Slide 1:

My Project is about protein synthesis which is closely linked to ribosome biogenesis

and cell proliferation.
The cell cycle duration depends on the time needed for mass accumulation via protein
synthesis.
So, if translation and proliferation rates are uncoupled, cells are not able to maintain
cell size and functions.
As ribosome production increases and translation capacity expands. But how is
ribosome biogenesis coordinated with the cell’s proliferative response in a single cell
cycle?

A study measured on mouses where they have knockdown all CDKs except CDK1,
proved that only with CDK1 cells could still proliferate. This is why I will mainly focus on
cell growth mediated translation, ribosome biogenesis, being all connected with CDK1.

As cell proliferation has a main role on G1 phase I will start by focusing on this phase.
As I said before, CDK1 was found to bind to all cyclins and regulate all cell cycle. So, my
question is what is CDK1 effect on G1 phase?

Slide 2:
My main goals are:
 to define how CDK1 affects translation and mRNA stability via LARP1.
 which interactor of CDK1, cyclins or Ringo C, helps CDK1 on this role?
 Finally, last focus will be on trying to conjugate Ringo C, LARP1 and CDK1 on cell
proliferation and ribosome biogenesis.

Slide 3:
Going back to Katharina’s finding we can see she has developed a Ribo-seq and have
identified that inhibition of CDK1, by RO3306, lead to a reduction on protein synthesis,
as we can see on Figure B.
On Figure C, we can see that that reduction was more pronounced on 5’TOP mRNAs.

Katharina also performed a phosphoproteomics on LARP1 and realized that inhibition

of CDK1 led to dephosphorylation of LARP1 on S444 and T449.
By polysome profile, Katharina could also check that when on CDK1 inhibition there is a
shift to monosome on 5’TOP mRNAs, which could mean that translation is more
affected on 5’TOP mRNAs.
Interestingly, the same is not seen when LARP1 is not present. Translation was not
affected on CDK1 absence, if LARP1 is also not present. Which could mean that for
CDK1 act on translation, LARP1 is requested.

Slide 4:
How does LARP1 functions?
LARP1 can function as a repressor of translation when mTORC1 is inactive, when there
are not enough nutrients.
When mTORC1 is active, LARP1 is phosphorylated and its DM15 region is released from
the 5’TOP motif. the eIF4F complex can bind and TOP mRNAs are translated. However,
when mTORC1 is inactive, LARP1 stays bind to the 5’TOP motif and its translation is
suppressed.

Slide 5:
On the other hand, LARP1 can stabilize mRNAs.
LARP1 by its PAM2 motif, which is between La-module and Rna Recognition motif, can
bind PABP and therefore bind to the 3’Poly(A)tail. It is also known that La-module can
also bind directly to the 3’Poly(A)tail. This will protect TOP mRNAs from deadenylation
and promote stabilization.

Slide 6:
And with this we come to the first Aim.
 As seen before, stability of mRNAs can be due to LARP1 and so I will test if
inhibition of CDK1 could also have a role on stability.
 I will generate LARP1 knockouts in another cell lines, specially RPE1 cells as they
are more physiological relevant, without mutations.
 Make LARP1 mutants on those phosphosites that Katharina found to be related
to CDK1.
 Confirm with those mutants if by changing phosphorylation sites where CDK1
acts, if there are changes on LARP1 to its other protein bindings, like PABP? TOP
mRNAs?
 Perform a PAR-Clip on LARP1 but upon CDK1 inhibition.

Slide 7:
So far, I have performed a few stability tests and I see little to no effects upon CDK1
inhibition. However:
 These are only 3 repeats as I started by having trouble on normalization and
within a set of primer, which after changing its problem was gone.
 Timm is working on the same experiments but under 16h of treatment and so
far, he also does not see any difference on stability.

Slide 8:
For the generation of LARP1 knockout, I started by finding the suitable guides for it.
We wanted next to the STOP codon, to the last exon, to ensure that if even some
functional domains are still translated, the protein will not work out due to disruption
near the C-terminus.
So far, we did not have any positive knockouts. Not many clones survived and when
performing a Western Blot, we could still see the protein expression.

Slide 9:
I have performed a polysome profile.
Upon RO3306, we can see a shift into the monosomes, which could indicate an
important role on CDK1 on translation.
We have tried out on Cyclin E, as being a proliferation gene, which could have an
interesting role, as it does not have a 5’TOP motif.
Slide 10:
My second Aim is:
 to find which CDK1 activator is needed for translation.
 analyze everything related to Ringo C, since it’s a protein not well studied.
 Produce Ringo C mutants.

Slide 11:
By polysome profile, Katharina compared the effect of knocking down CDK1.
Katharina could see that upon CDK1 inhibition, there was a repression on translation as
we can observe by a reduction in polysomes.
Interestingly, also Ringo C knockdown caused a reduction on translation.

Slide 12:
And so, what is known about Ringo C?
Complete activation of CDK1 requires its binding to cyclins, a phosphorylation on its T-
loop and removal of phosphorylation sites.
However, if CDK1 binds Ringo C, no phosphorylation is required.

If we investigate this image, we can see that when CDK1 binds Cyclins it phosphorylates
this specific sequence. However, when binding Ringo C it phosphorylates other
sequences.

And so, I would like to study which effect this complex of CDK1-Ringo C can have on cell
proliferation and further on Ribosome Biogenesis.
For this, getting to know this complex’s substrates would help to identify its specific
functions.
If we take away Ringo C, it should then not alter proliferation because there are still
cyclins present, but if it would change translation then we would know that Ringo C
plays a role on translation.

Slide 13:
So, I have tried to perform a degron system on Ringo C.
We have 4 guides which will guide the Cas9 enzyme to the correct location on the DNA
sequence.
Cas9 enzyme will cut the sequence on the specific region and the cell afterwards, will
start its repair mechanisms. To avoid deletion or wrong modifications on the DNA
sequence, we have inserted a Repair template.

This system has also a V5-tag which allow us to delete and purify the protein using
specific antibodies for this tag. It is also combined to a degron which allows for a rapid
and reversible degradation of Ringo C.

I already transfected the cells, and they are currently growing on a 6 well plate.
Slide 14:
Finally, my final Aim is to conjugate LARP1, Ringo C, CDK1 roles on Ribosome
biogenesis.
So, on the G1 phase, its known to have 2 checkpoints.
The first checkpoint, senses growth factors, and if those are not enough, cells will exit
the cell cycle into a quiescence state or G0 state.
The second checkpoint, senses nutrients and so its related to mTOR pathway.

Let’s start with the Retinoblastoma Tumor Supressor, important for the cell cycle
progression which is phosphorylated by CyclinD with CDK4 e CDK6. Afterwards, will
become hyphophosphorylated by Cyclin E-CDK2. It will exit from its complex with E2F
transcription factors and progress into S phase.

 And so, I want to study how LARP1 and Ringo C mutants can impact the length
of G1 growth checkpoint by using a FUCCI system.
 Analyze cell cycles in each cell line by synchronization (starvation G1, mitotic
shake off G1, nocodazole M)