A genetic screen for maternal-effect suppressors of decapentaplegic identifies the eukaryotic translation initiation factor 4A in Drosophila

Abstract

The Dpp signaling pathway is essential for many developmental processes in Drosophila and its activity is tightly regulated. To identify additional regulators of Dpp signaling, we conducted a genetic screen for maternal-effect suppressors of dpp haplo-insufficiency. We screened approximately 7000 EMS-mutagenized genomes and isolated and mapped seven independent dominant suppressors of dpp, Su(dpp), which were recovered as second-site mutations that resulted in viable flies in trans-heterozygous with dpp(H46), a dpp null allele. Most of the Su(dpp) mutants exhibited increased cell numbers of the amnioserosa, a cell type specified by the Dpp pathway, suggesting that these mutations may augment Dpp signaling activity. Here we report the unexpected identification of one of the Su(dpp) mutations as an allele of the eukaryotic translation initiation factor 4A (eIF4A). We show that Su(dpp)(YE9) maps to eIF4A and that this allele is associated with a substitution, arginine 321 to histidine, at a well-conserved amino acid and behaves genetically as a dominant-negative mutation. This result provides an intriguing link between a component of the translation machinery and Dpp signaling.

Figure 1.

Mutagenesis screen of maternal-effect suppressors of dpp^H46. Isogenized Canton-S males were mutagenized and outcrossed to a line containing the TM3, Sb¹ Ser¹ third chromosome balancer. The progeny females with TM3, Sb¹ Ser¹ in the F₁ were individually crossed to dpp^H46 Sp¹ cn¹/CyO-23 males to test for maternal-effect suppression of dpp^H46 haplo-insufficiency. The presence of dpp^H46 survivors, which were recognized by the absence of CyO-23 and the presence of Sp¹ dominant markers in the F₂, would indicate suppression. Mutations were recovered by crossing the males of dpp^H46 survivors to appropriate balancer strains. The CyO-23 contains a second copy of the dpp^Hin region on the standard CyO balancer. A total of 6982 mutagenized chromosomes were scored.

Figure 2.

Cuticle phenotypes and amnioserosa cells of Su(dpp) mutants. Larval cuticles (A, C, E, G, I, and K) and amnioserosa cells of stage 10 embryos as revealed by immunostaining with anti-Krüpple antibody (B, D, F, H, J, and L). All the embryos are shown as lateral views with anterior to the left and dorsal up except in K, which was a dorsal view. (A and B) Wild-type cuticle and amnioserosa cells. (C and D) Su(dpp)YE2 homozygous embryos with segmentation defects in cuticle (C) and an increased number of amnioserosa cells. (E and F) Su(dpp)YE5 homozygous embryos with mild segmentation defects in cuticle (E) and an increased number of amnioserosa cells. (G and H) Su(dpp)YE9 homozygous embryos show severely defective head skeletons and mild segmentation defects in cuticle (G) and an increased number of amnioserosa cells. (I and J) Su(dpp)YE10 homozygous embryos most commonly exhibit a deletion of the A4 denticle band (I) and an increased the number of amnioserosa cells. (K and L) Su(dpp)YE31 homozygous embryos exhibit defects in the dorsal epidermis ranging from a dorsal hole (K) to completely dorsally open (not shown). These embryos have a decreased number of amnioserosa cells (L).

Figure 3.

Fine meiotic mapping of the Su(dpp)YE9 by P-element recombination. The Su(dpp)YE9 was recombined with l(2)[k09923] and crossed to a set of different P elements. Female flies with the genotype of Su(dpp)YE9 l(2)[k09923]/P element were crossed with w⁻ males. We scored the w⁻ recombinant flies in the next generation for those that had lost both copies of the mini-white⁺ gene, resulting from a recombination event between l(2)[k09923] and the P element. These w⁻ recombinants were then individually crossed with dpp^H46 Sp¹ cn¹/CyO-23 males to test for suppression and lethality. (A) P elements utilized for the fine meiotic mapping of Su(dpp)YE9. They are within the 1.1-Mb region that we had narrowed down by male recombination mapping. The number of w⁻ recombinants obtained, the results of suppression of dpp^H46/+ lethality, and the location of Su(dpp) YE9 relative to each P element are indicated. In the case of chic[k13321], 34 of the 37 w⁻ recombinants maintained the ability to suppress dpp^H46/+ and the remaining 3 did not. These data indicate that Su(dpp)YE9 was proximal to chic[k13321]. In the case of EP(2)2273, all the w⁻ recombinants (120 in total) retained the suppression, suggesting that Su(dpp)YE9 is distal to EP(2)2273. (B) A scheme to illustrate the location of the P elements and the distance between them. The large arrows indicate the mapping data for Su(dpp)YE9, which was narrowed down to a 74-kb region in 26A1-B3.

Figure 4.

SNP mapping of the Su(dpp)YE9. (A) The P-element strains such as vri[k05901] and l(2)k06502) contain A at SNP-JL2 and are designated as T1. T2 strains such as Canton-S [from which Su(dpp)YE9 is derived] contain C at this position. The sequences surrounding SNP-JL2 are shown, and the single different nucleotide between the these two stains is underlined. The two “3′-mismatched” primers at the SNP-JL2 locus are JL2mis5(A) and JL2mis3(C), as indicated by long arrows. Each primer matches to only one strain, but not the other at the 3′ terminal nucleotide. When combined with two genomic primers, JL26F2 and JL26R2, DNA fragments of 380 or 180 bp will be amplified (see materials and methods). The first two lanes in the agarose gel are PCR amplification from Canton-S (T2) and vri[k05901] (T1) genomic DNA. Only one band was amplified from a genome homozygous at the SNP-JL2 locus and this band had the same size with either T1 or T2, reflecting the polymorphic variant of the original chromosomes. The rest of the lanes are examples of the w⁻ recombinants. (B) Summary of the SNP mapping data. Three types of recombination events assumed to have occurred in the white⁻ recombinants are indicated. The data from the suppression test and the corresponding genotype of these white⁻ recombinants are shown in the table (see text). These results indicate that Su(dpp)YE9 is located to the distal side of SNP-JL2. (Bottom) Su(dpp)YE9 was narrowed down to a 33-kb region as indicated.

Figure 5.

Identification of Su(dpp)YE9 as a point mutation of gene eIF4A. (A) Structure of eIF4A protein is shown. The eight highly conserved amino acid sequence motifs are indicated. The N-terminal, helicase, and RNA-recognition domains are indicated. Amino acid sequence is shown for motif V and its flanking regions. The Su(dpp)YE9 locus had a single amino acid change at position 321 of the conceptual eIF4A protein from arginine (R) to histidine (H). This amino acid is next to the highly conserved motif V of DEAD-box-containing proteins. (B) Alignment of the highly conserved surrounding region of motif V of eIF4A protein from human to yeast. The numbers indicate the position in each primary amino acid sequence. The position of R-to-H change is indicated at the top.