Complex Network of Coupled Interactions

in Gene Expression

T. Maniatis & R. Reed, Nature 416:499-506, 2002

 Topics

1. Coupling between transcription and alternative splicing

2. Coupling between splicing and RNA export

3. Regulation of RNA stability

4. RNA interference

5. MicroRNAs expression, processing and function in the

regulation of gene expression

 Evidence for Co-transcriptional Splicing

EM analysis of chromosomal spreads

Biochemical analysis of chromosomal attached and released RNA

Identification of SR-like proteins as CTD binding proteins

Colocalization of phosphorylated Pol II in nuclear speckles

CTD dependent recruitment of splicing factors to nascent transcripts

Enhancement of in vitro splicing by phosphorylated Pol II

Promoter-dependent alternative splicing

Proudfoot, N.J. TIBS 25:290-293, 2000

Electron Microscopic Analysis of Chromosomal Spreads:
Evidence for Co-transcriptional Splicing

Beyer and Osheim, Genes Dev. 2:754-765, 1988

 Targeting of Splicing Factors
to Nascent Transcripts Depends
on Pol II CTD

Cell lines were constructed to

express wt and CTD-truncated
α-amanitin-resistant Pol II

Endogenous Pol II was inhibited by

α-amanitin

Nascent transcripts (pem) was

detected by in situ hybridization

Splicing factors were localized by

using specific antibodies

Results show that CTD is required

for the recruitment of splicing factors
to the site of transcription

Misteli and Spector, Mole. Cell 3:679-705, 1999

Promoter-dependent Alternative Splicing

Cramer et al., Mole. Cell 4: 251-258, 1999

 Slow Pol II Induces Alternative Splicing

wt Pol II
(α-amanitin sensitive)
hC4 Pol II (R749H)
(α-amanitin resistant)

Mata et al., Mole. Cell 12:525-532, 2003

 Evidence for Co-transcriptional
Capping and Polyadenylation

Nascent transcripts at ~30 nts after initiation are capped

Quanyl transferase and methylase both bind to phopshorylated CTD

CPSF binds to TFIID; both CPSF and CstF also bind to CTD

Deletion of CTD inhibits polyadentylation in transfected cells

Enhancement of in vitro 3’ cleavage by CTD

Poly A signal is required for transcriptional termination

Proudfoot, N.J. TIBS 25:290-293, 2000

Model for Coupling Transcription to mRNA Processing

Co-transcriptional capping

Co-transcriptional spliceosome
assembly

Co-transcriptional polyadenylation.

Transcriptional termination
enhanced by polyadenylation

N. Proudfoot, TIBS 25:290-293, 2000

 Topics

1. Coupling between transcription and alternative splicing

2. Coupling between splicing and RNA export

3. Regulation of RNA stability

4. RNA interference

5. MicroRNAs expression, processing and function in the

regulation of gene expression

Splicing-derived mRNAs are More Efficiently Exported

A. Spliced mRNA is exported more

efficiently in injected Xenopus
oocytes.

B. Isolation of RNP complex containing

spliced mRNA or a mRNA
assembled in extracts.

C. Purified mRNP is more efficiently

exported than assembled
mRNA-protein complexes.

Luo and Reed, PNAS 96:14937-14942, 1999

Couple Splicing to mRNA Export

Pol II

TREX

UAP56
3’ss Aly
Spliceosome
Cap
5’ss

EJC
Cap Aly (A)n
TAP
Nucleus

Cytoplasm
 Topics

1. Coupling between transcription and alternative splicing

2. Coupling between splicing and RNA export

3. Regulation of RNA stability

4. RNA interference

5. MicroRNAs expression, processing and function in the

regulation of gene expression

 Circular mRNA in vivo

eIF4F (= eIF4E, eIF4A, and eIF4G)

PABP

AAAAAA m7Gppp 5’UTR AUG

3’UTR UAA

Implications: PolyA binding protein is required for efficient translation

RNA decay in many cases are translation-dependent
Deadenylation will result in decapping

Tharun and Parker, 1997

Model for translation stimulation by 5'-3' interactions

Initiation

ATG
5' UTR

CAP
4E
Elongation
Recycling 4G

3 PABP PABP PABP

eRF
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
eRF1

STOP

3' UTR
 mRNA decay pathways in eukaryotic cells

m7G AUG UAA AAAAAAAAAlong

Deadenylation-indep. Endonucleolytic
decapping Cleavage (Rnase L ?)
m7G PolyA shorting
(PAN)
AUG UAA UAA AAAAAAAAAlong m7G AUG UAA AAAAAAAAAlong

Decapping 3’ to 5’ decay
mG
7
AUG UAA AAAoligo

Decapping
(Dcp1p) 3’ to 5’ decay
m7G 5’ to 3’ decay

AUG UAA AAAoligo

5’ to 3’ decay m7G AUG UAA

(Xrn1p)

AUG UAA AAAoligo

Tharun and Parker, 1997

 Decapping Mechanisms

A. Positive and negative effect on the decapping enzyme

Dcp1p
-
+
Mrt1p Mrt3p Pab1p

m7Gppp AUG UAA AAAAAAlong

B. Nonsense-mediated recruitment of the decapping enzyme

Dcp1p + Upf1 Upf2

Upf3
AUG UAA UAA AAAAAAlong
ppp
m 7G

Tharun and Parker, 1997

 The Exosome and its Target

Rep42 Rrp4

Rrp45 Rrp41

Rrp43 3’- 5’ Rrp40

exoriboucleases Chen, C-Y., et al., Cell 107:451-464, 2001

Rrp44 Csl4
Rrp46 Mtr3

KSRP TTP

m7Gppp ARE AAAAAA

3’-5’ degradation
van Hoof and Parker, Cell 99:347-350, 1999
 Nonsense-mediated RNA Decay: Where does It Occur?

