Origins and Mechanismsof miRNAs and siRNAs
Present by: Mozhdeh Mirahadi1
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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Outlines
• IntroductionIntroduction • RNA silencing • Definition of RNA interference• Mechanism of RNA interferenceMechanism of RNA interference• Argonaute• siRNAs; Sources of siRNA Precursors• RISC• Posttranscriptional Silencing by siRNAs• MicroRNAs• MicroRNA Biogenesis• Posttranscriptional Repression by miRNAs• ConclusionConclusion 3
Introduction
RNA i( RNA Interference )
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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 MechanismDefense against Infection by viruses, etc
As a defense mechanism to protect against transposons and other insertional elements
Genome Wide RegulationRNAi plays a role in regulating development and genome maintenance.
30% of human genome regulated
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RNAi:Silencing in Cenorhabditis elegans
dsRNA administrated to worms can permeate and affect the entire body causing a systemic RNA-interference
RNAi studies represents a means of identifying partial or complete loss-of-function phenotypes, possibly leading to the identification of gene function.
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Cenorhabditis elegans
RNAi can be induced in C. elegans in three simple ways:Injection of dsRNA into the worm gonads
Soaking the worms in dsRNA solution
Feeding the worms engineered bacteria producing dsRNA
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Mechanism of RNAi
RNA i( RNA Interference )
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The Players In Interference
RNAsiRNA: dsRNA 21-22 nt.
miRNA: ssRNA 19-25nt. Encoded by non protein coding genome
RISC: RNA induced Silencing Complex, that cleaves mRNA
EnzymesDicer : 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 termini• b. 3’-hydroxyl termini• c. 2/3-nucleotide 3’ overhangs
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siRNA design
21-23nt
2-nt 3' overhangs ( UU overhangs )
G/C content: 30-50%.
No basepair mismatch
Synthesised siRNA should not target introns, the 5′and 3′-end untranslated regions (UTR), and 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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Generation of small interference RNA
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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 siRNA
Function 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.
And also miRNA help to regulate gene expression, particularly during induction of heterochromatin formation serves to downregulate genes pre- transcriptionally (RNA induced transcriptional silencing or RITSRITS)
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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 ribonuclease
Enzyme 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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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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Summary of Players
Drosha and Pasha are part of the “Microprocessor” protein complex (~600-650kDa)
Drosha and Dicer are RNase III enzymes
Pasha is a dsRNA binding protein
Exportin 5 is a member of the karyopherin nucleocytoplasmic transport factors that requires Ran and GTP
Argonautes are RNase H enzymes
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Mechanism of RNA interference
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Mechanism of RNA interference
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Figure 1. Core Features of miRNA and siRNA SilencingFigure 1. Core Features of miRNA and siRNA Silencing 22
Argonaute: At the Core of RNA Silencing
The Argonaute superfamily can be divided into three separate subgroups:
the Piwi clade that binds piRNAs,
the Ago clade that associates with miRNAs and siRNAs,
third clade that has only been described thus far in nematodes.
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Figure 2. A Diversity of siRNA SourcesFigure 2. A Diversity of siRNA Sources 24
RISC Assembly and siRNA Strand Selection
Although single-stranded siRNAs can load directly into purified Argonaute proteins, the double-stranded siRNAs that are generated by Dicer cannot and rely instead upon siRISC assembly pathways (Figure 2).
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Figure 3. Mechanisms of siRNA Silencing26
Amplification of siRNA
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siRNAs Can Induce Heterochromatin Formation
siRNAs are not restricted to posttranscriptional modes of repression. In 2002, siRNAs were shown to induce heterochromatin formation in S. pombe, consistent with earlier reports of transcriptional gene silencing (TGS) in plants.
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Illustration of miRNA processing
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Another View
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MicroRNA Biogenesis
MicroRNAs in the plant and animal (Figure 4)
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Figure 4. Biogenesis of miRNAs and Assembly into miRISC in Plants and Animals
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MicroRNA Associations
miRNA strand
miRNA* strand
In Drosophila
in humans, C. elegans, and Drosophila indicates
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Posttranscriptional Repression by miRNAs
The miRNA acts as an adaptor (Figure 5)
The degree of miRNA-mRNA complementarity has been considered a key determinant of the regulatory mechanism.
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Figure 5. Possible Mechanisms of miRISC-Mediated Repression35
Conclusions
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
The effect is non-stoichiometric; small amounts of dsRNA can wipe out an excess of mRNA (pointing to an enzymatic mechanism)
ssRNA does not work as well as dsRNA
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Any guestion?
Thank you!
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