RNA Interference

RNA Interference
Department of Animal Science
National Chung Hsing University
Pin-Chi Tang
1
Outline
RNA processing in eukaryote
Small RNA
Mechanism of RNA interference
Application of RNAi
2
The Analysis of Gene Function
Production of transgenic animals
Deletion of gene from genome
Gene targeting
RNA interference
3
The Central dogma of living Cells-1
4
RNA processing in eukaryote
5
Transcription in Eukaryotes
Biology, 6th ed., 2000
6
Transcription in Eukaryotes
Prokarytoes
Eukaryotes
a single RNA
polymerase
multiple different RNA
polymerases
RNA polymerase
binding directly to
promoter sequences
RNA polymerases need
to interact with a variety
of proteins
7
Classes of genes transcribed by eukarytoic RNA polymerase
8
Transcription in eukaryotes
Formation of a polymerase II
transcription complex
TBP: TATA binding protein
TAF: TBP-associated factors
D: TFIID
B: TFIIB
F: TFIIF
E: TFIIE
H: TFIIH
At least 5 TFs are required for
initiation of transcription by RNA
polymerase II in reconstituted in vitro
system
The Cell, 1st ed., 1997 9
Transcription in eukaryotes
Transcription of polymerase III genes
The Cell, 1st ed., 1997 10
Transcription in eukaryotes
Regulation of transcription in eukaryotes
Promoters and enhancers
Regulatory proteins
Eukaryotic repressors
Activators
Chromatin structure
DNA methylation
The Cell, 1st ed., 1997 11
Transcription in eukaryotes
Processing of rRNA
The Cell, 1st ed., 1997 12
Transcription in eukaryotes
Site of a.a. attachment
Processing of tRNA
in prokaryotes and eukaryotes
The Cell, 1st ed., 1997 13
Transcription in eukaryotes
Processing of tRNA - Modification of bases
The Cell, 1st ed., 1997 14
Transcription in eukaryotes
Aligns mRNA on the ribosome during translation
Processing of eukaryotic mRNAs
The Cell, 1st ed., 1997 15
Transcription in eukaryotes
polyadenylation signal
Stability
Translation
Regulation
Formation of the 3’ends of eukaryotic mRNAs
The Cell, 1st ed., 1997 16
Transcription in eukaryotes
Splicing of eukaryotic pre-mRNAs
The Cell, 1st ed., 1997 17
Transcription in Eukaryotes
18
Small RNA
19
RNA world
before the evolution of DNA
The discovery of catalytic RNA molecules
Non-protein-coding RNA molecules have
been identified
Acting alone
or
with proteins, such as RNPs
(ribonucleic proteins)
20
Short interfering RNAs (siRNAs)
Micro RNAs (miRNAs)
regulate gene expression
at a post-transcriptional level
in a sequence-specific manner
21
Phenotypes of chimeric CHS transgenotes and
variations among flowers on single plants
A control (parental) V26 flower is
shown along with four different CHS
(chalcone synthase) transgenotes.
Four representative flowers are
shown in a row for each of four
transgenotes, identified at the left
of each row.
Plant Cell 2:279-289, 1990
22
Heritability and variation among progeny of CHS transgenotes
back-crossed to V26 (Parental).
Plant Cell 2:279-289, 1990
23
M
15mm 30mm 40mm 53mm 58mm 58mm
Developmental pattern of expression of endogenous CHS messages
in violet flowers from control plants.
Plant Cell 2:279-289, 1990
24
Lane 1: molecular weight marker
Lane 2: the undigested probe
Lane 3: a 40-mm corolla control
RNA
Lanes 4 through 9: contain RNase
protected RNA from
corollas of 218.38 flowers
15 mm, 30 mm, 40 mm,
53 mm, and 58 mm (two
samples) in length,
respectively.
Lane 10: is a tRNA negative control
E: the endogenous CHS-protected
fragment at 96 bases
I: the introduced CHS-protected
RNA at 157 bases
P: the radiolabeled probe at 208
bases
Developmental pattern of expression of introduced and endogenous
CHS messages in white flowers of transgenote218.38.
Plant Cell 2:279-289, 1990 25
Comparison of steady-state CHS
message levels in violet and white
flowers from transgenote 218.41.
Lane 1: molecular weight standard
Lanes labeled "2“: RNase protections of
RNA isolated from three separate
40-mm-long violet revertant
corollas
Lanes labeled "3“: RNase protections of RNA
isolated from three separate 40mm-long white corollas
E: the position of the protected fragment for
the endogenous CHS transcript
I: the position of the protected fragment
for the introduced CHS transcript.
