RNA interference

Presented by:

C.SWORNA KUMARI

M.PHIL BIOTECHNOLOGY

Outlines

Introduction Introduction RNA silencing

Definition of RNA interference

Discovery of RNAi

Mechanism of RNA interferenceMechanism of RNA interference

Applications of RNA interferenceApplications of RNA interferenceTherapeutic applications

Other applications

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

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.

What is RNA interference (RNAi)?

“The Process by which dsRNA silences gene expression...” Degradation of mRNA or translation inhibition

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

Nobel prize winners in the C. elegans field Sidney Brenner

John Sulston

Robert Horvitz

Andrew Fire

Craig Mello

RNAi was found to work in many diverse species

Fungi

Trypanosomes

Insects

Zebrafish

Mice

Mechanism of RNAi

RNA i( RNA Interference )

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

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

Generation of small interference RNA

miRNA

Originate from capped & polyadenylated full length precursors (pri-miRNA)

Hairpin precursor ~70 nt (pre-miRNA) Mature miRNA ~22 nt (miRNA)

Illustration of miRNA processing

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)

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

Dicer’s domains

1 4 32 2

Dicer is a ribonuclease (Rnase III family) with 4 distinct domainsDicer 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.

RNA i( RNA Interference )

STEPS INVOLVED IN RNA INTERFERENCE

RNA interference

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing (PTGS), and quelling.

STEP 1

• dsRNA is processed into sense and antisense RNAs • 21-25 nucleotides in

length • have 2-3 nt 3’ overhanging

ends • Done by Dicer (an RNase

III-type enzyme)

Step 2

The siRNAs associate

with RISC (RNA-

induced silencing

complex) and

unwind

Step 3

the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs.

Step 4

RISC cuts the mRNA approximately in the middle of the region paired with the siRNA

The mRNA is degraded further

What are sense and antisense RNA?

Messenger RNA (mRNA) is single-stranded, called "sense" because it results in a gene product (protein).

5´   C U U C A  3´     mRNA3´   G A A G U  5´     Antisense RNA

What are sense and antisense RNA?

Antisense molecules interact with complementary strands of nucleic acids, modifying expression of genes.

5´   C U U C A  3´     mRNA3´   G A A G U  5´     Antisense RNA

Applications of RNAi

RNA i( RNA Interference )

Therapeutic uses of RNAi

Hematology (blood)

Oncology (cancer)

Stem cell biology

Infectious diseases

Hematology (blood)

Hematologic disorders result fromLoss of gene function

Mutant gene function

Absent gene function

RNAiMay be used to create models of disease processes

Could help to develop pharmacologic and genetic therapeutic targets

Oncology (cancer)

Targeting of oncogenesDominant 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

Stem cell biology

Mouse researchKnock 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

Infectious Diseases

Virus targetingRNAi – inhibit cellular and viral factors of disease

RNA transcriptase is RNAi target

Inhibition of replication

Main goalRender cells resistant to infectious organisms

Hepatitis C

Infects ~200 million people worldwide

Often fatal

2002, Anton McCaffrey and Mark Kay at Stanford University

Injected "naked" RNA strands into the tail veins of mice

RNAi treatment controlled the virus in mice

Silencing genes in HIV

AIM:

Silence the main structural protein in the virus, p24, and the human protein CD4.

Hit the virus where it counts by eliminating a protein it needs to reproduce or cause infections.

Respiratory infections

RSV ( RESPIRATORY SYNCTIAL VIRUS), infects almost every child by the age of two

Short strands of "naked" RNA

Controlled the virus in mice

Clinical trials are ongoing

Macular degeneration

Macular degeneration is the leading cause of adult blindness

Excess VEGF which leads to sprouting of excess blood vessels behind the retina & obscuring vision.

The new RNAi drugs shut down genes that produce VEGF. The drug can be injected directly into the eyeFirst clinical trial: 24 patients, launched in 2004. Two months after being injected with the drug, 6 of the patients had significantly clearer visionOther patients' vision had at least stabilized More extensive trials are ongoing

Huntington’s disease

Ideal candidate for RNAi therapy

Disease caused by protein, that affects more than 30,000 people in the U.S. alone.

We would want to shut down the expression of the gene coding for the abberant protein

2004, Beverly Davidson and colleagues at the University of Iowa Davidson treated mice with Huntington's

Other uses of RNAi

Testing Hypotheses of Gene Function

Target Validation

Pathway Analysis

Studying cell division

Gene Redundancy

Functional Screening

Gene Redundancy

In many cases, eliminating the expression of a single gene in higher eukaryotes can be tolerated even if that gene product functions in a critical pathway. This is because many critical cell functions are accomplished by more than one gene product.

When one gene product is eliminated, the redundant gene product compensates to allow the cell or animal to survive. Identifying redundant genes could be achieved by co-transfecting siRNAs and assaying for a given phenotype.

Evaluating each of the candidate genes alone to ensure that they only cause the cell cycle defect when reduced in combination with the target gene would help pinpoint the most likely redundant gene

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

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

Advantage of RNAi

Downregulation of gene expression simplifies "knockout" analysis.

Easier than use of antisense oligonucleotides. siRNA more effective and sensitive at lower concentration.

Cost effective

High Specifity

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

Importance of RNAi

Powerful for analyzing unknown genes in sequenced genomes.

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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