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Class notes BI644 Biology of Ageing Week 7-8
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Class notes for BI644 Biology of Ageing Week 7-8
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7.1RNA Pol III Inhibition Increases LifespanTORC1 Inhibition Extends Lifespan in a Number of Species
We know that TORC1 inhibition extends lifespan in lots of different species by inhibiting the TOR
pathway. The key part of the TOR pathway is the TORC1 kinase. It is regulated by lots of different
nutrients and growth factors (in metazoa) and it can be inhibited by
using rapamycin.
There are several things downstream of the TOR pathway, but this
section is going to focus on polymerases. There are 3 nuclear RNA
polymerases – Pol I, Pol II and Pol III. Pol II is responsible for
transcribing the majority of mRNA in the cell, i.e., RNA that will go on
to encode protein coding genes. Pol I is responsible for transcribing
ribosomal RNA and Pol III transcribes 5S ribosomal RNA and the tRNAs.
All of these together make up the protein synthesis machinery; the
TOR pathway is a protein synthesis driving machine – it wants to drive
growth and protein synthesis.
The polymerases are multiple subunit complexes and some of the subunits are conserved between
the polymerases, but some are also unique to the polymerase.
RNA Polymerase III Is Required for Worm Development
This was studied in C. elegans and by another lab in flies.
As expected from a gene that is important in driving growth, if you knock an RNA polymerase III
expression down in a worm, there will be a problem in embryonic development and growth in
general.
In this experiment, they took one subunit of RNA polymerase III,
called C160 and is called RPC-1 in worms and dC160 in flies. This is a
subunit of RNA polymerase III that is involved in DNA binding.
Without the subunit, there is no polymerase function. They used
RNAi in the worms to knock down this subunit. If you have control
RNAi, the worms develop normally – the top images show lots of
adult worms. However, if you treat the worms with RPC-1 RNAi and
you knock down Pol III function, then the worms do not develop
properly – the bottom images show hardly any adult worms on the plate – and this is because they
removed a key component that is required for growth.
If you treat the worms with RPC-1 RNAi then
you don’t completely remove expression of
the gene but there is a dramatic reduction.
This was shown using a technique called
quantitative PCR, which allows you to
measure in real time the amount of RNA that
is being produced for a gene of interest.
If you fed worms RNAi against RPC-1 and knocked down the activity of RNAi polymerase III, then you
could extend lifespan.
, There was also collaboration with another lab which conducted the same experiment in yeast and
flies. He showed that using a chronological lifespan in
yeast, as time went on, if you knocked down the same
subunit of RNA polymerase II by treating them with
RPC160-6 (C160 subunit), then the yeast cells were
surviving for longer – chronological lifespan in yeast
was extended if RNA polymerase III was knocked down.
This was also shown in flies – RNA Pol III
knockdown reduces the amount of the Pol III
subunit RNA that is being produced then there
is an increase in the lifespan of the flies.
There is an evolutionary conserved response to
lifespan – there is an extension in yeast, worms
and flies – if you knock down the polymerase.
Inhibition in the Gut is Sufficient to Extend Lifespan
They were also able to see which tissues were involved in Pol III
activity. In worms, you can knock down a gene in a specific tissue by
using a special genetic and RNAi technique. It involves taking a
strain of worms that is deficient in RNAi everywhere, and then you
reintroduce RNAi capability only in certain tissues. A similar method
can be used in flies.
They found that if they did a gut specific Pol III knockdown in worms
of in flies, there was an extension to lifespan. The effects of Pol III
seem to be working in the gut.
In flies, this can be done more specifically, and you can knock down
genes specifically in different cell types. They knocked down RNA
Pol III in the intestinal stem cells then there is an extension of
lifespan.
Pol III Inhibition Promotes Gut Health
They started to wonder whether there was something special about
the gut that promotes longevity in this way. They began by looking at the permeability of the gut
with age.
One thing that happens as worms or flies get older, is that the gut becomes more permeable – lots
of small holes begin appearing in it, becomes leakier and starts to fall apart. This can be measured
using an assay called a smurf assay. You can feed the worms or flies a blue dye that is essentially
blue food colouring. If they are young and the gut is
really healthy, they have a score of zero and most of
the dye is found in the middle of the gut. But as they
become older and the gut becomes more
permeable, the worms become bluer and bluer as
there are holes in the middle of the gut and the blue
dye is taken up.
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