Transcript ESTs to genome

Alternative Splicing
1
Eukaryotic genes
Splicing
Mature mRNA
2
The mechanism of RNA
splicing
3
The mechanism of splicing
5’ splice site
1
1
Branch point
CAG GTRAGT
A
-OH
A
3’ splice site
2
A
1
2
YYYYYYYYYNCAG G
2
4
Alternative splicing
1
2
50-70% of mammalian
genes
Mature
splice
variant I
1 23
3
4
Can be specific to
tissue, developmentalstage or condition
(stress, cell-cycle).
Alternative
Splicing
4
13
4
Mature
splice
variant II
5
Some types of alternative
splicing
Exon skipping
Alternative Acceptor
Alternative Donor
Mutually exclusive
Intron retention
6
Sex determination in fly
7
Sex determination in fly
8
Sex determination in fly
9
Many variants in one gene
10
DSCAM
11
Antibody secretion
12
Antibody secretion
immunoglobulin μ heavy chain
13
Tissue specific alternative splicing
14
Detection of alternative splicing
By sequencing of RNA
 Old methods (1995-2007) – ESTs
 New methods:

– Splicing-sensitive microarrays
– RNA-seq
15
Expressed Sequence Tags (ESTs)
AAA
AAA
AAA
AAA
AAAAAAAAA
mRNA
RT
AAAAAAAAA
TTTTTTTTTT
cDNA
Cloning
Vector
16
EST preparation
5’ EST
Picking a clone
3’ EST
Random-primed
EST
Average size of EST ~450bp
17
Alignment of ESTs to the
genome
DNA
EST
EST
EST
EST
EST
EST
8 million public human ESTs, collected over >10 years (NCBI)
18
Splicing microarrays
19
Massive sequencing of RNA (RNA-seq)
20
RNA-seq on multiple tissues
21
Wang et al Nature 2008
Splicing regulation
22
Tissue specific alternative
splicing
How is this process regulated?
23
Regulation of alternative
splicing
Splicing Enhancers/Silencers
 Specifically bind SR proteins

24
Model for ESE action
SR
brain
Y(n)
AG
Weak splice site
Exon
Exonic Splicing
Enhancer (ESE)
25
SR proteins structure
26
27
Discovery of ESEs
Exon
Silent mutations
can cause exon skipping
28
Regulators of splicing
Signal
transduction
ISE
SR proteins
(Splicing
factors)
ESE/ESS
ISS
• Complex regulation usually exists
• Hard to find intronic elements
• For most alt exons – regulation unknown
29
How can we break the regulatory
code?
1. Comparative genomics
 2. High throughput methods

30
Comparative genomics:
Use the mouse genome to
find sequences that regulate
alternative splicing
31
Human-mouse comparisons
32
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




33
Conservation of near introns
34
(from VISTA genome browser, http://pipeline.lbl.gov)
Collection of exons
Human DNA
AF010316
AF217965
AF217972
BE614743
BE616884
AI972259
35
Finding the mouse homolog
Mouse DNA
Human DNA
AF010316
AF217965
AF217972
BE614743
BE616884
AI972259
243
Alt.
1753
Const.
36
Conservation in the intronic
sequence near exons
Mouse DNA
Human DNA
AF010316
AF217965
AF217972
BE614743
BE616884
AI972259
243
Alt.
1753
Const.
37
Results
Constitutive exons
Constitutive exons
17%
83%
Alternative exons
Flanking
conserved
introns
Alternative exons
77%
23%
~100 bp from each side of the 38exon
Conservation of introns
39
Alternative splicing regulatory
sequences?

Could serve as binding sites for splicing
regulatory proteins
40
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)

41
Functional elements in the
human genome

5% of the human genomic sequence is
considered functional
42
Functional elements in the
human genome
Composition of functional 5% genomic sequence
Unknown
50%
Coding exons
30%
UTR and
promoters
20%
43
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 least 2 Million bases in the human genome
might be involved in alternative splicing regulation.
 >1%
of all functional DNA in the
genome regulates alt splicing!
44
How can we break the regulatory
code?
1. Comparative genomics
 2. High throughput methods

45
CLIP-seq
Ule et al, Science 2003:
340
sequences
Licatalosi et al, Nature 2008: 412,686 sequences
46
Nova, a brain-specific splicing
regulator
Ule et al, Science 2003: 340 sequences
47
48
Ule et al, Science 2003: 340 sequences
Extracting the regulatory motifs
49
The power of deep sequencing (2008)
50
Mutations causing aberrant splicing
Exon
~15% of all point mutations
linked to genetic disorders
involve splicing alterations
51
Mutations causing aberrant splicing: SMN
52
Summary – alt splicing
Increases the coding capacity of genes
 We have 25,000 genes but much more
protein isoforms

53
RNA EDITINA
54
RNA EDITING
55
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”.

56
Mode of operation: A-to-I
editing
Editing performed
by ADAR enzymes
(dsRNA specific
adenosine deaminases)
Double strand
RNA is required
A-> G
57
Mechanism of RNA-editing (A-to-I)
58
Functions of RNA editing
Defense against dsRNA viruses
 Also involved in endogenous regulation

59
60
Functional consequences of RNA
editing
Splicing
Protein change
RNA stability

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).
61
Finding RNA-editing sites

Theoretically easy :
find mismatch
between genome to
RNA




Huge number of
sequencing errors
Mutations
Duplications
SNPs
Signal drowns in
noise
62
Computational approach for
identification of editing sites
Alignment of ESTs to genome
 Find potential intramolecular dsRNA
 Data cleaning

63
Levanon et al, Nature Biotech 2004
Intramolecular dsRNA
RNA
Exon
Intron
64
Levanon et al, Nature Biotech 2004
ESTs to genome
65
Levanon et al, Nature Biotech 2004
•dsRNA regions
66
Levanon et al, Nature Biotech 2004
•dsRNA regions
•Masking EST’s ends
67
Levanon et al, Nature Biotech 2004
•dsRNA regions
•Masking EST’s ends
•Masking poor sequence regions
68
•dsRNA regions
•Masking EST’s ends
•Masking poor sequence regions
•Removing known genomic SNPs
69
Levanon et al, Nature Biotech 2004
•dsRNA regions
•Masking EST’s ends
•Masking poor sequence regions
•Removing SNPs
•Collecting candidates
70
Levanon et al, Nature Biotech 2004
Results
DNA
RNA
(ESTs)
71
72
Levanon et al, Nature Biotech 2004
73
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
74
Levanon et al, Nature Biotech 2004
And the obligatory next generation sequencing study…
(Li, Levanon et al, Science 2009)
Editing sites
in non-repetitive
regions
75
Connection between editing and splicing
ADAR gene (editing enzyme)
Negative feedback loop
76
Evolution of a new exon
77
Summary – alt splicing and RNA editing
Increases the coding capacity of genes
 We have 25,000 genes but much more
protein isoforms

78