Alberto Kornblihtt-Enfermedades raras de la piel
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Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
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Alberto Kornblihtt-Enfermedades raras de la piel
- 1. FUNDACIÓN RAMÓN ARECES Simposio internacional Enfermedades raras de la piel DNA damage and alternative splicing Alberto Kornblihtt IFIBYNE-UBA-CONICET Depto. Fisiol. Biol. Mol. Cel. Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires - Argentina
- 2. Alternative splicing 1. It is more a rule than an exception. It is estimated to affect the expression of nearly 95% of human genes. 2. It explains how a vast protein diversity is achieved with a limited number of genes. 3. Mutations that affect alternative splicing regulatory sequences (splicing enhancers and silencers) are a widespread source of human disease. 4. Alternative splicing regulation not only depends on the interaction of splicing factors with their target sequences in the pre-mRNA but is coupled to RNA polymerase II (pol II) transcription.
- 3. Desmodus rotundus (vampire bat) Dorsal root ganglia N N C C Trigeminal ganglia >43 ºC >30 ºC NOXIOUS HEAT INFRARED SENSING PAIN FOOD TRPV1 (ION CHANNEL) Julius group: Gracheva et al. Nature 2011
- 4. 3' NH2 COOH ED A (ED I) ED B (ED II) IIICS 120 aa 64 aa 95 aa 89 aa 0 aa 90 aa91 aa 5' RGD Human fibronectin Kornblihtt et al. PNAS 1983 Kornblihtt et al. EMBO J 1984 Vibe Pedersen et al. EMBO J. 1984 Kornblihtt et al. EMBO J. 1985 Gutman et al. FEBS Lett. 1986 Gutman et al. PNAS 1987
- 5. Zipursky lab: Schmucker et al. Cell 2000 Drosophila DSCAM gene: 38,016 different variants
- 6. ESEsuboptimal 3’ splice site SF2/ASF + SF2/ASF (SR protein) GAAGAAGAG Fibronectin EDI exon pre-mRNA ttgcctaacagACA…
- 7. ESEsuboptimal 3’ splice site SRSF1 SRSF1 (SR protein) Fibronectin EDI exon pre-mRNA ttgcctaacagACA…
- 8. ESE3’ splice site optimized by mutation GAAGAAGAG EDI exon is made constitutive pre-mRNA tttccttacagACA…
- 9. ...agCTGTCACAATATCCAGGAAGAAGAGAATATCTGTGGACATGCATGgt... ESE MUTATION Exon skipping ESE weak 3’ splice site SR protein pre mRNA
- 12. Pol II elongation DNA damage Histone marks G9a alternative splicing Mammaliancells Talk plan
- 13. Splicing is mostly co-transcriptional Guigó lab: Tilgner et al. Genome Res. 2012 Beyer and Osheim Genes Dev. 1988
- 14. Beyer and Osheim Genes Dev. 1988
- 15. Neugebauer lab: Carrillo Oesterreich et al., Cell 2016
- 16. Transcription Capping Splicing Polyadenylation Post-transcriptional pre-mRNA processing Co-transcriptional pre-mRNA processing CAP CAP An Pol IIPol II CAP An Capping Splicing Polyadenylation Transcription Primary transcript
- 17. But… co-transcriptionality does not necessarily mean coupling
- 18. Pol II Pol II Concurrent reaction Coupled reaction Modified from Lazarev and Manley, RNA 2007
- 19. Splicing and alternative splicing are coupled to RNA polymerase II transcription Cramer et al. PNAS 1997 Cramer et al. Molecular Cell 1999
- 20. Modes of coupling changes in pol II elongation rate (kinetic coupling) and/or recruitment of processing factors to RNA polymerase II (CTD), chromatin or nascent RNA (recruitment coupling)
- 21. Slow elongation, higher exon inclusion
- 22. Weak 3’SS Strong 3’SS Exclusion Fast elongation/no pauses pol II SF1 U2AF 65U1 U2AF 35 First come, first served (where first served means first committed) U1 U2AF 35U2AF 65SF1 U1 U2AF 35U2AF 65SF1 pol II U1 U2AF 35U2AF 65SF1 U1 U2AF 35U2AF 65SF1 Inclusion Slow elongation/ with pauses Weak 3’SS Strong 3’SS Kadener et al. EMBO J. 2001 Nogués et al. JBC 2002 de la Mata et al. Mol. Cell 2003 Fededa et al. Mol. Cell 2005 Alló et al. NSMB 2009 Muñoz et al. Cell 2009 de la Mata et al. RNA 2010 Dujardin et al. Mol. Cell 2014
- 23. The role of pol II elongation was confirmed by the use of a slow RNA polymerase II
