Figure 1.
Identification of Photoshop Mutants
(A) FLP/MARCM system used to generate GFP-labeled homozygous mutant tracheal cell clones in heterozygous animals. Heterozygous cells have mutations (asterisk) in trans to a GAL80 transgene (triangle) that inhibits expression of a tracheal-specific, btl-GAL4-driven GFP transgene on another chromosome (not shown). All cells also carry a heat-inducible FLP transgene on another chromosome (not shown). Following S phase of the cell cycle (S), FLP-mediated recombination (R) at the FRT, and M phase (M), homozygous mutant tracheal cells are segregated that lack the GAL80 transgene and express GFP (green), distinguishing them from homozygous GAL80 sister cells and nonrecombined heterozygous cells.
(B) Anteriors of L3 larvae with wild-type (WT; y w) control clones (top) or photoshop mutant 14J clones (bottom). Larvae were photographed next to each other to facilitate comparison. Note strong GFP signal in 14J clones but barely detectable signal in control clones. Bar, 0.5 mm.
(C) GFP signal is increased throughout tracheal system in photoshop mutant 25G larvae. A wild-type (y w FRT19A/Y; btl-GAL4, UAS-GFP) control larva is shown above, and a hemizygous 25G/Y photoshop mutant (25G/Y; btl-GAL4, UAS-GFP) larva is shown below. Bar, 1 mm.
(D) GFP signal is increased throughout tracheal system in photoshop 32AP larvae. Above is a control heterozygous (32AP/+; btl-GAL4, UAS-GFP/+) larva showing weak GFP signal throughout tracheal system. In the middle is a 32AP/Df mutant female (32AP/Df; btl-GAL4, UAS-GFP/+) larva showing enhanced GFP signal throughout. Below is a 32AP/Y mutant male (32AP/Y; btl-GAL4, UAS-GFP/+) larva: GFP signal is indistinguishable from that observed in a 32AP/Df larva, indicating 32AP is an amorphic (null) allele. Bar, 1 mm.
(E) Effect of a photoshop mutation on a DsRed transgene. Photoshop 14J tracheal clone in y w 14J FRT19A/ w FRT19A, FLP122 larva carrying btl-GAL4 and UAS-DsRed1 transgenes. Two unicellular tracheal tubes are indicated by brackets; 14J− clone has higher DsRed1 signal than neighboring wild-type (14J+) cell. Bar, 25 μm.
(F) Effect of photoshop 25G mutation outside the tracheal system. A mixture of wild-type and 25G/Y mutant animals carrying da-GAL4, UAS-CD8:GFP is shown. GFP signal is increased throughout all tissues of the 25G mutants. 25G genotype of high-signal larvae was confirmed by PCR. Bar, 2 mm.
(G) Effect of photoshop 32AP mutation in epidermis. A wild-type larva is shown above, and below is a 32AP/Y mutant larva (bottom) carrying e22c-GAL4, UAS-nls:DsRed2, which expresses a nuclear localized DsRed2 in epidermis and other epithelial tissues. Animals were photographed next to each other. The nls:DsRed2 signal is enhanced in the epidermis of the 32AP/Y mutant. Bar, 1 mm.
(H) Effect of photoshop mutants on GFP RNA levels. Quantitative real time RT-PCR was done on the GFP transcript in wild-type (y w FRT19A/Y), 25G/Y, and 32AP/Y L3 larvae carrying one copy of da-GAL4 and UAS-CD8:GFP. Results of replicate reactions were individually normalized to parallel reactions on rp18LA transcript. Values shown are mean ± 2 standard errors of the mean normalized to value in wild type.
Figure 2.
Photoshop Mutations Are Alleles of Upf2, Upf1, and Smg1
(A) Mapping of photoshop mutations 14J and 29AA. Shown are genetic maps of the X chromosome (top) and ct–v interval (middle) with visible markers (top) and SNP markers (middle) used to map lethality associated with 14J and 29AA mutations. Lines beneath maps show regions of 14J and 29AA X chromosomes (top) or 14J chromosome (middle) that were hemizygous-male viable, localizing 14J between XC35 and XC40 markers. Bottom panel shows predicted genes in mapped interval. Genes are alternately shaded black and white for clarity.
