Patrocles: a database of polymorphic miR-mediated gene regulation in vertebrates

(1)

Patrocles: a database of

polymorphic miR-mediated gene

regulation in vertebrates

Denis Baurain

Samuel Hiard

Wouter Coppieters

Carole Charlier

Michel Georges

(2)

Polymorphic miR-mediated

gene regulation

AAAAAAAAA…. 3’-UTR mature miR miRNP Pri -miR nucleus Pre -miR Host gene ? Exportin5 cytoplasm Drosha complex Dicer Helicase mRNA miR/miR* miRNP



Targets (1)



miRs (100s)



Silencing machinery

(overall effect)

DNA

Sequence

Polymorphisms: DSPs



Considerable sequence space is devoted to miR-mediated gene regulation

(targets, miRs, silencing machinery)



DSPs in silencing components are likely to contribute to (complex)

phenotypic variation including disease



Proof in animals: Texel sheep, Clop et al. (2006) Nat. Genet. 38:813-818



Suggestions in humans: Sethupathy & Collins (2008) TIG 24:489-497

(3)

Polymorphic miR-mediated

gene regulation

AAAAAAAAA…. 3’-UTR mature miR miRNP Pri -miR nucleus Pre -miR Host gene ? Exportin5 cytoplasm Drosha complex Dicer Helicase mRNA miR/miR* miRNP



Targets (1)



miRs (100s)



Silencing machinery

(overall effect)

DNA

Sequence

Polymorphisms: DSPs



Considerable sequence space is devoted to miR-mediated gene regulation

(targets, miRs, silencing machinery)



DSPs in silencing components are likely to contribute to (complex)

phenotypic variation including disease



Proof in animals: Texel sheep, Clop et al. (2006) Nat. Genet. 38:813-818



Suggestions in humans: Sethupathy & Collins (2008) TIG 24:489-497

http://www.patrocles.org/

Mining public databases for

SNPs and other DSPs in the

3 sequence compartments

(4)

Patrocles - Overview

Patrocles

miRBase

miRs 8nt motifs

UCSC

alignments

Ensembl

3’-UTRs SNPs

SymAtlas

gene expr.

GEO

DGV

HapMap

1000 genomes

CNVs gene expr. genotypes allele freqs

Literature

8nt motifs miR expr. CNVs eQTL machinery

(5)

Patrocles - Overview

Patrocles

miRBase

miRs 8nt motifs

UCSC

alignments

Ensembl

3’-UTRs SNPs

SymAtlas

gene expr.

GEO

DGV

HapMap

1000 genomes

CNVs gene expr. genotypes allele freqs

Literature

8nt motifs miR expr. CNVs eQTL machinery

Updates and Synchronization...



Currently, 7 species



human



chimp



mouse



rat



dog



cow



chicken

(6)

target sites in 3’-UTRs target site motifs SNPs in 3’-UTRs 3’-UTRs 2,674,395 (12.4%) 4,072,176 (15.5%) sequence space 19,595 30,290 (9.0%)

conserved L NOT X-targets

57,154 64,010 (22.4%)

conserved X NOT L-targets

9,436 10,425 (27.7%)

conserved X AND L-targets

31,416 37,700 X AND L-targets 455,620 661,187 X OR L-targets 219,392 375,054 L-targets 267,644 323,833 X-targets 58 59 X AND L-octamers 948 1,164 X OR L-octamers 466 683 L-octamers 117 170 miR* 484 676 miR 540 540 X-octamers 111,178 (87.8%) 114,305 (83.9%)

known ancestral allele

126,589 136,159 total 21,634,548 26,261,732 sequence space 21,911 24,319 genes mouse human

(7)

57,154 64,010 (22.4%)

9,436 10,425 (27.7%)

(8)

Friedman et al. (2009) Genome Res. 19:92-105

Targets – Methods



2 collections of 8nt motifs



X-targets: 540 8nt motifs (mammals)

conserved in 3’-UTRs, putative miR target sites

Xie et al. (2005) Nature 434:338-345



L-targets: 683 8nt motifs (human)

rc(2-8nt)+A from mature miRs in miRBase

Lewis et al. (2005) Cell 120:15-20



2 collections of 7nt motifs (from L-targets)



7mer-A1



7mer-m8

(9)

57,154 64,010 (22.4%)

9,436 10,425 (27.7%)

(10)

57,154 64,010 (22.4%)

9,436 10,425 (27.7%)

(11)

Targets - Concordance between

X and L target site motifs

540 8mers 577 7mers 554 6mers 683 8mers 1265 7mers 1448 6mers

91%

40%

(12)

Targets - Conserved vs.

(13)

1. human: A ...TTTGGTG

A

AACCAAC... => ancestral allele

human: G ...TTTGGTG

G

AACCAAC... => derived allele

chimp ...TTTGGTG

A

AACCAAC... => sibling species

2. rat ...TTTGGTG

A

AACAAAC...

mouse ...CTTGGTG

A

AACAAAC...

3. dog ...TTTGGTG

A

AACTAAC...

cow ...TTTGGTG

A

AACTAAC...

(3/3) TTTGGTG

A

(3/3) TTGGTG

A

(3/3) TGGTG

A

AA

(3/3) GGTG

A

AAC

(2/3) not in dog/cow gtg

a

aacc

(2/3) not in dog/cow tg

a

aacca

(2/3) not in dog/cow g

a

aaccaa => hsa-miR-29b-2*

(2/3) not in dog/cow

a

aaccaac

Targets

(14)

1. human: A ...TTTGGTG

A

AACCAAC... => ancestral allele

|||||||||

3'-GAUUCGGUGGUACACUUUGGUC-5' => hsa-miR-29b-2*

|||.|||||

human: G ...TTTGGTG

G

AACCAAC... => derived allele

chimp ...TTTGGTG

A

AACCAAC... => sibling species

2. rat ...TTTGGTG

A

AACAAAC...

mouse ...CTTGGTG

A

AACAAAC...

