Nonsense-mediated mRNA decay effectors are essential for zebrafish embryonic development and survival

Abstract

The nonsense-mediated mRNA decay (NMD) pathway promotes rapid degradation of mRNAs containing premature translation termination codons (PTCs or nonsense codons), preventing accumulation of potentially detrimental truncated proteins. In metazoa, seven genes (upf1, upf2, upf3, smg1, smg5, smg6, and smg7) have been identified as essential for NMD; here we show that the zebrafish genome encodes orthologs of upf1, upf2, smg1, and smg5 to smg7 and two upf3 paralogs. We also show that Upf1 is required for degradation of PTC-containing mRNAs in zebrafish embryos. Moreover, its depletion has a severe impact on embryonic development, early patterning, and viability. Similar phenotypes are observed in Upf2-, Smg5-, or Smg6-depleted embryos, suggesting that zebrafish embryogenesis requires an active NMD pathway. Using cultured cells, we demonstrate that the ability of a PTC to trigger NMD is strongly stimulated by downstream exon-exon boundaries. Thus, as in mammals and plants but in contrast to invertebrates and fungi, NMD is coupled to splicing in zebrafish. Our results together with previous studies show that NMD effectors are essential for vertebrate embryogenesis and suggest that the coupling of splicing and NMD has been maintained in vertebrates but lost in fungi and invertebrates.

FIG. 1.

NMD factors are ubiquitously expressed and maternally provided during zebrafish embryogenesis. (A to P) mRNA expression patterns of NMD factors in embryos at two- to eight-cell stages revealed by antisense probes. Sense controls are shown on the right. Bars, 200 μm.

FIG. 2.

Upf1 is essential for zebrafish embryonic development and survival. (A to I) Lateral views of embryos injected with two different morpholinos (MOs) directed against either a splice site or the translational initiation codon (Start-site) of the upf1 transcript. (B to D and F to H) Representative phenotypes observed in Upf1 morphants at 1 dpf (mild to strong phenotypes). (A and E) Embryos injected with the corresponding 5-bp mismatch control MOs. Upf1 morphants exhibit extensive necrosis in the central nervous system (empty arrowheads), impaired eye development (filled arrowheads), abnormal somite morphogenesis (black arrows), and perturbation in the yolk sac extension (red arrows). (I) Uninjected wild-type embryo. Percentages of surviving embryos at 5 dpf are indicated below the panels. The total numbers of injected embryos are indicated in parentheses. Bar, 200 μm. (J to M) Lateral close-up views of heads and tails of Upf1 morphant and uninjected embryos shown in panels G and I, respectively. Arrows and arrowheads are as described above. (N to Q) Coinjection of human GFP-UPF1 mRNA rescued Upf1 morphants yielding embryos with weak and intermediate phenotypes and an increase in embryonic survival at 5 dpf. Note that only embryos showing a GFP signal were included in the analysis. (R and S) Western blot analysis of Upf1 in protein lysates from zebrafish embryos injected with Upf1 MOs or the respective controls. Protein lysates from human HeLa cells, zebrafish AB9 fin fibroblast cells, and uninjected wild-type embryos were analyzed in parallel. α-Tubulin served as a loading control. The migration of zebrafish Upf1 is consistent with its predicted size (122 kDa), which is slightly lower than that of human Upf1 (124 kDa).

FIG. 3.

Upf2, Smg5, Smg6, and Smg7 are essential during zebrafish embryogenesis. (A to O) Lateral views of embryos injected with antisense MOs directed against either translation initiation codons (Start-site) of upf1, upf2, upf3a, smg5, and smg6 mRNAs or splice sites of smg7 and upf3b pre-mRNAs. (A to G) Representative phenotypes observed in morphant embryos at 1 dpf. (H to N) Embryos injected with the corresponding 5-bp mismatch control MOs. (O) Uninjected wild-type embryo. Percentages of surviving embryos at 5 dpf are indicated below the panels. The total numbers of injected embryos are indicated in parentheses. Arrows and arrowheads are as described for Fig. 2. Bars, 200 μm.

FIG. 4.

Depletion of Upf1 suppresses NMD in vivo. (A to J) Expression of slc24a5 mRNA in wild-type and gol^b1 embryos injected with upf1 Splice-site or Start-site MOs (B to E and G to J). Uninjected embryos are also shown (A and F). Empty arrowheads indicate detectable expression levels in the retinal pigment epithelium. Filled arrowheads show detectable expression in the melanophores. There is no detectable slc24a5 mRNA in uninjected gol^b1embryos (F). slc24a5 mRNA expression is partially restored in golden embryos injected with the Splice-site (G and H) or the Start-site (I and J) upf1 MO. Percentages of embryos displaying the phenotype shown in the panel are indicated in the top right corner. The total numbers of injected embryos are indicated in parentheses. Bars, 200 μm.

FIG. 5.

Upf1 depletion restores the expression levels of the PTC-containing slc24a5 mRNA. (A and B) Expression of slc24a5 mRNA was analyzed by quantitative real-time PCR in uninjected wild-type and gol^b1 embryos as well as in embryos injected with Upf1 MOs (Start-site or Splice-site) or the corresponding 5-bp mismatch control MOs. slc24a5 mRNA levels were normalized to that of β-actin, which served as an internal control. These normalized values were then divided by those observed in wild-type embryos. In the lower panels, the products obtained after quantitative PCR amplification were analyzed on 0.8% agarose gels. The numbers on the right indicate the positions of DNA size markers in kilobases.

FIG. 6.

Splicing-dependent NMD in zebrafish. (A) Schematic representation of the GFP-β-tubulin reporters used in this study. The positions of PTCs and of the natural stop codon (STOP) are indicated. IN, intron. (B to D) Fish cells transiently expressing GFP-β-tubulin-WT or the indicated PTC reporters were mock treated (−) or incubated with cycloheximide (+ CHX, 100 μg/ml) for 45 min. Total RNA samples were isolated and analyzed by Northern blotting using a probe specific for GFP. The numbers below the lane numbers (relative increases) indicate the levels of GFP-β-tubulin transcripts normalized to those of the transfection control GFP mRNA. For each reporter, these values were set to 1 in mock-treated cells. (C) Normalized mRNA reporter levels relative to those of the wild-type reporter, which was set to 100. Mean values and standard deviations from three independent experiments are shown. (E and F) Fish cells were transiently transfected with a mixture of two plasmids, one expressing the GFP-β-tubulin reporters with or without PTC as indicated and another expressing GFP. Plasmids encoding SMG6 wild type (WT) or mutant (DDAA) were included in the transfection mixtures, as indicated. (F) Reporter mRNA levels were normalized to that of the GFP mRNA. For each reporter, these normalized values were set to 1 in the absence of SMG6. Mean values and standard deviations from three independent experiments are shown.