(B) Upf2 mutations in photoshop mutants 14J, 29AA, and 25G. 29AA (AAG to TAG) and 25G (TGA to AGA) are point mutations that disrupt Upf2 protein sequence as indicated, whereas 14J is a deletion of nucleotides 1682–1695 (TCCTGCCCTATCTC) that disrupts the coding sequence at codon 562. Filled boxes, coding sequence; open boxes, UTRs; arrow, direction of transcription; bracket, extent of Upf2 genomic fragment in rescue transgenes, extending from ~600 bp upstream of Upf2 mRNA to 78 bp downstream of polyadenylation site.
(C) 3′ end of Upf2 coding sequence showing effect of 25G mutation, which converts stop to arginine codon and extends coding sequence 15 residues. Below is an alignment of C-termini of Drosophila melanogaster (D.m.) Upf2 and homologs from human (H.s.), C. elegans (C.e.), and Saccharomyces cerevisiae (S.c.). Similar residues shaded grey.
(D) Mapping of photoshop mutations 13D and 26A. Lethality associated with 13D and 26A mutations mapped between markers XA46 and XA48, an interval that includes Upf1.
(E) Mutations in Upf1 in 13D and 26A. Grey box, region homologous to domain in S. cerevisiae Upf1p required for interaction with Upf2p.
(F) Mapping of photoshop mutation 32AP. Lines beneath each map indicate regions of 32AP chromosome that did not enhance GFP expression in hemizygous males, showing that 32AP maps between XA24 and XB9, an interval containing Smg1. Df(Smg1)exe2B is an ~46-kb deficiency that uncovers Smg1 and the photoshop phenotype of 32AP.
(G) Mutation in Smg1 in 32AP. 32AP also contains a silent mutation (not shown) in codon 1755 (GCC to GCT). Grey box, Smg1 kinase domain.
Figure 3.
Effect of Photoshop Mutations on Steady State Levels of an Adh Transcript Containing a Nonsense Mutation
Left, agarose gel analysis of PvuII-digested products of RT-PCR of Adh RNA extracted from heterozygous Adhn4/Adh+ adult males that were wild-type (WT) for NMD genes (lanes 1 and 2), hemizygous for Smg132AP (lanes 3 and 4), or hemizygous for Upf225G (lanes 5 and 6). Adhn4 is a nonsense mutation that also disrupts a PvuII site that divides the 671-bp PCR product into 484-bp and 187-bp fragments. For each genotype, two independent RNA samples were analyzed.
Right, quantification of Adhn4 and Adh+ RNA levels by capillary electrophoresis of PvuII-digested RT-PCR products. Similar results were obtained by directly sequencing the same RT-PCR products and quantitating the level of each product from the chromatogram.
Table 1.
Transgenes Tested for Enhanced Expression in Photoshop Mutants
Figure 4.
Effect of a Photoshop Mutation on Expression of GFP Transgenes
Pairs of larvae of genotypes Upf225G/Y; e22c-GAL4, UAS-nls:DsRed2/UAS-GFP (Upf225G mutant, left in each panel) and w/Y; e22c-GAL4, UAS-nls:DsRed2/UAS-GFP (Upf2+ control, right in each panel). The DsRed2 (internal control) transgene contains an SV40 3′ UTR and was the same in all larvae, whereas the GFP test transgene differed in reporter and 3′ UTR sequences as indicated. DsRed channel (A–G) shows effect of Upf225G on the internal control DsRed2 transgene. GFP channel (A′–G′) shows effect of Upf225G on the GFP test transgenes indicated. Photographic exposures were the same and images were processed identically to facilitate comparison. Similar results were obtained for at least two independent insertions of each eGFP-variant transgene. The ratio of GFP expression for each transgene in Upf225G versus Upf2+ larvae was quantitated by GFP fluorescence measurements of micrographs of pairs of Upf225G and Upf2+ larvae, and the fluorescence ratio (average ± standard deviation, n = 2 independent insertions for each eGFP transgene) is shown. (The expression ratio of the DsRed2 internal control construct in Upf225G versus Upf2+ larvae was 4.7 ± 0.7). Only GFP transgenes with an SV40 3′ UTR were enhanced in Upf225G mutants, independent of the intron in this UTR. Transgenes with a 3′ UTR derived from hsp70 were not enhanced. eGFP, enhanced GFP reporter; + I, with added synthetic intron; ΔI, SV40 intron deleted; pUAST.h, pUAST with hsp70 3′ UTR replacing SV40 3′ UTR. Bar, 1 mm.
