ABSTRACT: Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein with cold shock domains, involved in regulation of messenger RNA stability and translation. To address the biological role of Unr, we inactivated the unr gene by homologous recombination in mice and embryonic stem (ES) cells. Embryos deficient for Unr die at mid-gestation, and the main phenotypic defects observed, growth deficiency and absence of neural tube closure, suggest a role of Unr in the balance proliferation/differentiation during early development. Here, we report that in Unr-null ES cell cultures, we observed a greater proportion of partially differentiated colonies, together with dispersed, refractile cells with stellate morphology, reminiscent of primitive endoderm (PrE) cells. DNA microarray, immunostaining, and RNA analyses revealed that Unr-null ES cells express a set of PrE markers, including the GATA6 transcription factor, a key inducer of PrE. Although Unr-deficient cells did not downregulate the pluripotency regulators Oct4, Nanog and Sox2, they grew more slowly than the wild-type lines, and their clonogenicity was lower. Silencing of Unr by RNA interference in ES E14 (129 genetic background) resulted in similar phenotypic and molecular changes as those observed in unr-/- ES cells (C57Bl/6 background). Finally, we show that ectopic expression of Unr in unr-/- ES cells partially reverses the endoderm-specific gene expression and the differentiation phenotype. We propose that Unr prevents the differentiation of ES cells into PrE, by controlling the stability of GATA6 mRNAs, since the decay of GATA6 mRNAs is increased in the absence of Unr. In summary, these results indicate that an essential function of Unr is to stabilize ES cells in a pluripotent state by repressing PrE gene expression. Keywords: Transcriptome analysis of wild-type and unr K.O. ES cells. We have compared the transcriptome of wild-type and unr-/- ES cells. Heterozygous unr+/- mice were intercrossed, and unr+/+ and unr-/- ES cell lines were established from individual blastocysts. Total RNA were prepared from unr+/+ and unr-/- cells, either under growing conditions, or two or 24 hours after gamma-irradiation. Two or three replicates per condition.
