Microrna Innovations and the Origin of Novel Cell Types

MicroRNA innovations and the origin of novel cell types

A. Tanzer1,2,3, P.F. Stadler1,2,4, G.P.Wagner3

1Bioinformatics Group, Department of Compute Science, University of Leipzig, Germany 2Institute for Theoretical Chemistry, University of Vienna, Austria [email protected] 3Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06511, USA 4The Santa Fe Institute, Santa Fe, USA

Bled, Feb 20, 2009 Regulatory RNAs in an Eukaryote Cell

pre−miRNA translational inhibition Protein vRNP Drosha RNAi RISC Ribosome pri−miRNA tRNA vRNA + vault proteins CAJAL BODY rRNA Introns NUCLEOLUS mRNA mRNA rRNA miRNA NUCLEUS Splicosome snoRNA Dicer pre−rRNA miRNA RNAse_MRP pre−mRNA snRNA scaRNA pre−miRNA Y_RNA + Ro Proteins 7SL_RNA + ? 7SL_RNA Y_RNA pre−tRNA RNAse_P vRNA Y RNP SRP tRNA Regulatory RNAs in an Eukaryote Cell

pre−miRNA translational inhibition Protein vRNP Drosha RNAi RISC Ribosome pri−miRNA tRNA vRNA + vault proteins CAJAL BODY rRNA Introns NUCLEOLUS mRNA mRNA rRNA miRNA NUCLEUS Splicosome snoRNA Dicer pre−rRNA miRNA RNAse_MRPpre−mRNA snRNA scaRNA pre−miRNA Y_RNA + Ro Proteins 7SL_RNA + ? 7SL_RNA Y_RNA pre−tRNA RNAse_P vRNA Y RNP SRP tRNA What are MicroRNAs?

◮ small non-coding RNAs (ncRNAs) ◮ regulate gene expression ◮ target DNA and mRNA ◮ part of protein complexes Maturation of miRNAs

A G U A A U U U A G U A U G A U G C U A A U U U U A ◮ G C C G miR-92-2 G A U primary-precursor micro RNA C miR-106a G U U U U _ U U U G A C U G U _ A U C G U U _ A U A _ A C G _ C C C _ U G G _ A C C G _ G U A G _ G G A U A UG G _ U U _ U A C G C _ U G G G _ C A U A C G U U G A G C U A C C A C A G C A C G G C U C G A C _ U C A U A C C A C G G C C U (pri-miRNA) G G A A U A G U U G U C U C U A C A G C G A _ _ A U G U A _ _ C U C A C U _G A C G U C A _ C C U U A A G GC U A _ U U AUAA A C U A U A G _ A AC A U U C G _ U A G U G U U A G U C A A A A C G U C U A A A C G U G U C G G U A U A U A A U U U U A U _ C G A C U U A C A A U U A G U U U U G C A G A U U U G C AGUUC A G CGU A U A U A G A G G U G A C C C U G U A C U A G U A G G G U U A U U U U A U G U U C A G A U U C G G A C C U CCU C A G U U U A A U U A CA A C U G U G A G C A C C U G G A U C G A A A U A A A A U A G C U A A U A C A U U U G G U A G U U A miR-18 C U A U A C A ◮ U G U G U C U A C A U A U A A U U G G A C miR-19b-2 C G G A C A U U A U U A C G U C G C C A U G G A U C C C U CU G C Cleavage I: Drosha G U G A C A U G C U G C U A A G C G A A C U G C C A A U A A U A C U U C U G AGA U A A U G A A G G A A C A A A A C A G U U U A U U C U C G U C A UG U ◮ A A U A _ A G A G UC G UA A C precursor miRNA (pre-miRNAs) G A A A U C G G U C G A C _ U A _ U U C A U _ A G C _ G U U U A C G G U A A U U U _ A C G A A C C C A C C A C C U A U G A U G A U U C ◮ G A U G U G G U C U G G G C A U C export to cytoplasm (exportin-5) G C G A A U U U A G C G C U U G C A A G U U G U A U _ G U A G A _ G A _ _ G G U _ A _ U C _ G G G _ C miR-20 _ U _ A C A C G G A G A A A U U A C U G C U A U C ◮ U U U A Cleavage II: Dicer G G C ◮ mature miRNA (miRNAs) Maturation of miRNAs

G C U A ◮ C G primary-precursor micro RNA A U G G A G A U U A A U (pri-miRNA) A U U A G C U G C A U ◮ A G Cleavage I: Drosha A U A U G C U A G C ◮ C G precursor miRNA (pre-miRNAs) U G U A A A C G A U G G ◮ U A export to cytoplasm (exportin-5) G C C G A U G C GU A ◮ A U Cleavage II: Dicer G C U AU G C A G U U ◮ mature miRNA (miRNAs) A U G Maturation of miRNAs

