Transcript RNAi - University of Maryland, College Park

The Mechanisms and Applications of RNAi
Gwendolyn Bishop, Mary Pham, Everett Oliver, Rebecca Smith, Sabbie Sandhu, Ashley Wong
University of Maryland, College Park
BCHM 465
Prof. Kahn
APPLICATIONS
INTRODUCTION
1. Functional Genomics:
RNA interference can be used as a tool for determining gene function. According to the Central Dogma of
molecular biology, DNA is transcribed into RNA which is then translated into protein. Proteins and some RNAs
are the functional components within a cell, thus the ability to selectively destroy RNA allows researchers to
effectively repress gene expression.
RNA interference (RNAi) is a molecular biology mechanism where the presence of
certain fragments of double-stranded RNA (dsRNA) interferes with the expression of a
particular gene which shares a sequence with the dsRNA that is homologous to that
gene.
• The revolutionary finding of RNAi resulted when plant scientists in the USA and the
Netherlands tried to produce petunia plants with improved flower colors by introducing
additional copies of a gene encoding a key enzyme for flower pigmentation into petunia
plants. When the scientists ended up with fully or partially white flowers they discovered
that both types of genes, the endogenous and the newly introduced transgenes, had been
turned off.
• A few years later plant virologists made a similar observation. In their research they
surprising observation that plants carrying only short regions of viral RNA sequences not
coding for any viral protein showed enhanced tolerance or even resistance against virus
infection. They concluded that viral RNA produced by transgenes can also attack incoming
viruses and stop them from multiplying and spreading throughout the plant. They called this
phenomenon “virus-induced gene silencing” or simply “VIGS”. These phenomena are
collectively called post transcriptional gene silencing.
• Many laboratories around the world searched for the occurrence of this phenomenon in
other organisms. Scientists A. Fire and C. Mello injected double stranded RNA into C.
elegans and noticed a potent gene silencing effect. They coined the term RNAi.
Previous methods of studying gene function have involved manipulation of DNA. A typical experiment required
the generation of random mutants by exposing a group of organisms to mutagens or through the use of DNA
inserts. The few that expressed a phenotypic change relevant to a given study were then selected and the
genetic location of the sequence change would be determined. The advent of RNAi-based gene silencing,
along with its use of genome sequence information, has dramatically changed how these tests are done. RNAi
is a form of reverse genetics, meaning researchers can systematically pick genes rather than beginning with
mutants and then searching for the genes affected. One major advantage of this method is that the genetic
location of any given “mutation” is predetermined, which removes a large portion of previous labor.
Additionally, all genes can be analyzed. If a gene is vital, a mutant might not survive long enough to be
noticed, let alone studied. With RNAi, researchers can pick a chromosome and systematically knock out
genes as they appear sequentially.
Various methods exist for inserting dsRNA into target organisms. One method is direct injection which, though
currently used for medical applications, is not the best option for high throughput research. Viruses can also
inject an appropriate duplex. There are two ways of expressing dsRNA in bacterial vectors: by encoding a
hairpin structure or by use of a dual promoter that expresses both the sense and the anti-sense strand. In an
experiment conducted on C. elegans in 2000, dual promoters where inserted into E. coli plasmids. The worms
that ate these bacteria contained the dsRNA for several days and it was carried on to their progeny that were
produced within this time period. The embryos were taped as they developed and any changes relative to wild
type phenotype were recorded.
MECHANISM
FUTURE APPLICATIONS
A lot of research is currently being conducted investigating the use of RNAi as a future
cancer therapeutic. Results from in vitro and in vivo animal studies look promising. This
method is appealing due to the specificity of RNAi in silencing target genes without
affecting other genes. As more genes involved in causing cancer are being discovered
and sequenced the efficiency of RNAi increases. RNAi regulates gene expression thus
having the capability to inhibit expression of protein encoding genes involved in cancer.
The ability of RNAi to specifically silence targeted genes makes it a potentially highly
effective method of treating cancer.
Research is being conducted to design specific siRNA that targets telomerase.
Telomerase is an enzyme that produces telomeres which are tandem repeats of DNA
(TTAGGG) located at the ends of chromosomes. Under normal cell division telomeres
shorten with each round of cell division because DNA polymerase cannot incorporate
nucleotide bases in the 3’ 5’ direction, and thus cannot replace the 5’ RNA primer with
DNA, resulting in a loss of genetic information. Consequently with each replication,
telomere sequences are shortened. When telomeres reach critical shortening, DNA
sensing molecules are activated and initiate an intracellular mechanism that leads to
cell cycle arrest and replicative senescence. Telomerases increase telomere length,
thereby enabling cancerous cells to evade senescence and allows enhanced replicative
potential resulting in virtual immortality. Inhibition of telomerase activity prevents
telomere extension, potentially causing replicative senescence and apoptosis of
cancerous cells. Altering the telomerase will hinder cancer development and essentially
make the cancerous cells susceptible to “old age”. The future use of siRNA is
appealing since there are very few or no effects on normal diploid cells. However, only
in vitro and animal studies have been conducted so far.
MECHANISM
In Non-mamlian Species:
A. Introduction of dsRNA triggers the RNAi Pathway.
B. Dicer (cytoplasmic Nuclease) cleaves the dsRNA,
thus produces siRNA(21-23bp)
C. siRNA unwinds and assembles into RISC (RNA Induced Silencing
Complex).
