New insight of telomerase trafficking

Telomere and telomerase

As you all might know Telomere is the repetitive DNA locates at the end of
chromosome and it serves to maintain chromosome integrity, preventing
illegitimate recombination and end-to-end joining. The telomere has the end
replication problem, which means some telomere sequence is lost after each round
of DNA replication. If this loss is not compensated, the telomeres will reach a
critically short length, triggering the cell to enter apoptosis.

Telomerase is a ribonucleoprotein that maintains the length of telomere. The

minimal components for active telomerase are telomerase reverse transcriptase
TERT and RNA component TR. The telomerase reverse transcriptase catalyzes the
adding of those repetitive DNA to telomere using the RNA motif within the
telomerase as a template.

As you can see from here, this is the template region of hTR for the telomerase
reverse transcriptase to catalyze the adding of the DNA repeat to the telomere 3’end
to extend it.

Why are we studying telomerase?

The majority of adult somatic cells do not have appreciable telomerase activity and
telomeres gradually shorten, limiting cell division ability, however in the majority of
human cancer cells, telomerase is activated and provides the sustained proliferative
ability to these cells. So an understanding of telomerase biology has important
implications for both cancer and aging.

Trafficking pattern of telomerase

Telomerase is not always found at telomere for the whole cell cycle but specifically
in S phase, which is the time when DNA replicates and telomeres is synthesized. It
appears that telomerase activity can be controlled by cell cycle regulated telomerase
trafficking. So studying the trafficking of telomerase can help us to understand the
biogenesis and the function of the enzyme.

I will explain the trafficking of telomerase at other phases of cell cycle later but right
now we are focusing on S phase. Here is some pictures showing how it is look like in
S phase that telomerase is at telomere.
 Here is some picture using the combine immunofluoresence and FISH
technique to show the trafficking of hTR in the HeLa cell line. All the cells are
synchronized in S phase using a doubleThymidine blocking methold.

BrdU is a synthetic nucleoside that can be incorporated into the newly synthesized
DNA of replicating cells so it’s a good indicator for S phase. The red panel here
shows either hTR or hTERT staining in the cell, as you can see they accumulated to
form couple foci in the cell. The green panel shows multiple telomere foci in the cell
staining for the numerous number of the chromosome ends. This is the merge of the
hTR and telomere panel, from the arrow you can see hTR localize to telomere during
S phase of the cell.

Important factors

There are multiple factors that can affect the trafficking of telomerase.

…..

In my research the first question I ask is whether telomere shelterin protein TPP1
can recruit telomerase.

Telomere shelterin protein and telomerase recruitment

There are six core proteins forming a complex with the telomeric DNA at the end of
chromosome. The TRF1, TRF2, RAP1, POT1, Tin2 and TPP1 and they protect the
telomere end from being damaged.

Tin2 and TPP1 are considered the core of the 6 protein complex and the deletion of
any of them will make the complex hard to form.

This is a structure of TPP1, it has the hypothesized Oligonucleotide binding fold,

which we consider to be important in telomerase interaction, the RD domain for
recruitment of POT1, the TID domain for interaction with TIN2.

TPP1 exhibit little or no DNA binding activity so it does not bind directly to
telomere. Instead, TPP1 link to telomere via either TIN2 or POT1.

TPP1 protects telomere. The knockdown of TPP1 results in an increase formation of

TIF the telomere dysfunction induced focus.

TPP1 regulates the other shelterin proteins. For example, it promote the formation
of TIN2-TRF2 and the TRF1-TRF2 complex. It is essential for the POT1 to localize to
telomere and control their binding affinity and thus regulate the length of telomere
as well.
So we can tell that TPP1 is very important in telomere protection and shelterin
protein formation, but little was known about TPP1 telomerase reaction and its role
in telomerase recruitment.

Eladio’s paper

Previous research in budding yeast has revealed that Est3 protein, an protein that
share high structural and functional similarity of TPP1, interact with the telomerase
reverse transcriptase.

