Alternative splicing in Alternative splicing in human tissueshuman tissues

John Castle

July 20, 2007Rosetta / Merck

Seattle, USA

AcknowledgementsAcknowledgements

Rosetta/MerckJason JohnsonChris Armour Ronghua ChenPhil Garrett-EngeleAmit KulkarniLee LimChris RaymondJyoti Shah

CollaboratorsTom Cooper (Baylor)

(Data not shown)Guey-Shin WangDuyen Tran

Chaolin Zhang (summer intern from CSHL)Publications citedSpellman and Smith, 2006Boutz et al., 2007 (Ares and Black labs)Zhu, B. et al., 2005 (Gulick lab)

Needs for alternative splicing at a Needs for alternative splicing at a pharmaceutical companypharmaceutical company

Biomarker identificationIdentify transcript structures expressed in a tissue,

including novel isoformsReport regulation of known isoforms for use in pathway

analysis or as drug targets

RequirementsRequirementsRobust, high-confidence, high-throughput profiling

– Array patterns, amplification, sequencing

Visualization and analysis tools for profiling dataInterpretation, including isoform function and pathways

Discovery-mode junction arrays (Refseqs only):multiple samples help, the events you don’t monitor hinder interpretation

Junction Probe

X06989

NM_201414

NM_000484

Predictions from residuals (model-observed)

Junction

Tis

sues

Log probe intensity

Junction

Melanoma

Lung Carcinoma

Brain

Fetal BrainBrain-Amygdala

400300200100

Brain samples

6 7 95

RT-PCR Primers

mixture withnon-Refseqnot predicted(no probe)

Gene = APP

Some probe strategies and limitation for Some probe strategies and limitation for microarray patternsmicroarray patterns

Exon arrays – Easiest to design and interpret– Miss substantial fraction of known AS events 5’SS, 3’SS, intron retentions

Junction arrays– Less control over probe position– Single probe events difficult to interpret (e.g. 3-nt 3’SS vs. 300 nt inserted exon), complicating validation– More known and unknown events detected, but accordingly more difficult to interpret– May want to use exon-to-intron junction probes as controls for “half-hybridization” in intensity-based experiments

Junction + exon arrays– 2x expense, but more markers of same event– Different probe lengths affect intensities (not ratios)

Splicing event arrays (mutually exclusive probes)– Coverage of more splicing events from ESTs– Need extra probes to estimate gene-level expression– Can’t detect novel splicing events– Unmonitored splicing events/mixtures more easily confound analysis

Junction + exon + mutually exclusive probes for known events– Expensive– Most complex data analysis– Best balance of discovery and monitoring (one data set for both)– Enables reciprocal analysis

All methods– False predictions created by saturated probes, dim probes, constant probes, specific & non-specific cross-hybridization– Splicing predictions can be confounded by strong gene-level regulation or complex isoform mixtures– Read-out differentially regulated splicing events – difficult to accurately quantitate isoforms– More samples help

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

1 10 *12atg1 10 *12atg

Brain

Fetal Brain

Colon

400300200100

Junction probe for double exon drop

Junction + Exon + Isoform-monitoring probesJunction + Exon + Isoform-monitoring probes

1 10 *12atg1 8 *7atg

Analysis tools for alternative splicing Analysis tools for alternative splicing profiling dataprofiling data

Confounding factors include• Inaccurate estimates of the gene expression• Spurious measurements, such as from cross-

hybridization, bad probes, and high background

Needed output• A confidence value (e.g., a p-value)• Expression changes, including both:

– Exon regulation– Splicing event regulation (e.g., % of total gene expression)

• Several measurement types, including:– Novel transcript script structure– Individual regulated exon (may be a minor component)– Differentially expressed alternative splicing event

SyntheticSynthetic data show how dark and saturated data show how dark and saturated can lead to compressed ratioscan lead to compressed ratios

These probes compress ratios and can erroneous values:• Probes with high background • Probes always dim• Probes near saturation

If not filtered, these appear to be novel splicing predictions.

Real data showing single probe predictions Real data showing single probe predictions with high cross-hyb potentialwith high cross-hyb potential

Gene level consistent across samples, but relatively low

All alternative isoforms show low expression in both samples and no regulation.

One probe shows differential expression with intensities much higher than the gene expression, suggesting cross-hybridization.

We have an 0-for-8 validation rate of these predictions.

What transcript structure is expressed?What transcript structure is expressed?

Large prediction, but change in isoform ratios is meaningless.

The RefSeq isoform (exon 17) is not expressed, just alternate form (#2).

looks like a splicing prediction, but isn’t

Probes with high background and constant-intensity probes create false predictions.

The cDNA lacking exon 17 should be used for screening.

No alternative splicing

Exon 10 is a novel cassette exon. The use of multiple probes increases the confidence of this prediction.

The exon 10 is upregulated ~10-fold.

