1. Towards a new gene definition: the hindrance of Alternative Splicing Laura Fontrodona Montals Mòdul 4: Genòmica i Proteòmica Màster Oficial de Genètica-UAB

2. Background

3. Ben-Dov et al., 2008 High–throughput methods used for AS analysis A. Transcript/genomic alignmentsB. Splicing-sensitive microarrays

4. Molecular basis of Splicing Mechanism Splice signals that delineate the proper exon-intron boundaries. 5’ splice site (5’ss) 3’ splice site (3’ss) Branch site (BS) Polypyrimidine tract Kim et al., 2007

5. Molecular basis of Splicing Mechanism Regulation of Splicing (both constitutive and alternative). Cis-acting factors: ESEs, ISEs, ESSs, ISSs Trans-acting factors: the spliceosome ( 5 diferent RNPs and more than 150 proteins) Wang et al., 2007

6. Origin and evolution of Alternative Splicing: Comparative genomics

7. Types of Alternative Splicing A.Exon skipping B.Alternative 3’ss selection C.Alternative 5’ss selection D.Intron retention AS is believed to be a major source for the phenotypic complexity in higher eukaryotes. Exon skipping exhibits a gradual increase in its relative prevalence along the eukaryotic tree, suggesting it is the more important event in shaping phenotypic complexity. Kim et al., 2007

8. Do the same evolutionary forces apply to Alternative and Constitutive Splice Exons? Comparative genomics analysis Kim et al., 2007 Exons that are alternatively spliced in both human and mouse are more conserved than constitutive exons.

9. Do the same evolutionary forces apply to Alternative and Constitutive Splice Exons? Comparative genomics analysis Kim et al., 2007 The sequence conservation extends into the flanking introns of conserved alternative cassette exons for 80 to 100 nucleotides compared to constitutive ones.

10. Do the same evolutionary forces apply to Alternative and Constitutive Splice Exons? Comparative genomics analysis Kim et al., 2007 The type of the 5’ss dictates constitutive or alternative splicing and also the level of inclusion/skipping in alternative splicing.

11. Do the same evolutionary forces apply to Alternative and Constitutive Splice Exons? Comparative genomics analysis Kim et al., 2007 Alternative exons are shorter and are flanked by longer introns than constitutively spliced ones.

12. Do the same evolutionary forces apply to Alternative and Constitutive Splice Exons? Comparative genomics analysis Kim et al., 2007 Exonic Splicing Regulatory sequences (ESR) are significantly more conserved in alternatively spliced exons. A maximum of 60% of identity A maximum of 80% of identity

13. Do the same evolutionary forces apply to Alternative and Constitutive Splice Exons? Comparative genomics analysis Kim et al., 2007 Exclusion or inclusion of symmetrical exons would mantain the reading frame of the downstream exons.

14. Origin of alternative cassette exons Kim et al., 2007

15. Origin of alternative cassette exons The inclusion level may serve as an indicator of the age of alternatively spliced exons and, consequently, of their origin and evolution Alternative cassette exons with low inclusion levels probably originated from de novo exonization of intronic sequences. Alternative cassette exons with high inclusion levels probably originated from exons that were previously constitutively spliced.

16. What is a gene? From the abstract elements of heredity of Mendel and Morgan to the present-day ORFs

17. “One thing only I know, and that is that I know nothing” Socrates “The more I know, the less I understand” A. Einstein

18. Problems with the current definition of a gene o Gene regulation o Overlapping and spliced genes o Overlapping o Splicing o Trans-splicing/tandem chimerism o Parasitic and mobile genes o The large amount of “junk DNA” under selection

19. The ENCODE view: dispersed genome activity o Unannotated transcription TARs (Transcriptionally Active Regions) o Unannotated and alternative TSSs TSSs (Transcription Start Sites)

20. Gerstein et al., 2007 Unannotated and alternative TSSs Many known protein genes have alternative TSSs that are sometimes >100 kb upstream of the annotated transcription start site.

21. The ENCODE view: dispersed genome activity o Unannotated transcription TARs (Transcriptionally Active Regions) o Unannotated and alternative TSSs TSSs (Transcription Start Sites) o More alternative splicing o Dispersed regulation

22. Gerstein et al., 2007 Dispersed regulation Many regulatory elements actually reside within the first exon, introns, or the entire body of a gene.

23. Toward an updated definition of a gene Gerstein et al., 2007

24. Take-home messages

25. A. Why do cells tolerate noise instead of turning down the volume? o Some truncated mRNA molecules, which are generated as a result of AS, are translated into proteins. o There are little evidences to indicate whether this alternative splicing isoforms have a role as functional proteins or whether they are merely “noise” of the splicing machinery. Take-home messages B. Sequencing of genomes and transcripts from more organisms from a broad range of lineages will further our understanding of the alternative splicing mechanism.

26. C. The classical view of a gene as a unit of hereditary information aligned along a chromosome, each coding for one protein, has changed dramatically over the past century. And now the ENCODE project has increased the complexity still further. Take-home messages

27. Bibliography - International Human Genome Sequencing Cosortium. Initial sequencing and analysis of the human genome. Nature, Vol 409. February 2001 - Eddo Kim, Amir Goren, and Gil Ast. Alternative splicing: current perspectives. BioEssays, 2007 - Manuel Irimia, David Penny, and Scott W. Roy. Coevolution of genomic intron number and splice sites. Trends in Genetics, Vol 23. April 2007 - Rotem Sorek. The birth of new exons: Mechanisms and evolutionary consequences. RNA, Vol 13. 2007 - Mouse Genome Sequencing Consortium. Initial sequencing and comparative analysis of the mouse genome. Nature, Vol. 420. December 2002 - Rotem Sorek, Ron Shamir, and Gil Ast. How prevalent is functional alternative splicing in the human genome?. Trends in Genetics, Vol. 20. February 2004 - Douglas L. Black. Mechanisms of Alternative pre-messenger RNA Splicing. Annual Reviews Biochemistry, 2003

28. Bibliography - Martha J. Fedor. Alternative Splicing Minireview Series: Combinatorial Control Facilitates Splicing Regulation of Gene Expression and Enhances Genome Diversity. Journal of Biological Chemistry, Vol. 283. January 2008 - Claudia Ben-Dov, Britta Hartmann, Josefin Lundgren, and Juan Valcárcel. Genome-wide Analysis of Alternative Pre-mRNA Splicing. Journal of Biological Chemistry, Vol 283. January 2008 - M. Gerstein, C. Bruce, J. Rozowsky, D. Zheng, J. Du, J. Korbel, O. Emanuelsson, Z. Zhang, S. Weissman, and M. Snyder. What is a gene, post-ENCODE? History and updated definition. Genome Research, 2007

29. Thank you for your attention!!