In human being, TPP1 is the first telomeric shelterin protein to be discovered to

interact with telomerase.

This is a paper came out from our lab showing that TPP1 together with Tin2, is
required to recruits telomerase to telomere.

Depletion of TPP1 or TIN2

This is one figure from the paper from our lab showing a depletion of TPP1 or Tin2
result in a loss of telomerase to localize to telomeres.

Remember that we shown before, in cancer cell, the majority of both components of
hTR and hTERT will go to telomere and here we are showing the hTR in Red, the
telomere in Green and the merge of these two panels showing in yellow are the foci
of hTR-telomere colocalizations.

As you can see, after either knocking down the TPP1 or Tin2 (which can also reduce
the TPP1’s contact to telomere (point to the western)), telomerase RNA component
still forms foci in the cell, but they are not going to telomere anymore.
Quantitatively, hTR-telomere association significantly dropped after knocking down
of either protein.

Although TPP1 sounds very promising, in this experiment we cannot exclude the
possibility that Tin2 being factor for recruitment of telomerase because when TPP1
was knocked down, the TIN2 level also dropped.

The OB fold of TPP1 is required for the association…

Last figure showed that either TPP1 or TIN2 or both are required for localization of
telomerase. Further evidence from that paper shown that the in TPP1, it was the OB
fold that is required for telomerase recruitment.
Can TRF2-TPP1-OB recruit…

Think of a way to introduce my research. (11/10/11)

Here is a model showing telomerase trafficking pathway. In human cancer cells, hTR

So in my research I asked two questions of telomerase trafficking from two systems.

The first one I focus on here to ask if telomere shelterin protein TPP1 can recruit
telomerase and the second one I focus on here to ask why mouse telomerase RNA is
not accumulating in Cajal body.

The trafficking of each of the telomerase components to Cajal bodies and telomere
appears to be interdependent. For example, the localization of hTR to Cajal body and
telomere required hTERT and the newly discovered telomerase holoenzyme,
TCAB1. And the trafficking of hTERT to telomere needs hTR as well.

Cajal body is consider to be a site for telomerase assembly because both of hTR and
hTERT can be found at Cajal body. What’s more, the overexpression of hTERT in
normal cell line (fibroblast or smooth muscle cells) result in accumulation of hTR in
Cajal bodies.

hTR is recruit to telomere specifically in S phase, but hTERT can be at telomere

outside S phase?

Trafficking pattern of telomerase

Here is some picture using the combine immunofluoresence and FISH technique to
show the trafficking of hTR in the HeLa cell line.

The DAPI stain for the nucleus. The red panel here shows the hTR staining in the
cell, as you can see it accumulated to form couple foci in the cell. The green panel
shows Coilin antibody staining, a marker for Cajal body. As you can see from the
merge of the previous blue and red and green channels hTR colocalize with Cajal
bodies in the nucleus.

For the second panel, BrdU is a synthetic nucleoside that can be incorporated into
the newly synthesized DNA of replicating cells so it’s a good indicator for S phase. As
you can this cell has a BrdU staining, which means the cell is in S phase. The second
picture shows the hTR and the third picture shows the telomere staining in the cell.
As you might know human cell has 23 pair of chromosomes and each chromosome
has 4 telomere ends so there should be many telomere foci in each cell. As you can
see this cell is forming multiple telomere foci as well. This is also the merge of the
hTR and telomere panel, as you can see hTR localize to telomere during S phase of
the cell.
 and BrdU is the staining maker for DNA replication which is S phase. hTR can be
seen colocalizing with Coilin labeled Cajal bodies during most all the cell cycle, and
in S phase, here indicated by BrdU staining, a portion of hTR can go to a subset of
telomere.

Why hTR goes to a subset of telomere?

Important factors in telomerase trafficking

Telomerase Trafficking in mouse

So now we know that telomerase RNA component can go to Cajal body and telomere
in S phase. Then we try to utilize a different system, the mouse system to study
telomerase trafficking.

