Download Piwi-interacting RNAs and the role of RNA interference

Piwi-interacting RNAs and the role of RNA interference
Anthony DiMatteo
Senior Comprehensive Paper
Catholic University of America
Abstract:
There is a well-understood pathway for RNA silencing used in plants and animals which
involves small interfering RNAs (siRNAs), which uses non-coding RNA and is approximately
21 base pairs long. Recently another pathway has been discovered termed piwi-interacting
RNAs (piRNAs). They are approximately 30 base pairs in length. It is linked to the Argonaute
family of proteins. The RNA and protein complex is called the piwi-interacting RNA complex
(piRC), which exhibits DNA helicase activity. Genetic analysis shows that Piwi, which is a
protein of Drosophila melanogaster, and its mouse orthologs, Miwi, Mili, and Miwi2, are
heavily involved in spermatogenesis. Repeat-associated small interfering RNAs (rasiRNAs)
have been identified in Drosophila and seem to also function with the piwi Argonaute protein.
RasiRNA seems not to require the proteins responsible for making miRNA and siRNA, Dicer-1
and Dicer-2, respectively. The Dicer proteins digest dsRNA to produce miRNA and siRNA in
the cytoplasm. piRNAs and piRC complexes have been detected in rat, mouse, and human.
piRNA functions through a pathway completely distinct from the siRNA interference pathway.
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Introduction
RNA interference (RNAi) has provided a very powerful research tool, and one that
promises a vast application in the treatment of various pathologies. Since its discovery in 1998
its usefulness has continued to grow, along with a greater understanding of the mechanisms
behind its function. RNAi is used as a silencing pathway to decrease, or in some cases eliminate,
the expression of a specific gene. Two pathways are currently well understood, with a third,
recently discovered pathway being explored. Small interfering RNAs (siRNAs), which originate
from double stranded RNA precursors, act as targeting molecules, and microRNAs (miRNAs)
which derive from hairpin-shaped precursors are encoded in the genome as small noncoding
RNA genes. Both act through a similar pathway to silence the gene expression. A third pathway
through the use of Piwi-interacting RNAs (piRNAs) has been recently discovered in mammals
with similar RNAs discovered in Drosophila.1 piRNAs are single stranded RNA fragments
transcribed from repeat regions of the genome originally thought not to be transcribed.1
Associated Proteins
When double stranded RNA (dsRNA) enters a cell it is processed into small 19 to 21
basepair fragments by the Dicer enzyme, which is an enzyme that recognizes dsRNA, then acts
as a molecular ruler and cleaver. Its structure sheds some light on how it functions, which is
depicted in Figure 1. This is the structure of dicer from Giardia intestinalis determined by x-ray
crystallography, which is a simpler version of the human dicer.2 It contains two domains, RNase
III and Piwi Argonaute Zwille (PAZ). The PAZ domain binds the end of dsRNA, and is
separated from the two RNase III domains by 65Å, the same length as 25 base pairs. The RNase
III domains then cleave the dsRNA into a 25 nucleotide fragment.
2
Figure 1: The structure of the Dicer enzyme, displaying how it
measures dsRNA, then cleaves it into 25 nucleotide fragments.2
siRNAs are part of an effector nuclease which targets homologous RNAs for degradation.
This complex is referred to as the RNA-induced silencing complex (RISC). It is made up of a
group of proteins which use the siRNA as a guide, presumably identifying the substrate through
Watson-Crick base-pairing. The proteins in this complex are members of the Argonaute protein
family, which are defined as having a PAZ and PIWI domains. A schematic of the structure of
RISC is shown in Figure 2.3 The Argonaute PAZ domain most likely holds the 3' end of siRNA,
providing the proper orientation for recognition and cleavage of mRNA. PIWI contains the
active site for cleaving the mRNA, shown by the scissors in the schematic.3
3
Figure 2: Schematic depiction of RISC.3
Significance of RNAi 4
The discovery of RNAi has opened up a new dimension of research in various areas of
medical research and has the potential to transform medicine. Areas of particular interest are in
macular degeneration, hepatitis C, Huntington’s disease, HIV, respiratory infections, and cancer.
