self-splicing intronrnas: ribozymes, parasites and agents ... · the “tetrahymena ribozyme” and...
TRANSCRIPT
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 1
1
Self-splicing intron RNAs:
ribozymes, parasites
and agents of genomic change
Anna Marie Pyle
Yale University
2
The Pyle lab
Structure and
Mechanistic Function of
Group II Introns
3
exon 1 intron exon 2
splicing
+
functional gene discarded intron
A typical gene encoding one of your proteins (a eukaryotic gene):
exons
1 2 3 4 5 6 7 8 9 10
What is an intron?When a gene is transcribed into RNA,
it is not ready for action3it must be spliced
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 2
4
Introns are not always junk*
• Sometimes they are junk
• But often they are useful:
• They can encode additional proteins
• Encode small regulatory RNAs (miRNAs, RNAi)
• Splicing sequence can control gene expressi on
• Encode genomic parasites (mobile genetic elements,
like group I and group II introns)
5
There are different classes of intron
and different strategies for their release
Group I intron
self-splicing
Group II intron
self-splicing
Nuclear splicing
spliceosomal
tRNA splicing
enzymatic
6
The autocatalytic group I and group II introns:
Classified originally by Michel, Jacquier and Dujon3
They noticed that two types of introns could be categorized into families
based on similarities in sequence and secondary structure
Michel et al. (1982) Biochimie 64, 867
Group I
U
A A A C
C U
U
U
G
G
C
A
U
A A G
U
U A
A
U
U U
C A A
U U A EB S 2 C A
C
C A
C A
U U A U
A
U
A
A U G
G U
U G
U A
C C A
A U
A U A C
A U G
E BS 1
α
A A U
A A A U A
A U A A
U A U A
U A U
U U A U U
A U U A
U A U A A
U U
C A U
A U
G U A
U A
A A A
U
A U
U G U A
U U U
A
A
U
G
A
A A A C
U
U U A U
A
G U
U U G
G
A A
A G U A G
A U A
C
A A A
A U
C
G U
G A
U G
A
U
G
A
U
A A G
C
A C
C A
C A G
G
A A
G
A
G
A
G
A
G
U
U
U U
C
A
U U
A
A
U
G
U
A A
A
A
U
U
U
C
C
C U
A G
A
A A A
A A
G G G G
G
U
U
U U
C
C C U
U U
C A
A G
G A
A A
A A A C
U U
U
U U
U
U
A
A A
A
A A
A A
A A
A A U
U U
U U
U
G
A
U
C G
A
U
G
A U
C
A
U A
U A
U
A
U G
A
C
C
U
A U
A
U
A G
G U
A U U
α
A
A
A A
A A
U
G
A
C
C C
G C
A
U A U
A A
U
A U A
U U
U
U U U
A
U A
A A U
A A
A
A U
U U
A
U G
G U A A A
U
U
A
U
A A
A U U
U A
U A A A
U A C
U A U U
U A
U U U A
U G A
U A
A A
A C A
G
A
G
A
A A
G U C
U
I
II
I B S
1
2
G
G A
U
C
G
A
U
C
G
A
U
C
G
A
G
G
A
U
C
U U
U
G
G U
U A C
G
U A
U
5 ' e xo n
3 '
exo n
G
G
G G
A
A
A
A
U
C
G
A
C
G
A U
C
G
A
U
C
G
A
U
C
G
A U
C
G A A
A
IV
V
VI
III
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
U
U A
A
U A
A U
U
U
A
G G G
A
U
U
A U
A
U A
U
A U
A U
A
U
A U
U
U U
A
G A
A
U A
G A
U A
A A
A
C
A
U
C
G
A U
G
A U
U
U
A A
G G
U A
U A
A
A
A A
A
A U
U
U C
U
U
U
A
A
C
G
A
U U
U U
U
C
C C
G G
A A
A
G
U
A A
G U
U
A C G A G
C G
U
G
A A
A A
G
A C
G C
U
G A G U U G C U
G C
U
A U
C
A U C
C
A
U G
A
A
A A
A A G U
U U C
G
a
u
G
C G
G G
G
1 36 nu cl .
1 18 n uc l.
ε
ε
Group II
ai5γγγγ group II
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 3
7
Discovery of self-splicing by an intron:
Tom Cech and Arthur Zaug were investigating
transcription and splicing of ribosomal RNA
genes in the ciliate protozoan
Tetrahymena thermophila
picture from Molecular Probes, Inc.
Major observation: the group I intron spliced
from pre-rRNA in the controls. Only factors
required: Mg2+ and guanosine (no proteins!)
