FIGURE 3.

RNA interference with the CeY14 gene results in embryonic lethality but does not cause accumulation of poly(A)⁺ mRNA in the nuclei. (A) CeY14 predicted protein and human Y14 protein sequences were compared using the GAP program (GCG10 software), and the alignment was generated using PRETTYBOX (GCG10 software). Identical residues are highlighted in black, and the dsRNA fragment used for RNAi corresponds to the solid line above the sequence. (B) RNA interference with the CeY14 gene results in late embryonic lethality (a,b). Wild-type embryonic developmental stages are shown for comparison (c) comma, (d) 1.5-fold, (e) twofold. Each embryo is ~50 μm in length. (C) The effectiveness of RNAi was determined by examining the level of the residual transcripts following dsRNA injections by RT–PCR with specific primers, as described previously (Longman et al. 2000). CeY14 mRNA is specifically depleted in RNAi-treated animals (lane 4) compared with wild-type animals (lane 2); whereas the level of a control mRNA, corresponding to CeNXF1/Tap1, is unaffected (lanes 1,3). The figure shows a negative of an ethidium bromide-stained agarose gel. (M) 100-bp ladder DNA size marker. (D) Depletion of CeY14 does not result in nuclear accumulation of poly(A)⁺ mRNA. Injected animals and control N2 animals were fixed and subjected to RNA in situ hybridization as described. In each panel, the intestine is the predominant tissue characterized by the presence of large nuclei. No accumulation of poly(A)-containing RNAs was observed in nuclei of intestines of RNAi-treated animals (indicated by arrows in b). (a) The corresponding DAPI-stained nuclei. Poly(A)⁺ RNA distribution in intestines of wild-type, untreated worms are shown in d (nuclei are indicated by arrowheads), and corresponding DAPI- stained nuclei are shown in c. Bar, 25 μm.