Macular degeneration is the leading cause of adult blindness in the developed world,
affecting more than 1.65 million people in the United States alone. It results from a growth of
blood vessels in the macular region of the retina. Some cases are caused by a mutation in the
Fibulin 5 gene, which is involved in the production of blood vessels. Researchers aim to shut off
the expression of this gene with RNAi. This disease is particularly attractive to treatment with
RNAi since the RNAi drugs can be delivered directly to the diseased tissue through injections to
the eye. These RNA strands are not packaged or protected in any way, so they are referred to as
“naked” RNAi drugs. In this example they can easily reach their intended target intact since
traveling through the bloodstream is not necessary, which could potentially break them down.
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Various viruses are also the target of RNAi research, such as Hepatitis C, respiratory
syncytial virus, also known as the common cold, and HIV. Each virus comes with its own set of
challenges, yet it is hoped that each will be defeated with RNAi. In each case there are particular
genes of interest that if their expression can be eliminated, the replication of the virus, therefore
the spread of the infection, can be treated. The delivery method for the RNA is a challenge in
each case. It is hoped to create a treatment for the common cold and other respiratory infections
that can be directly inhaled. Also, a potential treatment for HIV is extracting stem cells from a
patient’s marrow, altering it to be resistant to HIV through the use of RNAi, then transfusing
these back into the patient where they will develop into healthy immune system cells.
RNAi treatment for Huntington’s disease is also being eyed optimistically. Huntington’s
disease, a genetic disease, affects a patient throughout his or her lifetime. However, the delivery
method of RNA for a prolonged period of time, such as the patient’s lifetime, is a problem.
Some researchers have used viruses to deliver the RNA throughout a patient’s lifetime, thereby
continuously shutting down the expression of the bad gene.
Cancer seems like a promising candidate for RNAi, since if the expression of a bad gene
can be shut off, the growth of a tumor can be halted. The delivery of effective RNAi drugs to a
tumor site is quite difficult, so right now RNAi therapy is sought after as a way to reduce
multiple drug resistance, which is caused by the protein P-glycoprotein, a member of a family of
membrane pumps with ATP-binding cassette domains. It forms a membrane pump that pumps
an assortment of small molecules out of the cell, including chemotherapy agents, before they can
have any effect. The use of this RNAi treatment with existing chemotherapy regiments can form
a very powerful attack against cancerous cells.
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The scope of RNAi as a treatment option is extremely wide. It is an area of great
promise, but a great deal of research is still needed in this area. The biggest hurdle will be the
successful delivery of RNAi drugs to their effective site, since the fragile naked RNA will be
broken down before reaching most sites within the body.
RNAi 5
First, the discovery of RNAi and its usefulness must be explored. Early research in the
RNAi field began when single stranded DNA was used in an attempt to silence gene expression.
This was referred to at the time as post-transcriptional gene silencing. The researcher’s goal was
to silence the par-1 gene in C. elegans in order to assess its function. It was found that when
injecting antisense RNA the gene’s expression was disrupted. However, it was also discovered
that when injecting sense par-1 RNA expression was also disrupted. Nuclear run-on
experiments displayed that a homologous transcript was made, but was rapidly degraded in the
cytoplasm.
The most powerful discovery was made in 1998 by the research team led by Andrew
Fire. He discovered that when injecting double stranded RNA into C. elegans the silencing was
extremely efficient, much more efficient than when using just the single stranded antisense or
sense RNA. The effect of dsRNA was so potent that even a few RNA molecules were capable of
silencing homologous gene expression in the organism. Additionally, injecting dsRNA into the
gut of C. elegans silenced gene expression in the entire organism and the first generation
offspring.
Fire’s initial research was on the expression of the unc-22 gene, a gene which encodes an
abundant but nonessential myofilament protein. Semiquantitative data was collected regarding
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the relationship of unc-22 activity and phenotype of the organism. A decrease in unc-22 results
in a severely twitching phenotype, and the complete elimination of unc-22 results in the
appearance of muscle structural defects and decreased motility. The phenotype produced by
interference using unc-22 dsRNA was highly specific and the progeny of these organisms
displayed behavior identical to loss-of-function mutations in unc-22. The effects on three other
genes were explored and the same effects were displayed when injected with dsRNA for that
gene.
