Control of mRNA decapping by positive and negative regulatory elements in the Dcp2 C-terminal domain

Abstract

Decapping commits an mRNA to complete degradation and promotes general 5' to 3' decay, nonsense-mediated decay (NMD), and transcript-specific degradation. In Saccharomyces cerevisiae, a single decapping enzyme composed of a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2) targets thousands of distinct substrate mRNAs. However, the mechanisms controlling this enzyme's in vivo activity and substrate specificity remain elusive. Here, using a genetic approach, we show that the large C-terminal domain of Dcp2 includes a set of conserved negative and positive regulatory elements. A single negative element inhibits enzymatic activity and controls the downstream functions of several positive elements. The positive elements recruit the specific decapping activators Edc3, Pat1, and Upf1 to form distinct decapping complexes and control the enzyme's substrate specificity and final activation. Our results reveal unforeseen regulatory mechanisms that control decapping enzyme activity and function in vivo, and define roles for several decapping activators in the regulation of mRNA decapping.

Keywords: decapping activators; decapping enzyme; mRNA decapping; positive and negative regulation.

FIGURE 1.

Identification of Dcp1, Edc3, Pat1, and Upf1-interacting domains of Dcp2. (A) Mapping of Dcp1, Edc3, and Pat1-interacting domains of Dcp2. (B) Mapping of Upf1-interacting domains of Dcp2. Dcp2 fragments fused to Gal4(DB) or Gal4(AD) were assayed for interactions with Gal4(AD)-Dcp1, Edc3, and Pat1 (A) or Gal4(DB)-Upf1 (B), respectively. A and B include schematic representations of the Dcp2 domain structure and the Dcp2 fragments used in the experiment. Shaded bars indicate interacting fragments, whereas white bars indicate fragments that failed to interact. The minimal binding sites for Dcp1, Edc3, Pat1, and Upf1 are bracketed by dashed lines. In A and B, blue colony color indicates an interaction and white colony color indicates no interaction. Constructs highlighted with a black diamond indicate Dcp2 segments that interact with Upf1, but not Pat1, or vice versa.

FIGURE 2.

Identification of the conserved Edc3, Pat1, and Upf1-binding Motifs in the C-terminal domain of Dcp2 and a summary of the interacting domains involving Dcp2 and Dcp1, Edc3, Pat1, and Upf1. (A) Multiple sequence alignments of Dcp2's Edc3-, Upf1-, and Pat1-binding motifs with the respective conserved sequences from other fungal Dcp2 orthologs. Amino acids are colored based on physicochemical properties ([h] hydrophobic, [l] aliphatic, [p] polar, [s] small, [t] turn-like, [u] tiny). (B) Protein domains involved in interactions between Dcp1 and Dcp2, and between Dcp2 and Edc3, Pat1, or Upf1. Schematic representations of the domain structures for each factor are shown. Interactions between factors are indicated by red lines with arrowheads, and domains implicated in each interaction are marked by straight blue lines. For Dcp2, E-BD indicates Edc3-binding domain, U-BD indicates Upf1-binding domain, and P-BD indicates Pat1-binding domain.

FIGURE 3.

The Dcp2 C-terminal domain harbors an inhibitory element that targets the Dcp2 catalytic domain and subjects the decapping enzyme to negative regulation. (A) Northern analysis of the consequences of incremental deletions from the C-terminus of Dcp2 on YRA1 pre-mRNA decay in dcp2Δ and edc3Δdcp2Δ cells. (B) Northern analysis of the effects of amino acid substitutions in the catalytically important residues on dcp2-N245 decay activity in dcp2Δ and edc3Δdcp2Δ cells. In A and B, schematic representations of Dcp2 domain structure and dcp2 alleles used in the experiment are shown on top and the positions of the Edc3, Upf1, and Pat1-binding sites are indicated by dashed lines. Black dots in the Nudix domains shown in B demark positions of amino acid substitutions. Representative Northern blots were hybridized with random-primed probes specific for YRA1 pre-mRNA, ade2-1 mRNA, and the SCR1 transcript, with the last serving as a loading control. The relative levels of YRA1 pre-mRNA or ade2-1 mRNA derived from the Northern blots are depicted as bar graphs at the bottom of each panel. The data were the average of at least three independent experiments and were normalized to the respective cells harboring the wild-type DCP2 allele (labeled 1 in red). The red error bars indicate standard deviations. The red stars indicate the P-values from paired two-tailed Student's t-tests ([*] P < 0.05, [**] P < 0.01).

