General decapping activators target different subsets of inefficiently translated mRNAs

Abstract

29The Dcp1-Dcp2 decapping enzyme and the decapping activators Pat1, Dhh1, and 30 Lsm1 regulate mRNA decapping, but their mechanistic integration is unknown. We analyzed 31 the gene expression consequences of deleting PAT1, LSM1, or DHH1, or the DCP2 C- 32 terminal domain, and found that: i) the Dcp2 C-terminal domain is an effector of both negative 33 and positive regulation; ii) rather than being global activators of decapping, Pat1, Lsm1, and 34 Dhh1 directly target specific subsets of yeast mRNAs and loss of the functions of each of 35 these factors has substantial indirect consequences for genome-wide mRNA expression; and 36 iii) transcripts targeted by Pat1, Lsm1, and Dhh1 exhibit only partial overlap, are generally 37 translated inefficiently, and, as expected, are targeted to decapping-dependent decay. Our 38 results define the roles of Pat1, Lsm1, and Dhh1 in decapping of general mRNAs and 39 suggest that these factors may monitor mRNA translation and target unique features of 40 individual mRNAs. 41 3 65 decapping of general wild-type mRNAs (Parker, 2012), and NMD-specific regulators (Upf1, 66 Upf2, and Upf3) are required for decapping of nonsense-containing mRNAs (He and 67 Jacobson, 2015b, Nicholson and Muhlemann, 2010). Edc3 manifests the most fastidious 68 substrate specificity, being required for decapping of only the yeast YRA1 pre-mRNA and 69 RPS28B mRNA (He et al., 2014, Dong et al., 2007, Badis et al., 2004. All of these decapping 70 activators are conserved from yeast to humans, but their precise functions in mRNA 71 decapping regulation are largely unknown. The two major functions proposed for yeast 72 decapping activators, translational repression and decapping enzyme activation (Parker, 73 2012, Nissan et al., 2010, Coller and Parker, 2005), are still controversial (Sweet et al., 2012, 74 Arribere et al., 2011). 75 Yeast decapping activators exhibit highly specific interactions with each other and with 76 the decapping enzyme. Pat1 interacts with both Dhh1 and the Lsm1-7 complex (He and 77 Jacobson, 2015a, Sharif et al., 2013, Sharif and Conti, 2013, Nissan et al., 2010, Bouveret et 78 al., 2000, Wu et al., 2014), Upf1, Upf2, and Upf3 interact with each other (He et al., 1997), 79 and Edc3 interacts with Dhh1 (He and Jacobson, 2015a, Sharif et al., 2013). Pat1, Upf1, and 80 Edc3 also interact with specific binding motifs in the large C-terminal domain of Dcp2 (He and 81 Jacobson, 2015a, Harigaya et al., 2010). These interaction data and additional observations 82 led us to propose a new model for regulation of mRNA decapping (He and Jacobson, 2015a) 83 in which different decapping activators form distinct decapping complexes in vivo, each of 84 which has a unique substrate specificity that targets a subset of yeast mRNAs. To test 85 aspects of this model, and to further understand the roles of Pat1, Dhh1, and the Lsm1-7 86 complex in general mRNA decapping we have analyzed the effects of deletions of the PAT1, 87 5 LSM1, or DHH1 genes and the large Dcp2 C-terminal domain on transc...

Discovery of cellular substrates of human RNA decapping enzyme Dcp2 using a stapled bicyclic peptide inhibitor

“…It is possible that binding of CP21 to Dcp2 specifically disrupted presentation of these DDX6-associated RNAs to the decapping complex (Figure 7). In a study of yeast Dcp2 active site vs C-terminal mutants, differential stabilization of RNA substrates by different mutations was observed, providing precedent for this hypothesis (He et al, 2018).…”

Pat1 activates late steps in mRNA decay by multiple mechanisms

“…Pat1 works together with Dcp1/Dcp2 and the Lsm1-7 complex to promote bulk 5’-3’ decay on numerous transcripts in yeast (12). Our work reveals how Pat1 interacts with and activates separate steps in this pathway.…”

“…A mechanistic description of how the Pat1/Lsm1-7 complex is recruited to target mRNAs remains poorly understood. In budding yeast, Pat1/Lsm1 bind the 3’ end of the mRNA and promote degradation of inefficiently translated mRNAs (12, 30). In S. pombe and higher order eukaryotes, however, an additional 1-3 uridines are added to a subset of transcripts after deadenylation but prior to decapping, which are enriched when Lsm1 is deleted (19, 20).…”

“…Structural and biochemical studies have demonstrated that Pat1 and Lsm1-7 form a heterooctameric complex that engages transcripts containing oligo(A) tails that result from deadenylation (27–30). Pat1 and Lsm1-7 are functionally linked, as they bind the 3’ end of mRNA and deletion of either Pat1 or Lsm1 causes an increase in steady-state levels of numerous overlapping transcripts (12, 30). In addition, deletion of Pat1 or Lsm1 results in an accumulation of deadenylated, capped mRNA intermediates, suggesting a block in decapping (21, 22, 31).…”

Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing

“…In eukaryotic cells, decapping enzymes play a major role in determining mRNA fate, and thus their activity is tightly regulated (15). The function of the cellular decapping enzyme DCP2, for example, is controlled by both cis-acting factors, such as autoinhibition by its C-terminal domain, and trans-acting proteins that regulate its activity on specific mRNA substrates (16)(17)(18). Like DCP2, VACV D10 contains a Nudix hydrolase domain (19), however it lacks the inhibitory C-terminal domain, supporting the hypothesis that D10 may function as a constitutively active decapping enzyme.…”