1. Zeevik Melamed & Dror Hollander Gil Ast Lab Sackler Medical School 06-06-2013 RNA Splicing & UCSC Genome Browser

2. Lectures Overview  Introduction to RNA splicing  UCSC genome browser  Hands-on session

3. RNA Splicing

4. Outline of the 1st part  Background - Determining gene architecture  Alternative splicing (AS)  Regulation of AS  Detection of splicing events  AS and disease

5. A Genomic View

6. exon 1 exon 2 3’ splice site 5’ splice site Polypyrimidine tract C\T {15-20} Branch site A Pre-mRNA mature-mRNA intron The Splicing Process GU

7. Outline of the 1st part  Background - Determining gene architecture  Alternative splicing (AS)  Regulation of AS  Detection of splicing events  AS and disease

8. Gene number Arabidopsis thaliana 25,500~ Drosophila melanogaster ~13,600 C. elegans 19,000~ Rice (Oryza sativa) ~50,000 Mouse ~24,000 Homo Sapiens ~24,000

9. Alternative Splicing Exon 19 Exon 20 Exon 21 Intron 19 Intron 20 pre-mRNA Splicing isoform 1Splicing isoform 2 Exon 19 Exon 20 Exon 21 mRNA Exon 19Exon 21

10. Sources of Biological complexity With a limited number of genes  Co & post-transcriptional modifications: Alternative splicing  Contradicts the central dogma of molecular biology: One gene – one protein

11. Alternative Splicing Events

12. Outline of the 1st part  Background - Determining gene architecture  Alternative splicing (AS)  Regulation of AS  Detection of splicing events  AS and disease

13. Regulation of Alternative Splicing Trans-acting regulatory proteins

14. PTB PTBP1, a splicing factor known to repress alternatively spliced exons in non-neuronal tissues

15. Expression of PTB and nPTB is anti-correlated across human tissues Ratio to reference pool nPTB is enriched in CNS tissues PTB is depleted. nPTB PTB Brain tissues

16. Outline of the 1st part  Background - Determining gene architecture  Alternative splicing (AS)  Regulation of AS  Detection of splicing events  AS and disease

17. Detection of splicing events

18. Outline of the 1st part  Background - Determining gene architecture  Alternative splicing (AS)  Regulation of AS  Detection of splicing events  AS and disease

19. FD – Familial Dysautonomia Riley-Day Syndrome  Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy that occurs almost exclusively in the Ashkenazi Jewish (AJ) population.  Carrier frequency of 1:31 in AJ population and 1:18 in those of Polish descent.  Abnormal development and survival of the sensory and autonomic nervous system with progressive depletion of sensory and autonomic neurons.  FD symptoms include gastrointestinal and cardiovascular dysfunction, vomiting crises, pain and temperature insensitivity, and recurrent pneumonia  50% of patients die before the age of 40 years.

20. FD mutation IVS +6T>C in >99.3% of disease-causing alleles which results in the skipping of exon 20 and consequently generates a frameshift, which yields a stop codon in the reading frame of exon 21.