Focused on splicing of a group I intron.
Note: splicing had been discovered a few years
earlier (Sharp, P.A. also Roberts, R.J.)
The image ca…
Kruger & Cech et al. Cell (1982) 31, 141
Zaug, Grabowski & Cech, Nature (1993) 301, 578
8multiple-turnover ribozyme
productsubstrate
Converting a self-splicing RNA
into a multiple-turnover enzyme:
self-splicing intron
Zaug & Cech, Science (1986) 231, 470
Cech, Ang. Chem. (1990) 29, 759
9
The “Tetrahymena ribozyme”
and the discovery of RNA catalysis
• Primary significance: a first example of RNA catalysis.
It was a first "ribozyme"
• Ribozyme activity was co-discovered by Sidney Altman:
the tRNA processing enzyme Ribonuclease P (RNAse P)
Tom Cech and Sid Altman shared the 1989 nobel prize for the discovery of ribozymes
Ribozymes are enzymes made entirely of RNA: RNA catalysts
B. Stearothermophilus RNAse P
Altman et al. Cell (1983) 35, 849
Altman et al. Science (1984) 223, 285
Pace et al. PNAS (2005) 102, 13392
Mondragon et al. Nature (2005) 437, 584
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 4
10
The “Tetrahymena ribozyme”
was a powerful tool
• Mechanisms of RNA catalysis
• The catalytic repertoire of enzymes
• RNA folding
• RNA structure
Provided some of the first insights into:
11
Mechanistic enzymology
of the “Tetrahymena ribozyme”
†
-Nuc
Nuc
O
P
O
O
O
base
OH
-
OH
base
O
O
O
O
base
P OO
O
OHO
Nuc O
HO
O
base
P OO
O
OHO
-
OH
base
O
O
OH
5'
3'
5' 5'
3'
+O base
O-O
H
O
3'
O OH
McSwiggen & Cech, Science (1989) 244, 679
Rajagopal & Szostak, Science (1989) 244, 692
Herschlag & Cech, Biochemistry (1990) 29, 10159
†
-Nuc
Nuc
O
P
O
O
O
base
OH-
OH
base
O
O
O
O
base
P OO
O
OHO
Nuc O
HO
O
base
P OO
O
OHO
-
OH
base
O
O
OH
5'
3'
5' 5'
3'
+
O base
O-O
H
O
3'
O OH
McSwiggen & Cech, Science (1989) 244, 679
Rajagopal & Szostak, Science (1989) 244, 692
Herschlag & Cech, Biochemistry (1990) 29, 10159
12
New insights into molecular recognition of RNA
Question: how does the ribozyme recognize RNA targets?
Answer: base pairing3.and something else3 E-S binding
energy was too tight to be base-pairing alone
What was the "something else"? Some new form of RNA interaction?
Hypothesis: the RNA backbone of substrate is recognized by the ribozyme
O
BB
O
O
OH
RO
PO O
OR
O
H
RO
PO O
OR
-2.2
C C C U UCG GG A GG
5'
5'
Ribozyme
-1.2 kcal/mol
by specific functional groups
in the ribozyme core
Pyle & Cech Nature (1991) 350, 628; Pyle, Murphy & Cech, Nature (1992) 358, 123
Szewczak & Strobel et al. Nat. Str. Biol (1998) 5, 1037
The RNA backbone
is recognized at specific positions3
reaction site
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 5
13
Latham & CechScience (1989) 245, 276
The Tetrahymena ribozyme clearly had
an "inside“ and an "outside" when one
examined patterns of protections
from solvent-based probes (hydroxyl radicals)
Celander & CechScience (1991) 251, 401
The Mg2+ dependence of "folding“ suggested a hierarchical process
The time-dependence of "folding“
was also measured by hydroxyl
radical footprinting. Revealed
an elaborate assembly process
Sclavi et al. JMB (1997) 266, 144
Sclavi et al. Science (1998) 279, 1940
Sclavi et al. JMB (1997) 266, 144
Sclavi et al. Science (1998) 279, 1940
Celander & Cech Science (1991) 251, 401
The Mg2+ dependence of "folding“ suggested a hierarchical process
Framing the “RNA folding problem”
Latham & Cech Science (1989) 245, 276
The Tetrahymena ribozyme clearly had an "inside"
and an "outside" when one examined patterns
of protections from solvent-based probes
(hydroxyl radicals)
The time-dependence of "folding“ was also measured by hydroxyl
radical footprinting. Revealed an elaborate assembly process
14
Zaug, Biochemistry (1993) 32, 7946
Group I intron diversity
P5abc ismissing
Tetrahymena Anabaena
Where are group I introns found? bacteria, viruses (bacteriophages),
microbial eukaryotes, plants, algae, fungi, 3 animals (anemone, coral)