Mechanism of action in RNA interference
The mechanism behind the function of RNAi interference was first investigated by a
team of researchers led by Phillip Zamore.6 They discovered that dsRNA acts as the director in
the specific degradation of mRNA. The dsRNA introduced into the cells were first processed
into 21 to 23 nucleotide fragments, and these fragments then acted as the guide for the splicing of
the mRNA. They performed many experiments which identified homologous endogenous
mRNA cleaved in the regions corresponding to the dsRNA.
With this information, the following mechanism was proposed and is illustrated in Figure
3. In the first step the dsRNA enters the cell and undergoes cleavage by the Dicer enzyme into 19
– 21 basepair duplexes with 2 nucleotide 3’ overhangs.7 As shown in the figure, Dicer appeared
to be made up of two major peptides each present as a dimer, but in light of the newly identified
structure of the enzyme this is no longer clear.2 The cleaved RNA fragments are referred to as
small interfering RNA (siRNA). Step two is the effector step, as the siRNA duplexes bind to a
nuclease complex which will perform the cleaving of the mRNA.7 This complex is named the
RNA-induced silencing complex (RISC), and targets a homologous transcript through base
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pairing interactions. RISC that is bound to double-stranded siRNA is inactive. A helicase must
unwind the siRNA in order for RISC to become active. The cleavage of the mRNA takes place
approximately 12 nucleotides from the 3’ terminus of siRNA.
Figure 3: Mechanism of RNAi with Dicer and RISC. 7
Due to the potency of the silencing effect, a third step, amplification, has been proposed.7
Something must happen which allows only a few dsRNA molecules to completely silence the
expression of a gene in some cells. Most likely, RISC has multiple turnover events, meaning it is
capable of acting upon multiple substrates while active.
RNAi interference in mammalian cells presented an interesting challenge, since it was
discovered that when transfecting with dsRNA more than 30 nucleotides, nonspecific
suppression of gene expression took place.7 This is a result of an antiviral response in
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mammalian cells, which is in place to protect the cells from infection by a virus. This is exactly
what the researchers were trying to do in the transfection, use a virus to deliver the dsRNA
directly to the cell. This pathway can be circumvented by directly inserting the siRNA with
fragment sizes less than 30 nucleotides, 21 basepair dsRNA with 2 nucleotide 3’ overhangs are
optimal. The potency of this approach in mammalian cells varies a great deal. The best results
show a 90% reduction in target mRNA and protein levels, however most cases do not yield
results with this much effective silencing.7
Piwi-interacting RNAs
The most recent development in the RNAi field is the discovery of piRNA, which is
believed to function in a completely distinct pathway from siRNA. These RNA fragments are
approximately 30 nucleotides long and are believed to be produced by the host cell, as opposed
to being manufactured by the siRNA pathway.1 They are part of a pathway that is perhaps
intrinsic to spermatogenesis.
The RNAs involved in piRNA interference interact with proteins related to those of
siRNA but are not identical.1 They are proteins of the Argonaute family. Certain proteins such
as Ago1 and Ago2 associate with siRNAs to form the complexes involved in associating with the
target mRNA and repressing its expression. Another subdivision of the argonaute proteins was
discovered to be distinct from Ago1 and Ago2 and does not associate with siRNA. The first
discovered protein of this subfamily of proteins is the Piwi protein of Drosophila melanogaster,
and appears to play a significant role in germline development. The mouse orthologs are Miwi,
Mili, and Miwi2. Genetic analysis indicates that these proteins are essential for spermatogenesis.
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The understanding of how these proteins regulate the germline was unclear until the discovery of
the small RNA that associates with them.1
Small RNA that partners with Piwi proteins was discovered by Lau et al.8 They
discovered testis-specific RNA in extracts from rat testis. In samples of a partially purified
ribonucleoprotein complex from the rat testis they discovered this RNA with sizes of 25 to 31
nucleotides, with sizes between 29 and 30 nucleotides being most prevalent. Lau et al purified
the RNA-protein complex through the use of conventional chromatography and they identified a
protein that is homologous to Riwi, which has been identified in rat. It also has a subunit which
is homologous to the human protein RecQ1, which is known to exhibit DNA helicase activity.
