Rnai

Post – transcriptional gene silencing

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PTGS in plants (Co-suppression)  Quelling in fungi  RNAi in animals

Co-suppression

What is RNAi A novel way to regulate gene expression

2006 Nobel Prize

What is RNAi RNA interference (RNAi) is a highly evolutionally conserved process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA), when introduced into a cell, causes sequence-specific degradation of homogolous mRNA sequences.

It was first discovered in 1998 by Andrew Fire and Craig Mello in the nematode worm Caenorhabditis elegans and later found in a wide variety of organisms, including mammals.

Mechanism of RNA interference  

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A. On entering the cell, long dsRNAs act as a trigger of RNAi process. B. It is first processed by the RNAse III enzyme Dicer in an ATP-dependent reaction. C. Dicer processes dsRNAs into 21-23 nt short interfering RNA (siRNA) with 2-nt 3' overhangs. siRNA can also be synthesized outside the cell and then be introduced into a cell. D. The siRNAs are incorporated into the RNA-inducing silencing complex (RISC) which consists of an Argonaute (Ago) protein as one of its main components. Ago cleaves and discards the passenger (sense) strand of the siRNA duplex leading to activation of the RISC. E and F. The remaining guide (antisense) strand of the siRNA guides RISC to its homologous mRNA, resulting in the endonucleolytic cleavage of the target mRNA

1995 

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First noticed that sense RNA was as effective as antisense RNA for suppressing gene expression in worm Guo S, and Kemphues KJ. C. elegans

Guo S, and Kemphues KJ. par-1, a gene required for establishing polarity in C. elegans embryos, enc . Cell. 1995 May 19;81(4):611-20.

First report…. No clue

Cell lineages and early divisions in C. elegans

Scoring for Embryonic lethality in RNA Injections

1998 

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First described RNAi phenomenon in C. elegans by injecting dsRNA into C. elegans which led to an efficient sequence-specific silencing and coined the term "RNA Interference". Fire et al. C. elegans Fire

et

al.

Potent and specific genetic interference by double-stranded RNA in

Caenorhabditis elegans. Nature. 1998 Feb 19;391(6669):806-11.

Effects of sense, antisense and mixed RNAs on progeny of Injected animals

Analysis of RNA-interference effects in individual cells

Fluorescence micrographs show progeny of injected animals from GFP-reporter strain PD4251. a–c, Progeny of animals injected with a control RNA (double-stranded (ds)-unc22A). a, Young larva, b, adult, c, adult body wall at high magnification. These GFP patterns appear identical to patterns in the parent strain, with prominent fluorescence in nuclei (nuclear-localized GFP–LacZ) and mitochondria (mitochondrially targeted GFP). d–f, Progeny of animals injected with ds-gfpG. Only a single active cell is seen in the larva in d, whereas the entire vulval musculature expresses active GFP in the adult animal in e. f, Two rare GFPpositive cells in an adult: both cells express both nuclear-targeted GFP–LacZ and mitochondrial GFP. g–i, Progeny of animals injected with ds-lacZL RNA: mitochondrial-targeted GFP seems unaffected, while the nuclear-targeted GFP– LacZ is absent from almost all cells (for example, see larva in g). h, A typical adult, with nuclear GFP–LacZ lacking in almost all body-wall muscles but retained in vulval muscles. Scale bars represent 20 mm.

Effects of mex-3 RNA interference on levels of the endogenous mRNA

Negative control

AS mex 3 RNA

Enbryo from Un injected parent

Ds mex 3 RNA

Interference contrast micrographs show in situ hybridization in embryos. The 1,262-nt mex-3 cDNA clone20 was divided into two segments, mex-3A and mex- 3B, with a short (325-nt) overlap (similar results were obtained in experiments with no overlap between interfering and probe segments). mex-3B antisense or dsRNA was injected into the gonads of adult animals, which were fed for 24 h before fixation and in situ hybridization (ref. 5; B. Harfe and A.F., unpublished observations). The mex-3B dsRNA produced 100% embryonic arrest, whereas .90% of embryos produced after the antisense injections hatched. Antisense probes for the mex-3A portion of mex-3 were used to assay distribution of the endogenous mex-3 mRNA (dark stain). four-cellstage embryos are shown; similar results were observed from the one to eight cell stage and in the germ line of injected adults. a, Negative control showing lack of staining in the absence of the hybridization probe. b, Embryo from uninjected parent (showing normal pattern of endogenous mex-3 RNA20). c, Embryo from a parent injected with purified mex-3B antisense RNA. These embryos (and the parent animals) retain the mex-3 mRNA, although levels may be somewhat less than wild type. d, Embryo from a parent injected with dsRNA corresponding to mex-3B; no mex-3 RNA is detected