Plant Cell 2:279-289, 1990
26
The phenomenon of
post-transcriptional gene silencing
(PTGS)
•PTGS is ubiquitous in both the animal and
plant kingdoms
•PTGS is responsible for important biological
functions
•As a tool for the knocking out of gene
expression in the field of functional genomics
27
co-suppression
Tomato
Polygalacturonase gene
Fruit ripening
Tobacco plant
Transformed with -1 ,3-glucanase gene
Decrease in mRNA
C. elegans
Introduction of antisense or sense RNA into
embryos
28
Quelling
Neurospora crassa - fungus
Transformation of albino-1 gene
expected
result
Carotene biosynthesis, and
intense orange phenotype
~30 % albino
29
Discovery of miRNAs
The first miRNA was discovered in C.
elegans by Victor Ambros and his group.
They describe the identification of the
first miRNA, lin4, and report the
sequence complementarily between lin-4
and the 3’
UTR of the lin-14 mRNA.
>cel-lin-4 MIMAT0000002
UCCCUGAGACCUCAAGUGUGA
(Cell,1993. 75, 843-854)
(by Dr. J. F. Lin)
30
Discovery of miRNAs
Lin4 and Lin14 were identified in
a genetic screen for defects in
the temporal control of postembryonic development.
Disrupt of Lin4 block L1L2
Disrupt of Lin14 promote L1L2
Lin14
Lin4
Life cycle of C.
elegans
Science 1984. 226, 409
(by Dr. J. F. Lin)
31
Discovery of miRNAs
Lin4 encodes a 22-nucleotide non-coding RNA
Lin4 is partially complementary to 7 conserved sites located in 3’
UTR of Lin14
Nature 2004 5:522
32
Gene transfer technology
Potent and unexpected responses
to foreign nucleic acids
The ability of some transgenes to
silence the expression of
homologous loci
33
Discovery of miRNAs
New Insight in Gene regulation
replication
DNA
Transcription
RNA
Translation
Protein
Genomic DNA silencing
-Methylation
Transcription control
-Template recognition (TF and promoter)
RNA half-life (RNA degradation)
-PolyA protection
-RNA binding protein
Post-transcriptional regulation
-microRNA
Protein half-life (Protein degradation)
-Ubiquitination
-SUMOlation
Protein sorting
Protein-Protein Interaction
(by Dr. J. F. Lin)
34
Effects of mex-3 RNA interference on levels of the endogenous mRNA
a.
b.
c.
d.
Negative control
Embryos from uninjected parent (showing normal pattern of endogenous mex-3 RNA
Embryos from a parent injected with purified mex-3B antisense RNA
Embryos from a parent injected with dsRNA corresponding to mex-3B
Nature 391:806-811, 1998
35
zebrafish
6hpf
10hpf
14hpf
Non-injected
Injected
Microinjection of ds-ntl affects distribution of the endogenous mRNA.
Ventral views are shown for the 6 hpf embryos. The other embryos are
viewed from the dorsal side with anterior to the left. Arrowheads mark
equivalent positions of the notochord.
Biochem. Biophysiol. Res. Commun. 263:156-161, 1999
36
Double-stranded RNA can be introduced experimentally to
silence target genes interested
Nature 418:244-251, 2002 37
dsRNA triggers an interferon response
dsRNA
Activation of RPK
Inactivation of EIF2a
Activation of the 2’
, 5’oligoadenlyate synthetase
RNase L activation
Non-specific suppression of translation
Apoptosis
38
dsRNA responsive protein kinase
Mechanism of PKR-induced apoptosis
(A) PKR activation regulates translational and transcriptional pathways
resulting in the specific expression of selected proteins that triggered
cell death by engaging with the caspase pathway.
Apoptosis 5:107-114, 2000
39
Mechanism of PKR-induced apoptosis
(B) Through an unknown mechanism,
upon PKR activation, FADD recruits
procaspase 8, activating it to its active
form, caspase 8 then in turn, activates
downstream caspases such as caspase 3,
6, 7, which cleave multiple targets
triggering cell death. Role of the caspase 9
pathway in these events is unknown.
Apoptosis 5:107-114, 2000 40
transgenic mouse embryos
Injected MmGFP dsRNA
Injected c-mos dsRNA
MmGFP dsRNA specifically abrogates the expression of MmGFP
in the MmGFP transgenic embryos
Nature Cell Biology 2:70-75, 2000
41
Injected MmGFP dsRNA
Injected E-cadherin dsRNA
Injection of E-cadherin dsRNA into the zygote reduce E-cadherin
expression and perturbs the development of injected embryos
Nature Cell Biology 2:70-75, 2000
42
Western blot analysis of E-cadherin expression
Nature Cell Biology 2:70-75, 2000
43
Effect of Mos and Plat dsRNA on the relative
abundance of Mos and Plat transcripts.