- 24. Drosophila RNA pol II wt Slow (C4) L N D A R D K T G L N D A H D K T G 741 Chen et al. J. Biol. Chem. 1996 Human RNA pol II wt Slow (hC4) L N D A R D K T G L N D A H D K T G 749 de la Mata et al. Molecular Cell 2003
- 25. RNAPII -amR EDI minigene endogenous RNAPII -amanitin RNAPII a-amR endogenous RNAPII EDI -am or wt slow de la Mata et al. Molecular Cell 2003
- 26. WT slow WT slow RT-PCR 4X The human slow pol II increases fibronectin EDI inclusion de la Mata et al. Molecular Cell 2003
- 27. Lis lab: Kwak et al. Science 2013 Precision nuclear run-on- sequencing (PRO-seq)
- 28. Slow elongation, higher exon inclusion 80% of elongation-sensitive alternative splicing events
- 29. Slow elongation, higher exon skipping 20% of elongation-sensitive alternative splicing events
- 30. U2AF65 CFTR exon 9 E9TG11/T5 ETR-3 Dujardin et al. Molecular Cell 2014
- 31. WT slow WT slow RT-PCR 4X WT slow WT slow 5X de la Mata et al. Molecular Cell 2009 FN E33 (EDI) CFTR E9 Slow Pol II Dujardin et al. Molecular Cell 2014
- 32. Camptothecin (CPT) inhibits while Trichostatin A (TSA) promotes elongation Padgett method camptothecin trichostatin A Dujardin et al. Molecular Cell 2014
- 33. Ctrl CPT RT-PCR FN E33 (EDI) CFTR E9 Ctrl CPT Camptothecin (CPT) Dujardin et al. Molecular Cell 2014
- 34. RT-PCR Ctrl TSA FN E33 (EDI) CFTR E9 Trichostatin A (TSA) Ctrl TSA Kadener et al., EMBO J. 2001 Nogués et al., J. Biol. Chem. 2002 Dujardin et al. Molecular Cell 2014
- 35. Mechanism?
- 36. Strong 3’SS DNA Fast elongation Inclusion pol II U2AF 35U2AF 65SF1 U1U1 U1U1 U2AF 35U2AF 65SF1 U2AF 35U2AF 65SF1 ETR-3 U2AF 35U2AF 65SF1 Slow elongation/pauses U2AF 35U2AF 65 SF1 Strong 3’SS DNA U2AF 35U2AF 65SF1 U2AF 35U2AF 65SF1 Skipping pol II U1 U1 ETR-3 U1 Strong 3’SS Strong 3’SS Dujardin et al. Molecular Cell 2014
- 37. changes in pol II elongation rate (kinetic coupling) Changes in the template chromatin structure that limit or facilitate elongation Modulation of pol II intrinsic activity (e.g. CTD phosphorylation, association to elongation factors)
- 38. UV light and alternative splicing
- 39. TRANSFECTION TRANSFECTION UV IRRADIATION UV IRRADIATION TRANSFECTION NO IRRADIATION 1.0 ± 0.1 8.2 ± 0.7 7.7 ± 0.5 The UV effect is not due to the damage of the DNA template in cis RT-PCR Muñoz et al. Cell 2009
- 40. initiation P P-TEFbTFIIH elongation DRB flavopiridol CTD phosphorylations regulate Pol II activities P P P P P P P (YSPTSPS)52 CTD P P P P Pol II Pol II Pol II RNA polymerase II Pol II Pol II Pol II Muñoz et al. Cell 2009
- 41. UV light activates pTEFb and causes CTD hyperphosphorylation pTEFB inhibitor (DRB) - + - + UV (40J/m2) - - + + Pol II O Pol II A Western blot Muñoz et al. Cell 2009
- 42. CTD Glu for Ser mutants CTD (YSPTSPS)30 (YSPTEPS)30 (YEPTSPS)30 Ser2----Ser5 Ser2----Glu5 Glu2----Ser5 Pol II Pol II Pol II Pol II Pol II Pol II Muñoz et al. Cell 2009
- 43. 3.0 2.0 1.0 Replacement of either Ser2 or Ser5 by Glu duplicates the effects of UV light on alternative splicing RT-PCR Muñoz et al. Cell 2009
- 44. CTD Ala for Ser mutant CTD (YSPTSPS)30 (YAPTAPS)30 Ser2----Ser5 Ala2----Ala5 Pol II Pol II Pol II Pol II Muñoz et al. Cell 2009
- 45. Replacement of Ser2 and Ser5 by Ala prevents the effects of UV light on alternative splicing RT-PCR Ser2-Ser5 Ala2-Ala5 UV (50 J/m2) - + - + 3.0 4.0 2.0 1.0 0 Pol II Muñoz et al. Cell 2009
- 46. Time (sec.) Untreated cells 1 hr post-UV 2 hr post-UV 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Relativefluorescence UV irradiation inhibits pol II elongation FRAP Muñoz et al. Cell 2009
- 47. Fast elongation Skipping Pol II Pol II Pol II Inclusion mRNA Pre-mRNA mRNA Pre-mRNA Pre-mRNA Pre-mRNA UV IRRADIATION Slow elongation Pol II Muñoz et al. Cell 2009
- 48. UV effect on alternative splicing: DNA damage? RNA damage? Protein damage?
