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Alternative Splicing. Splicing. Eukaryotic genes. Mature mRNA. The mechanism of RNA splicing. 5’ splice site. Branch point. 3’ splice site. 1. 2. CAG. GTRAGT. A. YYYYYYYYYNCAG. G. 1. 2. -OH. A. A. 1. 2. The mechanism of splicing. 1. 3. 4. 1. 2. 3. 4.

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the mechanism of splicing

5’ splice site

Branch point

3’ splice site

1

2

CAG

GTRAGT

A

YYYYYYYYYNCAG

G

1

2

-OH

A

A

1

2

The mechanism of splicing
alternative splicing1

1

3

4

1

2

3

4

Alternative splicing

1

2

3

4

Can be specific to tissue, developmental-stage or condition (stress, cell-cycle).

50-70% of mammalian genes

Alternative

Splicing

Mature

splice

variant I

Mature

splice

variant II

some types of alternative splicing
Some types of alternative splicing

Exon skipping

Alternative Acceptor

Alternative Donor

Mutually exclusive

Intron retention

antibody secretion1
Antibody secretion

immunoglobulin μ heavy chain

detection of alternative splicing
Detection of alternative splicing
  • By sequencing of RNA
  • Old methods (1995-2007) – ESTs
  • New methods:
    • Splicing-sensitive microarrays
    • RNA-seq
expressed sequence tags ests

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AAA

AAA

AAA

AAA

TTTTTTTTTT

Expressed Sequence Tags (ESTs)

mRNA

RT

cDNA

Cloning

Vector

est preparation

5’ EST

3’ EST

Random-primed

EST

EST preparation

Picking a clone

Average size of EST ~450bp

alignment of ests to the genome
Alignment of ESTs to the genome

DNA

EST

EST

EST

EST

EST

EST

8 million public human ESTs, collected over >10 years (NCBI)

rna seq on multiple tissues
RNA-seq on multiple tissues

Wang et al Nature 2008

tissue specific alternative splicing1
Tissue specific alternative splicing

How is this process regulated?

regulation of alternative splicing
Regulation of alternative splicing
  • Splicing Enhancers/Silencers
  • Specifically bind SR proteins
model for ese action

SR

brain

Y(n)

AG

Weak splice site

Exon

Exonic Splicing

Enhancer (ESE)

Model for ESE action
discovery of eses
Discovery of ESEs

Exon

Silent mutations

can cause exon skipping

regulators of splicing

SR proteins (Splicing factors)

Signal

transduction

ISE

ISS

ESE/ESS

Regulators of splicing
  • Complex regulation usually exists
  • Hard to find intronic elements
  • For most alt exons – regulation unknown
how can we break the regulatory code
How can we break the regulatory code?
  • 1. Comparative genomics
  • 2. High throughput methods
the mouse genome
The mouse genome
  • 100 million years of evolution
  • Average conservation in exons: 85%
  • Only 40% of intronic sequences is alignable
  • Average conservation in alignable intronic sequences: 69%
  • Average conservation in promoters: 77%
  • Function => evolutionary conservation
conservation of near introns
Conservation of near introns

(from VISTA genome browser, http://pipeline.lbl.gov)

collection of exons

BE616884

AI972259

Collection of exons

Human DNA

AF010316

AF217965

AF217972

BE614743

finding the mouse homolog

BE616884

AI972259

Finding the mouse homolog

Mouse DNA

Human DNA

AF010316

AF217965

AF217972

BE614743

1753

Const.

243

Alt.

conservation in the intronic sequence near exons

BE616884

AI972259

Conservation in the intronic sequence near exons

Mouse DNA

Human DNA

AF010316

AF217965

AF217972

BE614743

1753

Const.

