1.rna processing and regulation 2.network interactions in gene expression 3.rasl-dasl the genome:...
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1. RNA processing and regulation
2. Network interactions in gene expression
3. RASL-DASL the genome: approaches for systems biology
Topics
rRNA Processing Pathway: Involvement of A Series of Endo- and Exonucleases
45S18S 5S 28S
41S
20S 32S
28S
5S
18S
Maturation of tRNAs
• tRNA is transcribed by RNA Pol III.
• The 5’ end is generated by RNase P, an RNA enzyme.
• The 3’ end is generated by RNase D followed by post-transcriptional addition of CCA.
• Many nucleotides are modified, which are critical for matured tRNAs to function in translation.
• Some tRNA genes contain a single intron, which is removed byendonuclease and RNA ligase.
Discovery of Introns
Transcription Unit in Eukaryotes
5’ 5’ 3’3’
Exon (100 - 300 nts)
Intron (103 - 104 nts)
Transcription Polyadenylation
Promoter
Addition of a Cap to the 5’ End of Transcript
pppNpNp
ppNpNp
ppNpNp
ppNpNp
Gp
CH3-Gp
Pi
GTP
PPi
Transfer of methyl group to the cap
CBP80/20
Phosphatase
Guanyl transferase
Methylase
Polyadenylation signals
Cap AAUAAA GU-rich
160K30K
100K73K
Cleavage & polyadenylation specificity factor(CPSF)
64K
77K
50K
Cleavage stimulation factor(CstF)
PAP
Cleavage site
Keller and Minvielle-Sebastia, Curr. Opion, Cell Biol. 9:329-336, 1997
The Polyadenylation Pathway
•PAP stimulates cleavage by CPSF•Bound PAP adds A residues at a slow rate to the 3'OH group•Binding of poly(A) binding protein II accelerates A addition•PBBII plays a role in signaling poly(A) of about 200-250 A residues
From Mole. Cell Biol., Lodish et al., 2000
Consensus Splicing Signals
A/CAG GURAGU YNYURAY--Y10-20--YAG
exon exonintron
5’ splice site 3’ splice sitebranchpoint
polypyrimidine tract exonic enhancer
R: PurineY: PyrimidineN: Any base
exonic silencer
The Nuclear pre-mRNA Splicing Pathway
P P
A 2’HO
3’OH
P
pA
P pA 3’HO
Pre-mRNA
Lariat intermediate
Ligated exons Released lariat intron
Step I: 5’ splice site cleavage and branch formation
Step II: 3’ splice site cleavage and exon ligation
+
The Spliceosome Assembly Pathway
U1
U1 U2A
U6
U5U4
U2
U1
U4U6
U5
U2
ATP
E(Commitment Complex)
A(Pre-spliceosome)
B(spliceosome)
C(Activated Spliceosome)
U6
U5
U2mRNA
Nuclear ExportExon 1
Exon 1
Exon 2
Exon 2
Mechanistic Similarity between Splicing of Nuclear pre-mRNAs and Self-splicing of Group II Introns
Establishment of the Catalytic Core in the Spliceosome and Similarity to the Catalytic Core in Group II Introns
Nilsen, T.W. In RNA Structure and function, 1998
Human:
% of alternatively spliced genes
# of isoforms per gene
Worm:
59%
22%
3.17
1.34
Source: Nature 409:898, 2001
Alternative Splicing: A Genomic Issue
Different Types of Alternative Splicing
1. Balanced Splicing:
2. Alt. 5' Splicing:
3. Alt. 3' Splicing:
4. Exon Skipping:
5. Mutual Exclusion:
5'ss 3'ss
5'ss 3'ss5'ss
5'ss 3'ss3'ss
5'ss 3'ss3'ss 5'ss
5'ss 3'ss
21
VASE7 8
Embryonic brain
Adult brain
NCAM
AUGF
AUGM
2 3
(a) (b)
(c)
(e)
(d)
fruitless
FGFR-2
Binds KGF
Binds FGF
7(IIIa) 10IIIb IIIc
Thyroid
Neurons
34 5 6
A
A
msl-2
Calcitonin/CGRP
Examples of Biologically Important Alternative Splicing Events
Smith and Valcarcel, TIBS 25:381-388, 2000
Genomic DNA
1 12 1 48 1 33 1 2
Exon 4 Exon 6 Exon 9 Exon 17
TM
Protein
Dscam: An Exon Guidance Receptor with 38,016 Isoforms Generated by Alternative Splicing
Schmucker. D. et al., Cell 101:671-684, 2000