What types of genes are they in? nuclear rRNA, organellar rRNA, mRNA, tRNA
Haugen, Simon, Bhattacharya, Trends in Genetics (2005) 21, 111
15
Group I intron structure and catalytic mechanism
Adams & Strobel et al. Nature (2004) 430, 45
Stahley & Strobel, Science (2005) 309, 1587
Guo & Cech et al. Mol. Cell (2004) 16, 351
Crystal structure of the Azoarchus intron, with both exons
Adams & Strobel et al. Nature (2004) 430, 45
Stahley & Strobel, Science (2005) 309, 1587
Guo & Cech et al. Mol. Cell (2004) 16, 351
Crystal structure of the Azoarchus intron, with both exons
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 6
16
Group I intron mobility and reverse-splicing
Woodson & Cech, Cell (1989) 57, 335
Roman & Woodson, PNAS (1998) 95, 2134
The discovery of reverse-splicing led to new ideas about intron mobility
and mechanisms for intron dispersal to new sites and new organisms
translatedintron ORF
mRNA
E attacks
intronless allele
homologous
recombination
during DSBR
intronself-splices
A major mechanism for group I intron mobility
Chevalier & Stoddard Nucl.Acids Res . (2001) 29, 3757
The discovery of reverse-splicing led to new ideas about intron mobility
and mechanisms for intron dispersal to new sites and new organisms
Woodson & Cech, Cell (1989) 57, 335
Roman & Woodson, PNAS (1998) 95, 2134
translatedintron ORF
mRNA
E attacks
intronless allele
homologous
recombination
during DSBR
intronself-splices
A major mechanism for group I intron mobility
Chevalier & Stoddard Nucl.Acids Res . (2001) 29, 3757
17
Group I intron applications:
the engineering and application of trans-splicing ribozymes
Sullenger & Cech, Nature (1994) 371, 619
See applications:
"Ribozyme-mediated induction of apoptosis
in human cancer cells by targeted repair
of mutant p53 RNA", Shin et al.Molecular
Therapy (2004) 10, 365
"Efficient and specific repair of sickle
β-globin RNA by trans-splicing ribozymes
Byun et al. RNA (2003) 9, 1254
18
Group II Introns and the big picture:
Nuclear splicing
spliceosomal
1977
Group I intron
self-splicing
1982
Group II intron
self-splicing
1986
tRNA splicing
enzymatic
1983
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 7
19
Arnberg, Cell (1980) 19, 313
Halbreich, Cell (1980) 19, 321
First glimpse of group II introns:
• Arnberg and Halbreich surmised that these were circular intron RNAs
• They suggested that the circles might arise through some form of splicing reaction
Microscopy on yeast mitochondrial RNA rev ealed large, stable circles
20
Phil Perlman and Craig Peebles made in-v itro transcripts of an mRNA that contained a “group II” intron (in parallel
with Grivell, Arnberg, labs). When these were incubated with Mg2+, strik ing reaction products were observed
TLC of digested circ les showed a nuclease-
resistant “branched” nucleotide near the 3’-end
Primer extension showed
products were not s imple c irc les
190
490
A typical splic ing gel
from Liu et al. JMB (1997) 267,163
Proposed mechanism:
STEP 2STEP 1OHA
5' 3' 3'5'OH 3'OH
3'5'
lar iat intermediate lar iat
Proposed mechanism:
Peebles & Perlman Cell (1986) 44, 213
Van der Veen et al. Cell (1986) 44, 225
Arnberg et al. Cell (1986) 44, 235
Proposed mechanism:
Peebles & Perlman Cell (1986) 44, 213
Van der Veen et al. Cell (1986) 44, 225
Arnberg et al. Cell (1986) 44, 235
STEP 2STEP 1OHA5' 3'
3'5'
OH3'OH
3'5'
lariat intermediate lariat
Discovery of self-splicing by group II introns:
Phil Perlman and Craig Peebles made in-vitro transcripts of an mRNA that contained
a “group II” intron (in parallel with Grivell, Arnberg, labs)
When these were incubated with Mg2+, striking reaction products were observed
Primer extension showed products were not simple circles
190
490
A typical splicing gel from Liu et al. JMB (1997) 267,163
Proposed mechanism:
Peebles & Perlman Cell (1986) 44, 213
Van der Veen et al. Cell (1986) 44, 225
