Alberto R. Kornblihtt

Research Focus

Our research focuses on the regulation of alternative pre-mRNA splicing, with particular emphasis on the mechanisms that couple the splicing and transcription machineries. We study how changes in the rate of transcriptional elongation and or recruitment of splicing factors to the transcription apparatus affect alternative splicing and contribute to the generation of multiple protein variants from a single gene.

Our studies on the coupling of alternative splicing with transcription started with the discovery of the promoter effect on alternative splicing. Later we found that different promoters caused different elongation rates to RNA polymerase II and characterise a series of factors that affect splicing by modulating pol II elongation. The most direct evidence of the critical role of elongation was the use of a low RNA polymerase II mutant. When transcription is carried out by this mutant, inclusion of certain alternative cassette exons is increased, according to the “first come, first serve” model . In collaboration with another EURASNET laboratory we demonstrated that the slow pol II mutant is indeed slow in vivo, using imaging techniques that allow to measure pol II elongation in vivo and in real time.

In parallel, we found that when a gene contains two regions of alternative splicing, there is co-ordination between them although they are separated by thousands of base pairs. This co-ordination implies, for example, that mutations that stimulate the inclusion of an alternative exon at one of the regions, also cause higher inclusion levels of the alternative exon at the other region of the same gene. The co-ordination effect displays polarity, meaning that the effects of regions that are proximal to the promoter over regions that are distal is higher than in the opposite way. Consistently with the importance of pol II elongation in splicing, we found that the polar effect was critically dependent on the rate of elongation.

The carboxy terminal domain (CTD) of RNA polymerase II has been implied in the coupling of transcription with pre-mRNA processing. We found that the effect on alternative splicing of a splicing regulatory factor, the SR protein SRp20, depends on the presence of the CTD. In fact, when transcription is performed by a pol II molecule lacking the CTD, SRp20 cannot exert its role. This highlights the importance of the pol II molecular not only as a transcription machine but also as a pre-mRNA factory, capable of transcribing and processing the nascent mRNA.

We are currently studying the mechanisms by which DNA damage caused by UV irradiation affects alternative splicing. We are also developing small RNA tools to regulate alternative splicing through a recently discovered mechanism known as transcriptional gene silencing.

Publications

1. Muñoz, M.J., Pérez Santangelo, S., Paronetto, M.P., de la Mata, M., Pelisch, F., Boireau, S., Glover-Cutter K., Ben-Dov, C., Blaustein, M., Lozano, J.J., Bird, G., Bentley, D., Bertrand, E. and Kornblihtt, A.R. (2009). DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation. Cell 137, 708-720. Commented as Leading Edge in Cell 137, 600-602.
2. Schor, I.E., Rascovan, N., Pelisch, F., Alló, M. and Kornblihtt, A.R. (2009). Neuronal cell depolarization induces intragenic chromatin modifications affecting NCAM alternative splicing. Proceedings of the National Academy of Sciences 106, 4325-4330.
3. Pearson, J.L., Robinson, T.J., Muñoz, M.J., Kornblihtt, A.R., Garcia-Blanco, M.A. (2008). Identification of the cellular targets of the transcription factor TCERG1 reveals a prevalent role in mRNA processing. Journal of Biological Chemistry 283(12), 7949-61.
4. Boireau , S., Maiuri, P., Basyuk, E., de la Mata, M., Knezevich, A., Pradet-Balade, B., Backer, V., Kornblihtt, A. R., Marcello, A. and Bertrand, E. (2007). The transcriptional cycle of HIV-1 in real-time and live cells. Journal of Cell Biology 179, 291-304.
5. Caceres, J.F., Krainer, A.R., Kornblihtt, A.R. (2007). The pathology of pre-mRNA splicing: a meeting in the Italian Alps. Workshop on pre-mRNA processing and disease. EMBO Reports 8(8), 730-4.
6. Kornblihtt, A.R. (2007). Coupling transcription and alternative splicing. Advances in Experimental Medicine and Biology 623, 175-89.
7. De la Mata, M. and Kornblihtt, A. R. (2006). RNA polymerase II C-terminal domain mediates regulation of alternative splicing by SRp20. Nature Structural and Molecular Biology 13, 973-980.
8. Kornblihtt, A.R. (2006). Chromatin transcript elongation and alternative splicing. Nature Structural and Molecular Biology 13, 5-7.
9. Srebrow, A., Kornblihtt, A. R. The splicing-cancer connection. (2006). Journal of Cell Science 119, 2635-2641.
10. Kornblihtt, A. R. (2005). Promoter usage and alternative splicing. Current Opinion in Cell Biology 17, 262-268.
11. Fededa, J. P., Petrillo, E., Gelfand, M. S., Neverov, A. D., Kadener, S., Nogués, G., Pelisch, F., Baralle, F. E., Muro, A. F., and Kornblihtt, A. R. (2005). A polar mechanism coordinates different regions of alternative splicing within a single gene. Molecular Cell 19, 393-404.
12. de la Mata, M., Alonso, C. R., Kadener, S., Fededa, J. P., Blaustein, M., Pelisch, F., Cramer, P., Bentley, D. and Kornblihtt, A. R. (2003). A slow RNA polymerase II affects alternative splicing in vivo. Molecular Cell 12, 525-532. (News and Views in Nature Structural Biology 10, 876-878, 2003; selected as “exceptional”, factor 9, in Faculty of 1000.
13. Kadener, S., Cramer, P., Nogués, G., Cazalla, D., de la Mata, M., Fededa, J. P., Werbajh, S., Srebrow, A. and Kornblihtt, A. R. (2001). Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing. EMBO Journal 20, 5759-5768.
14. Cramer, P., Cáceres, J., Cazalla, D., Kadener, S., Muro, A. F., Baralle, F. E. and Kornblihtt, A. R. (1999). Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer. Molecular Cell 4, 251-258.

Key lab techniques: in vivo minigene analysis, transient transfections of mammalian cells, real time PCR, immunofluorescence, Chromatin Immunoprecipitation (ChIP).

Key lab reagents: reporter minigenes for alternative splicing.

Lab contact: Patricia Iwanczyszyn: patriciaiwan@fbmc.fcen.uba.ar

Lab website: www.fbmc.fcen.uba.ar/lfbm