SOAKING C. elegans WORKS ALMOST AS EFFECTIVELY AS INJECTING 

These images are from an experiment performed by Jeff Norman (in 1999), demonstrating the results of mex-3 in situ hybridization following an RNAi soaking protocol (for original methods, see Tabara et al. '98 Science 282: 430-31; specific protocol used in this experiment was obtained from K. Subrumaniam in Geraldine Seydoux's lab). The left panels show the wildtype pattern of endogenous mex-3 mRNA in untreated adults and embryos. The right panels show loss of mex-3 staining following soaking of L4 hermaphrodites overnight in mex-3 dsRNA. Endogenous mex-3 RNA is greatly reduced, although still faintly detectable; this experiment resulted in approximately 90% dead embryos. Although not as effective as directly injecting dsRNA, this approach is VASTLY EASIER and may be good enough for analysis of most maternally acting genes. Soaking as a delivery method, however, does NOT work for many other nematode species

2000 

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Reported processing of long dsRNA by Rnase III (Dicer) into shorter fragments of 21-23-nt intervals in Drosophila extracts Zamore et al. Drosophila Zamore et al. RNAi: double-stranded RNA directs the ATP-d . Cell. 2000 Mar 31;101(1):25-33.

RNAi Requires ATP

Denaturing agarose-gel analysis of 59-32P-radiolabeled Rr-luc mRNA incubated for the times indicated in an in vitro RNAi reaction with or without ATP, creatine phosphate (CP), or creatine kinase (CK), as indicated below each panel. Pretreated with hexokinase and glucose which converts ATP to ADP in lysate

RNAi Does Not Require mRNA Translation

A) Protein synthesis, as reflected by luciferase activity produced after incubation of RrlucmRNAin the in vitroRNAi reaction for 1 hr, in the presence of the protein synthesis inhibitors anisomycin, cycloheximide, or chloramphenicol, relative to a reaction without any inhibitor. (B) Denaturing agarose-gel analysis of 59-32Pradiolabeled Pp-luc mRNA after incubation for the indicated times in a standard RNAi reaction with and without protein synthesis inhibitors. The arrowhead indicates the position of full-length mRNA in the gel, and the bracket marks the position of stable, 59 cleavage products.

21–23 nt RNA Fragments Are Produced upon Incubation of dsRNA in Drosophila Embryo Lysate

An enlargement of the 17 to 27 nt region of a gel showing the products formed upon incubation of uniformly 32P-radiolabeled dsRNAs in lysate without and with ATP.

2001 

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Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains. C. elegans Bernstein et al. Role for a bidentate ribonuclease in the initiatio initiati Nature. 2001 Jan 18;409(6818):363-6.

2001   

First described RNAi in mammalian cells Mammals Elbashir et al. Duplexes of 21-nucleotide RNAs mediate RNA . Nature. 2001 May 24;411(6836):494-8.

2003 

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Short hairpin RNAs (shRNAs) induce sequencespecific silencing in mammalian cells. Mammals Paddison et al. Genes Dev. 2002 Apr 15;16(8):948-58 Sui et al Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5515-20 Paul et al. Nat Biotechnol. 2002 May;20(5):505-8 　

2003 

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First reported that siRNAs can be used therapeutically in whole animals. Mammals Song et al. RNA interference targeting Fas protects mice Nat Med. 2003 Mar;9(3):347-51. 　

