Catalytic RNAs

1. Catalytic RNAs The revenge of a mistreated molecule

2. The Modern Biology Dogma • DNA stores information • Proteins perform all the activity • RNA acts as intermediate between DNA and Proteins

3. The Discovery of Catalytic RNAs • Kruger, Cech et al Self-splicing RNA. Cell, 1982 • Gurrier, Altaman et al The RNA moiety of ribonuclease P. Cell, 1983

4. Group 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 cofactorhosted in a specialpocket

5. Group 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 cofactorhosted in a specialpocket • No external energy source is needed and the number of bonds is conserved throughout the reaction

6. Group 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 guanidinecofactorhosted in a specialpocket • No external energy source is needed and the number of bonds is conserved throughout the reaction  Therefore the reaction is fully reversible Cech, TR. Self-splicing of group I introns. Ann Rev. Biochem., 1990 Roman et al.. Group I reverse self-splicing in vivo. PNAS, 1998.

7. Hammerhead 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

8. Hammerhead 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 Cleavage  • 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

9. Hairpin 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

10. Hairpin 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 • Substrate should contain the consensus sequence (RBNGHY) • 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

11. Natural Ribozymes

12. Natural Ribozymes

13. The Ribozyme ID Card Cleavage domain and fold-stabilizing regions are largely independent Length ranging from 30 nt to 3000nt P-ester bonds cleavage and ligation No external source of energy required Divalent cations required The target is identified by sequence matching Kcat/Km up to 108 M-1min-1

14. Theoretical Implication The 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)

15. A closer look at the RNA World…

16. A closer look at the RNA World…  System’s reproduction rather than individual molecule’s replication.

17. Biotechnological Implication Sequence-specific activity

18. Biotechnological Application Anti-viral ribozymes Gene silencing Whatever one can think about! Sequence-specific activities Protein interfering Functional genomics

19. What’s so special about Ribozymes? Ribozymes are capable of both information storage and catalysis  Therefore, In vitro evolution suits perfectly to them!

20. Amplification step • RT-PCR (error-Prone) • T7 Transcription Mutation Mutant RNA Library Selection of the Best fitted Selection • Selection Parameters • Cleavage (Chakraborti,2004) • Binding (Joshi, 2003) • Ligation (Jaeger, 1999) • Folding (Luisi’s & Gallori’s groups) • Conditions Parameter • Temperature • pH • Ionic strength Then, what is in vitro evolution about? Individual RNA Molecule

21. Ribozymes in Practice 0. Vector Design 1. Encapsulation 2. Delivery 3. Vector release 4. Ribozyme expression 5. Co-localization 6. Cleavage and turnover.

22. Why should everybody love Ribozymes? • In vitro evolution • Easy synthesis • Turnover • Expression control • Target co-localization • Selectivity • Serum clearance • Cell up-taking • Ckat • Cell clearance and digestion Versus

23. Essential Bibliografy • For an historical approach to Ribozyme Kruger, Cech et al. Self-splicing RNA. Cell, 1982 Gurrier, 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 implications Marshall et al.Inhibition of gene expression with ribozymes.Cell.Mol.Neur,1994 Kijima et al. Therapeutic applications of ribozymes. Pharmacol.Ther., 1995 Sullenger et al. Rybozime trans-splicing. Nature, 1994 • Reviews Tanner NK. Rybozymes. FEMS Micr. Reviews, 1999

24. 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 introns

25. Group 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

26. Group 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  Therefore the reaction is fully reversible • No external energy source is needed and the number of bonds is conserved throughout the reaction 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