However, the low intensities on probes monitoring exon 10 suggest the +exon 10 isoform is a minor component.

A junction probe spanning exon 9-to-11 would enable a determination of the relative abundance.

Example of a potential biomarkerExample of a potential biomarker

Isoform composition change

-75

-50

-25

0

25

50

75

-75 -50 -25 0 25 50 75

RT-PCR semi-quantative (+/- 15)

Mic

roar

ray

R = 0.90R2 = 0.82

Validation of alternative splicing Validation of alternative splicing profiling of sample pairsprofiling of sample pairs

Eliminate suspect measurements, such as high background, from non-expressing genes, or that are not consistent with alternative splicing.

Validate by RT-PCR only changes in size greater than 3-nt.

Our Version 2 algorithm achieves >80% validation for call rate and has a good correlation (left). The algorithm is a reciprocal measurement, similar to Ule & Darnells and ExonHit’s, with basic additional filtering.

Version 3 (being tested) includes a p-value and additional filters.

Interpretation of splicing changesInterpretation of splicing changesCurrently in pathways:

- transcription factor to targetsNeed to add systems biology at a splicing level, such as:

- splicing factor to target exons- isoform specific nodes in pathways- isoform specific phenotypes & functions

Disease markers? Casual? Necessary?

KEGG MAPK Pathway

Need: controlled Need: controlled vocabulary for vocabulary for alternative splicing!alternative splicing!

miR-124

PTB nPTB

MEF2 genes

KTN1Additional

genes

Splicing changes, mediated through CU-rich elements

3’ UTR hexamer

Identified through shRNA experiments targeting PTB and nPTB, monitored on microarrays.

PTB and nPTB pathwayPTB and nPTB pathway

Spellman and Smith, 2006Boutz et al. Genes Dev. 2007

NeuronsGlial cells

Boutz et al. Genes Dev. 2007 (Ares and Black labs)

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

Ratio to reference

pool

nPTB is enriched in CNS tissues; PTB is depleted.

nPTB

PTB

Brain tissues

PTB and nPTB gene expressionPTB and nPTB gene expression

miR-124

PTB nPTB

MEF2 genes

KTN1Additional

genes

Splicing changes, mediated through CU-rich elements

3’ UTR hexamer

Identified through shRNA experiments targeting PTB and nPTB, monitored on microarrays.

PTB associated alternative splicingPTB associated alternative splicing

NeuronsGlial cells

Boutz et al. Genes Dev. 2007 (Ares and Black labs)

Zhu, B. et al. J. Biol. Chem. 2005

MEF2A, MEF2C & MEF2D have a 21 or 24-nt cassette exon, labeled exon β

MEF2 transcripts are alternatively spliced; changes MEF2 transcripts are alternatively spliced; changes lead to increased transcriptional activitylead to increased transcriptional activity

Exon β inclusion leads to increased activity in all MEF2 genes.

Zhu, B. et al. J. Biol. Chem. 2005

Brain

Adipose

Heart

MEF2A is alternatively spliced in normal tissuesMEF2A is alternatively spliced in normal tissues

Pool

The exon is 10-fold upregulated in heart. A junction probe would enable a determination as to whether it is a major form.

Brain

Adipose

Heart

Zhu, B. et al. J. Biol. Chem. 2005

MEF2D is alternatively spliced in normal tissuesMEF2D is alternatively spliced in normal tissues

The exon 21-nt long and the associated exon-probe (middle probe) performs poorly. The presence of junction probes permits the measurement.

Brain

Adipose

Heart

Zhu, B. et al. J. Biol. Chem. 2005

The mutually exclusive junction probe allows an estimate of the isoform change for exon 8. Change in isoform composition, versus brain:

Heart -80

Adipose -82

MEF2C is alternatively spliced in normal tissuesMEF2C is alternatively spliced in normal tissues

Paul L. Boutz et al. Genes Dev. 2007;

PTB nPTBMEF2Cexon β

KTN1exon 41

40 42 40 41 42

PTB knock-down leads to exon drop

PTB and nPTB gene expression correlate with target PTB and nPTB gene expression correlate with target alternative splice changes across normal tissuesalternative splice changes across normal tissues

PTB levels correlate with KTN1 exon 41 PTB levels correlate with KTN1 exon 41 expressionexpression

PTB associated alternative splicing,PTB associated alternative splicing, motif identification motif identification

miR-124 transcript targetsmiR-124 transcript targets

Transfect miR-124 and microarray profile

Lim et al, Nature, 2005

Hexamer p-valuegtgcct 1.60E-80tgcctt 3.30E-48agtgcc 8.50E-20gcctta 2.90E-17tgtgcc 4.90E-13gccttt 1.30E-10aagtgc 2.80E-10atgtgc 0.0001ccttac 0.003

… gene regulation in our body atlas

… 3’ UTR hexamers, using hypergeometric

overlap statistics

Compare genes downregulated to …

miR-124 targets include PTB; which was found down-regulated upon miR-124 transfection and whose 3’ UTR contains 5 hexamers; but not nPTB.

