Browsing by keyword "NMD"
Now showing items 1-4 of 4
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Dcp2 C-terminal Cis-Binding Elements Control Selective Targeting of the Decapping Enzyme by Forming Distinct Decapping Complexes [preprint]A single Dcp1-Dcp2 decapping enzyme targets diverse classes of yeast mRNAs for decapping-dependent 5’ to 3’ decay, but the molecular mechanisms controlling selective mRNA targeting by the enzyme remain elusive. Through extensive genetic analyses we uncover cis-regulatory elements in the Dcp2 C-terminal domain that control selective targeting of the decapping enzyme by forming distinct decapping complexes. Two Upf1-binding motifs target the decapping enzyme to NMD substrates, and a single Edc3-binding motif targets both Edc3 and Dhh1 substrates. Pat1-binding leucine-rich motifs target Edc3 and Dhh1 substrates under selective conditions. Although it functions as a unique targeting component of specific complexes, Edc3 is a common component of multiple complexes. Xrn1 also has a specific Dcp2 binding site, allowing it to be directly recruited to decapping complexes. Collectively, our results demonstrate that Upf1, Edc3, and Pat1 function as regulatory subunits of the holo-decapping enzyme, controlling both its targeting specificity and enzymatic activation.
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mRNA Decay Pathways Use Translation Fidelity and Competing Decapping Complexes for Substrate SelectionmRNA decay is an important step in gene regulation, environmental responsiveness, and mRNA quality control. One such quality control pathway, Nonsense-mediated mRNA Decay (NMD), targets transcripts whose translation terminates prematurely. However, the scope and the defining features of NMD-targeted transcripts remain elusive. To address these issues, we re-evaluated the genome-wide expression of annotated transcripts in yeast cells harboring deletions of the UPF1, UPF2, or UPF3 genes. The vast majority of NMD-regulated transcripts are normal-looking protein-coding mRNAs. Our bioinformatics analyses reveal that this set of NMD-regulated transcripts generally have lower translational efficiency, lower average codon optimality scores, and higher ratios of out-of-frame translation. General mRNA decay is predominantly mediated by decapping by the Dcp1-Dcp2 complex and 5' to 3' decay by Xrn1, but the exact mechanism of decapping regulation has remained largely unknown. Several in vitro and in vivo studies have revealed the importance of the C-terminal extension of Dcp2 and the identities of many decapping regulators that interact with the decapping complex. To better understand how decapping regulation is achieved by the C-terminal extension of Dcp2 we generated RNA-Seq libraries from a Dcp2 allele that lacks this portion of Dcp2 along with libraries from strains that contain single deletions of several decapping activators. Our transcriptome-wide results indicate that the C-terminal extension of Dcp2 is crucial for efficient regulation of decapping, and different decapping activators are responsible for targeting different sets of mRNAs. Considering the limited pool of Dcp1-Dcp2 in the cell decapping activators might be in competition for decapping complex binding. Collectively, our results yield valuable insights into the mechanism of substrate selection for mRNA quality control and decay in yeast.
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Poly(A)-Binding Protein Regulates the Efficiency of Translation TerminationMultiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a nonsense codon within an open reading frame (ORF) influences termination efficiency, we quantitate the production of prematurely terminated and/or readthrough polypeptides from 26 nonsense alleles of 3 genes expressed in yeast. The accumulation of premature termination products and the extent of readthrough for the respective premature termination codons (PTCs) manifest a marked dependence on PTC proximity to the mRNA 3' end. Premature termination products increase in relative abundance, whereas readthrough efficiencies decrease progressively across different ORFs, and readthrough efficiencies for a PTC increase in response to 3' UTR lengthening. These effects are eliminated and overall translation termination efficiency decreases considerably in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and also suggest that inefficient termination is a trigger for nonsense-mediated mRNA decay (NMD).
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Yeast Upf1 CH domain interacts with Rps26 of the 40S ribosomal subunitThe 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.
