Catalytic RNAsCatalytic RNAs

The revenge of a mistreated molecule

The Modern Biology DogmaThe Modern Biology Dogma

DNA stores information Proteins perform all the

activity RNA acts as intermediate

between DNA and Proteins

The Discovery of Catalytic The Discovery of Catalytic RNAsRNAs

Kruger, Cech et al

Self-splicing RNA.

Cell, 1982

Gurrier, Altaman et al

The RNA moiety of ribonuclease P.

Cell, 1983

Group I intronsGroup I introns The intron sequence is able to self-

splice from mitochondrial, plastid rRNA genes

The 3D structure aligns the exons sequences with the intron by an Internal Guide Sequence (IGS)

The reaction is initiated by the nucleophilic attack of the 3’ hydroxyl of an external guanidine cofactor hosted in a special pocket

The intron sequence is able to self-splice from mitochondrial, plastid rRNA genes

The 3D structure aligns the exons sequences with the intron by an Internal Guide Sequence (IGS)

The reaction is initiated by the nucleophilic attack of the 3’ hydroxyl of an external guanidine cofactor hosted in a special pocket

Group I intronsGroup I introns

O

O

O

O

O

O

OO

H

P

BASE

5'

BASE

P

HO

O

O

O

O

3'

-

-

-

-

3'

O

O

O

O

O H

P

BASE

5'

BASE

P

H

O O

O

O

O

O

O

O

OH

B

BOH..-

No external energy source is needed and the number of bonds is conserved throughout the reaction

Group I intronsGroup I introns The intron sequence is able to self-

splice from mitochondrial, plastid rRNA genes

The 3D structure aligns the exons sequences with the intron by an Internal Guide Sequence (IGS)

The reaction is initiated by the nucleophilic attack of the 3’ hydroxyl of an external guanidine cofactor hosted in a special pocket

No external energy source is needed and the number of bonds is conserved throughout the reaction

Therefore the reaction is fully

reversibleCech, TR. Self-splicing of group I introns. Ann Rev. Biochem., 1990

Roman et al.. Group I reverse self-splicing in vivo. PNAS, 1998.

Hammerhead RibozymeHammerhead Ribozyme It is the smallest

ribozyme known: only 40-50 nt

Fold consists in 3 helical regions

Cleavage occurs at GUH triplet (CUG)

Fold is stabilized by Stem II and III

It can be engineered to

perform a trans-activity

Hammerhead RibozymeHammerhead Ribozyme

It can be engineered to

perform a trans-activity

Birikh et al. The structure, function of Hammered ribozyme. Eur.J.Biochem., 1997

Marshall et al. Inhibition of gene expression with ribozyme.Cell.Mol.Neurobiol. 1994

It is the smallest ribozyme known: only 40-50 nt

Fold consists in 3 helical regions

Cleavage occurs at GUH triplet (CUG)

Fold is stabilized by Stem II and III

Cleavage

Hairpin RibozymeHairpin Ribozyme The fold is constituted by

4 stem regions The stems integrity (not

the sequence) is required for catalysis

Mg2+ is need for cleavage

It can be engineered to

perform a trans-activity

Hairpin RibozymeHairpin Ribozyme The fold is constituted by

4 stem regions The stems integrity (not

the sequence) is required for catalysis

Mg2+ is need for cleavage

Substrate should contain the consensus sequence (RBNGHY)

It can be engineered to

perform a trans-activity

The substrate specificity can be altered by changing stem I and II seq.

Walter et al. The Hairpin ribozyme. Curr.Opin.Chem.Biol. 1998

Hampel et al. The Hairpin ribozyme: development for gene therapy. Prog.Nucl.Acid Res.1998

Natural RibozymesNatural RibozymesGroup I Group II Rnase P Hammer

HeadHairpin Ribozyme

VS RNA Ribozyme

HDV Ribozyme

Tassonomic distribution

Eubacteria, Bacterioph.