In light of this, this small RNA was named Piwi-interacting RNA, piRNA, and the complex was
named the Piwi-interacting RNA complex, piRC. piRC exhibits adenosine triphosphate
dependent DNA helicase activity, most likely from the RecQ1 homolog.
The existence of piRNA was discovered when Lau and his team of researchers were
looking to identify possible complexes involved in transcriptional gene silencing (TGS). Two
pathways exist, one that acts in the transcriptional level and one that acts in the
posttranscriptional level. Posttranscriptional gene silencing destabilizes or inhibits the mRNA,
and TGS suppresses gene expression by altering chromatin conformation. At the core of each is
a small RNA that associates with a protein of the argonaute protein family. TGS was studied a
little, but the mechanism still proved difficult to decipher. The researchers decided to investigate
if TGS employed RNA fragments longer than the 21 to 23 nucleotide fragments of siRNA.
RNAs longer than 24 nucleotides have been linked to TGS and genomic repeats, which are often
not transcribed, in Arabidopsis, Tetrahymena, Drosophila, and zebra fish. These RNAs appear
to be enriched in the testis of Drosophila. This led the way to the team obtaining extract from rat
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testis. During separation on an ion-exchange column, a peak of RNAs larger than 22 nucleotides
was eluted. They argued that this suggested the existence of a new ribonucleoprotein complex.
Figure 4 displays the fractions collected during the separation, then run on a gel. The RNA was
end-labeled and run on a gel. The dashed boxes represent small RNAs that were purified,
converted to cDNA, and sequenced. The chart represents the findings of the cDNA sequences.
Fractions 15, 17, and 19 display considerable bands on the gel.
Figure 4: The results of rat testes extract eluted though a Q column.8
The RNAs collected in these fractions were characterized, and these particular fractions
of RNA turn out to derive from regions of the genome originally thought not to be transcribed.8
In fact, less than 1.1% matched annotated mRNAs. For the most part, this RNA was 25 to 30
nucleotides in length, with most between the sizes of 29 and 30 nucleotides. Northern blot
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analysis confirmed that the expression of these RNA is testis-specific, as displayed in Figure 5.
All of the RNA that did not match annotated RNA, such as miRNA, tRNA, rRNA, and snRNA,
was considered to be a member of this new class of small RNA, soon to be termed piRNA.
Figure 5: Northern blot analysis of rat tissue hybridized with body-labeled
RNA probes that correspond to small RNA sequences. This was performed for
chromosomes 6, 7, and 8. Finally, let-7 was the positive loading control.8
Next, the function of these RNAs was determined. To do this, their associated proteins
were purified. Lau and his team purified the proteins that associated with piRNA, and the peak
fractions from this process were resolved on a gel. The two bands on this gel were analyzed
using mass spectrometry, and they were identified as being Riwi and rRecQ1 proteins, shown in
Figure 6. In addition, Western blotting and coimmunoprecipitation confirmed that the small
RNA, Riwi, and rRecQ1 copurify. Since Riwi is a homologous to the Piwi protein, these RNA
were named Piwi-interacting RNAs, and the complex was called the piRNA complex.
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Figure 6: Proteins from the peak fraction of piRC run on a gel and silver
stained. Bands were cut from the gel and identified through mass
spectrometry. The bands were identified as labeled, Riwi and rRecQ1.8
The team led by Lau sought to understand the origins of piRNAs, so they investigated
from where on the genome the piRNAs were transcribed. A cDNA library from rat and mouse
testes was sequenced, which yielded 85,489 rat sequences and 95,423 mouse sequences.
Because some of the sequences represented more than one read of the genome, these
corresponded to 61,293 unique small RNA sequences in rat, and 65,681 in mouse. These
sequences were analyzed using a BLAST search. Many of the sequences were already annotated
and were excluded from the following analysis. Therefore, 40,698 unique sequences
representing 61,581 reads of rat and 43,332 sequences representing 68,794 reads of mouse were
confirmed to derive from the genomes. Next, an analysis was performed to locate the regions of
the genome that were transcribed to create these unknown sequences. They determined the
number of times a region of a given chromosome was transcribed to create piRNAs. Since there
were more reads than actual unique sequences, many of the loci of the genome must have been
transcribed multiple times. The number of hits each locus of the genome had were totaled.
