Research

Decapping commits an mRNA to complete degradation and is a key regulatory event in multiple cytoplasmic mRNA decay pathways including the general 5’ to 3’ decay, nonsense-mediated decay (NMD), and transcript-specific degradation. In the yeast Saccharomyces cerevisiae, a single decapping enzyme, comprised of a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2), and several decapping activators, including Upf1, Edc3, Dhh1, Pat1, and the Lsm1-7 complex, are required for mRNA decapping. Our current research seeks to define the functions and mechanisms of action of these yeast decapping factors in mRNA decapping.

 

The yeast Dcp1-Dcp2 decapping enzyme targets thousands of distinct substrate mRNAs for cap removal. However, the molecular mechanisms controlling this enzyme’s in vivo targeting specificity and temporal activation remain elusive. Our recent genetic experiments (He & Jacobson, RNA 21:1633-1647; He et al., eLife7: e34409, 2018) revealed that the large C-terminal domain of of Dcp2 encodes several key regulatory functions and appears to have a pivotal role in controlling the decapping enzyme’s targeting specificity and decapping activation. This Dcp2 domain contains an auto-inhibitory element and a set of conserved linear elements that promote the binding of specific decapping activators, including one Edc3-binding motif, two independent Upf1-binding motifs, and eight independent leucine-rich Pat1-binding motifs. These observations led us to formulate a new model for in vivo regulation of the yeast decapping enzyme. In this new model, the acitivity of the decapping enzyme is controlled by both negative and positive regulation. The inhibitory element in the C-terminal domain of Dcp2 keeps the enzyme in an inactive state. Different binding motifs in the Dcp2 C-terminal domain control the decapping enzyme’s targeting specificity and timing of activation. To test this model, we are currently investigating the roles of each Dcp2 regulatory element in decapping of different mRNA substrates and assessing whether the decapping enzyme forms distinct complexes with specific decapping activators in yeast cells.

 

In addition to the decapping enzyme, mRNA decapping also requires the function of specific regulators, generally known as decapping activators. For example, decapping of nonsense-containing mRNAs requires the Upf factors and decapping of general mRNAs requires Edc3, Dhh1, Pat1, and the Lsm1-7 complex. Two general functions, i.e., repressing mRNA translation and activating the decapping enzyme, have been proposed for some of these decapping activators, but convincing experimental evidence for these two functions is still largely lacking. To dissect the roles of decapping activators in mRNA decapping, we recently mapped the protein-protein interaction network for all known yeast decapping factors and identified the transcripts targeted by each of these factors experiments (Dong et al., Mol. Cell 25:559-573, 2007; He & Jacobson, RNA 21:1633-1647; He et al., RNA 23: 735-748, 2017; He et al., eLife7: e34409, 2018). These studies identified the decapping activators that interact directly with the C-terminal domain of Dcp2 and those that do not interact with Dcp2, and also revealed that rather than being global activators of decapping, Pat1, Lsm1, Dhh1, and Edc3 each target a specific subset of yeast mRNAs. Surprisingly, the Dcp2-interacting decapping activator Edc3 appears to only regulate two transcripts in the whole yeast transcriptome. Based on these results and additional observations, we hypothesize that decapping activators may generally monitor the kinetics of some specific steps in mRNA translation and target the kinetically unfavorable mRNAs for decapping. To test this hypothesis, we are currently analyzing the mRNP complexes formed by different decapping activators in yeast cells and also investigating the molecular features and the characteristics in translation for mRNAs targeted by each of these decapping activators.