Y14 and hUpf3b Form an NMD-Activating Complex

Gehring, Niels H.; Neu‐Yilik, Gabriele; Schell, Thomas; Hentze, Matthias W.; Kulozik, Andreas E.

doi:10.1016/s1097-2765(03)00142-4

Cited by 275 publications

(321 citation statements)

References 51 publications

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“…Surprisingly, these motifs exhibited 23% and 38% identity (38% and 57% similarity) to a UPF3b C-terminal motif, which is known to mediate binding to the EJC (Fig. 3A,B; Gehring et al 2003;Kunz et al 2006;Buchwald et al 2010). We therefore termed these motifs EBMs, or EBM1 and EBM2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Closer examination, however, revealed that this region does contain two short sequence motifs that are conserved. These motifs are related to the EBM found in the C terminus of the NMD factors UPF3a and UPF3b (Gehring et al 2003;Buchwald et al 2010).…”

Section: Discussionmentioning

confidence: 99%

“…SMG6DN, D14-3-3, DPIN, DEBM1, and DEBM2 carry deletions of the following amino acids, respectively: 1-576, 577-816, 1239-1419, 42-58, and 136-152. Plasmids for expression of HA-tagged NMD factors or MBP were constructed using the mammalian expression vector pCI-neo-lN (Gehring et al 2003) following standard procedures.…”

Section: Smg6-ejc Interaction Is Required For Nmdmentioning

confidence: 99%

“…The UPF3b C-terminal motif binds to the EJC (Gehring et al 2003;Buchwald et al 2010). Similarly, the conserved SMG6 N-terminal motifs confer direct binding to the EJC and were termed EJC-binding motifs (EBMs).…”

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confidence: 99%

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

Kashima¹,

Jonas²,

Jayachandran

et al. 2010

Genes Dev.

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Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that detects and degrades mRNAs containing premature stop codons (PTCs). In vertebrates, PTCs trigger efficient NMD when located upstream of an exon junction complex (EJC). Degradation of PTC-containing mRNAs requires the endonucleolytic activity of SMG6, a conserved NMD factor; nevertheless, the precise role for the EJC in NMD and how the SMG6 endonuclease is recruited to NMD targets have been unclear. Here we show that SMG6 interacts directly with the EJC via two conserved EJC-binding motifs (EBMs). We further show that the SMG6-EJC interaction is required for NMD. Our results reveal an unprecedented role for the EJC in recruiting the SMG6 endonuclease to NMD targets. More generally, our findings identify the EBM as a protein motif present in a handful of proteins, and suggest that EJCs establish multiple and mutually exclusive interactions with various protein partners, providing a plausible explanation for the myriad functions performed by this complex in post-transcriptional mRNA regulation.[Keywords: mRNA decay; exon junction complex; NMD; UPF1] Supplemental material is available at http://www.genesdev.org.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Smg6-ejc Interaction Is Required For Nmdmentioning

confidence: 99%

mentioning

confidence: 99%

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

Kashima¹,

Jonas²,

Jayachandran

et al. 2010

Genes Dev.

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“…Plasmids with high purity for mammalian cell transfection were prepared with a Maxiprep kit (Qiagen). Double tethering reporter pmirGlo-Ptight-2BoxB-2MS2: 2BoxB-2MS2 sequence was designed as follows: 5′-CTC- [37,38], synthesized, and PCR amplified and cloned to 3′-UTR of F-Luc in pmirGlo-Ptight vector reported [12] (NheI, SbfI; forward primer, 5′-ATACGCTAG-CCTCGACTAAGTCCAACTACTAAACTGGG-3′; reverse primer, 5′-GTATCCTGCAGGTTAGGATCTAATGAACCCGG-GAATACTG-3′).…”

Section: Plasmid Constructionsmentioning

confidence: 99%

YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA

et al. 2017

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Analysis of Nonsense‐Mediated mRNA Decay in Mammalian Cells

2012

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The nonsense-mediated mRNA decay (NMD) pathway acts to selectively identify and degrade mRNAs that contain a premature translation termination codon (PTC), and hence reduce the accumulation of potentially toxic truncated proteins. NMD is one of the best studied mRNA quality-control mechanisms in eukaryotes, and it has become clear during recent years that many physiological mRNAs are also NMD substrates, signifying a role for NMD beyond mRNA quality control as a translation-dependent posttranscriptional regulator of gene expression. Despite a great deal of scientific research for over twenty years, the process of NMD is far from being fully understood with regard to its physiological relevance to the cell, the molecular mechanisms that underpin this pathway, all of the factors that are involved, and the exact cellular locations of NMD. This unit details some of the fundamental RNA based approaches taken to examine aspects of NMD, such as creating PTC+ reporter genes, knocking down key NMD factors via RNAi, elucidating the important functions of NMD factors by complementation assays or Tethered Function Assays, and measuring RNA levels by reverse-transcription quantitative PCR.

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Y14 and hUpf3b Form an NMD-Activating Complex

Cited by 275 publications

References 51 publications

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

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

YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA

Analysis of Nonsense‐Mediated mRNA Decay in Mammalian Cells

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