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 cofactor hosted in a special pocket

5. 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 introns 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 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 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 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

9. Hairpin Ribozyme The fold is constituted by 4 stem regions The stems integrity (not the sequence) is required for catalysis Mg 2+ 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 Mg 2+ 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

11. Natural Ribozymes Group IGroup IIRnase PHammer Head Hairpin 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 nt40-50 nt50-60 nt154 nt85 nt Reaction Type P-ester bond Cleavage Ex. energy source None Recognition patterns Sequence recognition Sequence recognition and 3D Sequence recognition Cofactors & Ions requir. External GInternal AWaterMg2+X2+-- Proteins requirement None Structural proteins Ribonucleo- protein None Biological role rRNA maturation mRNA maturation tRNA maturation Self- cleavege Self- cleavage

12. Natural Ribozymes Group IGroup IIRnase PHammer Head Hairpin 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 nt40-50 nt50-60 nt154 nt85 nt Reaction Type P-ester bond Cleavage Ex. energy source None Recognition patterns Sequence recognition Sequence recognition and 3D Sequence recognition Cofactors & Ions requir. External GInternal AWaterMg2+X2+-- Proteins requirement None Structural proteins Ribonucleo- protein None Biological role rRNA maturation mRNA maturation tRNA maturation Self- cleavege Self- cleavage

13. The Ribozyme ID Card Kcat/Km up to 10 8 M -1 min -1 No external source of energy required The target is identified by sequence matching Length ranging from 30 nt to 3000nt P-ester bonds cleavage and ligation Divalent cations required Cleavage domain and fold-stabilizing regions are largely independent

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

17. Biotechnological Implication Sequence-specific activity

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

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. 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

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 Versus Serum clearance Cell up-taking C kat Cell clearance and digestion

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. Group 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

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 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