Crucial Role of Bysl in Mammalian Preimplantation Development as an Integral Factor for 40S Ribosome Biogenesis

The development of embryos injected with Bysl siRNAs was arrested at the 16-cell stage, resulting in a defect in differentiation toward trophectoderm. (A) Morphological alterations in siRNA-injected embryos were examined during blastocyst formation. Embryos were injected with 20 μM siEGFP-441, siOct3/4-670, or siBysl mixture containing 10 μM each of siBysl-845 and siBysl-976. Scale bar, 50 μm. (B) Cell numbers were determined periodically. Embryos were exposed to 10 μg/ml Hoechst 33342 and squashed under a coverslip, and the numbers of fluorescent nuclei were counted. Similar results were obtained in three independent experiments. **, P < 0.01; ***, P < 0.001. (C) The proliferative activity was assayed by BrdU incorporation. Untreated, without treatment with BrdU. Scale bar, 100 μm. (D) Marker gene expression was analyzed by RT-PCR at E0.5+3.25. (E) Confocal immunofluorescence imaging of cytokeratin EndoA with TROMA-1 antibody. Nuclei were stained with Hoechst 33342 (H33342). Fourfold-magnified views of the boxed areas are shown in the smaller, lower panels. Scale bar, 50 μm. (F) Immunofluorescent localization of Oct3/4 and Cdx2. Overlapping expression of Oct3/4 and Cdx2 in the nuclei appears white in the merged images. Scale bar, 50 μm. DIC, differential interference contrast.

Episomal expression of Bysl shRNAs inhibited proliferation of embryonic stem cells. (A) MGZ5 ES cells were transfected with 2 μg/ml of the pPPU6 episomal shRNA expression vector. One day following transfection, the cells were selected with 1 μg/ml puromycin. Seven days after transfection, undifferentiated ES cell colonies were visualized by staining for AP activity. Scale bar, 10 mm. (B) Morphological alterations in ES cells cotransfected with 1 μg/ml pHPCAG-Venus and 2 μg/ml pPPU6-shRNA. The cells were selected with 1 μg/ml puromycin and 100 μg/ml hygromycin B, and then photographed under epifluorescence at three days posttransfection. Scale bar, 200 μm. (C) Marker gene expression was analyzed by RT-PCR at 48 h posttransfection. Cdx2, Hnf4α, and brachyury are markers for trophoblast, endoderm, and mesoderm differentiation, respectively. ZHBTc4 ES cells (43) cultured in the presence of 1 μg/ml tetracycline for 24 h, MGZ5 ES cells cultured in the absence of LIF for six days, and embryoid bodies five days after plating were used as positive controls (P.C.) for Cdx2, Hnf4α, and brachyury, respectively.

Bysl localized to the nucleus and was concentrated in the nucleolus. (A) A Bysl-Venus fusion protein was expressed in COS-7 cells. The cells were fixed at one day posttransfection, immunostained for the nucleolar marker fibrillarin, and observed under a confocal laser scanning microscope. Venus alone diffusely distributed throughout the cytoplasm and nucleus (upper panels). H33342, Hoechst 33342. Scale bar, 20 μm. (B) Bysl-Venus mRNA transcribed in vitro was injected into fertilized eggs and photographed under epifluorescence one day later (upper panels), or injected into both blastomeres of two-cell stage embryos and photographed one day (middle panels) or two days (lower panels) later. Scale bar, 50 μm. (C) ZHBTc4 ES cells were transfected with a Bysl-Venus construct and cultured in the absence (undifferentiated; upper panels) or presence (differentiated into trophoblast lineage; lower panels) of 1 μg/ml tetracycline (Tc) for four days. Scale bar, 20 μm. DIC, differential interference contrast.

RNAi-resistant Bysl, which localizes to the nucleolus/nucleoplasm, overcame the proliferation inhibition caused by Bysl shRNAs. (A) The predicted structures of two shRNAs against Bysl. The three G-U mismatch mutations in the sense strand are presented in lowercase letters. (B) Silent mutations (underlined) were generated within each of the shRNA target sequences in Bysl-Venus to confer RNAi resistance. (C) MGZ5 ES cells were cotransfected with shRNA (2 μg/ml) and wild-type or silently mutated (sm) Bysl-Venus (2 μg/ml) expression vectors. The cells were photographed under epifluorescence at two days posttransfection. Note that smBysl-Venus was resistant to Bysl shRNAs. Scale bar, 200 μm. (D) Rescue of the knockdown phenotype by expression of RNAi-resistant Bysl. MGZ5 ES cells were first transfected with 2 μg/ml pHPCAG-ByslVenus or -smByslVenus followed by selection with 100 μg/ml hygromycin B. Four days after transfection, equal numbers of cells from each sample were subsequently transfected with 4 μg/ml pPPU6-shRNA followed by selection with 100 μg/ml hygromycin B and 1 μg/ml puromycin. The cells were stained for AP activity six days after the second transfection. Scale bar, 10 mm.

Deletion mutants of Bysl, which retain nucleolar localization, exerted a dominant negative effect. (A) Schematic representation of Bysl deletion mutants fused to Venus. Deletion mutants were generated based on the sequence similarity among humans, mice, and yeast (32.5% and 54.7% amino acid identity and similarity in total). (B) Western blot analysis of the mutant proteins expressed in COS-7 cells using anti-GFP fluorescent protein antibody (SC-8334; Santa Cruz Biotechnology). The deletion mutants were detected at the predicted molecular masses (MM), except the d1 mutant, which had an observed MM of 55 to 60 kDa. (C) Subcellular localizations of the mutants were determined in COS-7 cells. Note that d1 and d2 retained nucleolar localization. Scale bar, 10 μm. H33342, Hoechst 33342. (D) Functionalities of the deletion mutants were assessed by rescue of the knockdown phenotype in MGZ5 ES cells as described in the legend to Fig. 5D. (E) MGZ5 ES cells were transfected with 2 μg/ml pPPCAG constructs expressing the mutant proteins followed by selection with 1 μg/ml puromycin. The cells were stained for AP activity at six days posttransfection. Note that overexpression of d1 or d2 suppressed the proliferation of ES cells. (F) Summary of the properties of Bysl deletion mutants. The exact relationship between nucleolar localization and inhibitory effect is noted. Cy, cytoplasm; Np, nucleoplasm; No, nucleolus; NuSp, nuclear speckle-like; ND, not determined.