What is RNA interference?

Shooting down mRNA

BackgroundBackgroundWhat is it?What is it?Why use it?Why use it?The mechanism and processThe mechanism and process

RNAiRNAi

Plasmid

Virus

Jorgensen 1990van der Krol 1990

Gene injection (pigmentationEnzyme-petunias)Expectation: more red colorCo-suppression of transgene and endogenous gene.

Bill Douherty and Lindbo 1993Gene injection with a complete tobaccoetch virus particle.Expectation: virus expressionCo-suppression of transgene and virus particles via RNA.

Hamilton and Baulcombe 1998Identification of short antisense RNA sequences dsRNA?How?

Fire and Mello 1998Injection of dsRNA into C. elegansRNA interference (RNAi) or silencing

Ambros 1993 (2000)Identification of small RNA in C. elegans (micro RNA)

Shooting mRNA means RNA interference

What is RNA interference?

--Gene “knockdown”

--A cellular mechanism that degrades unwanted RNAs in the cytoplasm but not in the nucleus. Why?

--A way for the cell to defend itself.

Why use RNAi?

1. The most powerful way to inhibit gene expression and acquire info about the gene’s function fast

2. Works in any cell/organism

3. Uses conserved endogenous machinery

4. Potent at low concentrations

5. Highly specific.

The mechanism of small interfering RNAs (siRNAs)

What happens?

dsRNA is processed into shorter units (siRNAs) that guide the targeted cleavage of homologous

RNA.

RNA interference:

--A type of gene regulation

--Involve small RNA molecules

--Induce a double stranded RNA

The RNAi process

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

Gene regulation by small RNAs

Small temporal(St) RNAs prevent translation to stop gene expression quickly

siRNAs degrade mRNAto stop gene expression quickly

Dicer gene in C. elegans

--MicroRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression (down-regulation). --miRNAs are non-coding RNAs ( no proteins are made)--Stem-loop or hairpin loop intra-molecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA.

-your RNAi?

http://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.htmlhttp://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.html

BCR-ABL

RNAi

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Philadelphia chromosomechronic myelogenous leukemia (CML)acute lymphoblastic leukemia (ALL)

BCR

ABL BCR-ABL

ABL=tyrosine kinase activity BCR=substrate