Mouse telomerase also contains RNA component and the reverse transcriptase, we
call it mTR and mTERT.

mTR

……

This is very interesting because Cajal body has confirmed to be a very important
factor for telomerase trafficking to telomere and it might serve as delivering
telomerase to telomere.

Telomerase trafficking in mouse

So we have observed that hTR is accumulated in Cajal body in human cell line while
mTR is not accumulated in Cajal body in mouse cell line. So we asked the question
what is the determinant factor for telomerase RNA component to localize at Cajal
body.

Since we have 2 variable here, the different cell environment, different cell type give
out different environment; different RNA structure, one is human telomerease RNA
and one is mouse telomerase RNA. So we try to make it to be one variable here by
overexpressing hTR in the mouse cell line and stain for Cajal body to look for their
locations.
A

As you can see…..

As we mentioned before in human cancer cell line, the TERT component is needed
for hTR to localize to telomere. Previous research has already shown that mouse
TERT can bind with hTR and even expose higher telomerase activity than the mouse
TR and TERT combination. So hTR should have no problem to work with mouse
TERT to localize to telomere. And as we shown here hTR can also localize to
teloemre.

Human telomerase RNA

So in the same mouse cell line, hTR is able to reside in Cajal body while mTR is not,
means the difference of RNA structure is the key factor to determine whether
telomerase RNA can travel to Cajal body or not.

Here is the comparison of human telomerase RNA and Mouse telomerase RNA.

As you can see they are pretty much the same. The CR4-CR5 domain, the Box H/ACA
domain, the Pseudoknot Domain, the CR7 domain and the template, they are the
conserved regions through vertebrate.

The CR4-CR5 domain:

The CR7 domain:

All right in CR7 domain there is a CAB box UGAG here and it is considered to be
critical in hTR accumulation, 3’end processing, intranuclear localization of hTR and
Cajal body localization. As you can see human and mouse telomerase RNA shared
the same CAB box (Cajal Body box) here and the only difference in CR7 domain is
these two basepair differences, but they are considered not required for Cajal body
recruitment after mutational analysis.

And when you looked into the paper, it’s also shown that the localization to Cajal
body is Box H/ACA dependent. In this case, the difference between Box H/ACA in
hTR and mTR, even one nucleotide, might contribute to the non-Cajal body
colocalization.

Of course other structurual difference between hTR and mTR are also possible for
their difference in Cajal body localization. Such you can see the most obvious one
would be the 5’end, mTR only has 2 nucleotide while hTR has 45 nt upstream of the
template region and form a P1 stem, which is important for telomerase activity.
Telomerase Trafficking in Human

Shelterin protein’s role in recruitment.

Lately more and more evidence show up to point out that telomere shelterin protein
can regulate telomerase function.

TPP1’s role in telomerase recruitment.

TPP1 is one of the six shelterin protein of telomere. It’s shown to protect telomere
from eliciting the DNA damage response (Agueda, 2010. both MEFs and mice
depleted for TPP1 shoe induction of telomere damage foci TIFs).

TPP1 is reported to be required for telomerase recruitment to telomere (Eladio

2010, Agueda 2010). When you knockdown the TPP1 protein level in super
telomerase HeLa cells, hTERT associated telomeric DNA was reduced by 36%
compared with wild type using the Chip assay. The hTR-telomere association was
also reduced by 68%. The TPP1 deficient mouse cell line MEF also has a reduced
level of mTERT-chromatin binding.

RAP1

The role of TPP1 in regulation of telomerase trafficking

Previous study has shown that TPP1 OB fold interact with telomerase in vitro. The
recombinant human telomerase and the recombinant TPP1 or TPP1-OB can pull
down each other in immunoprecipitation experiments.

Also there is recent study show that human TPP1 can activate human telomerase to
enhance their activity, so as the mouse TPP1 to mouse telomerase. Basically what
they did is they made the chimeric complex of mouse POT1 and human TPP1, then
they discovered that the complex had enhanced telomerase activity then mouse
POT1 alone or mouse POT1 with mouse TPP1.