The Dcp1-Dcp2 decapping enzyme and the decapping activators Pat1, Dhh1, and Lsm1 regulate mRNA decapping, but their mechanistic integration is unknown. We analyzed the gene expression consequences of deleting PAT1, LSM1, or DHH1, or the DCP2 C-terminal domain, and found that: i) the Dcp2 C-terminal domain is an effector of both negative and positive regulation; ii) rather than being global activators of decapping, Pat1, Lsm1, and Dhh1 directly target specific subsets of yeast mRNAs and loss of the functions of each of these factors has substantial indirect consequences for genome-wide mRNA expression; and iii) transcripts targeted by Pat1, Lsm1, and Dhh1 exhibit only partial overlap, are generally translated inefficiently, and, as expected, are targeted to decapping-dependent decay. Our results define the roles of Pat1, Lsm1, and Dhh1 in decapping of general mRNAs and suggest that these factors may monitor mRNA translation and target unique features of individual mRNAs.

Nonsense-mediated mRNA decay (NMD) is generally thought to be a eukaryotic mRNA surveillance pathway tasked with the elimination of transcripts harboring an in-frame premature termination codon (PTC). As presently conceived, NMD acting in this manner minimizes the likelihood that potentially toxic polypeptide fragments would accumulate in the cytoplasm. This notion is to be contrasted to the results of systematic RNA-Seq and microarray analyses of NMD substrates in multiple model systems, two different experimental approaches which have shown that many mRNAs identified as NMD substrates fail to contain a PTC. Our recent results provide insight into, as well as a possible solution for, this conundrum. By high-resolution profiling of mRNAs that accumulate in yeast when the principal NMD regulatory genes (UPF1, UPF2, and UPF3) are deleted, we identified approximately 900 NMD substrates, the majority of which are normal-looking mRNAs that lack PTCs. Analyses of ribosomal profiling data revealed that the latter mRNAs tended to manifest elevated rates of out-of-frame translation, a phenomenon that would lead to premature translation termination in alternative reading frames. These results, and related observations of heterogeneity in mRNA isoforms, suggest that NMD should be reconsidered as a probabilistic mRNA quality control pathway that is continually active throughout an mRNA's life cycle.

mRNA 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.

Nonsense-mediated mRNA decay (NMD) plays an important role in eukaryotic gene expression, yet the scope and the defining features of NMD-targeted transcripts remain elusive. To address these issues, we reevaluated the genome-wide expression of annotated transcripts in yeast cells harboring deletions of the UPF1, UPF2, or UPF3 genes. Our new RNA-seq analyses confirm previous results of microarray studies, but also uncover hundreds of new NMD-regulated transcripts that had escaped previous detection, including many intron-containing pre-mRNAs and several noncoding RNAs. 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 and higher ratios of out-of-frame translation. NMD-regulated transcripts also have lower average codon optimality scores and higher transition probability to nonoptimal codons. Collectively, our results generate a comprehensive catalog of yeast NMD substrates and yield new insights into the mechanisms by which these transcripts are targeted by NMD.