The role of the PeBoW-complex in ribosome biogenesis and proliferation of mouse embryonic stem cells

The hallmark of embryonic stem (ES) cells is their ability for self-renewal (capability of unlimited cell division without the loss of pluripotency) as well as for differentiation into all cell types of the adult organism. One factor supposed to be involved in self-renewal is the rapid proliferation rate of ES cells, which is coupled to an unusual cell cycle distribution with the majority of cells in S-phase and a very short G1-phase. This is linked to the lack of a functional G1/S-phase checkpoint, which allows the cells to enter the S-phase almost directly after mitosis. Generally, cells have to closely coordinate growth and cell cycle progression during proliferation to prevent premature division. One important factor for cell growth is ribosome biogenesis. In mature cells, disruptions in ribosome biogenesis are directly linked to the cell cycle machinery by a p53-dependent activation of the G1/S-phase checkpoint, leading to an arrest of cells in G1-phase. During this work, the function of the proteins Pes1, Bop1 and WDR12, which were shown previously to be involved in ribosome biogenesis of mature cell lines, was investigated in mouse ES cells. Moreover, a putative crosstalk between ribosome biogenesis and proliferation of ES cells was assessed. A high expression of Pes1, Bop1 and WDR12 was observed in ES cells, which strongly decreased during in vitro differentiation. Localization of the proteins was predominantly nucleolar and the formation of a stable complex (PeBoW-complex), including all three proteins, was experimentally validated in mature mouse cells as well as in mouse ES cells. The function and stability of the proteins seems to be dependent on incorporation into the PeBOW-complex, as protein levels were interdependent on each other and no free, non-incorporated proteins were observed, except for WDR12. According to their nucleolar localization, depletion of Pes1 and Bop1 were shown to inhibit maturation of the 28S rRNA and thereby the large 60S ribosomal subunit. Further, impaired proliferation of ES cells was observed. Thus, the PeBoW-complex seems to be an essential factor for the rapid proliferation of ES cells and might therefore also be involved in self-renewal. However, first results suggest that the complex is not directly involved in the maintenance of pluripotency. No changes in the expression levels of pluripotency-genes like Nanog, KLF4 and Sox2 were observed. Moreover, alkaline phosphatase activity was equally detectable after depletion of Pes1 or Bop1 and no morphological changes within the ES cell colonies were observed. Impaired ribosome biogenesis is known to activate a p53-dependent checkpoint in mature cell lines, which leads to an arrest of cells in G1-phase. Treatment of mouse NIH3T3 cells with 5FU, a potent inhibitor of rRNA maturation, confirmed an activation of this checkpoint, leading to weak induction of the tumor suppressor p53, induction of the Cdk-inhibitor p21, an increase in active, hypo-phosphorylated Rb, and to accumulation of cells in the G1- and S-phase with an increase of cells in G1-phase. In contrast, ES cells showed strong induction of p53, but no induction of its target gene p21. The overall levels of Rb were strongly induced, but the ratio between inactive, hyper-phosphorylated Rb and active, hypo-phosphorylated Rb was not changed towards the active form. These results were observed upon 5FU treatment and upon depletion of Pes1 or Bop1. Hence, ribosomal stress does not lead to checkpoint activation via the p53-p21-Rb pathway in ES cells. Moreover, no robust accumulation of cells in G1-phase was observed. 5FU treated ES cells showed an accumulation of cells in S-phase instead. Whether this effect is regulated by the induced p53 needs further investigation. Overall, the results suggest that ES cells use different mechanisms as mature cells to coordinate their proliferation rate with ribosome biogenesis.