Alternative splicing
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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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Alternative Splicing

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Alternative splicing

Alternative Splicing


Eukaryotic genes

Splicing

Eukaryotic genes

Mature mRNA


The mechanism of rna splicing

The mechanism of RNA splicing


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


Sex determination in fly

Sex determination in fly


Sex determination in fly1

Sex determination in fly


Sex determination in fly2

Sex determination in fly


Many variants in one gene

Many variants in one gene


Dscam

DSCAM


Antibody secretion

Antibody secretion


Antibody secretion1

Antibody secretion

immunoglobulin μ heavy chain


Tissue specific alternative splicing

Tissue specific alternative splicing


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

AAAAAAAAA

AAAAAAAAA

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)


Splicing microarrays

Splicing microarrays


Massive sequencing of rna rna seq

Massive sequencing of RNA (RNA-seq)


Rna seq on multiple tissues

RNA-seq on multiple tissues

Wang et al Nature 2008


Splicing regulation

Splicing regulation


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


Sr proteins structure

SR proteins structure


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


Comparative genomics use the mouse genome to find sequences that regulate alternative splicing

Comparative genomics: Use the mouse genome to find sequences that regulate alternative splicing


Human mouse comparisons

Human-mouse comparisons


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


Conservation of introns

Conservation of introns


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


Alternative splicing

Functional elements in the human genome


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


Alternative splicing

Ule et al, Science 2003: 340 sequences


Extracting the regulatory motifs

Extracting the regulatory motifs


The power of deep sequencing 2008

The power of deep sequencing (2008)


Mutations causing aberrant splicing

Mutations causing aberrant splicing

Exon

~15% of all point mutations

linked to genetic disorders

involve splicing alterations


Mutations causing aberrant splicing smn

Mutations causing aberrant splicing: SMN


Summary alt splicing

Summary – alt splicing

  • Increases the coding capacity of genes

  • We have 25,000 genes but much more protein isoforms


Rna editina

RNA EDITINA


Rna editin g

RNA EDITING


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


Mechanism of rna editing a to i

Mechanism of RNA-editing (A-to-I)


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


Alternative splicing

ESTs to genome

Levanon et al, Nature Biotech 2004


Alternative splicing

  • dsRNA regions

Levanon et al, Nature Biotech 2004


Alternative splicing

  • dsRNA regions

  • Masking EST’s ends

Levanon et al, Nature Biotech 2004


Alternative splicing

  • dsRNA regions

  • Masking EST’s ends

  • Masking poor sequence regions


Alternative splicing

  • dsRNA regions

  • Masking EST’s ends

  • Masking poor sequence regions

  • Removing known genomic SNPs

Levanon et al, Nature Biotech 2004


Alternative splicing

  • 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)


Alternative splicing

Levanon et al, Nature Biotech 2004


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


Evolution of a new exon

Evolution of a new exon


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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