RNA Silencing

RNAi
Compiled by T S Lokeswari, 2013

Small untranslated regulatory RNAs Eukaryotes ncRNA Prokaryotes sRNA

Guillier, et al. Genes and Development 20:2338, 2006

Number of Publications
5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 1998 1999 2000 2001 2002 2003 2004 2005 2006

Entrez PubMed Search Terms: RNAi or siRNA or miRNA

Lecture Outline
Discovery General features siRNA
Role of translation

miRNA Problems

Focus on RNA interference - A users guide September 2006

Nature Genetics June 2006 Supplement

Early demonstration of RNAi in plants

391:806, 1998

No probe

No RNA

anti-sense ssRNA

dsRNA

In situ hybridization for mex-3 mRNA 4 cell embryos

In vitro transcription
Restriction digest
Blunt end 5 overhang 3 overhang

 RNA interference or RNAi is a remarkable process whereby small noncoding RNA silence specific genes

Small interfering RNAs inhibit expression of a homologous gene

RNAi mediated

siRNA
PTGS TGS VIGS

miRNA
Transcriptional

RNAi
RNA silencing pathways are triggered by 21-27 nt long small RNAs
Small interfering RNAs siRNA Repeat-associated small interfering RNAs rasi RNAs Micro RNAs miRNA Piwi-interacting RNA - piRNA

RNAi induction using long dsRNA only operates in plants and invertebrates Worms soak them in a solution of dsRNA, feed them bacteria expressing the appropriate construct In vertebrates, long dsRNA (>30 bp) induces on the IFN response including PKR, inhibits translation, and activation of RNase L, degrades mRNA

Novina and Sharp Nature 430:161 2004

RNA Interference: A Mechanism for Silencing Gene Expression

1. Small dsRNA fragments can silence the expression of a matching gene. This is RNA interference (RNAi), recently discovered in C. elegans.
a. Injecting dsRNA into adult worms results in specific loss of the corresponding mRNA in the worm and its progeny. b. RNAi also occurs in many other organisms, where it protects against viral infection and regulates developmental processes.

2. RNAi is highly specific and sensitive, with only a few molecules of dsRNA needed, making it an excellent research tool.

Dicer
Dicer generates RNAs with 2 nt 3 overhang and 5 phosphorylated terminus, both required for activity Fly Dicer requires ATP, human may not

RISC
RISC has helicase, exonuclease, endonucelase and homology searching proteins. Initial RISC is inactive until transformed into active form by unwinding of the siRNA duplex and loss of sense (passenger) strand Antisense (guide) strand defines specificity of RNAi

Processing of siRNA
Starting with dsRNA Which becomes guide strand in the RISC and which (passenger strand) is excluded?
Sequence and structure Strand with the less-tightly base pared 5 end is incorporated becomes guide strand

What is the endonuclease (Slicer) in RISC?

The ago1 mutant Arabidopsis develops abnormally because it does not produce an effector of silencing. The Argonaute genes were so named because the mutant plants look like an argonaute squid. Knew that Ago a RISC component
The Sainsbury Laboratory John Innes Centre Colney Lane Norwich, NR4 7UH, UK

Identification of Argonaute 2 as Slicer in humans

J. Liu et al., Science 305, 1437 -1441 (2004)

Published by AAAS

Fig. 2. Argonaute2 is essential for mouse development

J. Liu et al., Science 305, 1437 -1441 (2004)

Published by AAAS

Fig. 3. Argonaute2 is essential for RNAi in MEFs

J. Liu et al., Science 305, 1437 -1441 (2004)

Published by AAAS

Cytosolic players in siRNA and miRNA

Dicer (DCR)
Multi domain RNase III enzyme the cleaves dsRNA or stem-loop pre-miRNA into siRNA and miRNA

TRBP
TAR RNA Binding Protein, Cofactor for Dicer

RISC
RNA induced silencing complex

Argonaute (AGO)
PAZ domain binds the characteristic two-base 3' overhangs of siRNAs PIWI domain: dsRNA guided hydrolysis of ssRNA Ago2 is slicer in mammalian RISC Other Ago may function in miRNA silencing

Comparison of siRNA and miRNA

siRNA
miRNA Endogenous transcript Endogenous or exogenous dsRNA dsRNA mRNA cleavage ssRNA Translation inhibition and mRNA cleavage Imperfect complimentarity development Inhibit transposon and

Precursor

Structure
Function

perfect Target mRNA complimentarity

Biological
virus infection

Foreign DNA and Transgene

Foreign DNA and Transgene

Aberrant sense RNA RdRP dsRNA Dicer siRNAs Nature 2004, Vol 431, Sept.16:343

Heterochromatin formation and Transcriptional silencing

Self splicing miRNA

Mitrons :Short intronic hairpins RNA Ploymerase II or III pri-miRNA No need of Drosha Splicing machinery Lariat debranching enzyme pre-miRNAs Dicer Cell 130, July 13 2007: 89-100

Micro RNAs (MiRNAs) ~22NTs

RISC

miRNA
The miRNA are endogenous small RNA guides that repress the expression of target genes. Differ from siRNA in biogenesis not in functions, although mechanisms can be different. mRNA cleavage when complementarity is extensive, repress translation when not. lin-4 mutant worms had defects in timing of cell division. Encodes a small RNA that binds to and silenced lin-14 message. Lin-14 mRNA levels do not decline, but that may not always be the case. let-7 also found in other species.

miRNA
Abundant ssRNA from a few thousand to 40,000 molecules /cell Found in all metazoans 0.5-1% of genes siRNA targets genes from which it is derived in a sequence specific manner miRNA regulate separate genes and has imperfect complementarity May be 100s mRNA regulated by one miRNA Usually have many binding sites in each 3 UTR, and several different miRNA can target same 3 region. Combinatorial control

miRNA
Many miRNA are embedded in introns of protein encoding genes and are transcribed together with host genes. miRNA can be expressed in developmentally tissue specific fashion but may not be expressed in tissues where putative target sequences are.