Stops at position 39, 60-61, but

not at 101 and 141, render
mRNAs unstable.

NMD is detectable in both the

nucleus and the cytoplasm

Zhang, J. et al., RNA 4:801-815, 1988

Nonsense-mediated RNA Decay: The 50-55 nt Rule

Zhang, J. et al., RNA 4:801-815, 1988

 Exon Junction Complex and Non-
sense Mediated RNA Decay
A. The 50-55 nts rule for NMD
Stop Exon-exon junction

D: > 50-55 nts: NMD

< 50-55 nts: no effect

B. The 20-24 nts rule for post-splicing marker

Exon-exon junction

EJC

D = 20-24 nts
 NMD and Pilot Translation
Upf Upf

Cap EJC EJC (A)n

TAP TAP
Nucleus

Cytoplasm
Upf Upf stop Upf stop Upf stop

Cap EJC EJC (A)n Cap EJC EJC (A)n

Ribosome
stop Dcp1 stop Upf stop

Cap (A)n Cap EJC (A)n

Translation Degradation
 Topics

1. Coupling between transcription and alternative splicing

2. Coupling between splicing and RNA export

3. Regulation of RNA stability

4. RNA interference

5. MicroRNAs expression, processing and function in the

regulation of gene expression

 Tiny RNAs

siRNA: small interfering RNAs

miRNA: microRNAs
shRNA: small heterochromatic RNAs
 Amazing Effects of Tiny RNAs: A Brief History

• Post-transcriptional gene silencing in plant (1990)

• Induction of gene silencing by both sense and antisense
RNA in C. elegans (1995)
• More effective gene silencing by using dsRNA (1995)
• Interference of gene expression with synthetic siRNA (2001)
• Discovery of key factors in the RNAi pathway (Dicer, 2001)
• RNAi effect persistent across cells and animal generations
• Expression of many non-coding miRNAs in animal and plants
• miRNA and shRNA function in translational control, viral defense,
RNA decay, genomic stability, chromatin remodeling……
Post-transcriptional gene silencing (PTGS)
RNA interference (RNAi)

Sequence-specific RNA degradation

RNA silencing

Plant Cell 2:279-290, also 2:291-299, 1990

Detection of small RNAs in silencing plants

Andrew Hamilton Science 286:950

& Baulcombe DC Oct 29, 1999
mRNA is Cleaved in 21-23 Intervals by RNAi

Zamore P.D. et al., Cell 101:25-33, 2000

A Model for RNAi

Zamore P.D. et al., Cell 101:25-33, 2000

Dicer and RISC (RNAi-Induced Silencing Complex)

Hannon, G. J. Nature 418:244-251, 2002

 RNA silencing

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

Hannon, G. J. Nature 418:244-251, 2002

Gene Silencing by RNAi

Novina and Sharp, Nature 430:161-164, 2004

Why RNAi Does Not Work with Long dsRNA
in Mammalian Cells?
dsRNA >30 nt

Binding
and activation

Protein Kinase PKR 2’,5’-oligoadenylate synthetase

Phosphorylation Activation

eIF2α RNase L

Stall protein translation mRNA degradation

Small interfering RNA (siRNA)

19-nt duplex

• Sufficient to induce RNAi

• 3’-end insensitive to chemical modifications

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

Iamin A/C GL2 Pp-luc

j siRNA siRNA Buffer
Mr
205K
116K
97K
lamin A
66K
lamin C
55K
45K
36K

k
55K vimentin
45K

Elbashir, S.M. et al., Nature 411:494-498, 2001

RNAi Technology

• 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

Tuschl, T., Nat. Biotech. 20:446-448, 2002

Genome-wide Screens using RNAi

Hannon and Rossi, Nature 431:371-378, 2004

 Topics

1. Coupling between transcription and alternative splicing

2. Coupling between splicing and RNA export

3. Regulation of RNA stability

4. RNA interference

5. MicroRNAs expression, processing and function in the

regulation of gene expression

Cloning of Conserved miRNAs

Lee and Ambros, Science 294:862-864, 2001

 ~ 200 miRNAs cloned from Drosophila
C. elegans, mouse, human & Arabidopsis

Tuschl; Bartel; Ambros 2001

Science 15:853-864
Genes Encoding miRNAs and Conserved
Secondary Structure

Cullen B, Molecular Cell 16:861, 2004

Processing and Function of miRNA in Gene Expression

Novina and Sharp, Nature 430:161-164, 2004

Role of microRNAs in Plant and Animal Development
via Translational Control

Carringtom and Ambros, Science, 301:336-338, 2003

 Small heterochromatic RNAs (shRNAs)

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

1. Many reactions in the nucleus are mechanistically coupled.

3. Some proofreading mechanisms are operating to ensure the quality of

processed RNAs before they are exported out of the nucleus.

5. The EJC complex connects nuclear processing to translation and stability

of mRNA in the cytoplasm.

7. Small RNA is new paradigm for gene expression and regulation and
functions in a variety of pathways.

5. SiRNA is a powerful experimental tool in basic and clinical research and

applications.

6. MicroRNAs play fundamental roles in the regulation of gene expression.