Arnberg et al. Cell (1986) 44, 235
TLC of digested circles showed a nuclease-resistant “branched”
nucleotide near the 3’-end
21
†
-Nuc
Nuc
O
P
O
O
O
base
OH
-
OH
base
O
O
O
O
base
P OO
O
OHO
Nuc O
HO
O
base
P OO
O
OHO
-
OH
base
O
O
OH
5'
3'
5' 5'
3'
+O base
O-O
H
O
3'
O OH
Chemical mechanism of group II intron reactions:
Strikingly similar to mechanism of nuclear mRNA splicing by the spliceosome
Podar et al. MCB (1995) 15, 4466
Proposed mechanism:
Peebles & Perlman Cell (1986) 44, 213
Van der Veen et al. Cell (1986) 44, 225
Arnberg et al. Cell (1986) 44, 235
STEP 2STEP 1OHA
5' 3' 3'5' OH
3'OH
3'5'
lariat intermediate lariat
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 8
22
STEP 2
k2
k-2
OHA5' 3' 3'
5' OH3'OH
a
STEP 1
k1
k-13'5'
lar iat intermediate lar iat
Splicing can proceed hydrolytically
STEP 1
k1
b
A
5' 3'
H2O
3'5'
OH5'OH
STEP 2k2
k-2 3'5'
5'
3'
linear intermediate linear
There is an alternative mechanism for splicing: hydrolysis
Other important reactions catalyzed by group II introns
Splicing is highly reversible
Implications for fidelity of splicing, and for intron mobility
Implications for intron evolution, mobility and a powerful tool
Reviewed in: Lehman & Schmidt, Crit Rev Biochem Mol Biol (2003) 38, 249
Pyle & Lambowitz, RNA World, Ed 3. (2006), 469
23
Group II introns are retroelements
that attack duplex DNA:
Yang, Perlman & Lambowitz, Nature (1996), 381, 332
Pyle & Lambowitz, RNA World, Ed. 3 (2006), 469
They are the only known ribozyme
for which DNA is the biological target
24
Conservation of group II intron sequenceand secondary structure
from Cech, RNA World Ed. 1from Cech, RNA World Ed. 1
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 9
25
What is so interesting about group II introns?
• Evolution
• Chemical mechanism
• Specificity and targeting
• Folding
• Macromolecular architecture
• Structural dynamics
26
Catalytic engines for driving eukaryotic evolution
Retrotransposons
(i.e. LINE elements)
Eukaryotic spliceosomal
introns and machinery
Telomerase
Group II introns now in organellar
genes of plants, fungi, yeast and
in bacteria
~ 90%of yourDNA...
Retroviruses?
Ancestral group II
introns
Modern3.
Boeke, J. Genome Research (2003) 13, 1975
27
Group IIB
~900 nts
Group IIC Consensus:~400 nts
Group IIA ~700 nts
There are three classes of group II intron:
Where are group II introns found? eubacteria, archaea the organellar genes
of plants, fungi and yeast
Toor & Zimmerly, RNA (2001) 7, 1142
Group IIC Consensus:
~400 nts
Group IIA ~700 nts
Group IIB~900 nts
Toor & Zimmerly, RNA (2001) 7, 1142
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 10
28
U
AA
A
CC
U U
U
G
G
C
A
U
AA
G
U
U
A
A
U
U
U
C
A
A
U
U
AEBS2C
AC
C
A
CA
U U AU
A
U
A
AU
GG
UU
G
UA
CC
AA
UA U A C
A U G
EBS1
α
A A U A A A U A A U A A U A U A
U A U U U A U U A U U A U A UAA
UU
C
A
U
A
U
G
U
A
U
A
AA
A
U
A
UU G U
A
U
U
U
A
A
U
G
A
A
A
A
C
U
U UA
U
A
GU
UU G G
A
A
A
GU
AG
AU
AC
AAA
AU
C
GU
GA
U
G
A
U
G
A
U
AAG
C
AC
CA
CA
GG
AA
G
A
G
A
G
A
G
U
U
UU
C
A
UU
A
A
U
G
U
AA
A
A
U
U
U
C
C
CU
AG
A
AA
AA
A
GG
GG
G
U
U
UU
C
CC
UU
U
CA
A
GG
A
AA
AA
AC
UU
U
U U
U
U
A
AA
A
A A
A
A
AA
AA
U
UU
UU
U
G
A
U
CG
A
U
G
AUC
A
UA
UA
U
A
UG
A
C
C
U
A
U
A
U
AG
GUA
UU
α
A
A
A
A
AA
U
G
A
C
C C
GC
A
U
A
U
A
A
U
A
U
A
U
U
U
UUU
A
UA
AA
UA
A
A
AU
UU
A
U
G G U A AA
U
U
A
U
AA
AUUU A
UA
AA
UA
CU
AU
U
UA
UU
UA
UG
AU
AA
AA
CA
G
A
G
A
A A
GU
CU
I
I
IBS 1
G
GA
U
C
G
A
U
C
G
A
U
C
G
A
G
G
A
U
C
U
U
U
G
G
U
U
A
C
G
U
A
U
5'
exon
3'
exon
G
G
GG
A
A
A
A
U
C
G
A
C
G
AU
C
G
A
U
C
G
A
U
C
G
AU
C
G
AA
A
I
V
VI
II
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
U
UA
A
UA
AU
U
U
A
GG
GA
U
U
AU
A
UA
U
AU
A U
A
U
AU
U
U
UA
GA
A
UA
GA
UA
A A
A
C
A
U
C
G
AU
G
AU
U
U
AA
GG
UA
UA
A
A
AA
A
A
U
U
U
C
U
U
U
A
A
C
G
A
UU
U
U
U
C
C
C
GG
AA
A
G
U
A
A
GU
U
A C G A
GC
G
U
G
A A
AA
G
AC
GC
U
GAGUU G C U
GC
U
AU
C
A U C
C
A
UG
A
A
AA
AAG U
U UC
G
a
u
G
CG
GGG
136
nucl.