Drosophila Sex Determination Pathway
X:A ratio: 2:2 1:2
Sxl
+
Tra
Sxl off
tra (truncated) Tra-2
Dsx + Dsx
Negative regulator of male differentiation genes
Negative regulator of female differentiation genes
Tra/Tra-2SR
stop
stop
- Sxl
+ Sxl
Four Classes of RNA Binding Proteins implicated in Splicing Regulation
Family Name Examples Key Domain Required for Splicing
SR Proteins
SC35, ASF/SF2, 9G8,hTra2-, hTra2-, SRp20, 30c, 40, 46, 54, SRp55, 75, 86
RRM and RS
Yes
HnRNPs
hnRNP A/B, hnRNP F, H hnRNP I/PTB, nPTB
RRM, some with RGG boxes
No
KH-type
KSRP, Nova-1, PS1 KH
No
CELF Factors
CUG-BP1, 2, ETR-3,NAPOR
RRM
No
U2AF
SF2/ASFor
SC35
hnRNPA/B
3' splice site ESE ESS
Positive and Negative Influences of Splice Site Selection by Exonic Enhancers and Silencers
Dis. 5'ss Prox. 5'ss 3'ss
_ _
SC35
SC35
NE
+S100 +
Pre-mRNA
Prox.
Dist.
mRNAs
M
S100 hnRNP A1
hnRNP A1
1 2 3 4 5 6 7 8 9 10 11 12
SR and hnRNP proteins affect alternative splicing in opposite ways
Fu, X-D., et al., PNAS 89:11224-11228, 1992
Tissue-specific Alternative Splicing of the src pre-mRNA:Blockage of Splice Sites by PTB
Chou, M-Y., et al., Mole. Cell 5:949-957, 2000
Cell-specific Alternative Splicing of the FGFR2 Gene Establish an Autocrine Loop Critical for Development
7 8 9 10
KGFR (or FGFR7)
FGFR2
Fibroblastsother cells
Epithelial Cells
KGFRKGF
FGFR2 FGF
Myotonic Dystrophy: A Splicing Disease
Phenotype: Skeletal muscle hyperexcitability and progressive muscle wastingCause: CUG or CCUG expansion in 3' untranslated regions in DMPK or ZNF9 genesMechanisms: Defects in splicing of the muscle-specific chloride channel CIC-1
How?AAAAA
CUG(n)
CUG-BP1 (increased stability and nuclear localization)
DMPK
2 3 6 6b 7a 7 8
Stop
U/G(n) CIC-1
Nonsense-mediated mRNA decay & protein truncation
Reduced Cl conductance
Membrane hyperexcitability
Mankodi, A., et al., Mole. Cell 10:35-44, 2002Charlet-B, N., et al., Mole. Cell 10:45-53, 2002
Life, Sex, and WT1 Isoforms: Three Amino Acids Can Make All the Difference
(Hastie, Cell 106, 391, 2001)
Exon 9 Exon 10
CATACAG GTAAAACAA gtgcgtaaactt
K T S
c
- KTS
GC
+ KTS
+/- mice develop Frasier syndrome-/- mice die after birth with kidney defectsFunction as a transcriptional factorComplete male-to-female reversal Reduced Y-specific Sry expression
+/- mice are normal-/- mice die after birth with kidney defectsFunction in pre-mRNA processingUndifferentiated gonad
{
{
All cells express +KTS and -KTS isoforms;Double heterozygous mice are normal!
Hammes, A., et al., Cell 106:319-329, 2001
1. RNA processing and regulation
2. Network interactions in gene expression
3. RASL-DASL the genome: approaches for systems biology
Topics
Complex Network of Coupled Interactions in Gene Expression
T. Maniatis & R. Reed, Nature 416:499-506, 2002
Electron Microscopic Analysis of Chromosomal Spreads: Evidence for Co-transcriptional Splicing
Beyer and Osheim, Genes Dev. 2:754-765, 1988
Targeting of Splicing Factorsto Nascent Transcripts Depends
on Pol II CTD
Cell lines were constructed to express wt and CTD-truncated -amanitin-resistant Pol II
Endogenous Pol II was inhibited by -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
Hyperhosphorylated Pol II (IIo) Stimulates
Pre-mRNA Splicing in vitro
Hirose et al., Genes Dev. 13:1234-1239, 1999
Pol II by itself has no splicing activity
Hyperphosphorylated pol II (Iio), but not IIA, stimulates splicing in vitro
Pol II by itself has no splicing activity (b, lanes 1, 5).