Injection of siRNA duplex efficiently silences Fas gene expression in mouse hepatocytes. a,

Hepatocytes harvested 24 h after 3 injections of saline or Cy-5-labeled Fas(sequence Fas(sequence 1) siRNA were stained with albumin-FITC (albFITC) and analyzed by flow cytometry. A high proportion of hepatocytes take up the duplex siRNA, as indicated. b, RPA for Fas mRNA expression in hepatocytes from mice that were untreated (lane 1) or were injected 24 h earlier with saline (lane 2), GFP(sequence GFP(sequence 1) siRNA (lane 3) or Fas(sequence Fas(sequence 1) siRNA (lane 4). Silencing of Fas expression in mice treated with Fas siRNA is maintained 5 (lane 5) or 10 d (lane 6) later. Expression of other genes involved in the Fas pathway and housekeeping genes (L32 (L32 and Gapd) Gapd) were unaffected (names of the corresponding proteins are listed at right). Similar results were obtained in 3 independent experiments. The graph shows results of densitometric quantification of the Fas/ Fas/Gapdh ratios in 3 mice per condition. Fas mRNA levels in hepatocytes are significantly lower (*, P < 0.001) at all times in mice treated with Fas siRNA mice than in control mice. c, Fas immunoblot of lysates from hepatocytes obtained from untreated mice (lane 1), or 24 h after saline (lane 2), GFP siRNA (lane 3) or Fas siRNA (lane 4) injection, and 5 (lane 5) or 10 d (lane 6) after Fas siRNA injection. Mouse recombinant Fas and FasL proteins serve as positive (P) and negative (N) controls, respectively. Similar results were obtained in 3 independent experiments.

Fas gene silencing protects mice from fulminant hepatitis and hepatic fibrosis

a, Representative liver histology of ConA-induced hepatitis in mice injected with saline, GFP(sequence 1) siRNA and Fas(sequence 1) siRNA (n = 5 per group). Livers were stained with H&E 20 h after ConA injection. Original magnification 200. C. Representative liver histology 1 week after 6 weekly injections of ConA in mock-treated mice and mice injected with GFP(sequence 1) siRNA and Fas(sequence 1) siRNA (n = 3 per group). Original magnification 100. Livers of mice treated with Fas siRNA mice did not develop bridging fibrosis.

Survival advantage of mice injected with Fas siRNA as compared to saline or GFP siRNA

2004 

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First observed that siRNA silences gene at transcriptional level possibly through directing de novo DNA methylation. Human Morris et al. Small Interfering RNA-Induced Transcriptional Science. 2004 Aug 5

2004 

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First phase I clinical trial of siRNA drug for age-related macular degeneration (AMD) Human Acuity Pharmaceuticals

2006 

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Won Noble Prize in Physiology or medicine for discovering RNAi mechanism. Andrew Fire and Craig Mello

Initiation Step ATP ADP + ppi

ATP

DICER

KINASE

ADP + ppi RdRP

Effector Step • siRNA binding • siRNA unwinding • RISC activation

Dicer

siRNA 3’

5’

3’

5’

RISC • 2 RNA binding proteins • RNA/DNA Helicase • Translation Initiation Factor • RNA-Dependent RNA Polymerase (RdRP) • Transmembrane protein? C. elegans and plants – only systemic spread

Vectors expressing siRNAs U6

siRNA

H1

Vectors expressing siRNAs U6 Sense sequence

Sense sequence

Anti-sense Anti-sense sequence sequence Hair-pin loop

Vectors expressing siRNAs 

Plasmid-based vectors Transient nature  Low and variable transfection efficiency 

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Viral-based vectors Highly efficient  Better than plasmid-based 

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Pol II promoter-based plasmid vectors 

Tissue specific

siRNA Delivery 

in vitro Chemical transfection (Lipofectamine, Oligofectamine, TransIT-TKO, Siport Amine, Siport  Electroporation into parasites 

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in vivo Intramuscular injection  Hydrodynamic transfection into mammals 

Problems 

Silencing efficiency 

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Vector-based large dsRNA delivery

Systemic silencing

Therapeutic siRNAs siRNA target gene

Disease

p53 mutant K-Ras BCR-ABL MDR1 C-RAF Bcl-2 VEGF PKC-α Β-Catenin

Cancer

(Sioud, 2004)

Therapeutic siRNAs siRNA target gene

Disease

HIV-Tat HIV-Rev HIV-Vif, -Hef HPV-E6 and –E7 HBV-S1, -S2, -S, -X CCR5, CXCR4 CD4

Viral Infection

(Sioud, 2004)

Therapeutic siRNAs

siRNA target gene Fas receptor Caspase-8 TNF-α

Disease Acute Liver Failure Sepsis (Sioud, 2004)

The First Blue Rose (CSIRO)

How did they produce it?