Discovery Landmarks

Research Frontiers

Future Directions
 Expression of a Human Gene
Protein Coding Gene ncRNA Gene

DNA
Transcription Polyadenylation Transcription
exon intron

primary transcript / pre-mRNA

Splicing Processing
(nucleus)
Reverse
Transcription AAAAAAAAA Processing
mRNA Export
(cytoplasm)
Translation Degradation
Modification/
Protein
Folding, Modification, Complex
Transport, Complex Assembly
(cyt. or nuc.)
Assembly
Protein Complex
Degradation Degradation RNP
 Topics

1. Introduction to RNA processing in the nucleus

2. The pre-mRNA splicing pathway
3. Spliceosome assembly: snRNPs and protein factors
4. RNA catalysis
5. Alternative splicing: mechanisms and regulation
6. Biological consequences of regulated splicing
7. Global approaches to alternative splicing
rRNA Processing Pathway: Involvement of
A Series of Endo- and Exonucleases

18S 5S 28S
45S

41S

20S 32S

5S
18S 28S
 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 by

endonuclease and RNA ligase.
Discovery of Introns
 Transcription Unit in Eukaryotes

Transcription Polyadenylation

Exon (100 - 300 nts)

Promoter
5’ 3’ 5’ 3’

Intron (103 - 105 nts)

Addition of a Cap to the 5’ End of Transcript

pppNpNp

Phosphatase
Pi
ppNpNp
GTP
Guanyl transferase
PPi
GpppNpNp
Transfer of methyl group to the cap
Methylase

CH3-Gp ppNpNp

CBP80/20
 Polyadenylation signals

Cleavage & polyadenylation specificity factor

(CPSF)
Cleavage stimulation factor
(CstF)

73K
100K 50K
160K PAP
30K 77K
Cap AAUAAA GU-rich
64K

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
•PABII plays a role in signaling poly(A)
of about 200-250 A residues

From Mole. Cell Biol., Lodish et al., 2000

 Topics

1. Introduction to RNA processing in the nucleus

2. The pre-mRNA splicing pathway
3. Spliceosome assembly: snRNPs and protein factors
4. RNA catalysis
5. Alternative splicing: mechanisms and regulation
6. Biological consequences of regulated splicing
7. Global approaches to alternative splicing
 Consensus Splicing Signals

5’ splice site

branchpoint
polypyrimidine tract exonic enhancer

exonic silencer
3’ splice site
 The Nuclear pre-mRNA Splicing Pathway

2’ A
HO

Pre-mRNA P P

Step I: 5’ splice site cleavage

and branch formation

Lariat intermediate pA

P
3’
OH

Step II: 3’ splice site cleavage

and exon ligation

Ligated exons P + pA 3’ Released lariat intron

HO
 Topics

1. Introduction to RNA processing in the nucleus

2. The pre-mRNA splicing pathway
3. Spliceosome assembly: snRNPs and protein factors
4. RNA catalysis
5. Alternative splicing: mechanisms and regulation
6. Biological consequences of regulated splicing
7. Global approaches to alternative splicing
Requirement for U1 and U2 in Pre-mRNA Splicing in vitro
The Spliceosome Assembly Pathway
U1
Exon 1 Exon 2 E
(Commitment Complex)
ATP
U1 U2
A
A
(Pre-spliceosome)

U1 U6
B
U4 U5
(spliceosome)
U2

U4
U6 C
U5 (Activated Spliceosome)

U2
U6
U5
U2 Exon 1 Exon 2

mRNA
 SR proteins Function in Multiple Steps in
the Spliceosome Assembly Pathway

Promote U1 recognition Recruit U2AF complex to the 3’ splice site

of the 5’ splice site or compete against exonic silencing complex

U1 snRNP

70K
Inhibitory
SR SR Complex
Protein  U2AF65 U2AF Protein 
35
Exon  Py AG Exon 
Enhancer Inhibitor

Graveley, B. RNA 6:1197-1211, 2000

 SR proteins Promote Cross-intron interactions
and mediate the recruitment of U4/6.U5 tri-snRNP
U4/U6¥U5 tri­snRNP

27K
100K

SR
Protein

U1 snRNP

70K

U2AF65 U2AF
35
Exon 1  A Py AG Exon 2 
U2 snRNP

Graveley, B. RNA 6:1197-1211, 2000

 Topics

1. Introduction to RNA processing in the nucleus

2. The pre-mRNA splicing pathway
3. Spliceosome assembly: snRNPs and protein factors
4. RNA catalysis
5. Alternative splicing: mechanisms and regulation
6. Biological consequences of regulated splicing
7. Global approaches to alternative splicing
Mechanistic Similarity between Splicing of Nuclear
pre-mRNAs and Self-splicing of Group II Introns
 RNA Catalysis in the Spliceosome