1.
2.
3.
4.
5.
Uninjected oocytes
Oocytes injected with Mos dsRNA at t=20 h
Oocytes injected with Plat dsRNA at t=20 h
Oocytes injected with water at t=20 h
Uninjected oocytes at t=20 h
Development 127:4147-4156, 2000
44
Injected 106 molecules
Injected 105 molecules
1.
2.
3.
4.
5.
Oocytes injected with water
Oocytes injected with sense RNA
Oocytes injected with antisense RNA
Oocytes injected with dsRNA
Uninjected oocytes
Effect of Mos and Plat sense, antisense and dsRNA on the
relative abundance of Mos and Plat transcripts.
Development 127:4147-4156, 2000
45
A
B
Effect of Mos sense, antisense and dsRNA on MAP
kinase and MPF activities.
Oocytes were injected with 106 mol., or 105 mol. of either Mos sense, antisense
and dsRNA.
Development 127:4147-4156, 2000
46
Extracts from S2 cells transfected with
Casp9 dsRNA or Dicer dsRNA
Dicer participates in RNAi.
Nature 409:363-366, 2001
47
Biogenesis of miRNAs
Dorsha/ Dicer: RNase-III enzyme
Exportin5: Ran-GTP dependent
cargo transporter
RISC: RNA-induced silencing
complex
(Nature 2004 5:522 )
(by Dr. J. F. Lin)
48
Biogenesis of miRNAs
•Transcriptional regulation:
–Polymerase II transcription
–Three types of miRNA
•Intronic miRNAs
– Transcriptional regulated with same promoter of its host
gene
•Polycistronic cluster miRNAs
– Own promoter
•Intergenic miRNAs
– Own promoter
(by Dr. J. F. Lin)
49
Biogenesis of miRNAs
•Intronic miRNAs
(http://www.usc.edu/programs/pibbs/site/faculty/ying_s.htm)
(by Dr. J. F. Lin)
50
Mos inverted repeat
Different constructs affect the effect of RNAi
Biochem. Biophy. Res. Comm. 287:1099-1104, 2001
51
Different constructs affect the effect of RNAi
Biochem. Biophy. Res. Comm. 287:1099-1104, 2001
52
How does RNAi work?
Genetic and biochemical data indicate a
possible two-step mechanism for RNA
interference (RNAi):
an initiation step
an effector step
53
Guide sequences
A model for the mechanism of RNAi
http://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.html
Nature Reviews Genetics 2: 110-119, 2001
54
A model for the mechanism of RNAi from siRNA
55
miRNA processing and RNAi in mammals.
Gene Therapy 13:478-486, 2006
56
The RNAi pathway.
RISC: RNA induced silencing
complex
miRNP: RISC-like
ribonucleoprotein particles
57
Actions of miRNAs
Precursor miRNA
Dicer
21-23nt miRNA
Inhibit Translation
Cleavage of target mRNA Deadenylation of target mRNA
(by Dr. J. F. Lin)
58
A model for the mechanism of RNAi from miRNA
59
Application of RNAi
60
Design of dsRNA triggers
 Avoid regions of the mRNA which might bind RNA
regulatory proteins, such as 5’and 3’UTR;
UTR
 Avoid regions close to the start site (<100 nt):
between +100 (AUG as +1) to the stop codon.
codon
 23 nt, the consensus 5’
-AA[N19]UU-3’.
 Sequence of <70% , >30% GC , ideally 50%
 Avoid highly G-rich
 End with two 3’2-deoxythymidine residues
 Select 3-6 sequences per gene
 Perform a BLAST
61
Delivery of dsRNA triggers
 Transfection reagents
 Retroviral integration
 Transposon hopping
 Homologous recombination
 Random plasmid integration
 Feeding
62
The applications of RNAi
‧Investigation of gene function
high-throughput genetic screen
‧Potential therapeutic tool
63
RNA silencing pathways.
Nature Biotech. 21:629-630, 2003 64
Target sites for HBV transcripts
Schematic of U6 promoter
constructs
Predicted folding of HBVU6no.2
Nature Biotech. 21:639-644, 2003
65
HBsAg measurements in medium of shRNA-treated cultured cells
Nature Biotech. 21:639-644, 2003
66
Potential applications of RNAi in mammalians
Cancer Cell 2:17-23, 2002
67
The potential uses of RNAi in ES cells.
Gene Therapy 13:478-486, 2006 68
Schematic diagram to illustrate siRNA targets important for tumor-host interaction.
siRNA technology can be used to target molecules that are important for tumor
angiogenesis, invasion, metastasis and immune evasion.
Gene Therapy 13:464-477, 2006
69