FIGURE 4.

Mapping of the cis-inhibitory element in the C-terminal domain of Dcp2, and analyzing the effects of loss of this element on Dcp2 protein accumulation and mRNA decay function. (A) Northern analysis of the effects of small incremental deletions between amino acids 300–475 of Dcp2 on YRA1 pre-mRNA decay in edc3Δdcp2Δ cells. (B) Northern analysis of the effects of small internal deletions between amino acids 300–400 of Dcp2 on YRA1 pre-mRNA decay in edc3Δdcp2Δ cells. (C) Sequence alignment of the Dcp2 inhibitory element with conserved sequences from Dcp2 orthologs of several other yeast species. (D) Western analysis of the effects of C-terminal and internal deletions on Dcp2 protein expression in edc3Δdcp2Δ cells using triple-HA tagged dcp2 alleles. (E) Northern analysis of the consequences of the Dcp2 C-terminal N245 deletion on EDC1 mRNA and POR1 mRNA levels in control cells or cells harboring deletions of the EDC3, UPF1, PAT1, LSM1, or DHH1 genes. In A and B, schematic representations of Dcp2 domain structure and dcp2 alleles used in the experiment are shown on top and the positions of Edc3, Upf1, and Pat1-binding sites are indicated by dashed lines. The Dcp2 amino acid sequence required for cis-inhibitory activity is highlighted by a red bar. In A, B, and E, representative Northern blots were hybridized with random-primed probes specific for YRA1 pre-mRNA, EDC1 mRNA, POR1 mRNA, or the SCR1 transcript. The relative levels of YRA1 pre-mRNA, EDC1 mRNA, and POR1 mRNA derived from Northern blots are depicted as bar graphs of the corresponding panels. The data comprise the average of at least three independent experiments and were normalized to the respective cells harboring the wild-type DCP2 allele (labeled 1 in red). The red error bars indicate standard deviations. The red stars indicate the P-values from paired two-tailed Student's t-tests ([*] P < 0.05, [**] P < 0.01). In D, Western blots were probed with monoclonal antibodies against the HA epitope or Pgk1, with the latter serving as a loading control.

FIGURE 5.

The Dcp2 C-terminal domain plays an important role in both transcript-specific and general mRNA decapping. (A) Northern analysis of the consequences of the Dcp2 C-terminal N245 deletion on Edc3-mediated mRNA decay, NMD, and general mRNA decay in yeast cells harboring either deletions of the EDC3 or UPF1 genes or the respective wild-type genes. Schematic representations of the Dcp2 domain structure and the genomic R34-tagged or untagged dcp2-N245 allele used in the experiments are shown on top. Overexposed blots for CYH2 pre-mRNA and ade2-1 mRNA are indicated by an asterisk. (B) Northern analysis of the effect of restoring Edc3 interaction with dcp2-N245 on YRA1 pre-mRNA decay. Schematic representations of the Edc3 domain structure and EDC3 alleles tagged with either a basic or acidic leucine zipper used in the experiments are shown on top. V indicates the empty vector control. In A and B, representative Northern blots were hybridized to random-primed probes specific for the indicated transcripts with the SCR1 transcript serving as a loading control. The relative levels of each transcript derived from Northern blots are depicted in bar graphs on the right side of each panel. The data are the average of at least three independent experiments and are normalized to the cells harboring the wild-type DCP2 allele (A) or the empty vector (B) (labeled 1 or V in red, respectively). The red error bars indicate standard deviations. The red stars indicate the P-values from paired two-tailed Student's t-tests ([*] P < 0.05, [**] P < 0.01).

FIGURE 6.

Model for in vivo regulation of the yeast mRNA decapping enzyme. Details are described in the main text. In the Dcp2 images, N indicates the negative regulatory element. P1, P2, P3, and P4 indicate different positive regulatory elements, including the Edc3, Pat1, and Upf1-binding sites.