A B
U1 U2
U6
GA GAU
A UU UCA AAG
G C UGCAU A
U GG G CCU
UCCAU CAUA AUGAUGU C CC AUU
UC A GUUC A CGU
AGGUA GU UACUA C AGGU GU GG C
U A AAG
Exon 1 Exon 2 U A
UU U
U4

C D
C U
G A
U A
C G
C G
C A
C GU
U A
U A
U6
G C A
AC A CGUUUUACAAAGAGAUUUAUUUCGUUUU
A G A
UG GA C
UA GA G G G UUUUCCGUUUCUCUAAGCA n A
A
5’ U UGAUC
U
C r n
U5 NAG G
ACUAG A U C
r
y
y

AU U U
C
A
G U2 r AGC n n n r
U
r
y
G Domain 5
GC C U U
GUAGUA 5’ GUGCG y UUG n n n y G n
A
UG G A C AUCAU G
3’ A
G
r r r r r r
yA yyyy yy
3’ A Domain 6
 Topics

1. Introduction to RNA processing in the nucleus

2. The pre-mRNA splicing pathway
3. Spliceosome assembly: snRNPs and protein factors
4. RNA catalysis
5. Alternative splicing: mechanisms and regulation
6. Biological consequences of regulated splicing
7. Global approaches to alternative splicing
 Gene number and organismal
complexity
Fruit fly Nematode worm Human Plant

No. of genes: ~13,000 ~19,000 20-25,000 ~25,000

Alternative transcription, alternative splicing and

alternative polyadenylation can generate a huge
diversity of proteins from even a single gene
More than half of all human genes are alternatively spliced
 Many Different Ways to Splice
Intron retention Mutually exclusive exons

Alternative 5’ choices Combinatory exon selections

Alternative 3’ choices Alternative promoter and splicing

Exon inclusion/skipping Alternative splicing & polyadenylation

pA

pA
 Dscam: An Exon Guidance Receptor with 38,016
Isoforms Generated by Alternative Splicing

Genomic DNA
Exon 4 Exon 6 Exon 9 Exon 17

1 12 1 48 1 33 12

Protein

TM
These isoforms play
a role in wiring the
nervous system,
affecting whether
neurons connect to
each other.

Schmucker et al. Cell 2000, Wojtowicz et al. Cell 2004

 Sex Determination in Drosophila
X:A ratio: 2:2 + 1:2
+ Sxl
stop

Sxl  2 3 4 Sxl off 
­ Sxl

- Sxl
stop

Tra  1 2 Tra (truncated) 

Tra­2  + Sxl

Tra/Tra-2

+ Dsx  3 4 5 Dsx 

Negative regulator of Negative regulator of

male differentiation genes female differentiation genes
Multiple Classes of RNA Binding Proteins
implicated in Splicing Regulation

Family Name Family Members Key Domain Required for Splicing

SR Proteins SC35, ASF/SF2, 9G8, RRM and RS Yes

hTra2-α, hTra2-β,
SRp20, 30c ,38, 40, 46,
54, 55, 75, 86

hnRNP hnRNP A/B, hnRNP F, H RRM, some with No

proteins hnRNP I/PTB, nPTB RGG boxes
TIA-1, Elav, Fox-1, 2, 3

KH-type RNA KSRP, Nova-1,2, PSI KH No

binding factors

CELF Factors CUG-BP1, RRM No

CUB-BP2/ ETR-3,
NAPOR

MBNL MBNL 1, 2, 3 C3H zinc finger No

 Regulation of Alternative Splicing
via Enhancers and Silencers

Interactions across exon

­­ ­ ­
Interactions across intron Interactions across intron
+
+ +
++ + ­ ­
U2 SR A/B U1
U2AF

5’ ss BPS Py
ESE ESS 3’ ss
3’ ss 5’ ss
 SR and hnRNP proteins affect alternative
splicing in opposite ways

S100 + SC35 S100 + hnRNP A1

hnRNP A1 SC35
_ _
NE M

Pre­mRNA
Prox.

mRNAs
Dis. 5'ss Prox. 5'ss 3'ss
Dist.