CTD is necessary, but not sufficient for the effect (c, lanes 5-8, 12-25).
Promoter-dependent Alternative Splicing
Cramer et al., Mole. Cell 4: 251-258, 1999
Splicing-derived mRNAs are More Efficiently Exported
Luo and Reed, PNAS 96:14937-14942, 1999
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.
RNA Metabolism in the Nucleus: Coupling RNA Splicing to Nuclear Export
Cytoplasm
Nucleus
SR SR SR
SR SR SR
Aly AlyTAP
hGle2
hGle
p15
hDbp5
Partial orMutant pre-mRNA
Normal pre-mRNA
hnRNPs
Retainedand degraded
Retainedand degraded
Exported
introns Spliced mRNP
Exportcomplex
Reed and Magni, Nature Cell Biol 3:E201-204, 2001
Circular mRNA in vivo
m7GpppAAAAAA
eIF4F (= eIF4E, eIF4A, and eIF4G)PABP
5’UTR AUG
UAA3’UTR
Implications: PolyA binding protein is required for efficient translationRNA decay in many cases are translation-dependentDeadenylation will result in decapping
Tharun and Parker, 1997
Elongation
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
PABP PABP PABP
CAP
4E
4G
eRF3
3' UTR
5' UTRATG
STOP
eRF1
Initiation
Recycling
Model for translation stimulation by 5'-3' interactions
mRNA decay pathways in eukaryotic cells
AUG UAAm7G AAAAAAAAAlong
AUG UAAm7G AAAAAAAAAlong
m7G
AUG UAAm7G
AUG UAA AAAAAAAAAlongUAA
AUG UAAm7G AAAoligo
m7G
AUG UAA AAAoligo
AUG UAA AAAoligo
Deadenylation-indep.decapping
EndonucleolyticCleavage (Rnase L ?)
PolyA shorting(PAN)
Decapping(Dcp1p)
5’ to 3’ decay(Xrn1p)
3’ to 5’ decay5’ to 3’ decay
Decapping 3’ to 5’ decay
Tharun and Parker, 1997
Decapping Mechanisms
AUG UAA AAAAAAlong
m7Gppp
Dcp1p +UAAUpf3
Upf1 Upf2
AUG UAA AAAAAAlongm7Gppp
Dcp1p
Pab1p
-
Mrt1p Mrt3p+
A. Positive and negative effect on the decapping enzyme
B. Nonsense-mediated recruitment of the decapping enzyme
Tharun and Parker, 1997
The Exosome and its Target
Rep42
Rrp43
Rrp45
Rrp44
Rrp46 Mtr3
Rrp41
Rrp40
Rrp4
Csl4
3’- 5’ exoriboucleases
ARE
KSRP TTP
m7Gppp AAAAAA
3’-5’ degradationvan Hoof and Parker, Cell 99:347-350, 1999
Chen, C-Y., et al., Cell in press 2001
Nonsense-mediated RNA Decay: Where does It Occur?
Zhang, J. et al., RNA 4:801-815, 1988
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
Nonsense-mediated RNA Decay: The 50-55 nt Rule
Zhang, J. et al., RNA 4:801-815, 1988
Two Positional Rules
The 50-55 nts Rule for NMD
The 20-24 nts Rule for Post-splicing Marker
Stop Exon-exon junction
D: > 50-55 nts: NMD < 50-55 nts: no effect
Exon-exon junction
D = 20-24 nts
Exon-exon Junction Complex that Links Splicing, Export, and NMD
Kim, V.N. et al., Science293:1832-1836, 2001
Summary
1. All RNAs (rRNA, tRNA, and mRNA) are matured in a series of processing steps after transcription.
2. mRNA processing takes place in the spliceosome, a large step-wise assembled ribonucleoprotein machinery.
3. Alternative splicing is very common and plays an important role in development and disease.
4. Many reactions in the nucleus are mechanistically coupled. 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.