First Slide:

My topic is a coordination of protein synthesis and cell proliferation.

So, in order for the cell divide needs to first grow, it needs to have coupling between growth
and cell division. And cell growth is defined by the production of more proteins. How can a
cell make more proteins in a short time? In which cell cycle phase is this more important?
And its known to be more important in G1 and now we know there are 2 points. The second
point, the growth point, which is already know that is dependent on translation and on
mTOR. And because it is dependent on these 2, we are expecting to be dependent also on
the ribosome biogenesis. But we want to address this question: is ribosome biogenesis
really? Is it only mTOR giving this unput or is it CDK1 another giving the input?

Ribosome Biogenesis:
Once cells get a growth stimulus, cells get the signal to divide/proliferate. They need to
prepare, by making more proteins, have enough ATP. So it makes sense that having more
ribosomes then you can produce more at the same time. But to produce ribosomes you
need to used up some of that machinery. Because ribosome biogenesis is one of the events
that requires a lot of energy. To have more ribosomes, we need rRNA but it uses a different
polymerase. From the beginning we can not make a lot of those proliferation factors
because the machinery was already used up to to make ribosome proteins. By this first
phase of making ribosome proteins, in the second wave we would have the double or 4
times more the amount of ribosomes and then of course we have more capacity in general
to translate.
A: in the first wave they used up more energy to produce ribosomes and in the second wave
we can translate the proliferation genes.
B: they start by using what the cell has already but using it more efficiently on these
transcripts.
5´TOP mRNAs are not good translated targets. GAPDH would be a much better proliferation
mRNA. Ribosome proteins should only be made once we really need them, because they
should not use even more energy than necessary. Same for 5´TOP mRNAs.
Testing this hypothesis is not that easy. When we have starvation and then we give EGF, a
stimulus, do we see 2 waves? Where on the first wave is use more energy, we make more
ribosomes, and so in total the area under the curve of polysomes increases, because we
have suddenly more ribosomes. Cyclin D and E mRNA would not come in the polysomes. But
TOP transcripts they would shift into the polysomes. In the second wave, after x hours of
EGF, it would 1: stop? And somehow the TOP mRNAs are translated worse and they are
shifted out again or 2: would TOP mRNAs stay there and just the proliferation genes go
there?
Does this depend that this proliferation genes, depend on the amount of ribosomes? We can
alter by using a LARP1 mutant, where they cannot produce more ribosomes.

For this we cannot check just by inhibiting CDK1, because if we inhibit CDK1 it anyways stop
the cell cycle. We can only measure this by impairing this CDK1-dependent translation role.
And the substrates we do not know so well. We know that LARP1 is a substrate, 4EBP is a
substrate but we don’t know the other substrates. If its RingoC we could interfere with Ringo
C, if Ringo C does not have a particular effect in the cell cycle itself. Only by the translation
function we could alter Ringo C. Or we can use a LARP1 version that can no longer be
phosphorylated by CDK1.
We would have LARP1 phosphodeficient mutants, and no matter where the cell gets the
proliferation stimulus, the cell should not be able to produce new ribosome proteins, still
need to check this! And if not, then we can uncouple this 2 mechanisms and see if they have
an effect on the time they spend there. Or is the time the same because cells cannot react,
because they would react by CDK1 by that phosphorylation site but they cannot, so they
would spend the same time but they cannot make enough protein, so they would become
smaller and smaller. Or to keep the cell size but stay longer on that G1 phase.

Cell proliferation is not cell growth.

It is G1 phase and specially people know nowadays that there is a R point and a growth point
in G1. We basically expect that when on mTOR inhibition, there is an increase in the pre-S-
phase. Also with cycloheximide treatment the G1-ps is lenghted. We expect that when we
interfer with ribosome biogenesis we would also length G1-ps, or with Ringo C does it also
length that phase?
Ringo C is doing this while acting on ribosome biogenesis, and therefore in the end I need to
know the substrates, the target that CDK1 or Ringo C are addressing, like LARP1. And the
phosphorylation sites.
For larp1 we know that its probably on of those phosphorylation sites but for Ringo we do
not even know the other substrates.
We did phosphoproteomics upon RO and there we found LARP to be changed.
If we do a phosphoproteomics of Larp1 and of Ringo, then maybe we can find substrates
that work on both, like LARP1.

We work on CDK1, which is mainly on M phase, why look into G1?