D. Antisense siRNA strand then guides the RISC to complementary
RNA molecules.
E. RISC cleaves the mRNA.
F. This leads to specific gene silencing.
Mammalian Systems:
Since some mammalian cells mount a potent antiviral response upon
introduction of dsRNA longer then 30bp, researchers transfect cells
with 21-23bp siRNA’s thereby inducing RNA in these systems without
eliciting an antiviral response.
2. Macular Degeneration:
Macular degeneration is when the protein, vascular endothelial growth factor or VEGF, is overproduced in the
eye. An excess of VEGF causes a build up of blood vessels behind the retina leading to blurred vision and
possible blindness. In order to destroy the mRNA that codes for VEFG, dsRNA is injected into the whites of the
eyes which then leads to reduced blood vessel formation and the shrinkage of present blood vessels. The first
RNAi treatment trial for macular degeneration started in 2004, where a quarter of the participants saw a
significant improvement in their vision after two months. Presently, Acuity Pharmaceuticals, Inc. is sponsoring a
study with siRNA called Cand5 for the treatment of macular degeneration. They are currently in phase I where
20-80 people use the treatment to determine safety, side effects and proper dose amount. If research and trials
go according to plan an RNAi treatment for macular degeneration could be available for the public as soon as
2009.
Macular degeneration is one of the first diseases to test RNAi as a treatment, the reason being that the RNA
can be directly injected into the diseased tissue. When the RNA has to travel through the body to get to the
target site the RNA has a better chance of getting degraded or affecting the wrong gene.
C. elegans were fed E. coli that expressed dsRNA
homologous to a gene on chromosome III. The progeny
were observed for phenotypic changes.
Intraocular injections of RNAi targeting vascular endothelial
growth factor inhibited 60% neovascularization (green
fluorescence) in laser-induced rupture of retinal membranes,
which mimics the growth and leakage of blood vessels
behind the retina in AMD.
The use of siRNA decreases the expression of telomerases.
An additional future application of RNAi includes inhibition of viral infections, specifically HIV infection,
whereby proteins critical to HIV’s survival are targeted. Long strands of RNAi (approximately 500 bp in
length) often illicit an interferon response in the cells. Accordingly, the “Dicer” step of RNAi can be
bypass by using plasmid derived short strands of RNAi (siRNA). The siRNA’s, which are approximately
21 -25 base pairs, then destroy complementary HIV mRNA’s, thereby effectively silencing the correlated
gene sequences; ultimately resulting in inhibition of HIV replication and/or its ability to attach to immune
cells.
Due to HIV’s ability to mutate, multiple genes can be targeted at once to ensure inhibition of HIV’s ability
to make critical proteins. Namely, the HIV-1 cellular receptor CD4’s (controlled by the nef gene), the
envelope associated proteins (controlled by the env gene) and the capsid proteins (controlled by the
gag protein) as explained in Nature 418, 435-438 (25 July 2002). The pictorial representation to the
right shows a representation of a HIV gene sequence along with a typical plasmid whereby the plasmid
is used to derive the siRNAs that will target the proteins related to the gag and env gene sequences.
The graph is used to show lack of antigen activity at specific gene sequences as an indication of the
degree of inhibition of that gene, as noted by a study printed in Nucleic Acids Research, 2002, Vol. 30,
No. 22 4830-4835
BIBLIOGRAPHY
HIV gene sequences
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Napoli C., Lemieux C., and Jorgensen R. (1990) "Introduction of a chalcone synthase gene into Petunia
results in reversible co-suppression of homologous genes in trans". Plant Cell 2: 279-289.
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mechanism for meiotically reversible transgene silencing". Proceedings of the National Academy of
Sciences of the United States of America 91 (12): 5538-5542.
3.
Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C. (1998). "Potent and specific
genetic interference by double-stranded RNA in Caenorhabditis elegans". Nature 391: 806-11
4.
Gonczy, Echeverri, et al. “Functional genomic analysis of cell division in C. elegans using RNAi of genes
on chromosome III.” Nature 408 (2000): 331-336.
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Liu, Zhongchi. “Lecture 15: Functional Genomics II.” BSCI410. 6 Apr 2006.
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Liu, Zhongchi. “Lecture 16: Functional Genomics III.” BSCI410. 11 Apr 2006
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http://www.ambion.com/
8.
Howard, Ken. “Unlocking the money-making potential of RNAi” Nature
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Biotechnology 21(2003): 1441-1446
Bold horizontal bars indicate targeting by siRNA
10. National Science Foundation. Ed. Aguirre, Lauren. Nov. 2005.
11. WGBH Educational Foundation. 22 April 2006
Representative bacteria plasmid
by which siRNA’s are derived
using PCR and purification
methods.
12. http://www.pbs.org/wgbh/nova/sciencenow/
Lack of antigen activity indicates degree of gene inhibition
13. Shammas, Masood A., Hemanta Koley, Ramesh B Batchu, Robert C Bertheau, Alexei Protopopov, Nikhil
C Munshi, and Raj K Royal. "Telomerase inhibition by siRNA causes senescence and apoptosis in
Barrett's adenocarcinoma cells: mechanism and therapeutic potential." Molecular Cancer 4.24 (2005):
doi: 10.1186/1476-4598-4-24. 20 April 2006
14. http://www.molecular-cancer.com/content/4/24/1/
15. Nucleic Acids Research, 2002, Vol. 30, No. 22 4830-4835