Drosophila mutants show NMD pathway activity is reduced, but not eliminated, in the absence of Smg6

Abstract

The nonsense-mediated mRNA decay (NMD) pathway is best known for targeting mutant mRNAs containing premature termination codons for rapid degradation, but it is also required for regulation of many endogenous transcripts. Components of the NMD pathway were originally identified by forward genetic screens in yeast and Caenorhabditis elegans. In other organisms, the NMD pathway has been investigated by studying the homologs of these genes. We present here the first unbiased genetic screen in Drosophila designed specifically to identify genes involved in NMD. By using a highly efficient genetic mosaic approach, we have screened ∼40% of the Drosophila genome and isolated more than 40 alleles of genes required for NMD. We focus on alleles we have obtained in two known NMD components: Upf2 and Smg6. Our analysis of multiple alleles of the core NMD component Upf2 reveals that the Upf2 requirement in NMD may be separate from its requirement for viability, indicating additional critical cellular roles for this protein. Our alleles of Smg6 are the first point mutations obtained in Drosophila, and we find that Smg6 has both endonucleolytic and nonendonucleolytic roles in NMD. Thus, our genetic screens have revealed that Drosophila NMD factors play distinct roles in target regulation, similar to what is found in mammals, but distinct from the relatively similar requirements for NMD genes observed in C. elegans and yeast.

FIGURE 1.

Mosaic approach used to detect animals with mutations in genes affecting NMD. (A) y w FRT^19A/Y; e22c-GAL4 UAS-nlsDsRed2::SV40 3′UTR/+ L3 (nonmosaic) larva shows uniform, low-level DsRed expression in epidermal nuclei. (B) y w Upf2^25G FRT^19A/Y; e22c-GAL4, UAS-nlsDsRed2::SV40 3′UTR/+ (nonmosaic) L3 larva, taken at the same exposure as A, shows uniform, high-level DsRed expression in epidermal nuclei, due to stabilization of the reporter mRNA. (C) UAS-FLP expression in the early embryonic ectoderm driven by e22c-GAL4 catalyzes recombination in heterozygous mitotic cells, resulting in homozygous mutant and wild-type sister cells. Cells that have not undergone recombination remain heterozygous. Expression of an NMD-sensitive reporter UAS-nlsDsRed2::SV40 3′UTR, which encodes a nuclear-localized red-fluorescent protein, is driven by e22c-GAL4 in the larval epidermis. In heterozygous and the wild-type sister cells, the reporter mRNA is targeted for degradation by NMD and shows a low level of DsRed fluorescence (light red). When a gene required for NMD is mutated, the homozygous mutant cell is recognized by an increase in fluorescence (increased red color). (Circles) Centromeres; (rectangles) FRT sites; (*) EMS-generated mutations. (D) y w FRT^19A; e22c-GAL4, UAS-nlsDsRed2::SV40 3′UTR, UAS-FLP/+ mosaic L3 larva shows uniform DsRed expression in epidermal nuclei. (Yellow asterisks) Underlying tracheal nuclei that also express DsRed. These are easily distinguished from epidermal cells under the microscope. (E) y w Upf1^26A FRT^19A/y w FRT^19A; e22c-GAL4, UAS-nlsDsRed2::SV40 3′UTR, UAS-FLP/+ L3 larva shows mosaic increased DsRed expression in epidermal nuclei: Low-level DsRed expression is punctuated with brighter, homozygous mutant nuclei (yellow arrowheads). (F) y w Smg1^32AP FRT^19A/y w FRT^19A; e22c-GAL4, UAS-nlsDsRed2::SV40 3′UTR, UAS-FLP/+ L3 larva shows mosaic DsRed expression in epidermal nuclei: Low-level DsRed expression is punctuated with brighter, homozygous mutant nuclei (yellow arrowheads). Scale bars, (A,B) 1 mm; (D–F) 0.5 mm.

FIGURE 2.