Fungal and Plant

Ubiquitous Plants’ viroids

Plants’ viruses

Varkud plasmid

Hepatitis delta virus

Genomic distribution rRNA

mtRNA, plRNA

Individual gene

-- --mRNA of Neurospora

spp.

Random integration

Length From 100 to 3000 nt

From 100 to 2500 nt

300-400 nt 40-50 nt 50-60 nt 154 nt 85 nt

Reaction Type

P-ester bond Cleavage

P-ester bond Cleavage

P-ester bond Cleavage

P-ester bond

Cleavage

P-ester bond Cleavage

P-ester bond Cleavage

P-ester bond Cleavage

Ex. energy source

None None None None None None None

Recognition patterns

Sequence recognition

Sequence recognition

Sequence recognition

and 3D

Sequence recognition

Sequence recognition

Sequence recognition

Sequence recognition

Cofactors & Ions requir.

External G Internal A Water Mg2+ X2+ -- --

Proteins requirement

NoneStructural proteins

Ribonucleo-protein

None None None None

Biological role

rRNA maturation

mRNA maturation

tRNA maturation

Self-cleavege

Self-cleavege

Self-cleavage

Self-cleavage

Natural RibozymesNatural RibozymesGroup I Group II Rnase P Hammer

HeadHairpin Ribozyme

VS RNA Ribozyme

HDV Ribozyme

Tassonomic distribution

Eubacteria, Bacterioph.

Fungal and Plant

Ubiquitous Plants’ viroids

Plants’ viruses

Varkud plasmid

Hepatitis delta virus

Genomic distribution rRNA

mtRNA, plRNA

Individual gene

-- --mRNA of Neurospora

spp.

Random integration

Length From 100 to 3000 nt

From 100 to 2500 nt

300-400 nt 40-50 nt 50-60 nt 154 nt 85 nt

Reaction Type

P-ester bond Cleavage

P-ester bond Cleavage

P-ester bond Cleavage

P-ester bond

Cleavage

P-ester bond Cleavage

P-ester bond Cleavage

P-ester bond Cleavage

Ex. energy source

None None None None None None None

Recognition patterns

Sequence recognition

Sequence recognition

Sequence recognition

and 3D

Sequence recognition

Sequence recognition

Sequence recognition

Sequence recognition

Cofactors & Ions requir.

External G Internal A Water Mg2+ X2+ -- --

Proteins requirement

NoneStructural proteins

Ribonucleo-protein

None None None None

Biological role

rRNA maturation

mRNA maturation

tRNA maturation

Self-cleavege

Self-cleavege

Self-cleavage

Self-cleavage

The Ribozyme ID CardThe Ribozyme ID Card

Kcat/Km up to 108 M-1min-1

Kcat/Km up to 108 M-1min-1

No external source of energy required

No external source of energy required

The target is identified by sequence matching

The target is identified by sequence matching

Length ranging from 30 nt to 3000nt

Length ranging from 30 nt to 3000nt

P-ester bonds cleavage and ligation

P-ester bonds cleavage and ligation

Divalent cations required

Divalent cations required

Cleavage domain and fold-stabilizing regions are largely independent

Cleavage domain and fold-stabilizing regions are largely independent

Theoretical ImplicationTheoretical ImplicationThe discovery of catalytic RNAs and their physiological roles introduce a new level of control in gene expression

Introns transposition and gene inactivation

(Lambowitz et al. 1993) Splicing alteration and proteins defects

(Vader et al. 2002; Decatur et al. 2002) Plant pathology

(Smith et al. 1992; Wilson, 1993)

The discovery that RNA is capable of both information storage and catalysis, suggested its implication for the origin of life

The chicken and the egg dilemma

(The RNA world. Edited by Gesteland and Atkins. 1993; Schwartz, 1995; Joyce, 2002, Lazcano and Miller, 2003)

Eigen’s Hypercycle

(Eigen and Schuster, 1978, Cronhjort, 1995; Szathmary, 2002)

A closer look at the RNA World…A closer look at the RNA World…

A closer look at the RNA World…A closer look at the RNA World…

System’s reproduction rather than individual molecule’s replication.

