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23. New Roles for RNA.pdf

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McGill University
Biology (Sci)
BIOL 200
Richard Roy

DISCOVERY The End DISCOVERY The EndNaveen Sooknanan McGill Fall 2011 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 st Upon the discovery of the human genome near the 21 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 (RNA) pithway 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) 1Naveen Sooknanan McGill Fall 2011 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 2Naveen Sooknanan McGill Fall 2011  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 RNA i  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 RNA eifect on the 3Helicase Risc protein ADP +P RSC RISC polyA Helicase Risc protein ADP +P RSC RISC polyANaveen Sooknanan McGill Fall 2011 target gene Before these long dsRNA fragments carry out their RNA effict, they must be cleaved into smaller, reactive dsRNA fragments which are 21-23 nucleotides long  The whole process is ATP dependant and mediated by an enzyme known as Dicer  These dsRNA fragments are known as small interfering (si) RNA (or micro RNA, but we will talk
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