1. What is RNA interference?
Shooting down mRNA

2. RNAi
Background
What is it?
Why use it?
The mechanism and process

3. Plasmid
Virus

5. Bill Douherty and Lindbo 1993 Hamilton and Baulcombe 1998
Jorgensen 1990
van der Krol 1990
Gene injection (pigmentation
Enzyme-petunias)
Expectation: more red color
Co-suppression of transgene
and endogenous gene.
Bill Douherty and Lindbo 1993
Hamilton and Baulcombe 1998
Gene injection with a complete tobacco
etch virus particle.
Expectation: virus expression
Co-suppression of transgene
and virus particles via RNA.
Identification of short antisense RNA
sequences
dsRNA?
How?
Ambros 1993 (2000)
Fire and Mello 1998
Identification of small RNA in
C. elegans (micro RNA)
Injection of dsRNA into C. elegans
RNA interference (RNAi) or silencing

6. Shooting mRNA means RNA interference

7. 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.

8. 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.

9. The mechanism of small interfering RNAs (siRNAs)
What happens?
dsRNA is processed into shorter units (siRNAs) that guide the targeted cleavage of homologous RNA.

10. The RNAi process RNA interference: --A type of gene regulation
--Involve small RNA molecules
--Induce a double stranded RNA
The RNAi process

11. 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)

12. Step 2 The siRNAs associate with RISC (RNA- induced silencing
complex) and
unwind

13. Step 3
the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs.

14. Step 4
RISC cuts the mRNA approximately in the middle of the region paired with the siRNA
The mRNA is degraded further

15. Gene regulation by small RNAs
Dicer gene in C. elegans
siRNAs degrade mRNA
to stop gene expression
quickly
Small temporal(St) RNAs
prevent translation to
stop gene expression quickly

16. -your RNAi?
--MicroRNAs (miRNA) are single-stranded RNA molecules of about 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.

18.
BCR-ABL
RNAi

19. Philadelphia chromosome
chronic myelogenous leukemia (CML)
acute lymphoblastic leukemia (ALL)
ABL
BCR-ABL
BCR
ABL=tyrosine kinase activity
BCR=substrate