Biotechnological ImplicationBiotechnological Implication

Sequence-specific activity

Sequence-specific activity

Gene silencing

Protein interfering

Functional genomics

Anti-viral ribozymes

Whatever one can think about!

Sequence-specific activities

Sequence-specific activities

Biotechnological ApplicationBiotechnological Application

What’s so special about Ribozymes?What’s so special about Ribozymes?

Ribozymes are capable of both information storage and catalysis

Therefore, In vitro evolution suits perfectly to them!

Then, what is in vitro evolution about?Then, what is in vitro evolution about?

Individual RNA

Molecule

Mutant RNA Library

Selection Parameters

•Cleavage (Chakraborti,2004)

•Binding (Joshi, 2003)

•Ligation (Jaeger, 1999)

•Folding (Luisi’s & Gallori’s groups)

Conditions Parameter

•Temperature

•pH

•Ionic strength

Amplification step

1. RT-PCR (error-Prone)

2. T7 Transcription

Selection of the Best fitted

Ribozymes in PracticeRibozymes in Practice

0. Vector Design

1. Encapsulation

2. Delivery

3. Vector release

4. Ribozyme expression

5. Co-localization

6. Cleavage and turnover.

Why should everybody love Ribozymes?Why should everybody love Ribozymes?

In vitro evolution Easy synthesis Turnover Expression control Target co-localization Selectivity

Versus

Serum clearance Cell up-taking Ckat

Cell clearance and digestion

Essential BibliografyEssential Bibliografy For an historical approach to RibozymeKruger, Cech et al. Self-splicing RNA. Cell, 1982Gurrier, Altaman et al. The RNA moiety of ribonuclease P. Cell, 1983

Mehanisms and Structures details

Cech, TR. Self-splicing of group I introns. Ann Rev. Biochem., 1990

Scott et al.Ribozymes:structure and mechanism in RNA catalysis.TrendsBioch,1996

Theoretical implications

Roman et al. Group I reverse self-splicing in vivo. PNAS, 1998.Matsuura et al. Encoding introns. Genes Dev., 1997

Biotechnological implicationsMarshall et al.Inhibition of gene expression with ribozymes.Cell.Mol.Neur,1994Kijima et al. Therapeutic applications of ribozymes. Pharmacol.Ther., 1995Sullenger et al. Rybozime trans-splicing. Nature, 1994

ReviewsTanner NK. Rybozymes. FEMS Micr. Reviews, 1999

Group II intronsGroup II introns It is found in mitochondrial and

plastidial mRNA The 3D structure aligns the exons

sequences with the intron by sequence matching: Guide Sequences

The reaction is initiated by the nucleophilic attack of 2’ hydroxyl group of a highly conserved internal Adenine

Group II intronsGroup II introns It is found in mitochondrial and

plastidic mRNA The 3D structure alignes the exons

sequences with the intron by sequence macthing: Guide Sequences

The reaction is initiaded by the nucleophilic attack of 2’ hydroxyl group of a highly conserved internal Adenine

No external energy source is needed and the number of bonds is conserved throughout the reaction

O

O

O

O

O

O

OO

H

P

BASE

5'

BASE

P

HO

O

O

O

O

3'

-

-

-

-

3'

O

O

O

O

O H

P

BASE

5'

BASE

P

H

O O

O

O

O

O

O

O

OH

B

BOH..-

Group II intronsGroup II introns It is found in mitochondrial and

plastidic mRNA The 3D structure aligns the exons

sequences with the intron by sequence matching: Guide Sequences

The reaction is initiated by the nucleophilic attack of 2’ hydroxyl group of a highly conserved internal Adenine

No external energy source is needed and the number of bonds is conserved throughout the reaction

Therefore the reaction is fully

reversible

Michel et al. Structure and activities of group II introns. Ann.Rev.Biochem., 1995.

Qin et al. The architetural organization of group II introns. Curr.Opin.Struct.Biol., 1998