- 49. Bluescript plasmid Bluescript plasmid Reporter minigene Reporter minigene Co-transfection of irradiated DNA mimics cell irradiation Muñoz et al. submitted
- 50. TRANSFECTION NO IRRADIATION DNA damage is sufficient to elicit the alternative splicing response RT-PCR TRANSFECTION NO IRRADIATION Reporter minigeneBluescript Muñoz et al. submitted
- 51. Cyclobutane pyrimidine dimer (CPD) Pyrimidine (6-4) pyrimidone photoproduct (6-4PP) 6-4 PPCPD Muñoz et al. submitted
- 52. Repair of CPDs by the marsupial photolyase prevents the UV effect on alternative splicing CPD photolyase inactive active UV - + - + Muñoz et al. submitted 0.00 0.20 0.40 0.60 0.80 1.00 Inactive Active exon2a+/exon2a- Control UVC (6-4)PP photolyase inactive active UV - + - + TBX3
- 53. Repair of CPDs by the marsupial photolyase prevents the UV effect on alternative splicing Muñoz et al. submitted
- 54. Muñoz et al. submitted
- 55. The CPD photolyase abolishes the UV-triggered Pol II phosphorylatiion CPD photolyase inactive active UV - + - + Pol II O Pol II A Muñoz et al. submitted
- 56. 6-4 PP CPD UV XPC XPE Cen2 HR23B DDB1 XPE, DDB1 XPC Cen2 HR23B TFIIH RPA XPA XPF XPG DNA Pol ∂/e TFIIH XPC, Cen2, HR23B GGR UV CPD/6-4-PP CSA CSB XAB2 TCR Pol II XPE
- 57. Reporter minigene Reporter minigene Plasmid without promoter Plasmid with promoter Is transcription coupled repair (TCR) involved in the alternative splicing repsonse to DNA admage Muñoz et al. submitted
- 58. Without promoter With promoter Without promoter With promoter Transcription coupled repair (TCR) is not involved in the alternative splicing repsonse to DNA damage Muñoz et al. submitted
- 59. Transcription coupled repair (TCR) is not involved in the alternative splicing repsonse to DNA damage Muñoz et al. submitted
- 60. 6-4 PP CPD UV XPC XPE Cen2 HR23B DDB1 XPE, DDB1 XPC Cen2 HR23B TFIIH RPA XPA XPF XPG DNA Pol ∂/e TFIIH XPC, Cen2, HR23B GGR UV CPD/6-4-PP CSA CSB XAB2 TCR Pol II XPE
- 61. XP proteins are involved
- 62. Muñoz et al. submitted Inhibition of gap-filling DNA synthesis enhances the UV effect on splicing
- 63. Muñoz et al. submitted Inhibition of gap-filling DNA synthesis enhances Pol II hyperphosphorylation
- 64. Muñoz et al. submitted Inhibition of gap-filling DNA synthesis enhances ATR kinase activation
- 65. Muñoz et al. submitted Inhibition of ATR redeuces the UV effect on alternative splicing and Pol II phophorylation
- 66. DNA damage RNA Pol II hyperphosphorylation Decreased elongation Alternative splicing ? ? Muñoz et al. Cell 2009 DNA damage RNA Pol II hyperphosphorylation Decreased elongation Alternative splicing Muñoz et al. submitted CPD Damage repair ssDNA ATR activation
- 67. changes in pol II elongation rate (kinetic coupling) Changes in the template chromatin structure that limit or facilitate elongation Modulation of pol II intrinsic activity (e.g. CTD phosphorylation, association to elongation factors)
- 68. Alternative splicing regulation by chromatin
- 69. REPLICATION Kadener et al. EMBO J. 2001 Nogués et al. J. Biol. Chem. 2002 Trichostatin A (TSA) TSA Pol II Pol II
- 70. Figure 4-44b Molecular Biology of the Cell (© Garland Science 2008) H3 H3 H3 H3
