RNA interference
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Transcript of RNA interference
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RNA interference (RNAi)
MBB601 Advances in Plant Molecular Biology
Presented ByMr. Ekatpure Sachin
2016-21-022
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Outline• Introduction
-RNA silencing-Definition of RNA interference-Discovery of RNAi
• Mechanism of RNA interference• Generation of small interfering RNA• Small interfering RNA delivery methods• Applications of RNA interference
-Therapeutic applications-Other applications
• Conclusion
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Introduction
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RNA silencing
• Several terms are used to described RNA silencing; usually there are three phenotypically different but mechanistically similar phenomena:
1. Cosuppression or post-trascriptional gene silencing (PTGS) in plants
2. Quelling in fungi
3. RNA interference in animal kingdom
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Definition
• RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes
• RNAi targets include RNA from viruses and transposons
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Need for interference
• Defense Mechanism-Defense against Infection by viruses, etc-As a defense mechanism to protect against transposons and other
insertional elements
• Genome Wide Regulation-RNAi plays a role in regulating development and genome maintenance -30% of human genome regulated
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PTGS in plants: The discovery of Jorgensen and Napoli in 1990
• They were trying to make petunias more purple • Overexpression of petunia gene• Entered homologous RNA
Expected:more pigments
Observed:white sectors
Cosuppression:
Loss of mRNAs of both endo-and transgene
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Andrew Fire Craig C. Mellow
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Discovery
Inject worms with dsRNA corresponding to a gene (important
for muscle function) involved in wiggling (unc-22)
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Conclusion: dsRNA triggers potent and specific gene silencing
Inject worms with dsRNAcorresponding to a gene (important for muscle function)involved in wiggling (unc-22)
Discovery
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Mechanism of RNAi
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RNAi Overview• During RNAi Double-stranded RNAs cut into short double-stranded RNAs,
s(small) i(interfering) RNA's, by an enzyme called Dicer
• These then base pair to an mRNA through a dsRNA-enzyme complex. This will either lead to degradation of the mRNA strand
• Highly specific process• Very potent activity• So far only been seen in eukaryotes• Evidence 30% of genome is regulated by RNAi
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The components In Interference
• RNA-siRNA: dsRNA 21-22 nt.-miRNA: ssRNA 19-25nt. Encoded by non protein coding genome
• RISC: -RNA induced Silencing Complex, that cleaves mRNA
• Enzymes-Dicer : produces 20-21 nt cleavages that initiate RNAi-Drosha : cleaves base hairpin in to form pre miRNA; which is later
processed by Dicer
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siRNAs• Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNAi), a
form of post-transcriptional gene silencing
• RNAi: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradation
• A single base pair difference between the siRNA template and the target mRNA is enough to block the process.
• Each strand of siRNA has:a. 5’-phosphate terminib. 3’-hydroxyl terminic. 2/3-nucleotide 3’ overhangs
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miRNA• Originate from capped & polyadenylated full length precursors (pri-miRNA)
• Hairpin precursor ~70 nt (pre-miRNA) Mature miRNA ~22 nt (miRNA)
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Difference between miRNA and siRNAFunction of both species is regulation of gene expression.Difference is in where they originate.
siRNA originates with dsRNA.siRNA is most commonly a response to foreign RNA (usually viral) and is often 100% complementary to the target.
miRNA originates with ssRNA that forms a hairpin secondary structure.miRNA regulates post-transcriptional gene expression and is often not 100% complementary to the target.Also miRNA help to regulate gene expression, particularly during induction of heterochromatin formation serves to downregulate genes pre- transcriptionally (RNA induced transcriptional silencing or RITS)
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Dicer
• Loss of dicer→loss of silencing processing in vitro• Dicer homologs exist in many organisms including C.elegans,
Drosphila, yeast and humans (Dicer is a conserved protein)
RNase III-like dsRNA-specific ribonucleaseEnzyme involved in the initiation of RNAi. It is able to digest dsRNA into uniformly sized small RNAs (siRNA)
Dicer family proteins are ATP-dependent nucleases.Rnase III enzyme acts as a dimer
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Dicer’s domains
1 4 32 2
Dicer is a ribonuclease (Rnase III family) with 4 distinct domains:
1. Amino-terminal helicase domain
2. Dual Rnase III motifs in the carboxy terminal segment
3. dsRNA binding domain
4. PAZ domain (110-130 amino-acid domain present in protein like Argo, Piwi..);it is thought to be important for protein-protein interaction
RISC• RISC is a large (~500-kDa) RNA-multiprotein complex,
which triggers mRNA degradation in response to siRNA
• Unwinding of double-stranded siRNA by ATP independent helicase
• The active components of an RISC are endonucleases called argonaute proteins which cleave the target mRNA strand
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Illustration of miRNA processing
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Generation of siRNA’s
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Generation of small interference RNA
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siRNA Expression Vectors
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siRNA design• 21-23nt• 2-nt 3' overhangs ( UU overhangs )• G/C content: 30-50%.• No base pair mismatch
Synthesized siRNA should not target introns, the 5 and 3 -end untranslated regions (UTR)′ ′Sequences within 75 bases of the start codon (ATG)
BLAST : eliminate any target sequences with significant homology to other coding sequences.
