentioning

confidence: 99%

“…The EJC core consists of the four proteins eIF4AIII, Y14, MAGOH, and MLN51 (also known as Barentz) (Andersen et al 2006;Bono et al 2006), and additional factors associate with the EJC core in a dynamic way, including Pinin, SKAR, SRm160, RNPS1, Acinus, SAP18, UAP56, Aly/REF, and the two NMD factors UPF3b and SMG6 (Le Hir et al 2000Kashima et al 2010;Bono and Gehring 2011;Murachelli et al 2012). UPF3b binds the EJC core via its C-terminal low-complexity region (Gehring et al 2003;Buchwald et al 2010), and two conserved EJC-binding motifs (EBMs) were identified in the N-terminal part of SMG6 that interact with essentially the same surface on the EJC core as UPF3b (Kashima et al 2010). EJCs located in the 5 ′ UTR and coding sequence of an mRNA are disassembled by the elongating ribosome by a process that involves the ribosome-bound protein PYM (Gehring et al 2009b;Bono and Gehring 2011).…”

Section: Introductionmentioning

confidence: 99%

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

Metze

Herzog

Ruepp

et al. 2013

RNA

122

126

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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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“…Smg6 is thought to be recruited to target mRNAs by virtue of its 14-3-3-like domain, which can bind phosphorylated Upf1 (Fukuhara et al 2005). In mammalian cells, Smg6 is also recruited to target mRNAs through direct recruitment to the EJC (Kashima et al 2010). Smg6 function has primarily been analyzed in cell culture, and while some genetic analysis has been performed in C. elegans (Hodgkin et al 1989;Page et al 1999), a role for SMG-6 endonuclease function has not yet been analyzed in this organism.…”

Section: Introductionmentioning

confidence: 99%

Drosophila mutants show NMD pathway activity is reduced, but not eliminated, in the absence of Smg6

Frizzell¹,

Rynearson²,

Metzstein³

2012

RNA

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The nonsense-mediated mRNA decay (NMD) pathway is best known for targeting mutant mRNAs containing premature termination codons for rapid degradation, but it is also required for regulation of many endogenous transcripts. Components of the NMD pathway were originally identified by forward genetic screens in yeast and Caenorhabditis elegans. In other organisms, the NMD pathway has been investigated by studying the homologs of these genes. We present here the first unbiased genetic screen in Drosophila designed specifically to identify genes involved in NMD. By using a highly efficient genetic mosaic approach, we have screened~40% of the Drosophila genome and isolated more than 40 alleles of genes required for NMD. We focus on alleles we have obtained in two known NMD components: Upf2 and Smg6. Our analysis of multiple alleles of the core NMD component Upf2 reveals that the Upf2 requirement in NMD may be separate from its requirement for viability, indicating additional critical cellular roles for this protein. Our alleles of Smg6 are the first point mutations obtained in Drosophila, and we find that Smg6 has both endonucleolytic and nonendonucleolytic roles in NMD. Thus, our genetic screens have revealed that Drosophila NMD factors play distinct roles in target regulation, similar to what is found in mammals, but distinct from the relatively similar requirements for NMD genes observed in C. elegans and yeast.

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SMG6 interacts with the exon junction complex via two conserved EJC-binding motifs (EBMs) required for nonsense-mediated mRNA decay

Cited by 75 publications

References 38 publications

Yeast Upf1 CH domain interacts with Rps26 of the 40S ribosomal subunit

Yeast Upf1 CH domain interacts with Rps26 of the 40S ribosomal subunit

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

Drosophila mutants show NMD pathway activity is reduced, but not eliminated, in the absence of Smg6

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