- 71. H3K9ac H3K9ac H3K9ac H3K9ac H3K9ac H3K9ac H3K9me H3K9me H3K9me H3K9meH3K9me Neuron depolarization TSA Neuron differentiation Alternative chromatin, alternative splicing Schor et al. EMBO J. 2013Schor et al. PNAS 2009 Pol II Pol II
- 72. Misteli Lab: Luco et al. Science 2010 Kornblihtt et al. Nat. Rev. Mol. Cell Biol. 2013 Naftelberg et al. Annu. Rev. Biochem. 2015 Alternative chromatin, alternative splicing Epithelial cells Mesenchymal cells
- 73. G9a is a H3K9 dimethyltransferase Fiszbein et al. Cell Reports 2016
- 74. H3K9 methylation necessary for neruon differentiation Fiszbein et al. Cell Reports 2016 differentiation(%)differentiation(%)differentiation(%) control siG9a BIX
- 75. Beta III tubulin mergeG9a Fully differentiated neurons Immature neuron mature nueron Primary cultures of rat neurons Alfredo Cáceres
- 76. G9a is a H3K9 dimethyltransferase Its exon 10 is alternatively spliced Fiszbein et al. Cell Reports 2016
- 77. G9a E10 alternative splicing participates in a positive loop Fiszbein et al. Cell Reports 2016 C C H3K9me H3K9me H3K9me H3K9meH3K9me E10
- 78. Conclusions Pol II elongation regulates alternative splicing (kinetic coupling) Slow elongation can promote either exon inclusion or skipping, depending on the particular alternative splicing event DNA damage regulates alternative splicing through DNA repair Alternative chromatin / alternatvie splicing Alternative splicing regulates G9a nuclear localization in a positive loop that controls neuron differentiation
- 79. Valeria Buggiano Ezequiel Petrillo Micaela Godoy Herz Manuel Muñoz Luciana Giono Nicolás Nieto Moreno Adrián Cambindo Botto UV Gwendal Dujardin Celina Lafaille Pol II Luciano Marasco Ana Fiszbein Luciana Gómez AcuñaIgnacio Schor Mariano Alló
- 80. Laboratorio de Fisiología y Biología Molecular - Depto. De Fisiología, Biología Molecular y Celular-IFIBYNE-CONICET Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires - Argentina Past Mariano Alló* Gwendal Dujardin* Ezequiel Petrillo* Ignacio Schor* Manuel de la Mata Paula Cramer Guadalupe Nogués Sebastián Kadener Demián Cazalla Juan Pablo Fededa Nicolás Rascovan Soledad Pérez Santangelo Anabella Srebrow Present Manuel Muñoz Luciana Giono Celina Lafaille Valeria Buggiano Ana Fiszbein Luciana Gómez Acuña Micaela Godoy Herz Nicolás Nieto Moreno Luciano Marasco Adrián Cambindo Botto
- 81. Collaborations Ben Blencowe (Toronto) Laurent Corcos (Brest) Eduardo Eyras (Barcelona) Juan Valcárcel (Barcelona) Reinhard Lührmann (Göttingen) Carlos Menck (São Paulo) Funding HHMI (USA) EURASNET (EU) ANPCyT (Argentina) Universidad de Buenos Aires CONICET (Argentina)
- 83. Celula Nave (It happens in the body of time, where truth dances) (Ernesto Neto, 2005) Fiszbein et al. Cell Reports 2016
- 84. Misteli and Kornblihtt labs: Luco et al. Review in Cell 2011 Kornblihtt Nat. Rev. Mol. Cell. Biol. 2013 Naftelberg et al. Annu. Rev. Biochem. 2015
- 85. CTD (YSPTSPS)52 capping factors polyadenylation factors splicing factors ?? mRNA factory P P PP PP P
- 86. Drago (Xul Solar, 1927) de la Mata et al. Nat. Struct. Mol. Biol. 2006