Overview of introns and their structure

1. Overview of introns and their structure DNA Helix Mosaic featured on the cover of Nature, February 15, 2001 Mathieu Lajoie

2. Discovery of introns 1977 Research Groups working on adenovirus at MIT and Cold Spring Harbor reported that mRNAs were: « mosaic molecules consisting of sequences complementary to several non-contigous segments of the viral genome » Phil Sharp (MIT) & Rich Roberts (CSH) Nobel prize in Medecine 1993

3. Discovery of introns Ovalbumine is the protein contained in the white part of the eggs so it’s easy to get a lot of its mRNA. Chambon hybridized this mRNA with the gene DNA and got the following image by electronic microscopy 1978 Pierre Chambon demonstrated the existence of introns in the ovalbumine gene.

4. Discovery of introns 1978 Pierre Chambon demonstrated the existence of introns in the ovalbumine gene.

5. Discovery of introns 1978 Pierre Chambon demonstrated the existence of introns in the ovalbumine gene.

6. Classification of introns

7. Phylogenetic distribution Spliceosomal introns • Eukarya Location • Protein coding gene in nucleus Size • 25 nt over 500 000 nt (TRPM3 human chr 9) Adapted from: http://mips.gsf.de/proj/yeast/reviews/intron/major_classes.html

8. Sequence constrain: very limited Spliceosomal introns Splice site Splice site 5’ AGGUPuAGPy … PyPyPyPyPyAG 3’ consensus sequence (vertebrate)

9. Splicing mecanism Spliceosomal introns • Spliceosome: 5 snRNP + more than 760 proteins • snRNP = snRNA + 7 proteins • Needs ATP • Produce a lariat

10. Splicing mecanism Spliceosomal introns

11. Spliceosomal introns Exemple The dystrophin gene ~ 2400 kb contains 78 introns wich represent 99.5 % of his length. Only 0.5 % of the gene is coding !

12. Spliceosomal introns Utility ? • Could protects gene families against unequal recombination • Alternative splicing

13. Phylogenetic distribution tRNA introns • Eukarya • Archaea • Eubacteria Location • Nuclear tRNA genes Size • ~ 15 nt – 60 nt Splicing • Three protein enzymes: a site-specific endonuclease, a tRNA ligase, and a phosphotransferase

14. Phylogenetic distribution Group I introns • Fungal mitochondria • Plant mitochondria and chloroplasts • Nuclear genome of some protists and fungi Eubacterial & bacteriophage genomes. • Not found in vertebrate genes. Adapted from: http://mips.gsf.de/proj/yeast/reviews/intron/major_classes.html

15. Location Group I introns • Mitochondrial mRNA and rRNA genes • Chloroplastic rRNA and tRNA genes • Nuclear rRNA genes • mRNA genes of phage • tRNA genes of bacteria Adapted from: http://mips.gsf.de/proj/yeast/reviews/intron/major_classes.html

16. Structure Group I introns • Four blocks of conserved sequences in the catalytic core • No splice site consensus • Well defined secondary structure • Size range from 68 to 3000 nt • Most are over 400 nt Adapted from: http://mips.gsf.de/proj/yeast/reviews/intron/major_classes.html

17. Newer representation Old style drawing Exon seq. in lower case and boxed Shows how splice sites can be brought close together by “internal guide sequence”. splice site Conserved core Cr.LSU intron http://www.esb.utexas.edu/herrin/bio344/lectures/PT3_Splicing1rev.ppt

18. 3-D Model of Tetrahymena rRNA Intron Catalytic core consists of two stacked helices domains: 1. P5 – P4 – P6 –P6a (in green) 2. P9 – P7 – P3 – P8 (in purple) The “substrate is the P1 – P10 domain (in red and black), it contains both the 5’ and 3’ splice sites. http://www.esb.utexas.edu/herrin/bio344/lectures/PT3_Splicing1rev.ppt

19. Splicing mecanism Group I introns

20. Splicing mecanism Group I introns • Needs external G nucleotide cofactor

21. Splicing mecanism Group I introns • Needs external G nucleotide cofactor

22. Splicing mecanism Group I introns • Needs external G nucleotide cofactor

23. Splicing mecanism Group I introns • Needs external G nucleotide cofactor • Produce a linear intron

24. Splicing mecanism Group I introns • Needs external G nucleotide cofactor • Produce a linear intron • Most can splice without protein • Doesn’t need ATP • Splicing modulated by some proteins

25. Mobility Group I introns Group I intron Exon n Exon n+1

26. Mobility Group I introns Group I intron Exon n Exon n+1 ORF

27. Mobility Group I introns Group I intron Exon n Exon n+1 ORF « homing endonuclease »

28. Mobility Group I introns Group I intron Exon n Exon n+1 ORF « homing endonuclease » Gene with target sequence without group I intron Exon n

29. Mobility Group I introns Group I intron Exon n Exon n+1 ORF « homing endonuclease » Gene with target sequence without group I intron Exon n Exon n Exon n+1 ORF

30. Phylogenetic distribution Group II introns • Organellar genome of lower eukaryotes • Eubacteria • Archaebacteria Location • Organelle: mRNA • Eubacteria: mostly outside ORF • Archea: inside other introns (twintrons) Dai, L., Toor, N., Olson, R., Keeping, A., and Zimmerly, S. (2003). Database for mobile group II introns. Nucleic Acids Res. 31: 424-426

31. Phylogenetic distribution Group II introns • Organellar genome of lower eukaryotes • Eubacteria • Archaebacteria Location • Organelle: mRNA • Eubacteria: mostly outside ORF • Archea: inside other introns (twintrons) Dai, L., Toor, N., Olson, R., Keeping, A., and Zimmerly, S. (2003). Database for mobile group II introns. Nucleic Acids Res. 31: 424-426

32. 2D structure Group II introns

33. 2D structure Group II introns RT: Reverse Transcriptase X : Maturase D : DNA binding En: Endonuclease

34. Group II intronssplicing No protein is required in vitro Very similar to spliceosomal splicing

35. Group II intronssplicing Reverse transcriptase is required in vivo

36. Group IImobility

37. Group IImobility • Such process occurind at specific sites (highly efficient) • is called retrohoming • « Invasion » into unrelated sites (low frequencies) is called • retrotransposition

38. Group IIevolution Dai, L., Toor, N., Olson, R., Keeping, A., and Zimmerly, S. (2003). Database for mobile group II introns. Nucleic Acids Res. 31: 424-426

43. Group II evolution It is widely accepted that group II introns are the ancestors of slpiceosomal nuclear introns foung in higher eukaryote, including humans. • Common splicing mecanism • Structural similarities between group II introns and snRNA-intron-exon pairings in spliceosome. Dai, L., Toor, N., Olson, R., Keeping, A., and Zimmerly, S. (2003). Database for mobile group II introns. Nucleic Acids Res. 31: 424-426

44. Group II evolution LINE : 20 % of Human genome Dai, L., Toor, N., Olson, R., Keeping, A., and Zimmerly, S. (2003). Database for mobile group II introns. Nucleic Acids Res. 31: 424-426

45. Group IIevolution

46. Might group II intron confer an advantage to the host genome ? Group II evolution • For • Exon shuffling might creates new genes • Alternative splicing increase proteome (in Euglena ct) • Spread of genes contained in introns • Against • Genome instability & senescence (In Podospora mt) • Generation of scrambled genes (in Chlamydomonas)

47. Classification of introns http://mips.gsf.de/proj/yeast/reviews/intron/major_classes.html