Figure 5.
Effect of Photoshop Mutations on Expression of Endogenous Genes
(A) Results of quantitative real-time RT-PCR of indicated genes using RNA from y w FRT19A/Y (WT), Upf225G/Y, and Smg132AP/Y L3 larvae carrying one copy of da-GAL4 and UAS-CD8:GFP. Amplifications of replicate reactions were individually normalized to internal control reactions with rp18LA. Values shown are means ± 2 standard errors of the mean relative to the results with y w FRT19A/Y. GFP RNA levels in these larvae (see Figure 1H) are included for comparison. EK, photoshop-independent control gene defined by expressed sequence tag EK161155.
(B) Effect on tra RNA levels in females. Above are shown sex-specific splice patterns in the coding portion of the tra transcript. Males use splice pathway shown below line to produce traL, whereas females use both splice pathways to produce traL and traS. traS encodes a 197-residue protein, whereas traL contains an early termination codon and encodes a 37-residue protein. Arrows, position of PCR primers used to simultaneously amplify traL (364-bp product) and traS (189 bp). Bottom left shows RT-PCR on RNA from heterozygous Upf225G/+ and homozygous Upf225G female larvae; aliquots of reaction were taken at PCR cycles indicated. Note increased traL in Upf225G homozygotes. Bottom right shows quantification of results after 30 PCR cycles. Areas of the peaks are indicated, normalized to traS peak. These and similar experiments (not shown) indicate traL is increased ~10-fold in Upf225G mutants. This was greater than the value measured for Upf225G mutant males (~4-fold; Table S1; Figure 5A), perhaps due to sex-specific differences in NMD or differences in sensitivity of the assays.
Figure 6.
Effects of Photoshop Mutations on Tracheal System, Nervous System, and Eye Development
(A and B) Fluorescence (A) and brightfield (B) images of tracheal dorsal branch terminal cells in y w Upf214J FRT19A/y w FRT19A, FLP122; btl-GAL4, UAS-GFP/+ mosaic L3 larva. Homozygous Upf214J clone (arrowhead) expresses GFP at a higher level than contralateral heterozygous control cell (arrow), but clone has formed normal cytoplasmic branches (A) and a normal air-filled lumen in each branch (B). Bar in (A) (for [A] and [B]), 25 μm.
(C and D) Wild-type (WT) control clone (C) and homozygous Upf126A clone (D) in type IV da neurons labeled with ppk-GAL4, UAS-CD8:GFP. Both control and Upf126A clones show complex dendritic arborization fields typical of type IV da neurons, although in the latter the aborizations are easier to visualize because of increased GFP expression. Bar in (C) (for [C] and [D]), 100 μm.
(E and F) Control (y w) wild-type clones (E) and Upf113D clones marked with w− in adult eyes. Wild-type clones (E) are easily detected as w− patches (white areas, demarked with dotted lines) in the w+ (red) heterozygous eye, whereas only small w− clones or single w− cells are detected in the Upf113D heterozygous eye (F). Similar results were obtained with Upf214J (not shown).
(G and H) Similar experiment with Upf214J mutant except eye cells not part of the clone were eliminated by GMR-hid technique. Upf2+ control clones proliferate (G) to form an eye (slight eye roughness is common with GMR-hid technique), as do Upf214J clones (H), although the latter are somewhat smaller and rougher than the controls. Similar results were obtained with Upf126A and Upf113D (not shown).
Figure 7.
Model for Drosophila NMD Pathway Action on SV40 3′ UTR
In wild type (WT), SV40 3′ UTR enhances transgene expression (arrow). However, effect is partially offset by transcript degradation by NMD machinery, giving an intermediate level of expression ( + + ). In photoshop (NMD) mutants, transcript degradation is abrogated, resulting in strong enhancement by SV40 3′ UTR (thick arrow) and high level of expression ( + + + ). The hsp70 3′ UTR does not enhance expression (or alternatively promotes degradation) but is also not a target of the NMD pathway, giving a low level of expression (+) insensitive to photoshop mutants.