Fu, X-D., et al., PNAS 89:11224-11228, 1992

1 2 3 4 5 6 7 8 9 10 11 12
Tissue-specific Alternative Splicing

Non-neuronal cells (N1 skipping)

U1 U2
3 4
PT B
PTB P TB
P T B
KSRP
ISS N1 H p28 p50
U1
Neuronal cells (N1 inclusion)

U1 U2 U1 ISE U2
3 N1 4
KSRP
H p28 p50
nPTB
PTB ATP
 Recursive Splicing

5’ss 3’ss 5’ss 3’ss

5’ss 3’ss
 Topics

1. Introduction to RNA processing in the nucleus

2. The pre-mRNA splicing pathway
3. Spliceosome assembly: snRNPs and protein factors
4. RNA catalysis
5. Alternative splicing: mechanisms and regulation
6. Biological consequences of regulated splicing
7. Global approaches to alternative splicing
 Cell­specific Alternative Splicing of the FGFR2 Gene 
Establish an Autocrine Loop Critical for Development

FGFR2 FGF

FGFR2

Fibroblasts 7 8 9 10 Epithelial
other cells     Cells

KGFR (or FGFR7)

KGF KGFR
 Myotonic Dystrophy: A Splicing Disease

Phenotype: Skeletal muscle hyperexcitability and progressive muscle wasting

Cause: CUG or CCUG expansion in 3' untranslated regions in DMPK or ZNF9 genes
Mechanisms: Defects in splicing of the muscle-specific chloride channel CIC-1

How?
DMPK AAAAA CUG-BP1 (increased stability and
nuclear localization)

U/G(n) CIC-1

Stop

CUG(n) 2 3 6 6b 7a 7 8

Nonsense-mediated mRNA decay & protein truncation

Reduced Cl conductance

Membrane hyperexcitability

Mankodi, A., et al., Mole. Cell 10:35-44, 2002

Charlet-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)

All cells express +KTS and -KTS isoforms;

Double heterozygous mice are normal!
+/- mice develop Frasier syndrome

- KTS { -/- mice die after birth with kidney defects

Function as a transcriptional factor
Complete male-to-female reversal
Reduced Y-specific Sry expression
c
Exon 9 Exon 10
CATACAG GTAAAACAA gtgcgtaaactt
K T S

C G

{
+/- mice are normal
+ KTS -/- mice die after birth with kidney defects
Function in pre-mRNA processing
Undifferentiated gonad

Hammes, A., et al., Cell 106:319-329, 2001

Tissue-specific Ablation of SR Proteins in the Heart

A Protein RRM RRM RS

Not I Aat II EcoR I Xba I

Gene

Xba I Xba I Xba I

Targeting Neo TK
Construct
P1 P2 P3

B Week 3 Week 5 Month 10

WT KO WT KO WT KO

Ding et al., EMBO J 23, 885-896, 2004

 Postnatal Remodeling of Heart by
Regulated Alternative Splicing

A. Regulated splicing in development C. Elevated Ca2+ sparks
NLS
CaMKIIδ 13 14 15 16 17
Allele

δA δB δC

d1 d10 d20 d30 d52 d56 d30 d80

wt ko wt ko wt ko wt ko wt ko wt ko neg ko ko

δA
δB
δC

ASF/SF2 SC35

B. Differential cellular targeting by isoforms

δA δB δC
Xu et al., Cell 120, 59-72, 2005
 Topics

1. Introduction to RNA processing in the nucleus

2. The pre-mRNA splicing pathway
3. Spliceosome assembly: snRNPs and protein factors
4. RNA catalysis
5. Alternative splicing: mechanisms and regulation
6. Biological consequences of regulated splicing
7. Global approaches to alternative splicing
 We are Still in the Early Stage in
Understanding the Biology of Splicing

• Are all mRNA isoforms biologically important?

• How is alternative splicing regulated in development?

• How are the cell and tissue-specificity established?

• How does transcription affect alternative splicing?

• How is alternative splicing regulated by signaling?

• How may human diseases result from splicing defects

or misregulation of alternative splicing?
“Moist” biology screens for exonic
splicing enhancers and silencers

+ ­  

Enhancers (+) Silencers (-)

Computational Laboratory screen
screen

cells containing
candidate splicing silencers
splicing enhancer
sequences

analyze sequences to
test in
identify commonalities
the lab
and infer functions
Computational Approach to
Identifying
Splicing Enhancers
in the Human Genome

Fairbrother et al., Science 297:1007-1013

 RESCUE-ESE Predicted ESE Motifs
5A 3A
10 candidate Half similar to
5B 3B
known ESEs
enhancer
“motifs” 5C 3C All increase
identified
5D 3D exon inclusion
in human cells
5E 3E

14 of 30 HPRT exon skipping mutations disrupt ESEs E→X

Fairbrother, Yeh et al., Science 2002

 A Cell-Based Screen for Splicing Silencers

Test many random silencer candidates

1 2 3 Random “10-mers” (black)
embedded in exon 2

GFP exons

Some Sort out the

splice But some green cells
normally skip exon 2

Cell count
test2

Log (GFP intensity)

Then clone them

Analyze the sequences

to identify commonalities
and infer functions
 Results of ESS Screen
141 ESS 10-mers (133 unique)
Dissimilarity
6.0 5.0 4.0 3.0 2.0 1.0
FASS-ESS Possible
Group Trans-Factor

B hnRNP A1?

