Abstract
In Saccharomyces cerevisiae, mRNA transcripts with premature termination codons are targeted for deadenylation independent decapping and 5′ to 3′ decay in a quality control pathway termed nonsense-mediated decay (NMD). Critical factors in NMD include Upf1, Upf2, and Upf3, as well as the decapping enzyme, Dcp2/Dcp1. Loss of Upf2 or Upf3 leads to the accumulation of not only Upf1 and Dcp2 in P-bodies, but also of the decapping-activators Pat1, Dhh1, and Lsm1. An interaction between Upf1 and Dcp2 has been identified, which might recruit Dcp2 to the NMD decapping complex. To determine the nature and significance of the Dcp2-Upf1 interaction, we utilized the yeast two-hybrid assay to assess Upf1 interactions with various mRNA decapping factors. We find that although Dcp2 can interact with Upf1, this interaction is indirect and is largely dependent on the Edc3 protein, which interacts with the N-terminal domain of Upf1 at an overlapping, but not identical, site as Upf2. We also found that Pat1 has an independent two-hybrid interaction with the N-terminus of Upf1. Assessment of both reporter and endogenous NMD transcripts suggest that the decapping stimulators, including Edc3 and Pat1, as well as Edc1 and Edc2, are not essential for NMD under normal conditions. This work defines a larger decapping complex involved in NMD, but indicates that components of that complex are not required for general NMD and might either regulate a subset of NMD transcripts or be essential for proper NMD under different environmental conditions.
Highlights
An important mRNA quality control pathway in eukaryotic cells is the nonsense-mediated decay (NMD) pathway, whereby transcripts with aberrant termination codons are targeted for degradation
NMD targets mRNAs with premature termination codons (PTCs) that can arise by many mechanisms, including poor transcription fidelity, frameshift mutations, and inefficiently spliced intron-containing mRNAs that are transported to the cytoplasm [reviewed in 2]
In eukaryotes, including mammalian cells, Drosophila, Caenorhabditis elegans and yeast, the core NMD machinery is comprised of the factors Upf1, Upf2, and Upf3, which are all essential for NMD to occur in the cytoplasm [5,6, reviewed in 7]
Summary
An important mRNA quality control pathway in eukaryotic cells is the nonsense-mediated decay (NMD) pathway, whereby transcripts with aberrant termination codons are targeted for degradation. It is possible that these stimulators of decapping comprise the larger decapping complex that co-localizes with Upf in P-bodies and may regulate NMD in a redundant manner with other decapping-associated factors in yeast Since both Upf and Dcp2/Dcp are essential for NMD in yeast, we set out to understand the Upf interaction with the decapping enzyme and the broader decapping complex. The results obtained suggest that the decapping stimulators are not essential for NMD during normal growth conditions Given that both Pat and Edc associate with Upf, and that decapping-associated factors are localized to Upf1- and Dcp2-containing foci upon loss of Upf or Upf, it is possible that these decapping stimulators affect a subset of NMD transcripts or have essential roles for NMD under different conditions
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