The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1

Abstract

The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells.

mi RISC and the CCR 4– NOT complex silence mRNA targets independently of 43S ribosomal scanning

“…To this end, we overexpressed a DCP1 mutant that inhibits decapping in a dominant negative manner (DCP1* mutant; Chang et al , 2014; Appendix Table S1). Tethered Dm GW182, AGO1, and NOT1 degraded the F‐Luc‐5BoxB‐poly(A) reporter (Fig 2M, lanes 3, 5, and 7 versus 1).…”

Drosophila Bag-of-marbles directly interacts with the CAF40 subunit of the CCR4–NOT complex to elicit repression of mRNA targets

“…In this pathway, deadenylation is followed by decapping and 5'-to-3' exonucleolytic degradation of the mRNA body. We therefore performed tethering assays in S2 cells depleted of the decapping enzyme DCP2 and overexpressing a catalytically inactive DCP2 mutant (DCP2 E361Q), which inhibits decapping in a dominant negative manner (Chang et al 2014). In these cells, degradation of the F-Luc-5BoxB mRNA by tethered Bam or the Bam-N fragment was impaired ( Fig.…”

In Silico Identification of Potentially Functional Conserved Motifs in Two Components of the 5’ to 3’ mRNA Decay Pathway of Plants

“…The Dcp1/EVH1-like domain of DCP1 is composed by two α-helices flanking seven β-sheets. The first helix is required to interact with DCP2, and the NR-loop, located between the third and fourth β-sheets, increases decapping activity in vitro, but does not contribute in the physical interaction between DCP1 and DCP2 [25]. The peculiar array of the β-sheets of the PH variants allows the recognition of distinct Proline-rich sequences (PRS) [26].…”

“…In humans, XRN1 stays close to the decapping complex due to the interaction with the scaffold protein EDC4, which also interacts with DCP1 and DCP2. The interaction between the human XRN1 and EDC4 depends on the EDC4-binding motif (EBM) ( Figure 4A) located at the C terminus, that recognizes the α-helical domain of EDC4 [25]. EBM is specific of human XRN1 and it is not conserved in fruit fly, baker's yeast and plants.…”