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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