Pentamer enrichment adjacent regulated Pentamer enrichment adjacent regulated cassette exonscassette exons

cassette exon3’

pre-mRNA

5’

upstream (udif)

upstream intronic fraction (uif)

exon downstream intronic fraction (dif)

duif

dexon5exon3exon5

Motif enrichment in the 200-nt intronic region immediately upstream of cassette exons upregulated in frontal lobe identifies TCTCT.

TCTCT

CTCTC

CTTTC

TTTCT

CTGCT

TGCTT

TCTCT enrichment in intronic regions TCTCT enrichment in intronic regions upstream of upregulated cassette upstream of upregulated cassette

exons in human body tissuesexons in human body tissuesPTB TCTCT enrichment

Upregulation Downregulation-

Log10 ratio

The intronic region upstream of MEF2A The intronic region upstream of MEF2A exon β contains a conserved TCTCTexon β contains a conserved TCTCT

TCTCT-associated exons are upregulated with TCTCT-associated exons are upregulated with decreasing PTB but not nPTB expressiondecreasing PTB but not nPTB expression

Interpretation of splicing changesInterpretation of splicing changesCurrently in pathways:

- transcription factor to targetsAdd: systems biology at a splicing level

- splicing factor to target exons associations- isoform specific nodes in pathways- isoform specific phenotypes & functions

PTB MEFC2 w/ exon β

KEGG MAPK Pathway

Need: controlled vocabulary Need: controlled vocabulary for alternative splicingfor alternative splicing

Conclusions from PTB storyConclusions from PTB storyPTB and nPTB expression is anti-correlated across 50

human tissues.PTB expression is anti-correlated with the expression of a

set of known cassette exons.The TCTCT pentamer is enriched in the intronic region

upstream of the cassette exons.Low PTB expression results in TCTCT enrichment; there is

less correlation with nPTB.

Pathway interpretation suggested: PTB inhibits expression of cassette exons with TCTCT-rich upstream introns; nPTB is unable to inhibit at least a subset of them

Needs for alternative splicing at a Needs for alternative splicing at a pharmaceutical companypharmaceutical company

Biomarker identificationIdentify transcript structures expressed in a tissue,

including novel isoformsReport regulation of known isoforms for use in pathway

analysis or as drug targets

RequirementsRequirementsRobust, high-confidence, high-throughput profiling

– Array patterns, amplification, sequencing

Visualization and analysis tools for profiling dataInterpretation, including isoform function and pathways

Back-ups

cassette exon3’

pre-mRNA

5’

upstream (udif)

upstream intronic fraction (uif)

exon downstream intronic fraction (dif)

duif

TCTCT is enriched in the ~50-nt upstream of up-TCTCT is enriched in the ~50-nt upstream of up-regulated cassette exons regulated cassette exons

DownUp

Conclusions from PTB storyConclusions from PTB storyPTB and nPTB show anti-correlated expression across 50

human tissues.PTB and a set of known cassette exons show anti-

correlated expression.The TCTCT hexamer is enriched in the intronic region -70

nt to -30 nt upstream of the cassette exons.Low PTB expression results in TCTCT enrichment; there is

less correlation with nPTB.

Pathway interpretation suggested: PTB inhibits expression of cassette exons with TCTCT-rich upstream introns; nPTB is unable to inhibit them

Novel exon 11 is predicted by a cross-species sequence analysis.

These microarray data suggest that the exon is expressed and regulated.

Validation of bioinformatics-predicted Validation of bioinformatics-predicted transcript structurestranscript structures

Junction probes enable monitoring of Junction probes enable monitoring of alternative 3’ and 5’ splice sitesalternative 3’ and 5’ splice sites

Sample B

Sample A

Sample A

Sample B

1 19 *20atg

1 10 *12NM_123456

Isoform 2

Cassette Exon

Junction probeExon probe

*

atg

atg

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1 10 *12atg

1 10 *12atg

1 10 *12atg

Some probe strategies for microarray patternsSome probe strategies for microarray patterns

1 10 *12atg

All junctions

Use of all junction probes reveals Use of all junction probes reveals potential novel transcript structurespotential novel transcript structures

Exons 14 and 17 are not previously known to be cassette exons.

Exon 3 is a known cassette exon

Exon 8 is a known mutually exclusive exon

Examine probe intensity (brain) for probes spanning all possible junctions (e.g. exons 12, exons 13, exons 1 4, …)

1 atg

All junctions

Bottom line – no predicted changes in alternative splicing, rather a single isoform – of two known - is significantly upregulated.

probes w/high predicted

background

mutually exclusive exon

Exon predicted to cross-hyb

Sample B

Sample A

Sample B

Sample A

110A

*atg10B

Mutually exclusive exon pair

Real data showing several probes with high Real data showing several probes with high backgroundbackground