243

Alt.

results
Results

Alternative exons

Constitutive exons

Flanking conserved introns

~100 bp from each side of the exon

alternative splicing regulatory sequences
Alternative splicing regulatory sequences?
  • Could serve as binding sites for splicing regulatory proteins
motif searching
Motif searching
  • Top scoring hexamer in conserved downstream regions: TGCATG (9-fold over expected)
  • Not over-represented downstream to constitutive exons.
  • Binding site for FOX1 (splicing regulatory protein)
functional elements in the human genome
Functional elements in the human genome
  • 5% of the human genomic sequence is considered functional
impact of splicing regulatory elements
Impact of splicing regulatory elements
  • ~12,000 alt. spliced exons in the genome
  • 77% have conserved flanking intronic sequences
  • ~100bp conserved on each side
  • 12,000 exons * 100 bp * 2 introns * 0.77=2M bases
  • ==>At least2 Million bases in the human genome might be involved in alternative splicing regulation.
  • >1% of all functional DNA in the genome regulates alt splicing!
how can we break the regulatory code1
How can we break the regulatory code?
  • 1. Comparative genomics
  • 2. High throughput methods
clip seq
CLIP-seq

Ule et al, Science 2003: 340 sequences

Licatalosi et al, Nature 2008: 412,686 sequences

nova a brain specific splicing regulator
Nova, a brain-specific splicing regulator

Ule et al, Science 2003: 340 sequences

mutations causing aberrant splicing
Mutations causing aberrant splicing

Exon

~15% of all point mutations

linked to genetic disorders

involve splicing alterations

summary alt splicing
Summary – alt splicing
  • Increases the coding capacity of genes
  • We have 25,000 genes but much more protein isoforms
what is rna editing
What is RNA editing?
  • Alters the RNA sequence encoded by DNA in a single-nucleotide, site-specific, manner
  • If splicing is “cut and paste” editing is the “spelling checker”.
mode of operation a to i editing
Mode of operation: A-to-I editing

Editing performed

by ADAR enzymes

(dsRNA specific

adenosine deaminases)

Double strand

RNA is required

A-> G

functions of rna editing
Functions of RNA editing
  • Defense against dsRNA viruses
  • Also involved in endogenous regulation
functional consequences of rna editing

Protein change

Splicing

RNA stability

Functional consequences of RNA editing
  • In human, RNA editing is particularly pronounced in brain tissues, due to excess of ADAR expression in brain
  • Neural disorders (glioblastoma, epilepsy, ALS) are linked to changes in RNA-editing patterns
  • Editing levels vary in other tissues (minimal editing in skeletal muscle, pancreas).
finding rna editing sites
Theoretically easy : find mismatch between genome to RNA

Huge number of sequencing errors

Mutations

Duplications

SNPs

Signal drowns in noise

Finding RNA-editing sites
computational approach for identification of editing sites
Computational approach for identification of editing sites
  • Alignment of ESTs to genome
  • Find potential intramolecular dsRNA
  • Data cleaning

Levanon et al, Nature Biotech 2004

intramolecular dsrna
Intramolecular dsRNA

RNA

Exon

Intron

Levanon et al, Nature Biotech 2004

slide65

ESTs to genome

Levanon et al, Nature Biotech 2004

slide66

dsRNA regions

Levanon et al, Nature Biotech 2004

slide67

dsRNA regions

  • Masking EST’s ends

Levanon et al, Nature Biotech 2004

slide68

dsRNA regions

  • Masking EST’s ends
  • Masking poor sequence regions
slide69

dsRNA regions

  • Masking EST’s ends
  • Masking poor sequence regions
  • Removing known genomic SNPs

Levanon et al, Nature Biotech 2004

slide70

dsRNA regions

  • Masking EST’s ends
  • Masking poor sequence regions
  • Removing SNPs
  • Collecting candidates

Levanon et al, Nature Biotech 2004

results1
Results

DNA

RNA

(ESTs)

rna editing a source for human transcripts diversity
RNA-editing – a source for human transcripts diversity
  • >12,000 editing sites in >1,600 human genes
  • Vast majority of editing – in UTRs
  • Vast majority of editing – in Alu (repetitive)
  • A few editing sites in protein-coding regions

Levanon et al, Nature Biotech 2004

and the obligatory next generation sequencing study li levanon et al science 2009
And the obligatory next generation sequencing study…(Li, Levanon et al, Science 2009)

Editing sites

in non-repetitive

regions

connection between editing and splicing
Connection between editing and splicing

ADAR gene (editing enzyme)

Negative feedback loop

summary alt splicing and rna editing
Summary – alt splicing and RNA editing
  • Increases the coding capacity of genes
  • We have 25,000 genes but much more protein isoforms
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