118
nucl.
ε
ε
'
IBS 2
β'
β
ζ
ζ'
'
The yeast ai5γγγγ intron
Reviewed in: Lehman & Schmidt, Crit Rev Biochem Mol Biol (2003) 38, 249
Pyle & Lambowitz, RNA World, Ed. 3 (2006), 469
I
II
III
IV
V
VI
Functional anatomy of a group II intron
DOMAIN 1
• exon/substrate recognition
• subset of active-site motifs
DOMAIN 3
• allosteric effector
of catalysis
DOMAIN 5
• active-site center
DOMAIN 6
• branch-site
29
Group II introns are strikingly modular
Like the Tetrahymena ribozyme, these "group II ribozymes" have been useful tools
for dissecting mechanistic enzymology of the intron family,
and of ribozymes in general
Example: the ribozyme can cleave small, synthetic oligonucleotide substrates
that contain single atom changes, to probe mechanism
Single deoxynucl eoti de substituti ons showed that group II introns
have a transition-s tate that avidly cleaves DNA. Later it was shown
that DNA cleavage is the natural function of group II introns
Michels & Pyle, Biochemistry (1995) 34, 2695
Griffin & Pyle et al. Chem. Biol. (1995) 2, 761
The introns can be redesigned into many different types of multiple-turnover ribozymes
30
Probing the transition-state with group II ribozymes
-
O
P
O
O
O
OH
5'
OO-
O
O
3'
O OH
C
G
• In - line SN2 reaction
• Mg2+ is directly involved in reaction chemistry
• Scissile 2'-OH is not involved in chemical step
• Still unclear how the phosphoryl oxygens
are stabilized
2'3'
ste
p 1
ste
p 2
Sontheimer & Piccirilli, Genes and Dev. (1999) 13, 1729
Gordon & Piccirilli, RNA (2000) 6, 199
Mg2+
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 11
31
Enzymology explains the unprecedented specificityof group II ribozymes
Group II introns react with exceptionally high specificity (choosing the correct
sequence in a sea of incorrect sequences)
A D13/D5 ribozyme
Xiang et al. Biochemistry (1998) 37, 3839
32
Group II intron ribozymes as model systems
for studying the RNA folding problem
33
Asuitable construct was needed
for folding studies
• Reacts with multiple-turnover
• Folded state is conformationally
homogenous
• Catalyzes a single, defined reaction
• Contains all the critical
functional domains
The D135 ribozyme
Swisher et al. EMBO J (2001) 20, 2051-2061
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 12
34
Swisher et al. EMBO J (2001) 20, 2051-2061
κ'
λ
λ
β'
κ
U
AAACCU
U
U
G
G
C
A
U
AAG
UU
AA
U
UUC
AA
UU
AEBS2
CA
CCACAU
U A
EBS1
CAU
AU
GUA
UA
UAUU G U
A
UUU
U
GA
AAACU
G
U
G U
G
A
U
CA C
CC
AGG
AA
G
G
G
G
U
U
UU
CA
UU
A
A
U
GU
AA
A
A
U
U
U
C
C
CU
AG
A
AAA
AA
GG
G
G
U
U
UU
C
CC U
UU
CA
A
GG
A
AA
AAAC
UU
U
U U
U
U
A
AA
A
A A
AA
AA
AA
U
UU
UU
U
G
A
U
CG
A
U
G
AUC
A
UAUA
U
A
UG
A
U
AU
U
AG
GUAUU
α
A
A
AA
AAUG
A
C C
GC
AUA
UA
A
UAUAUUU
UUU
A
UAA
AUA
A
A
AU
UU
UG
GU
A
AA
U
U
UA
A
AUU
UA
UAA
AU
AC
UA
UU
UAU
UU
A UG
AU
AA
AA C
AG
AG