Plasterk Cell 124:877, 2006

The structure of human pri-miRNAs

Du, T. et al. Development 2005;132:4645-4652

Processing of miRNA
Long primary Pol II transcript (pri-miRNA) Cleaved by Drosha, nuclear RNase III endonuclease to establish one end of the miRNA (pre-miRNA)
Also need dsRNA binding protein Pasha (flies) DGCR8 (humans)

The pre-miRNA exported from the nucleus by Exportin 5 Cut by Dicer miRNA Strand with the less-tightly base pared 5 end becomes mature miRNA, other strand becomes miRNA* and degraded Worms and mammals only one Dicer and it makes miRNA and siRNA. Flies have one for each.

Players in miRNA biogenesis

Drosha
Nuclear RNase III enzyme. Initiates miRAN biogenesis by cleaving pri-miRNA into premiRNA

Pasha
Partner of drosha is a dsRNA binding protein. Human DGCR8

Exportin-5
Nuclear transmembrane protein that transports pre-miRNA form nucleus to cytoplasm. Works in conjunction with GTP-Ran

Cullen Nature Immunology 7:563 2006

lncRNA functions

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.

Macular Degeneration Acuity Pharmaceuticals, Inc.

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.

In vivo applications of RNAi

Highly specific
Silence a single nucleotide difference in a dominant negative allele

Resistance not (less) a problem

Can design new RNAi if a mutation arises and original targeted sequence is changed

Problems
Stability Delivery Toxicity

Off Target Effects

Global, due to induction of innate immune responses Cross reactive, due to sequence homology with other mRNA sequences Not easy to recognize unless global gene expression studies performed. Good to have multiple target sequences Recently found in flies

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
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. 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. 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.

INTRODUCTION

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 potentFUTURE gene silencing effect. They coined the term RNAi. 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

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 siRNAs thereby inducing RNA in these systems without eliciting an antiviral response.

An additional future application of RNAi includes inhibition of viral infections, specifically HIV infection, whereby proteins critical to HIVs 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 siRNAs, which are approximately 21 -25 base pairs, then destroy complementary HIV mRNAs, 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 HIVs ability to mutate, multiple genes can be targeted at once to ensure inhibition of HIVs ability to make critical proteins. Namely, the HIV-1 cellular receptor CD4s (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

The use of siRNA decreases the expression of telomerases.

HIV gene sequ Represe ence ntative

C. elegans were fed E. coli that expressed Bold dsRNA horizontal homologous to bars a gene on indicate chromosome Lack of
1. 2. 3. 4. 5. 6. 7. 8. 9. 14. 15.

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. Dehio C. and Schell J. (1994). "Identification of plant genetic loci involved in a post transcriptional mechanism for meiotically reversible transgene silencing". Proceedings of the National Academy of Sciences of the United States of America 91 (12): 5538-5542.

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 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. Liu, Zhongchi. Lecture 15: Functional Genomics II. BSCI410. 6 Apr 2006. Liu, Zhongchi. Lecture 16: Functional Genomics III. BSCI410. 11 Apr 2006 http://www.ambion.com/ Howard, Ken. Unlocking the money-making potential of RNAi Nature Biotechnology 21(2003): 1441-1446

10. National Science Foundation. Ed. Aguirre, Lauren. Nov. 2005. 11. WGBH Educational Foundation. 22 April 2006 12. http://www.pbs.org/wgbh/nova/sciencenow/

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 http://www.molecular-cancer.com/content/4/24/1/

Nucleic Acids Research, 2002, Vol. 30, No. 22 4830-4835

Intraocular injections of RNAi targeting vascular endothelial growth factor inhibited 60% neovasculariza
BIBLIOGRAPHY

Mechanism of miRNA suppression of gene expression

Transcription mRNA degradation Translational repression
1 Initiation 2 Elongation 3 Termination 4 Release

Co-translational degradation of the nascent peptide

Western blot for LIN-14 protein Transcription same (run-on) RNA levels ~ same Lin-4 miRNA expressed at end of L1

Fig. 1. miRNAs target the initiation step of translation

No eIFs or Met-tRNAimet

Humphreys, David T. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 16961-16966
Copyright 2005 by the National Academy of Sciences

Scanning model of Cap-dependent Translation Initiation

eIF5B

E P A

E P A

Ftima Gebauer & Matthias W. Hentze Nature Reviews Molecular Cell Biology 5, 827-835 (2004)

Other Studies
Both 5 cap and 3 poly(A) tail are necessary but not sufficient for miRNA repression of translation EMCV IRES
Uses everything that canonical translation initiation does except eIF4E Resistant to miRNA

New model
Block in translation initiation Sequestration in P-bodies In some cases this may lead to mRNA decay