Dhx34 and Nbas function in the NMD pathway and are required for embryonic development in zebrafish

Abstract

The nonsense-mediated mRNA decay (NMD) pathway is a highly conserved surveillance mechanism that is present in all eukaryotes. It prevents the synthesis of truncated proteins by selectively degrading mRNAs harbouring premature termination codons (PTCs). The core NMD effectors were originally identified in genetic screens in Saccharomyces cerevisae and in the nematode Caenorhabditis elegans, and subsequently by homology searches in other metazoans. A genome-wide RNAi screen in C. elegans resulted in the identification of two novel NMD genes that are essential for proper embryonic development. Their human orthologues, DHX34 and NAG/NBAS, are required for NMD in human cells. Here, we find that the zebrafish genome encodes orthologues of DHX34 and NAG/NBAS. We show that the morpholino-induced depletion of zebrafish Dhx34 and Nbas proteins results in severe developmental defects and reduced embryonic viability. We also found that Dhx34 and Nbas are required for degradation of PTC-containing mRNAs in zebrafish embryos. The phenotypes observed in both Dhx34 and Nbas morphants are similar to defects in Upf1, Smg-5- or Smg-6- depleted embryos, suggesting that these factors affect the same pathway and confirming that zebrafish embryogenesis requires an active NMD pathway.

Figure 1.

Dhx34 and Nbas are conserved between zebrafish and human. Schematic representation of the zebrafish Dhx34 and Nbas proteins. The shaded boxes represent the regions of sequence conservation within proteins; the level of conservation is indicated. Functional domains associated with ATP-dependent RNA helicases in Dhx34 (DEXDc, HELICc, PFAM:HA2 and PFAM:DUF 1605) are indicated. Predicted functional domains of the Nbas protein are indicated.

Figure 2.

Dhx34 and Nbas are ubiquitously expressed during zebrafish embryogenesis. Wholemount in situ hybridization against dhx34 and nbas using antisense riboprobes revealed ubiquitous expression of dhx34 and nbas transcripts (seen as purple staining) during early zebrafish development. No significant staining was detected using the control sense probes.

Figure 3.

Dhx34 is required for proper embryonic development in zebrafish. (A) Translation block and splice site MOs against zebrafish Dhx34 cause a distinctive range of developmental phenotypes at 24hpf characterized by shortening of the anteroposterior axis, somite malformation and significant reduction in head size. Morphants injected with the mismatch control MOs are indistinguishable from uninjected wild-type embryos. (B) Co-injection of Dhx34 translation block MO and human full-length DHX34 mRNA rescued the morphant phenotype in 73.8% of the injected embryos at 24 hpf. Expression of the mRNA alone has no overt effect on development. The numbers beside each panel indicate the percentage of embryos with the observed phenotype. Numbers in brackets correspond to the total of analysed embryos.

Figure 4.

Nbas is essential for zebrafish embryonic development. (A) Injections with translation block and splice site MOs against zebrafish nbas reproduced the developmental phenotype observed in Upf1 and Dhx34 morphants. (B) Co-injection of Nbas translation block MO and human full length NAG/Nbas mRNA rescued the morphant phenotype in the majority of the embryos at 24 hpf. Expression of the mRNA alone had no effect on embryonic development. The numbers beside each panel represent the percentage of embryos showing each phenotype and in brackets is the total number of the analysed embryos.

Figure 5.

Both Dhx34 and Nbas are required for the in vivo degradation of the nonsense slc25a5 transcript in zebrafish. Expression of slc25a5 mRNA was measured in wild-type (A) and golden^b1 mutant (B) uninjected embryos, or embryos injected with indicated MOs, by real-time RT–PCR. The level of slc25a5 transcript was normalized to dct mRNA. The normalized values were then divided by values for uninjected samples (no MO) and expressed as relative mRNA levels.