A
A A
GU C
U
GA
U
A
C
U
C
G
G
GG
AA
A
A
U
C
G
A
C
G
AU
C
G
A
U
C
G
U
C
G
G
AAA
C
G
U
C
U
G
CG
CG
A
GG
GA
U
U
AU
A
UA
U
AU
A U
A
U
AU
UUU
A
GA
AU
A
GA
UA
AAA
C
A
U
C
G
AU
G
U
U
U
A
GG
UA
UA
A
A
AA
AU
U
UC
U
U
UA
A
C
A
UU
UU
U G
A
U
AA
GU
U
C G AGC
GUG
A A
A
G
AC
GCU
GAGUU
G C U
ε
β
ζζ'
A
A
A
AA
A
AA
AA
AA
A
AAA
U
U
U
UU
U
UU
U
UU
UUU
U
U
UU
GG
G
GG
GG
G
GG
CC C
C
C
α'
ε'
A
θ'
θ
A
AAA A
AA
A
A
A
A
AA
AC
C
C
A
A
A
A
A
A
A
G
A
GAA
5
410
395
371
356
260
233
210
195
76
95
160
36
599662
617
840
590
425
AG C
U
C
U UCG
λ'G
C
GA
3'5'
D3
D5
D1
Blue = 2x - 4.4x protection
Green = 4.5x - 8x protection
Red = 8x -10x protection
1 2 3 T1
D135 is 40% protected:highly compact
35Adapted from: Woodson et al. JMB (1997) 273, 7; JMB (2000) 296, 133
primary structure
random coil
RNA can have a complex folding landscape
monovalent
ions
K+
divalent
ions
Mg 2+
secondary structure
mix of conformers
tertiary structure
properly folded
Mg 2+ Mg 2+
misfolded tertiary structure
off-pathway species
36
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12 14
time (min)
fraction p
rote
cte
d
Representative region: 110-116
k fold = 1-2 min-1
k collapse = 2 min-1
Su et al. JMB (2003) 334, 639
Su & Waldsich, Nucl. Acids Res (2005) 33, 6674
14
45
73
111
149
172
0 3 5 10
20
30
40
60
90
12
0
15
03
00
60
0
T1
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12 14
time (min)
fraction p
rote
cte
d
• All 32 protections appear synchronously
• Protections not observed until ~30s
• Timescale for protections relatively slow
• Lack of hierarchy among protections
• There are no subdomains that fold early
The D135 group II ribozyme folds directlyto the native state
Representative region: 110-116
k fold = 1-2 min-1
k collapse = 2 min-1
D135 collapses and folds to the native state slowly and directly,
without being caught in a "kinetic trap".
It has the simplest, most accurate folding pathway described to date
Su et al. JMB (2003) 334, 639
Su & Waldsich, Nucl. Acids Res (2005) 33, 6674
14
45
73
111
149
172
0
3 5 10
20
30
40
60
90
12
0
15
0
30
0
60
0
T1
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 13
37
Conclusions for the folding pathway:
K+ Mg 2+
obligateintermediate
fast fastslow
X
• Folding is slow, and reversible
• Pathway is direct, no unproducti ve intermedi ates
• Acquires native state at same rate as many other ribozymes
(they appear to fold fast, but have traps)
• The on-pathway intermedi ate requires high [Mg2+]
• There are no stable submoti fs in the entire intron (no P5abc)
38
Group II intron ribozymes as model systems
for studying RNA tertiary structure
39
Group II introns can react in pieces:
Jarrell et al. MCB (1988) 8, 2361
Franzen et al. NAR (1993) 21, 627
Pyle, Biochemistry (1994) 33, 2716
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 14
40
NN
NH