◮ primary-precursor micro RNA 5’ A C T (pri-miRNA) G C A ◮ Cleavage I: Drosha G T G A ◮ precursor miRNA (pre-miRNAs) A G G ◮ C export to cytoplasm (exportin-5) A C T T ◮ G Cleavage II: Dicer T 3’ ◮ mature miRNA (miRNAs) Functions of miRNAs

Cytoplasm: post-transcriptional gene scilencing (PTGS) ◮ mRNA degradation (RNAi) ◮ translational repression

Nucleus: transcriptional gene silencing (TGS) ◮ DNA methylation ◮ heterochromatin remodelling histone methylation

Nucleus: transcriptional gene activation (RNAa) ? ◮ binding to promoter regions Functions of miRNAs

Cytoplasm: post-transcriptional gene scilencing (PTGS) ◮ mRNA degradation (RNAi) ◮ translational repression

Nucleus: transcriptional gene silencing (TGS) ◮ DNA methylation ◮ heterochromatin remodelling histone methylation

Nucleus: transcriptional gene activation (RNAa) ? ◮ binding to promoter regions Functions of miRNAs

Cytoplasm: post-transcriptional gene scilencing (PTGS) ◮ mRNA degradation (RNAi) ◮ translational repression

Nucleus: transcriptional gene silencing (TGS) ◮ DNA methylation ◮ heterochromatin remodelling histone methylation

Nucleus: transcriptional gene activation (RNAa) ? ◮ binding to promoter regions Evolutionary origin of prominent ncRNA families Chordata Craniata Y RNA7SK ? U7 Cephalochordata Metazoa RNAse MRP Urochordata telomerase RNA most snRNAs Echinodermata

vault RNAs ? Hemichordata Eukarya Protostomia Choanoflagellata Fungi snoRNA C/D snoRNA H/ACA Microsporidia Alveolates Stramenophiles Rhodophyta Green Plants other protists LUCA Archea

rRNA Bacteria gRNA in Kinetoplastids only tRNA microRNA in multicellular animals and plants only ? RNAseP tmRNA 7SL/SRP small bacterial RNAs Molecular Evolution of selected miRNAs

mir-10, mir196, iab-4 in the Hox gene mir-17 cluster cluster

I−B A A A A A I−1 I−A B B B B B I−X I−C C C C C C ? D D D D D mir−196 II II−D Danio rerio Tetraodontiformes Latimeria Mammalia Heterodontus mir−10 iab4 Mammalia Teleostei A A I B B C C II D D Deletions ancestral teleost ancestral sarcopterygian 20 is copy of 17

93 is copy of 17 cluster duplication Branchiostoma ? A 18 is copy of 17 B Petromyzon marinus C ancestral gnathostome 18 deletion D 17 19 Cenorhabditis briggsae Cenorhabditis elegans Tribolium Apis mellifera Anopheles Drosophila 20 92 19b ancestral vertebrate 19b is copy of 19 106b Strongylocentrotus Ciona 93 19−3 25

Tanzer and Stadler. J Mol Biol. 2004; 339:327-35 Tanzer et al. J Exp Zoolog B Mol Dev Evol. 2005; 304:75-85 Expansion of The Metazoan MicroRNA Repertoire

Sm Cb Ce Am Tc Bm Ag D.sp. Sp Od Ci Cs Dr Tn Tr Xt Gg MdRn Mm Bt Cf Pt Hs

4 44 23 1 2 2 6 1 2 tandem 2 dupl. 4 1 1 40 6 4

1 5 1 1 non-local 40 2 3 1 1 dupl. 71 13 1 1 2

56 24 11 1 1 innovations 1 0 1

18 1 1

Hertel et al., BMC Genomics.2006;7:25 Expansion of The Metazoan MicroRNA Repertoire

Dr Tn Tr Xt Gg MdRn Mm Bt Cf Pt Hs

tandem 6 1 dupl. 2 2

40 6 4 non-local dupl. 1 5 1

1 40 2 3 1 1

2 innovations

56 24 11

Hertel et al., BMC Genomics.2006;7:25 Genomic Complexity

Mattick JS, The Journal of Experimental Biology 210, 1526-1547 MicroRNAs in Developmet

Sm Cb Ce Am Tc Bm Ag D.sp. Sp Od Ci Cs Dr Tn Tr Xt Gg MdRn Mm Bt Cf Pt Hs

4 44 23 1 2 2 6 1 2 2 4 1 1 40 6 4

1 5 1

1 40 2 3 1 1 71 13 1 1 2

56 24 11 1 1 1 0 1

18 1 1

chicken: http://geisha.arizona.edu/, zebraﬁsh: http://exiqon.com/ MicroRNAs in Developmet

Sm Cb Ce Am Tc Bm Ag D.sp. Sp Od Ci Cs Dr Tn Tr Xt Gg MdRn Mm Bt Cf Pt Hs

4 44 23 1 2 2 6 1 2 2 4 1 1 40 6 4

1 5 1

1 40 2 3 1 1 71 13 1 1 2

56 24 11 1 1 1 0 1

18 1 1 mir1: heart, somites

mir10b: limb buds

chicken: http://geisha.arizona.edu/, zebraﬁsh: http://exiqon.com/ MicroRNAs in Developmet