3. dog ...TTTGGTG

A

AACTAAC...

cow ...TTTGGTG

A

AACTAAC...

(3/3) TTTGGTG

A

...

(2/3) not in dog/cow g

a

aaccaa => hsa-miR-29b-2*

(2/3) not in dog/cow

a

aaccaac

Targets

Patrocles SNPs - Methods

CNC DNC not cons. P (CC) DC conserved ? der anc site \ allele +S +W7C / S7C

(15)

Targets

Patrocles SNPs - Results

mouse human 56 37 837 741 S 2,065 2,290 3,295 1,944 P 7,573 8,545 11,244 9,006 CNC 7,250 7,732 10,328 7,392 DNC 496+65 951+102 959+58 1,546+50 DC+CC 17,505 19,657 26,719 20,679 total Lewis Xie Lewis Xie pSNP class

# destructions

=

# creations

(16)

Targets - Patrocles SNPs

Evidence for purifying selection

SNP shuffling in 3’-UTR sequence space with preservation of trinucleotide context

(17)

Targets - Patrocles SNPs

Evidence for purifying selection

SNP shuffling in 3’-UTR sequence space with preservation of trinucleotide context

human - DC



possible elimination of SNPs affecting conserved targets



22 to 35% in human



53 to 67% in mouse



Chen & Rajewsky (2006) Nat. Genet. 38:1452-1456



depletion of SNPs in conserved miR target sites when compared to

(18)

Targets

Prioritization for lab validation



most interesting pSNPs are



pSNPs destroying conserved target sites



pSNPs creating target sites in anti-targets



to yield a phenotype, target and miR have to be

expressed in the same tissue (at the same time)



co-expression plots for human and mouse



target genes: SymAtlas



miRs: Landgraf et al. (2007) Cell 129:1401-1414



two different kinds of plots



comparing miR and target



comparing miR host gene (if any) and target

miR target

(19)

pSNPs - Co-expression plots

rs34542287 A/G [0.985/0.015] Destroyed Conserved target site miR-9 vs. actin-binding LIM protein 1

ACCA[A]AGA

rs28399411 G/A [0.994/0.006] Destroyed Conserved target site

miR-32 vs. Axonal membrane protein GAP-43 TGTGC[A]AT

mature miR counts

[+] direct evidence

[–] gross tissue mapping

host gene expression

[+] perfect matching of tissues

[–] indirect evidence

(20)



pri-miRs (stem-loops) from miRBase



pDSPs altering miR

sequence

 SNPs (de-)stabilizing interaction (seed, mature non-seed)



pDSPs altering miR

concentration

 SNPs altering processing efficiency (anywhere in stem-loop)

 CNVs encompassing miR genes

 human: http://projects.tcag.ca/variation/

 mouse: She et al. (2008) Nat. Genet. 40:909-914

 rat: Guryev et al. (2008) Nat. Genet. 40:538-545

 eQTL (or allelic imbalance) corresponding to host genes (only human)

 Morley et al. (2004) Nature 430:743-747

 Cheung et al. (2005) Nature 437:1365-1369

 Ge et al. (2005) Genome Res. 15:1584-1591

 Stranger et al. (2005) PLoS Genet. 1:e78

 Pant et al. (2006) Genome Res. 16:331-339

 Dixon et al. (2007) Nat. Genet. 39:1202-1207

 Goring et al. (2007) Nat. Genet. 39:1208-1216

 Spielman et al. (2007) Nat. Genet. 39:226-231

 Stranger et al. (2007) Nat. Genet. 39:1217-1224

Polymorphic miRs - Methods

A 5’ 9 8 7 6 5 4 3 2 miR seed target site 1 2 3 4 _{5 6 7 8} Targeted mRNA 1 5’ Pri-miR Host gene ? Pri-miR Host gene ?

(21)

Polymorphic miRs – Results

n.d. 85 affected miRs n.d. 78 eQTL miRs hosted in eQTL genes 0 256 affected miRs 0 158 CNVs miRs in CNVs 79 146 other 6 26 mature non-seed 4 12 seed 89 184 total 71 136 affected miRs SNPs in pre-miRs 466 676 pre-miRs mouse human

(22)

Polymorphic miRs – Results

Duan et al. (2007) Hum. Mol. Genet. 16:1124-1131

T

G

e.g., hsa-miR-125a

SNP in seed (+8) blocks processing

of pri-miR to pre-miR

(23)



manually curated list of 52 gene products involved in

RNA-mediated gene silencing



3 broad compartments



1. miR biogenesis: 4 (+4)



2. RISC/mRNP: 12 (+2)



3. P-bodies: 27 (+3)



pDSPs altering machinery gene

sequence



SNPs (non-synonymous, stop/frameshift, splicing site)



pDSPs altering machinery gene product

concentration



CNVs encompassing machinery genes (human, mouse, rat)



eQTL corresponding to machinery genes (human)

Silencing machinery – Methods

1

2

3

(24)

Silencing machinery – Results

n.d. 21

machinery genes identified as eQTL

0 17 affected genes 0 17 CNVs machinery genes in CNVs 52 42 splicing sites 2 45 stops / frameshifts 73 151 non-synonymous 127 237 total 35 49 affected genes SNPs in machinery genes 51 52 genes mouse human