The regulated phosphorylation of ribosomal protein (rp) S6 has attracted much attention since its discovery in 1974, yet its physiological role has remained obscure. To directly address this issue, we have established viable and fertile knock-in mice, whose rpS6 contains alanine substitutions at all five phosphorylatable serine residues (rpS6). Here we show that contrary to the widely accepted model, this mutation does not affect the translational control of TOP mRNAs. rpS6 P−/− mouse embryo fibroblasts (MEFs) display an increased rate of protein synthesis and accelerated cell division, and they are significantly smaller than rpS6 P+/+ MEFs. This small size reflects a growth defect, rather than a by-product of their faster cell division. Moreover, the size of rpS6 P−/− MEFs, unlike wild-type MEFs, is not further decreased upon rapamycin treatment, implying that the rpS6 is a critical downstream effector of mTOR in regulation of cell size. The small cell phenotype is not confined to embryonal cells, as it also selectively characterizes pancreatic ␤-cells in adult rpS6 P−/− mice. These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose tolerance.

TOP mRNAs are translationally controlled by mitogenic, growth, and nutritional stimuli through a 5-terminal oligopyrimidine tract. Here we show that LiCl can alleviate the translational repression of these mRNAs when progression through the cell cycle is blocked at G 0 , G 1 /S, or G 2 /M phases in different cell lines and by various physiological and chemical means. This derepressive effect of LiCl does not involve resumption of cell division. Unlike its efficient derepressive effect in mitotically arrested cells, LiCl alleviates inefficiently the repression of TOP mRNAs in amino acid-deprived cells and has no effect in lymphoblastoids whose TOP mRNAs are constitutively repressed even when they are proliferating. LiCl is widely used as a relatively selective inhibitor of glycogen synthase kinase-3. However, inhibition per se of this enzyme by more specific drugs failed to derepress the translation of TOP mRNAs, implying that relief of the translational repression of TOP mRNAs by LiCl is carried out in a glycogen synthase kinase-3-independent manner. Moreover, this effect is apparent, at least in some cell lines, in the absence of S6-kinase 1 activation and ribosomal protein S6 phosphorylation, thus further supporting the notion that translational control of TOP mRNAs does not rely on either of these variables.