Figure 4. Genetic interactions between the Dcp2 Edc3-, Upf1-, and Pat1-binding motifs, or between the Edc3-binding motif and Edc3, that affect mRNA decapping.

(A) Two-hybrid assays examining the effects of different combinations of element deletions on Edc3, Upf1, and Pat1 binding to Dcp2. Allele schematics and two hybrid analyses are as in Figure 1B. (B) Northern analyses of the consequences of simultaneous loss of the Dcp2 Edc3-binding motif and leucine-rich motifs. See also Figure 4—figure supplement 1 and Figure 4—figure supplement 2. (C) Northern analyses of the consequences of simultaneous loss of the Dcp2 Upf1-binding motifs and leucine-rich motifs, and the roles of single Dcp2 Edc3 or Upf1-binding motifs. See also Figure 4—figure supplement 3 and Figure 4—figure supplement 4. (D) Northern analyses of the consequences for Edc3 and Dhh1 substrates caused by loss of the Dcp2 Edc3-binding motif and trans deletion of EDC3. See also Figure 4—figure supplement 5. Northern analyses as in Figures 1 and 2. Two-hybrid analyses as in Figure 1.

Figure 4—figure supplement 1. Simultaneous loss of the Edc3-binding motif and the leucine-rich motifs has synergistic effects and causes substantial stabilization of both Edc3 and Dhh1 substrates.

(A) Schematics of dcp2 alleles harboring different combinations of deletions of the Edc3-binding motif and the leucine-rich Pat1-binding motifs. Each of the dcp2 alleles contains a triple HA-tag at its N-terminus and was integrated at the DCP2 genomic locus for phenotypic analysis of mRNA decay. Specific element deletions are marked by filled triangles. (B) Northern analyses of the consequences of simultaneous loss of the Dcp2 Edc3-binding motif and leucine-rich motifs. The northern blots shown here are the same as those in Figure 4B but contain matched loading control SCR1 blots. In all blots, lower case letters denote SCR1 blots duplicated for clarity of presentation.

Figure 4—figure supplement 2. Simultaneous loss of the Edc3-binding motif and the leucine-rich motifs has synergistic effects and causes substantial stabilization of both Edc3 and Dhh1 substrates.

Bar graphs of average ± SEM for the northern analyses depicted in Figure 4B. The relative levels of each decapping substrate in different strains were determined from two or three independent experiments, with one representative blot for each transcript shown in Figure 4B. In each case, the relative RNA levels in different mutants were determined by comparison to the levels of the same transcripts in wild-type cells.

Figure 4—figure supplement 3. Simultaneous loss of the Upf1-binding motifs and the leucine-rich motifs has no synergistic effects on mRNA decapping, and a single Edc3- or Upf1-binding motif alone can promote efficient decapping of Edc3 or nonsense-mediated mRNA decay (NMD) substrates.

(A) Schematics of dcp2 alleles harboring different combinations of deletions of the Upf1-binding motifs, the Edc3-binding motif, and the leucine-rich Pat1-binding motifs. Each of these dcp2 alleles contains a triple HA-tag at its N-terminus and was integrated at the DCP2 genomic locus for phenotypic analysis of mRNA decay. Specific element deletions are marked by filled triangles. (B) Northern analyses of the consequences of simultaneous loss of the Dcp2 Upf1-binding motifs and leucine-rich motifs, and the roles of single Dcp2 Edc3- or Upf1-binding motifs. The northern blots shown here are same as those in Figure 4C but contain matched loading control SCR1 blots. In all blots, lower case letters denote SCR1 blots duplicated for clarity of presentation.

Figure 4—figure supplement 4. Simultaneous loss of the Upf1-binding motifs and the leucine-rich motifs has no synergistic effects on mRNA decapping, and a single Edc3- or Upf1-binding motif alone can promote efficient decapping of Edc3 or nonsense-mediated mRNA decay (NMD) substrates.

Bar graphs of average ± SEM for the northern analyses depicted in Figure 4C. The relative levels of each decapping substrate in different strains were determined from three or four independent experiments, with one representative blot for each transcript shown in Figure 4C. In each case, the relative RNA levels in different mutants were determined by comparison to the levels of the same transcripts in wild-type cells.

Figure 4—figure supplement 5. Loss of the Edc3-binding motif and trans deletion of EDC3 have additive effects on decapping of both Edc3 and Dhh1 substrates.

Bar graphs of average ± SEM for the northern analyses depicted in Figure 4D. The relative levels of each decapping substrate in different strains were determined from two to four independent experiments, with one representative blot for each transcript shown in Figure 4D. In each case, the relative RNA levels in different mutants were determined by comparison to the levels of the same transcripts in wild-type cells.