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in Gene Expression
4. RNA interference
wt Pol II
(α-amanitin sensitive)
hC4 Pol II (R749H)
(α-amanitin resistant)
CPSF binds to TFIID; both CPSF and CstF also bind to CTD
Co-transcriptional capping
Co-transcriptional spliceosome
assembly
Co-transcriptional polyadenylation.
Transcriptional termination
enhanced by polyadenylation
4. RNA interference
Pol II
TREX
UAP56
3’ss Aly
Spliceosome
Cap
5’ss
EJC
Cap Aly (A)n
TAP
Nucleus
Cytoplasm
Topics
4. RNA interference
3’UTR UAA
Initiation
ATG
5' UTR
CAP
4E
Elongation
Recycling 4G
STOP
3' UTR
mRNA decay pathways in eukaryotic cells
Decapping 3’ to 5’ decay
mG
7
AUG UAA AAAoligo
Decapping
(Dcp1p) 3’ to 5’ decay
m7G 5’ to 3’ decay
Dcp1p
-
+
Mrt1p Mrt3p Pab1p
Rep42 Rrp4
Rrp45 Rrp41
Rrp44 Csl4
Rrp46 Mtr3
KSRP TTP
3’-5’ degradation
van Hoof and Parker, Cell 99:347-350, 1999
Nonsense-mediated RNA Decay: Where does It Occur?
Exon-exon junction
EJC
D = 20-24 nts
NMD and Pilot Translation
Upf Upf
Cytoplasm
Upf Upf stop Upf stop Upf stop
Ribosome
stop Dcp1 stop Upf stop
Translation Degradation
Topics
4. RNA interference
Sense/viral mRNA
RdRP (present in plants and nematodes
but not in fly and animal cells)
dsRNA
Dicer
siRNA
AGO2
RISC*
AGO1
P
mRNA
Transitive RNAi in Plants and Nematodes
Binding
and activation
Phosphorylation Activation
eIF2α RNase L
19-nt duplex
Tuschl 2001
G & D 15:188
Silencing of Nuclear Envelope Proteins
Lamin A/C in Hela Cells by Synthetic RNAi
Iamin A/C GL2 Pp-luc
siRNA siRNA Buffer
a bc
de f
g hi
k
55K vimentin
45K
• Synthetic siRNA
• Expression of hairpin from a
pol III promoter
• Improved delivery: transfection or
Infection with Adeno, AAV, &
Retroviruses
• In vitro transcribed dsRNA and
Then processed with Dicer
4. RNA interference
Tetrahumena
Two classes of small RNAs Cell 2002
EMBO 2002 in silencing plants: 110:689; 701
21:4671
111:803
21-22 nt - siRNA
24-26 nt - shRNA
S. pombe
Science 297 (2002)
1831; 1833; 2233
Summary of Lecture 2
7. Small RNA is new paradigm for gene expression and regulation and
functions in a variety of pathways.