We are interested in this connection and we found that CDK1 regulates translation and
ribosome biogenesis so we were wondering if it makes sense that a cell which wants to
proliferate a lot it needs to have high CDK1 activity for its cell cycle and at the same time if
this also regulates translation, we can couple this 2 things.
We want to test this by looking into the substrates of CDK1, and see if we alter them, do we
alter the speed of proliferation?
We are thinking specially about the G1 checkpoint because translation places a role in there,
so the question is if cdk1 in parallel? For mTOR and CDK1 is important that what we found so
far, seems to be different from mTOR. It’s always different phosphosites on Larp and on
mTOR, also what CDK1 is phosphorylating on 4EBP is different on where mTOR
phosphorylates. It would be a parallel, other mechanisms to activate the same.

Slide 4:
CDK1 has stronger effects on TOP mRNAs, and this was dependent on LARP1.
Since CDK1 is a kinase, the direct connection will be that CDK1 probably phosphorylates
LARP1, so we already checked for this and on phosphoproteomics we found LARP1 to be the
phosphorylated and reduced in these 2 sites.
We generated mutants that can no longer be phosphorylated on these sites, and we also
included the 440, because it’s close by. Usually, kinases cannot phosphorylate one site, they
try to use another site next to it. So, we have a triple mutant, deficient for all those 3 sites
and we have a triple mutant mimetic for these sites.
RPE1 because they only have telemerase activity not transformed.
U20S have good checkpoints.
Hela do not have the G1 checkpoint normal, since Rb is mutated. It is not ideal to use Hela if
I want to check cell cycles.

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Slide 5: translation

La-module can directly bind the PolyA tail without PABP. This helps to keep the length of the
PolyA tail. In one hand it protects the PolyA tail from being degraded and on the other hand
PABP can interact with the 4G complex and activates translation.
So LARP1 have a function on stability and translation.

So LARP1, via recruiting PABP and then the PolyA tail or directly LARP1 to the PolyA tail.
Depending where LARP1 binds to the mRNA it can be activated or repressed.
LARP1 does not only binds to the first C on the cap but also binds to downstream, to the end
of the 5’UTR and it would then activate translation. Binding at the end of the 5’UTR, but
releasing the cap, this would be activating translation, and this would be mediated by the
TOP motif, via La-module.

Inhibit translation by binding directly the cap, via DM15

activates translation by recruiting mTOR to TOP mRNAs and not binding anymore to the cap
but to the end of the 5’UTR.
Either LARP1 binds to the gene on the first C of the cap, then its inhibitory, this is when
mTOR is inhibited. If mTOR is not inhibited, it relases the GC on the cap, but now LARP1
binds…..by recruiting mtor, recruiting translation factors and switch to activator.

Slide 6: stability

LARP1 can bind via PABP to the PolyA tail, stabilze the RNAse, but all mRNAs not only top.
LARP1 can also bind PolyA tail itself, thereby protecting the poly tail length.
These effects of stability are more pronounced on TOP mRNA.

It is known when we inhibit mTOR it changes how LARP1 interacts with RNA, how LARP
interacts with other protein factors. If we inhibit CDK1-dependent phosphorylation sites,
does it change RNA binding? Does it change protein binding? Inhibiting CDK1 itself did not
change mRNA binding abilities of LARP. If we inhibit mTOR we enhance RNA binding.
If we inhibit CDK1, does it change binding of PABP to LARP? Since it is stabilizing Poly(A)tail
length, does it change the interaction of LARP?

By PAR-CLIP it is known on LARP1 upon mTOR inhibition is already known. but we also want
to see what happens upon CDK1 inhibition.
CDKS usually phosphprylate serine/threonine where a proline comes afterwards, then any
aminoacid and then lysine or ariginine.
But cdks are not that strict, so they can proliferate if there is only serine, threonine and a
proline or even phosphorylate others.

activate CDK1 to have the translation role but not activate the mitotic substrates. By
activating CDK1 via Ringo we can do that. If the translational substrates are Ringo-CDK1
substrates they have this sequence: serine, threonine, proline, x, thyrosine or ariginine or
tryptophane.
By activating cdk1-ringo substrates complexes we can specifically activate translation
substrates. We can get a switch on substrates. Like this the translational targets don’t need
to compete with the mitotic substrates.

Is CDK1-RINGOC changing translation? If we take away rINGO c It should not alter

proliferation because cyclins are still there. but if translation is alter, we know that ringo
plays a role. Is ringo together with CDK1? which substrate?