Screens to identify genes required for NMD on X and 3R. In the P₀, males carrying the appropriate FRT are mutagenized by treatment with EMS and mated en masse to reporter-containing females. In the F₁ generation, candidate NMD-mutant L3 larvae are identified by mosaic epidermal reporter expression, collected, and individually mated to flies containing marked FRTs. F₂ larvae are scored for transmission of the mosaic-expression phenotype, collected, and crossed to balancer-containing animals to establish stocks. (Middle column) Numbers of animals identified in each generation. (e22RF) Genotype e22c-GAL4, UAS-nlsDsRed2::SV40 3′UTR, UAS-FLP.

FIGURE 3.

Characterization of Upf2 alleles. (A) Mutations in Upf2. Alleles shown above the gene model are hemizygous lethal; those below the gene are hemizygous viable. 2-8A contains two mutations: a late glutamine to stop (black), as well as a serine-to-phenylalanine missense mutation (gray). This latter change is in a nonconserved domain of Upf2, and we suspect it does not contribute to the Upf2 defect. (Filled boxes) Coding sequences; (open boxes) 5′ and 3′ UTRs; (blue) bipartite Upf1 binding domain; (green) Upf3 binding domain; (purple) MIF4G domains (the third one of which overlaps the Upf3 binding domain); (open arrow) direction of transcription. (B) Effect of viable Upf2 mutations on levels of the PTC-containing transcript Adhⁿ⁴ (Q83Stop). Values are the proportion of Adhⁿ⁴ transcript relative to Adh⁺ transcript normalized to Upf2^25G, in which Adhⁿ⁴ is completely stabilized.

FIGURE 4.

Molecular and phenotypic characterization of Smg6 alleles. (A) Mutations in Smg6. (Filled boxes) Coding sequences; (open boxes) 5′ and 3′ UTRs; (blue) 14-3-3-like protein binding domain; (green) PIN endonucleolytic cleavage domain; (open arrow) direction of transcription. (B) Alignment of Smg6 PIN domains. These highly conserved domains are representative of PIN domains found in Smg6 and other PIN-domain-containing proteins (Takeshita et al. 2007). (Dm) D. melanogaster; (Hs) Homo sapiens; (Ce) C. elegans. (*) Invariant catalytic residues; (gray shading) residues conserved in all three species; (red shading) aspartic acid residue altered to a valine in Smg6^2a. (C) Effect of Smg6 mutations on levels of the PTC-containing transcript Adhⁿ⁴ (Q83Stop). Values are the proportion of Adhⁿ⁴ transcript relative to Adh⁺ transcript normalized to Upf2^25G, in which the Adhⁿ⁴ is completely stabilized. Error bars indicate SD, n = 2. (D) Effect of Smg6 mutations on levels of the endogenous NMD target tra in males. Levels for Smg6 alleles are normalized to FRT^82B/Df animals; Smg1^32AP/Y and Upf2^25G/Y are normalized to FRT^19A/Y. Error bars indicate SEM, n = 3. (E) Percentage survival to adulthood of Smg6 mutants. Each of the alleles are in trans to an Smg6 deficiency. The percentage is calculated relative to balancer-containing siblings. Error bars: 95% confidence interval of the binomial distribution. (F) Microarray analysis of relative magnitude of NMD defects observed in Upf2^25G, Smg1^32AP, and two Smg6 alleles, Smg6^2a and Smg6²⁹². For each mutant genotype, we calculated transcript expression relative to its respective wild-type control. To compare the effect of different NMD genes with transcript regulation, we first extracted targets that increased more than twofold in all four of our NMD-mutant conditions, giving a list of 87 genes (Supplemental Table S1). We then compared the magnitude of the increase observed in Smg1^32AP, Smg6^2a, and Smg6²⁹² with that observed in Upf2^25G. (Black bars) Transcripts that increased more in Upf2^25G relative to transcripts in Smg1 or Smg6; (gray bars) transcripts that increase more in Smg1 or Smg6 alleles relative to Upf2^25G.