C hnRNP H?

G hnRNP A1?

Wang et al. Cell 119, 831-845, 2004.

Approaches to Alternative Splicing

Use of exon-exon junction probes

Use of exon-specific probes

A B C

Genomic tiling
Profiling Splicing Using the Splice Junction Approach

Clark T. A. et al., Science 2002 296:907-910

RASL: RNA-mediated Annealing Selection and Ligation

Barcode 1 T7

T3
Barcode 2 T7
Exon 2
Cap Exon 1 Exon 4 (A)n
intron
Exon 3

Selection

Exon 1 Exon 2 Exon 4 (A)n

dTn-Biotin

A Exon 1 Exon 3 Exon 4 (A)n

dTn-Biotin

A
Ligation

PCR

Hybridization to the universal barcode array

 Cell Type Isoform Signatures

A B

LNCaP
PC-3
Splicing pattern &
Isoform ID PC3(M) LNCaP(M) Ps Spl. Index PCR primers
CASP2-1090 2413 2168
CASP2-1091 5105 516 7E-07 3.15
LAPC4 C21ORF18-0662 461 956
C21ORF18-0661 2240 1044 1E-05 2.12
SFRS7-1190 9347 4323
LNCaP SFRS7-1189 15460 18377 3E-05 1.36
TRPM2-0453 278 575
TRPM2-0452 378 381 5E-05 1.04
IMPDH2-0146 773 444
RWPE IMPDH2-0145 6731 9248 5E-05 1.26
RPL18A-0846 10440 12434
RPL18A-0845 677 341 3E-04 1.24
MCCC2-0035 2571 2995
MCCC2-0034 2423 1009 3E-04 1.49
FLJ20130-1174 1114 1297
FLJ20130-1173 1151 341 4E-04 1.98
BHC80-0965 1013 1623
BHC80-0966 1215 4083 5E-04 1.07
Up in all MYO6-0041 1285 3147
prostate MYO6-0040 11224 2317 5E-04 2.43
ATP5O-0648 12344 13413
cell lines ATP5O-0647 3194 968 5E-04 1.84
LGALS8-2089 2545 1657
LGALS8-2088 571 1057 7E-04 1.51
XBP1-0834 3951 5076
XBP1-0833 1190 569 1E-03 1.43
CANX-0782 12437 9583
PC3 CANX-0781 5040 6038 2E-03 0.64
SNRP70-1182 1479 1249
DU145 SNRP70-1181 732 1077 2E-03 0.80
BHC80-0952 551 1990
BHC80-0951 2366 3700 3E-03 1.21
SF3B3-0768 2666 3702
SF3B3-0767 3482 5710 4E-03 0.24 X
NR3C1-1281 11988 1438
NR3C1-1280 350 460 4E-03 3.51
HRMT1L1-0583 425 294
HRMT1L1-0584 5007 1708 5E-03 1.02

SFRS2-1108 2878 2651 X

SFRS2-1107 9708 11413 0.015 0.35
BAX-0114 1663 2648
BAX-0115 4249 3276 0.019 1.06
AMACR-0297 622 2669
Down in all AMACR-0298 742 2543 0.021 0.32
prostate NDUFV3-0461 10139 8560
NDUFV3-0462 4741 4991 0.045 0.32
cell lines SFRS15-0321 4214 2729
SFRS15-0322 3754 2554 0.384 0.07
 Validation of Prostate Cancer Isoform Markers by
Laser Capture Microdissection
A B
2
No change

CACNA1D Tx change
Normal
Splicing Change

Spl change
1.5
MAPT Both change

a b c
1

0.5
Tumor
AMACR

0 d e f
0 0.5 1 1.5 2

Transcript Change

C N1 T1 N2 T2 N3 T3 N4 T4

MAPT-1061
MAPT-1060

CACNA1D-2039
CACNA1D-2038

AMACR-2094
AMACR-2095

CD81
 Summary of Lecture 1

1. All RNAs (rRNA, tRNA, and mRNA) are matured in a series of

processing steps after transcription.

4. mRNA processing takes place in the spliceosome, a large step-wise

assembled ribonucleoprotein machinery.

6. The chemistry of mRNA splicing is likely catalyzed by ribozyme.

8. Alternative splicing is very common in higher eukaryotic cells and is

regulated by a large number of protein factors including SR proteins.

5. Alternative splicing plays an important role in development and disease.

6. Experimental and computational approaches are used to systematically

search for regulatory elements in the human genome.