Approximately two thirds of the piRNA matched a specific locus completely, and some loci had
multiple reads.8 The team reports the maximum being 149 reads for a single locus.
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Additionally, the remaining one-third of the reads, each of which mapped to multiple loci, were
analyzed by normalizing the number of reads by the number of genomic hits. This normalized hit
count was assigned equally to all the loci. For example, if a piRNA has four equal genomic hits,
each will contribute a quarter of a count to each of its four loci. The counts were integrated into
bins and plotted across each chromosome. 94% of the counts fell into 100 genomic clusters.
Figure 7 displays the results of this analysis.
Figure 7: A. Histogram of number of piRNA reads mapping to various clusters on the chromosomes. B.
Zoom view of clusters 1, 15, and 26. Note that cluster one does not have overlapping reads, as clarified
by this higher resolution view. Bars above and below the histograms designate regions orthologous to
mouse regions that also produce piRNAs.8
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It is known that siRNAs originate from dsRNA or foldback structures. However,
piRNAs mapped exclusively to either the plus or minus genomic strand.8 There was no evidence
of a double stranded RNA or foldback structure as a precursor. Cluster 1 in figure 5A does
appear to map to the same region on the plus and minus strand, but in fact the hits map to regions
juxtaposed to each other, as shown in the zoom view in figure 5B. Lau reports that the two
regions are separated by approximately 100 to 800 base pairs. He hypothesizes that perhaps this
separation between the two regions suggests that transcription originates in this gap region and
continues in a divergent, bidirectional manner. However, this is not confirmed. Lau compared
the sequences of the mouse clusters to the rat clusters and found that many of them were
homologous to each other. They had very similar strand specificity and abundances. These
similar regions of homology are marked by the bars above and below the histograms in figure 7.
Figure 8 displays Northern blot analysis to confirm that piRNAs derive solely from one
of the two genomic strands. No bands showed up when the sense strands was probed, unlike the
anti-sense strand.
Figure 8: Evidence that piRNAs derive from one of the two genomic strands.
This is a northern blot analysis with probes to clusters 4 and 6. Let-7 is the
loading control.8
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Faint bands showed up when the anti-sense human, and mouse, testes RNA was probed.
It would be expected that bands would have been detected for these samples, however due to
mutations in the sequences the probes were unable to hybridize sufficiently. Through a
conservation analysis, it was determined that sequence identities of piRNAs are only weakly
conserved among different species. Orthologous rat and mouse clusters were identified, and rat
residues were counted based on the number of rat piRNA reads. Then the estimated substitution
rates and insertion/deletion rates were calculated. It is expected that the high rate of substitutions
created enough sequence differences to prevent detection on the Northern blot. The piRNAs
with the greatest number of reads had the lowest nucleotide insertion or deletion rates. Figure 9
displays these analyses. The higher rate of conservation hints at evolutionary favorability for
these more abundant piRNAs.
Figure 9: Histograms from the piRNA conservation analysis. The first is the analysis of
nucleotide substitution rate per piRNA reads per nucleotide. The second is the analysis of
nucleotide insertion/deletion rate per piRNA reads per nucleotide.8
Characterization of piRNA binding proteins
The function of piRC must be assessed. Lau states that it was characterized by
considering the known functions attributed to RecQ and Ago family members.8 Human RecQ1
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is an ATP-dependent DNA helicase, and the Ago family of proteins is heavily involved in
cleaving RNA in the mechanisms discussed earlier. ATP-dependent helicases operate by
mechanisms that are not yet fully understood. However, the structures of multiple helicases have
been determined and have shed some light upon the way in which they function. As shown in
figure 10, four domains typically make up a helicase. At the beginning, in the absence of ATP,
both the A1 and B1 domains bind single-stranded DNA.9 The binding of ATP triggers
conformational changes in the A1 domain which releases the bound DNA and closes the space
between the two domains, thereby sliding A1 along the DNA. The enzyme catalyzes the
hydrolysis of ATP to ADP and Pi and the space between the domains reopens, which pulls the
DNA from domain B1 toward domain A1. By repeating this process the enzyme continues to
translocate down the DNA strand, displacing the opposite DNA strand as it progresses. The
progression of the single stranded DNA through the helicase can be observed by the movement
of the two black dots on the strand as they move further to the right.