H 2N
Sugar
O 6
N7
0 .00
0 .01
0 .02
0 .03
0 .04
0 .05
0 .06
0 .07
k o
bs (
min
-1)
D5 µM( )
0 1 2 3 4 5 6 7 8
D5(µM)
ko
bs
min
-1
C
G A G C C G U A U
C U U G G C A U G
G
U
UC
G
G
G
A A
A
A
A
C
C
A
5'
3'
Domain 5: the heart of the active-site
Single-atom changes to D5
Konforti & Pyle et al. Molecular Cell (1998) 1, 433
Abramovitz & Pyle el al. Science (1996) 271, 1410
OBase
OHO
P
O
- O
G
Km = 1 µM
I
Km = 55 µM
H
- S
H
41
binding face
]
Different functionsfor the two sides of D5:
catalytic
locus
points of tertiary
contact:
(1)
(2)
Mg2+ site
(Tb3+, NMR)
chemical face
42
Nucleotide analog interference mapping
The assay:
O
BASE
XO
O
P
OO
-S
5'
3'
Incorporate phosphorothioate linkages
to perform
+AHO
3'
5'
+ 5'A
Nucleotide Analog Interference
Mapping and Suppression (NAIM, NAIS)
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 15
43
Transcription, with random
incorporation of phosphorothioates
Selection step:
reaction with 32-P D56
( )
Position of
phosphorothioate
interference
Position of
deoxynucleotide
interference
dGTPαSGTPαS
dGG
PAGE
Iodine cleavage at
phosphorothioate linkages
Nucleotide Analog
Interference
Mapping (NAIM)
Strobel, S.A., Eckstein, F.Ryder & Strobel, Methods in Enzymology (2000) 317, 92
Fedorova et al.Handbook of RNA Biochemistry (2005), 259
44
The current map of important atoms
κ'
U
AAACC
U U
U
G
G
C
A
U
AAG
UUA
AU
UU
CA
AU
UA
EBS2C
ACCACA
U U A
EBS1
CAU
AU
GUA
UA
UAU
U G UAUUU
U
GA
AAACU
G
U
G U
G
A
U
CA C
CC
A GG
AA
G
G
G
G
U
U
UU
C
A
UU
AA
U
G
U
AAA
A
U
U
U
C
C
CU
AG
A
AA
A AA
GG G
G
U
U
UU
C
CC
U U U
CAA
GG
A
AA
A AAC
UU
U
U U
U
U
A
AA
A
A A
AA
AA
AAU
UU
UU
U
G
A
U
CG
A
U
G
A UC
A
UA
UA
U
A
UG
A
U
AU
U
AG
GU
AUU
α
A
AA
A
AAUG
A
C C
GC
AUAUAA
UAUAUU
U
UUU
A
UA
AA
UA A
A
AU
UU
UG G U
A
AA
U
U
UA
A
AUUU A
U AA A U
A C UA
UU
UA
U U U AUG
AU
AA
A AC
A G
AG
A
A A
G U C U
I
II
G
GA
U
C
G
A
U
C
G
A
U
C
G
A
G
G
A
U
C
UUU
G
GU
UAC
G
UAU
5'
3'
G
G
GG
AA
A
A
U
C
G
A
C
G
AU
C
G
A
U
C
G
U
C
G
AU
C
GA
AA
IVU
C
A
G
U
C
A
G
C
A
G
U
C
A
G
U
UA A
UA
AU U
U
A
GG
GA
U
U
AU
A
UAU
AU
A U
A
U
AU
U
UU
A
GA
A
UA
GA
UA
A AA
C
A
U
C
G
AU
G
U
U
U
A
GG
UA
UA
A
A
AA
AU
U
UC
U
U
UA
A
C
G
A
UU
UU
U GG
A
U
AA
GU
U
C G AG
CG
UG
A A
A
G
AC
GC
U
GAGUU G C U
GC
U
AU
CA U
C
C
A
UG
A
AA
A
AAG U
U UC
G
G
CG
GG Gε
β
ζ
ζ'A
A
A
A CC
G G
UU
UU
U
U
UAA
A
A
AA
A
AA
AA
AA
A
AAA
U
U
U
UUU
UU
U
UU
UUU
U
U
UU
GG
G
GG
GG
G
GG
C C CC
C
α'
β'
ε'
A
η'
η
θ'
θ
A
BC
C1
C2
D
D'D''
D'''
D2a
D2b
D3
OptionalORF
A
AAA A
AA
A
A
A
A
AA
AC
C
C
A
A
A
A
A
A
A
A
G
GA C
A
GAA
IBS1
IBS2
γ/γ'
κ
III
V
VI
i
A
B
C
λ
λ
λ'*
**
**
*
*
-7-deaza A
-2,6-Diaminopurine
-N6-Me A
-2’-modifications
-inosine
-phosphorothioate
-2-Aminopurine
*Boudvillain & Pyle, EMBO J. (1998) 17, 7091
Fedorova & Pyle, EMBO J. (2005)
• First step effects only
• These can be used as a guide for mutagenesis and NAIS...
