Zeevik Melamed & Dror HollanderGil Ast LabSackler Medical School

03-05-2011

RNA Splicing,UCSC Genome Browser & Galaxy

Lectures Overview

Introduction to RNA splicing UCSC genome browser & Galaxy Hands-on session

RNA Splicing

Outline of the 1st part

Background - Determining gene

architecture

Alternative splicing (AS)

Regulation of AS

Detection of splicing events

AS and disease

A Genomic View

exon 1 exon 2

3’ splice site

5’ splice site Polypyrimidine tract

C\T {15-20}

Branch site

A

Pre-mRNAmature-mRNA

intron

The Splicing Process

Outline of the 1st part

Background - Determining gene

architecture

Alternative splicing (AS)

Regulation of AS

Detection of splicing events

AS and disease

Gene number

Arabidopsis thaliana25,500~

Drosophila melanogaster~13,600

C. elegans 19,000~

Rice (Oryza sativa) ~50,000

Mouse~24,000

Homo Sapiens~24,000

Alternative Splicing

Exon 19Exon 20

Exon 21Intron 19

Intron 20

pre-mRNA

Splicing isoform 1 Splicing isoform 2

Exon 19Exon 20

Exon 20

Exon 21

mRNA

Exon 19 Exon 21

Sources of Biological complexity

With a limited number of genes

Post-trancriptional modifications:

Alternative splicing

Contradicts the central dogma of molecular biology:

One gene – one protein

Alternative Splicing Events

Outline of the 1st part

Background - Determining gene

architecture

Alternative splicing (AS)

Regulation of AS

Detection of splicing events

AS and disease

Hollander et al. 2010

Regulation of Alternative Splicing

Trans-acting regulatory proteins

PTBPTBP1, a splicing factor known to repress alternatively spliced exons in non-neuronal tissues

Expression of PTB and nPTB is Expression of PTB and nPTB is anti-correlated across human tissuesanti-correlated across human tissues

Ratio to reference pool

nPTB is enriched in CNS tissues PTB is depleted.

nPTB

PTB

Brain tissues

Outline of the 1st part

Background - Determining gene

architecture

Alternative splicing (AS)

Regulation of AS

Detection of splicing events

AS and disease

Detection of splicing events

Outline of the 1st part

Background - Determining gene

architecture

Alternative splicing (AS)

Regulation of AS

Detection of splicing events

AS and disease

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 .

FD mutationIVS +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.