Figure 10: Helicase mechanism9
Figure 11A and 11B display that ATPase and DNA unwinding activity followed the
rRecQ1 protein of piRC. In 11A, samples were collected in fractions from the purification on a
Superdex-200 column. Riwi, rRecQ1, and piRNAs purified together in the column in the same
fractions.8 11B displays fractions that contained rRecQ1 and displayed ATPase activity.
Fractions were incubated with radiolabeled ATP. Free phosphate produced by the ATPase was
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separated from unhydrolyzed ATP through the use of thin-layer chromatography. Also, by
using a complimentary substrate to a piRNA, cleavage activity was assayed. The results are
displayed in figure 11C. The fractions were incubated with a 17 base pair duplex, and then run
on a gel. The faster migrating samples indicate that the DNA duplex was unwound on the gel.
Figure 11: A. The top gel depicts the visualization of the proteins, and the second
gel depicts end-labeled RNAs. B. Fractions that contained rRecQ1 displayed
ATPase activity. C. This histogram displays the DNA unwinding activity. The
faster migrating bands on the gel are evidence of unwound DNA.8
Putting the entire mechanism together yields a schematic shown in, Figure 12. The left side of
the figure displays the creation of antisense RNA transcripts, while the right side displays the
production of sense transcripts. The center region serves as the promoter for both regions. It is
still unclear how the transcripts are processed into the 25 to 31 nucleotide fragments. The
piRNAs associate with the Piwi and RecQ1 homologs which forms piRC.
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Figure 12: The understood elements of the piRNA interference mechanism, including
transcription of the piRNA and its assembly with Piwi and RecQ1 to form piRC.1
Conclusion
The discovery of RNAi has yielded an exciting glimpse into an organism’s expression of
genes. RNAi is also being used as a powerful research tool and may be the key to finding cures
for many diseases. The application of RNAi is vast and there is no telling where future
developments might lead using this new technology. The discovery of piRNA is an interesting
development in RNAi. Perhaps piRNA is part of a cell’s innate ability to silence particular genes
at crucial moments of spermatogenesis. piRNA is a distinct classification of small RNAs, as it
has a larger size than siRNA and miRNA, and it associates with different proteins. The piRC is
the protein complex to which piRNA binds. Also, piRNA does not originate from double
stranded or hairpin-shaped precursors, whereas siRNA and miRNA do. piRNA clearly play a
role in spermatogenesis, however more research is needed to fully understand its role in
spermatogenesis and the control of the germline.
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References
(1) Carthew, R. Science 2006, 313, 305-306.
(2) MacRae, I.; Zhou, K.; Li, F.; Repic, A.; Brooks, A.; Cande, W.; Adams, P.; Doudna, J.
Science 2006, 311, 195-198.
(3) Song, J.; Smith, S.; Hannon, G.; Joshua-Tor, L. Science 2004, 305, 1434-1437.
(4) Nova: Science Now. “The RNA Cure?”
http://www.pbs.org/wgbh/nova/sciencenow/3210/02-cure.html.
(5) Fire, A.; Xu, S.; Montgomery, M.; Kostas, S.; Driver, S.; Mello, C. Nature 1998, 391, 806-811.
(6) Zamore, P.; Tuschl, P.; Sharp, P.; Bartel, D. Cell 2000, 101, 25-33.
(7) Hannon, G.; Seto, A.; Kim, J.; Kuramochi-Miyagawa, S.; Nakano, T.; Bartel, D.; Kingston,
R. Nature 2002, 418, 244-251.
(8) Lau, N.; Seto, A.; Kim, J.; Kuramochi-Miyagawa, S.; Nakano, T.; Bartel, D.; Kingston, R.;
Science 2006, 313, 363-367.
(9) Berg, J.; Tymoczko, J.; Stryer, L. Biochemistry, 6th Ed., pg 797.
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