45
dope with modified
phosphorothioates
Nucleotide analog interference suppression
Ryder & Strobel, Methods in Enzymology (2000) 317, 92
Fedorova et al. Handbook of RNA Biochemistry (2005), 259
5'
AHO+ 3'
A + 5'
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 16
46
Reaction with Iodine
Nucleotide Analog Interference Suppression (NAIS)
wild-type exD123 transcript
Selection: branchwith modified D56
Mutant exD123 transcript
MU
PAGE
WT
Position ofinterference
Position ofinterference suppression
47
U 3'UCU
G
AU
U
U
UU
G
G
GG
AAA
AG
G
GUG
A
CACC
G
C
5'
UC
A
GCG
CGCG
C
GA
5'3’
5115
818
844
836
823
ε-ε’
D5
Boudvillain & Pyle, EMBO J. (1998) 17, 7091
Boudvillain & Pyle et al. Nature (2000) 406, 315
The λ-λ’ interaction is mediated
by two minor groov e triples:
N
N
N N
O
O
HNH A 115
OC 837-2'-hyd roxyl
O
HO
H
O
R O
-OP
HN
N
N
NO
N
N
O
O
O
O
O
O
OR-O
OR
O
O -
O
G836
H
H
NH
O
PP
C825
H
H
H
N
NH
N
N
NO
N
N
O
O
O
O
O
O
O-O
OR
-O
O
ORO
H
H
C837
H
PG824
H
N
H
P
N
H
NH
N
N
N
O
O
O
O
O
RO
-O
HH
N H
G5
P
48
These have been used to confirm all the NAIS results thus far.....
let's use them for D6
DOMAIN 6: where is the branch-point located in the catalytic core?
Short-range crosslinkers
HN
NN
NHN
N
S
H2
N
S
O
RNARNA
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 17
49
A network of functional constraints knits the intron together
"Coordination
Loop"
DeLencastre & Hamill et al. NSMB (2005) 12, 626
50
A three-dimensional model of the ai5γ intron
β-β'
26
α-α'
5'exon
160
IB
ID(i)
ζζζζ -ζζζζ '
κκκκ -κκκκ '
ID(iii)
coordination
loop
ID(iv)
EBS2-IBS2
ID2a
ID3(i) ID3(ii)
Domain 6
Domain 5
273
264
I(ii)
I
DeLencastre & Hamill et al. NSMB (2005) 12, 626
D5 and the site of catalysis are located in a cleft that is anchored by regions of D1.
D6 fits alongside, inserting the branch-site ribose
51
A close-up view of the active-site:
We've learned that there is one active-site region for a group II intron ribozyme
Our model is a work in progress, a tool for guiding the biochemistry on group II introns.
It is subject to constant refinement as new constraints emerge
DeLencastre & Hamill et al, NSMB (2005) 12, 626DeLencastre & Hamill et al. NSMB (2005) 12, 626
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 18
52
Group II introns:
the future
53
Generalit y of “branching reaction s” : they may be quite common
Group II introns
(and spliceosome)
"Group I-like“ capping
ribozyme (GIR)
Ty1 retrotransposon
and retroviruses?
see Pyle, Science (2005) 309, 1530
C
Nielsen et al. Science (2005) 309, 1584
Menees, Science (2004) 303, 240
54
Group II introns as tools and therapeutics
Sullenger et al. Mol Therapeutics (2005), 11, 687
Articles by Lambowitz, A.M. and Perlman, P.S.
• Targeted gene disruption
• Expressi on of novel or repaired proteins
• Trans-splicing repair
Domain 4
ORF for maturase
(or new proteins)
Self-splicing intronRNAs:
ribozymes, parasites and agents of genomic change
Prof. Anna Marie Pyle
The screen versions of these slides have full details of copyright and acknowledgements 19
55
Group II introns and the proteins
that love them
• Although they are autocatalytic RNAs, group II introns depend on a host
of proteins in-vivo for many of their functions
• Encoded protein: maturases and mobility factors
• Nuclear-encoded genes: Mss116 and other ATPases
• The numerous proteins important for group II intron function in plants
• Ideal systems for tracking the interplay between RNA and protein
in the evolution and development of catalysis
see Pyle & Lambowitz, The RNA World Ed. 3, 2006
56
Special thanks
Members of the Pyle Lab, past and present
Philip S. Perlman
Alan Lambowitz
Michael Brenowitz
Tom Cech
Scott Strobel
National Institutes of Health
Howard Hughes Medical Institute
National Science Foundation
Searle Family Foundation
Beckman Foundation
Yale University
Columbia University
57