Fig. 5.

Cpeb1b regulates cytoplasmic polyadenylation of shha mRNA. (A) Confocal imaging shows colocalization of GFP-Cpeb1b and mCherry-Tent2 in the cytoplasm of HEK293T cells. (Scale bar, 10 μm.) (B) Confocal imaging shows that GFP-Cpeb1b colocalizes with mCherry-Tent2 in the condensates in the notochord of 16 and 22 hpf embryos but not in the hypoblast of 8 hpf embryos. (Scale bar, 10 μm.) (C) Outline of the PAT assay. Cytoplasmic RNA extracted from the embryos was subjected to G/I tailing. The complementary DNA was synthesized by reverse transcription using the poly(G/I) tailed RNA as template. Then, the specific PCR products were synthesized by specific primers and detected by gel electrophoresis. GS: gene-specific products, PA: poly(A) tail-included products. (D) Examination of the poly(A) length of shha mRNA in the LD cpeb1b MO-injected WT and cpeb1b mutant embryos (elimination of maternal effects) at different developmental stages by PAT assay. The PCR products were detected by gel electrophoresis. The red arrowheads denote smear bands from poly(A) PCR products. (E) Examination of the poly(A) length of rps18 mRNA in the LD cpeb1b MO-injected WT and cpeb1b mutant embryos at different developmental stages. The red arrowheads denote smear bands from poly(A) PCR products.