self-splicing intronrnas: ribozymes, parasites and agents ... · the “tetrahymena ribozyme” and...

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





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

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1 36 nu cl .

1 18 n uc l.

ε

ε

Group II

ai5γγγγ group II





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





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





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





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





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:




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:




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:




STEP 2STEP 1OHA

5' 3' 3'5' OH

3'OH

3'5'

lariat intermediate lariat





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





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





28

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118

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ε

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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+





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





34

Swisher et al. EMBO J (2001) 20, 2051-2061

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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





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





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)





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'





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





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





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)





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

self-splicing intronrnas: ribozymes, parasites and agents ... · the “tetrahymena ribozyme” and...

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