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.