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siRNA delivery methods
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• Effective methods for the delivery of small RNA to allow a sufficient silencing effect in the target organ(s) and/or cells are yet to be developed
• In particular, toxicity and side effects of RNAi must be well characterized and limited
• Therefore, careful design and selection of target sequence and quantification of the effect on the expression of target protein and mRNA are essential for success of gene interfering approaches
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High-pressure injection
“High-pressure injection” was the first strategy to demonstrate successful delivery of siRNA in vivo
A large volume (1–2mL) of saline containing unmodified siRNA is injected intravenously into the tail vein of mice within very short time (in less than 7 sec), which presumably results in the siRNA molecules being forced into several organs mainly the liver, kidney and to a lesser degree the lung
Certainly, such an approach seems to be impossible in human subjects (1000 mL saline solution containing siRNA per 10 kg of weight)
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• Delivery of siRNA to tissue is a problem both because:
– The material must reach the target organ –And it must enter the cytoplasm of target cells
• RNA cannot penetrate cellular membranes, so systemic delivery of siRNA is unlikely to be successful
• RNA is quickly degraded by RNAse activity in serum and even siRNA chemically modified to be more stable has a half-life of only a few hours at most
Problem
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• For these reasons, other mechanisms to deliver siRNA to target cells has been devisedThese methods include:
• Viral delivery • The use of liposomes or nanoparticles • Bacterial delivery• Chemical modification of siRNA to improve stability
Solution
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Viral delivery• Viral delivery has been used extensively in gene therapy to deliver DNA to target
cells • There are 5 main classes of viruses used in the delivery of nucleotides to cells:
• Retrovirus • Adenovirus • Lentivirus• Baculovirus• Adeno-associated-virus (AAV).
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Liposome's and nanoparticles• Liposomes and nanoparticles have been known as an alternative to viral delivery systems.
• Unmodified siRNA has a half-life of less than 1 hour in human plasma and siRNA is rapidly excreted by the kidneys.
• Liposomes and nanoparticles can act as envelopes to protect the siRNA from metabolism and excretion, but can also carry specific molecules designed to target the siRNA to specific tissue types
• Liposomes such as Lipofectamine have been used to carry siRNA into cells• Nanoparticles such as the cationic polymer, polyethyleneimine (PEI) have also been used to
successfully deliver siRNA to target cells
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Bacterial delivery• Bacterial delivery using nonpathogenic bacteria has been used to silence genes in a
process known as transkingdom RNA interference (tkRNAi)
• Generally, the shRNA is produced in bacteria that invade and release the RNA into eukaryotic cells (hence the term transkingdom)
• The bacteria can also be engineered to carry shRNA encoding DNA plasmids
• The advantages of this system include:
-Safety-Ability to control the vector using antibiotics
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Chemical modification• Finally, chemical modification of siRNA has been used to improve stability and prevent
degradation by serum RNAase
• Importantly, these modifications must obviously not affect the RNA interference activity of the siRNA
• One of the most common modifications is the use of locked nucleic acid residues (LNA).
A methylene bridge connects the 4.C with the 2.O in LNA residues. This modification increases the stability of oligonucleotides in serum, without reducing the gene silencing effect
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Applications of RNAi
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Hematology (blood)
• Hematologic disorders result from– Loss of gene function– Mutant gene function– Absent gene function
• RNAi– May be used to create models of disease processes– Could help to develop pharmacologic and genetic therapeutic targets
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Oncology (cancer)
• Targeting of oncogenes– Dominant mutant oncogenes, amplified oncogenes, viral
oncogenes• Define role of signaling molecules in tumor-creation• Improvement efficacy of chemotherapy and radiotherapy• Tumor regression through creation of potentially new mode
of chemotherapy
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Stem cell biology
• Mouse research-Knock out tumor-suppression gene in mouse embryonic stem cell
-Observe tumor phenotype
-Positive correlation between extent of Trp 53 (suppression gene) inhibition and severity of disease
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Infectious Diseases
• Virus targeting-RNAi – inhibit cellular and viral factors of disease-RNA transcriptase is RNAi target-Inhibition of replication
Main goal-Render cells resistant to infectious organisms
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Applications of RNA interference in crop improvement
Long et al., 2011, Genetics 189: 1093–1102.
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Conclusion of RNA i
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RNA interference characteristics
• dsRNA needs to be directed against an exon, not an intron in order to be effective
• Homology of the dsRNA and the target gene/mRNA is required
• Targeted mRNA is lost (degraded) after RNAi
• ssRNA does not work as well as dsRNA
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Advantage of RNAi• Down regulation of gene expression simplifies "knockout" analysis.• Easier than use of antisense oligonucleotides. siRNA more effective and sensitive
at lower concentration.• Cost effective • High Specificity • Middle region 9-14 are most sensitive• With siRNA, the researcher can simultaneously perform experiments in any cell
type of interest• Can be labelled• Ease of transfection by use of vector
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Importance of RNAi • Powerful for analyzing unknown genes in sequenced genomes.
• More efforts are being undertaken to target every human gene via siRNAs
• Faster identification of gene function
• Gene therapy: down-regulation of certain genes/ mutated alleles
• Cancer treatments– knock-out of genes required for cell proliferation– knock-out of genes encoding key structural proteins
• Agriculture
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Thank You
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