Sm Cb Ce Am Tc Bm Ag D.sp. Sp Od Ci Cs Dr Tn Tr Xt Gg MdRn Mm Bt Cf Pt Hs

4 44 23 1 2 2 6 1 2 2 4 1 1 40 6 4

1 5 1

1 40 2 3 1 1 71 13 1 1 2

56 24 11 1 1 1 0 1

18 1 1 mir1: heart, somites

mir10b: limb buds mir140:spinal cord mir140: cartillage

chicken: http://geisha.arizona.edu/, zebraﬁsh: http://exiqon.com/ MicroRNAs in Developmet

Sm Cb Ce Am Tc Bm Ag D.sp. Sp Od Ci Cs Dr Tn Tr Xt Gg MdRn Mm Bt Cf Pt Hs

4 44 23 1 2 2 6 1 2 2 4 1 1 40 6 4

1 5 1

1 40 2 3 1 1 71 13 1 1 2

56 24 11 1 1 1 0 1

18 1 1 mir1: heart, somites

mir122: liver

mir10b: limb buds mir140:spinal cord mir140: cartillage

chicken: http://geisha.arizona.edu/, zebraﬁsh: http://exiqon.com/ MicroRNAs in Developmet

Sm Cb Ce Am Tc Bm Ag D.sp. Sp Od Ci Cs Dr Tn Tr Xt Gg MdRn Mm Bt Cf Pt Hs

4 44 23 1 2 2 6 1 2 2 4 1 1 40 6 4

1 5 1

1 40 2 3 1 1 71 13 1 1 2 ?

56 24 11 1 1 1 0 1

18 1 1 mir1: heart, somites

mir122: liver

mir10b: limb buds mir140:spinal cord mir140: cartillage

chicken: http://geisha.arizona.edu/, zebraﬁsh: http://exiqon.com/ Novel Cell Type in Eutherian Mammals: Endometrium

◮ inner layer if the uterus ◮ implantation and decidualisation ◮ differentiation induced by progesterone and estrogen

adapted from: Kobayashi, A and Behringer, R R, Nat Rev Genet (2003), 4:969-980 MicroRNA expression analysis: Microarray

1 1 0.91

◮ majority expressed upon 0.8

homon treatment 0.64 0.6 ◮ vertebrate miRNAs act in 0.54 both diff. and undiff cells 0.46 0.4 ◮ eutherian and primate 0.36 miRNAs highly upregulated in diff. cells 0.2 0.09

0 0 vertebrates eutherians primates total MicroRNA expression analysis: Microarray

1 1 0.91

◮ majority expressed upon 0.8

homon treatment 0.64 0.6 ◮ vertebrate miRNAs act in 0.54 both diff. and undiff cells 0.46 0.4 ◮ eutherian and primate 0.36 miRNAs highly upregulated in diff. cells 0.2 0.09

0 0 vertebrates eutherians primates total MicroRNA expression analysis: Microarray

1 1 0.91

◮ majority expressed upon 0.8

homon treatment 0.64 0.6 ◮ vertebrate miRNAs act in 0.54 both diff. and undiff cells 0.46 0.4 ◮ eutherian and primate 0.36 miRNAs highly upregulated in diff. cells 0.2 0.09

0 0 vertebrates eutherians primates total Functional study: miRNA knock downs

mirVana miRNA microarray RNAup target predictions

0.8

0.6

0.4

0.2

0 100 150 200 250 300 bp in 3’UTR

Gene regulatory network in endometrial stromal cells

miR206 miR342 miR494

ER PGR

miR181 PR GDNF GZF1 BSF SP1

HoxA11 ? HoxA10 ETS1 miR486 miR188

Hmx3 EP3 EPR BiP p53 CALD1 miR500 AHSG miR185 EP4 miR30 EMX2 FKBP52 HoxA11OS Î²3Int Wnt4 CyclinD3 CEBP Decorin Laminin FOXO1A

Ihh SSEA3 Ptch EGFR CyclinE pRB p27 IGFBP1 p15 p57 LIF PRL IGFBP TIMP3 EBAF Somatostatin CNR1

FST BMP2 COUPTFII Muc1 Cox2 epiregulin cochlin IGFBP3 IRG1 sFRPR Msx1 STAT3 OsM Htype1 IL1Î± HBEGF amphiregulin

PGs PAI Summary

◮ miRNA are cool ◮ evolution of miRNAs still an ongoing process ◮ waves ob innovation - major transition in body plan formation ◮ novel miRNAs expressed in novel tissues ◮ ncRNA repertoire linked to complexity of organism ◮ are we ribo-organisms? Summary

IZBI, Univerity of EEB, Yale TBI, University of Leipzig: University: Vienna: Peter Stadler Günter Wagner Ivo Hofacker Jana Hertel Vincent Lynch Christoph Flamm Stephan Bernhard Deena Emera Stefan Washietl Alexander Vargas Ulli Mückstein