BIOL 200 Lecture Notes - Bioinformatics, Schizosaccharomyces Pombe, Neural Development

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6 Apr 2012
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Naveen Sooknanan McGill Fall 2011
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New Roles for RNA:
Through the discovery of these dsRNA molecules capable of carrying out chromosomal
rearrangement functions, it opened up a whole new window of research into new roles for RNA
within the cell
RNA actually has very important functions apart from being adaptors or reading
sequences in protein synthesis
This leads to a “chicken or egg” problem; which came first, RNA or DNA/proteins
While DNA is capable of faithfully keeping genetic information, it is not able to self-
replicate itself so as to transmit information to daughter cells
Proteins, on the other hand, are excellent for catalyzing these processes (translation,
transcription, etc.) but lack the ability to store genetic information
o It is therefore unlikely that DNA or proteins were the
predecessors of life
RNA seems to be the medium which, as we have seen in some
of our examples, are able to store genetic information (RNA
viruses) and transmit copies to daughter cells (self-replication)
Therefore, it seems like life all began with RNA, which is able
to fold up with secondary structure and carry out catalytic activities
o Peptidyl transferase (23S rRNA) and self-splicing RNA (type 1 introns), just to
name a few
Thus the primordial world is called by many as an “RNA world” where RNA were the
primary molecules
Upon the discovery of the human genome near the 21st century, scientists
were beginning to think that they were reaching “the end of science”
This was due to the highly complex techniques and technological
advancements made in the few years before 2000
Just when we thought we knew it all, two studies of a worm known as C. elegans
changed the way we thought about RNA
The first experiment was conducted with the objective of looking at early
embryonic processes.
o It was found that maternal products were disposed of in the embryo
by injecting double stranded DNA into C. elegans
o These double stranded RNA (dsRNA) structures were capable
of wiping out gene activity entirely through a process known
as the RNA inhibition (RNAi) pathway
o Dr. Fire and Dr. Mello, who conducted this series of
experiments, were awarded the Nobel Prize for Physiology
and Medicine in 2006
Simultaneously, a procedure was going on in another laboratory
which was trying to study another RNA based silencing method,
now known as micro RNA (miRNA)
Naveen Sooknanan McGill Fall 2011
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o This mechanism involves small double stranded RNA molecules, which, when
injected into C. elegans, could regulate the expression of other RNAs, while not
wiping out their expression entirely
o Dr. Ambros, Baucombe and Ruvkun (who worked with plants) were all awarded
the Albert Lasker award, which is the American Nobel Prize, in 2004
These two experiments, in effect changed the way we see the biological world
Looking at the first experiment in more detail, the injection dsRNA into the C. elegans (it doesn’t
matter where), will cause wipe out the activity of a specific gene
Each dsRNA corresponds to the shutting down of a different gene (which is the RNA
target)
For unknown reasons, dsRNA does not work well in neurons, as can be seen when
looking at fluorescent gene transcripts of C. elegans
o In the picture below, the target gene products are pretty much everywhere, but
when dsRNA is injected, all that are left are the gene products in neurons
o These target proteins are tagged with GFP
Also, this effect only lasts for one generation, meaning it does not propagate. It is,
however, highly conserved in all C. elegans
This experiments is not only limited to C. elegans now. Soon
after the discovery was made, everyone tried injecting
dsRNA into all kinds of different animals
o In flies, injection of a dsRNA which corresponds to a
red eye phenotype will produce a mutant phenotype with white eyes
This dsRNA is known as the trans gene
The dsRNA is now known to be an ssRNA which folds onto itself in a
hairpin structure
Upon much more research, some more important aspects were found regarding this
phenomenon:
It works post-transcriptionally, there is no effect on the gene, only the mRNA, and
therefore the gene product, disappears
o Therefore, dsRNA affects other RNAs
dsRNAs are capable of recognizing similar regions which are found in entire gene
families
o Since these family members contain similar sequences, dsRNA could bind to
them all
Only in C elegans (nobody knows why), the effect is systemic, meaning that the dsRNA
can spread from cell to cell
o Injecting the organism with dsRNA causes the very same organism to have the
inhibition effect
o Feeding the organism the dsRNA will cause the progeny (daughter organisms) to
become affects, and not the organism itself
The dsRNA could enter the system in a variety of ways. We already discussed feeding
and injection, but soaking the organism in a dsRNA solution would also work, affecting
only the progeny
Naveen Sooknanan McGill Fall 2011
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The effect of dsRNA is catalytic, meaning picogram amounts of dsRNA could wipe out
milligram amounts of gene products
o This means that somewhere along the process there is an amplification factor
This brings up a question: why does RNA interference even exist? Although there are only
hypotheses at this point, here are some theories:
It was perhaps a means of defending the cell against RNA based viruses
o Since the RNA gene of a virus is probably folded up into secondary structure, the
cell could recognize it as foreign and generate a response to have it degraded
It could also be a means of protecting important genes against transposable elements
o Transposons insert themselves randomly, but could sometimes hit a regions
downstream of a promoter, causing them to get transcribed
o The transposon could insert itself into either orientation, causing the formation of
two transposon mRNAs which are complementary to one another (called sense
and antisense)
This will be discussed in more detail in a bit
o If these two mRNAs hybridize (bind together) the cell will recognize them and
have them degraded through RNAi
This hybridization usually happens by forming a stem loop structure, as
we will describe next
A snapback construct of RNA will cause the formation of a double stranded DNA molecule with
a hairpin structure
This happens when two identical target sequences, one
frontwards and one backwards, are located downstream of
a promoter within an expression vector (we learned about
these before)
o They are linked together by a region of the gene
known as the linker
o There two oppositely oriented genes are known as trans genes and are reverse
complementary to each other
The gene will transcribe both sequences in order, and since they are reverse
complementary to each other, the molecule will “snap back” onto itself forming a hairpin
structure with a dsRNA region
Another method of expressing these dsRNA molecules is by placing two
trans genes on top of each other, meaning they are already base paired with
each other, into an expression vector. This is known as convergent
transcription
Located upstream (on the 5’ end) of each target sequence is a T7 promoter (also learned
before) which can actively transcribe both strands in the presence of β
galactosidase
The result is the production of two separate, reverse complementary RNA
strands which can bind together post-transcriptionally and form a dsRNA
with no hairpin structure
o If these are fed to C. elegans, is causes the RNAi effect on the