ongoing dna transfer from the mitochondrial to the nuclear genome miria ricchetti (mricch@...

Ongoing DNA transfer from the mitochondrial to the nuclear genome

Miria Ricchetti (mricch@ pasteur.fr)

Bioinformatic and Comparative Genome Analysis CourseHKU-Pasteur Research Center, Hong Kong, China

August 17-29, 2009

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1. The genomes of eukaryote cells

The genomes of eukaryote cells

chloroplasts (in plants)

mitochondria

nucleus

Endosymbiont theory

Mitochondria and chloroplasts are relics of free-living bacteria that formed a symbiotic association with the

precursor of the eukaryotic cell

Origin of mitochondria and chloroplasts

EUCARYOTES

Algae

Plants

Fungi

ProtozoaSlime moulds

Gram-positive bacteria

Spirochetes

Green photosynthetic

bacteria

EUBACTERIA

Methanogens

Extreme halophiles

Thermoacidophiles

Sulfolobus

Thermoplasma

ARCHAEBACTERIA

UNIVERSAL ANCESTOR

Animals

Purple photosynthetic

bacteria

Mitochondria

Cyanobacteria

Chloroplasts

Origin of mitochondria and chloroplasts

Mitochondria generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy.

Mitochondria are also involved in other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth.

Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction, and may play a role in the aging process.

Mitochondria

McBride HM et al. 2006. Curr. Biol. 16: R551-600;Bossy-Wetzel E, et al 2003. Curr Opin Cell Biol 15, 706-16..

View of mitochondria within a cell

Chloroplasts, also called plastids, are organelles found in plant cells and other eukaryotic organisms that conduct photosynthesis. During photosynthesis, chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH.

Chloroplasts

Eberhard S. et al. 2008. Ann. Rev. Geneti. 42 463-515Puthiyaveetil S, Allen JF. 2009, Proc Biol Sci. 276:2133-45

85,779 bp

Mitochondrial genome

Saccharomyces cerevisiae

Nuclear genome

Homo sapiens 16,554 bp2.9 x 109 bp

1.2 x 107 bp

Genomes size

Reclinomonas americana

size mt genome (kbp)

protein-coding genes

67

Plasmodium falciparum 3

Homo sapiens 13

average 12-24

Saccharomyces cerevisiae 17

16.5

85.8

Smallest free-living -proteobacterial genomeBartonella henselae < 2000 1600

Arabidopsis thaliana 367

69

6

(protist)

and most vertebrates

(parasitic protist)

Mt genome size

45

Mitochondrial Genome Products

The mitochondrial genome:

• encodes some proteins but most proteins for organelle function are encoded by chromosomal genes and imported from cytoplasm

• encodes most of RNA components of protein synthesis (mRNA, rRNA, tRNA)

in Saccharomyces cerevisiae mt proteins are coded:

about 400 in the nucleus(200 are of bacterial origin)

17 in the mt

Genes coding for mitochondrialfunction and propagation

Mitochondrial genome products (in humans)

Protein Complex Encoded by Encoded by Mitochondrial NuclearGenome Genome

__________________________________________________________________

Oxidative phosphorylationNADH dehydrogenase 7 subunits >41 subunitsSuccinate CoQ reductase 0 subunits 4 subunitsCytochrome b-c1 complex 1 subunit 10 subunitsCytochrome c oxidase complex 3 subunits 10 subunitsATP synthase complex 2 subunits 14 subunits

Protein synthesis apparatus 2 rRNAs none 22 tRNAs none (13 mRNAs)

Ribosomal proteins none (~80 in total)

Other mitochondrial proteins none All, e.g., mitochondrial

DNA pol, RNA pol,other enzymes,

structural proteins

• Circular

• Much smaller than nuclear genome with a compact genetic organization

(human 16.5 kb, yeast 85 kb, melon 2500 kb)

• in humans:

• ~ 10 identical molecules per mitochondrion

(100s-10,000s mitochondria per cell)

• 37 genes ; no introns (but present in S. cerevisiae)

• 13 are protein-coding genes (respiratory complex)

• 24 are non-coding RNA genes

2 ribosomal RNAs, 22 transfer RNAs

Mitochondrial Genome

Genetic Organization of Human Mitochondrial DNA

ND1

ND2

CO1

CO2

CO3ND3

ND4L

ND4

ND5

CYB

16S rRNA

23S rRNA

ATPase 8

HSTRAND

LSTRAND

D-loop

H strand synthesis H strand transcription

L strand transcription

L strand synthesis

ND6

16.5 kb

DNSA

7

ATPase 6

Genes encoding proteins rRNA genes tRNA genes

Genetic organization of S. cerevisiae mitochondrial DNA

CO3

par

15S rRNA

CO1

ATPase 8ATPase 6

var

21S rRNA

CO2

85.7 kb

ATPase 9

Cyt

ochr

ome

b

Exons

Noncoding

Introns

Mitochondria and human diseases

Mitochondrial dysfunctions are responsible for several human diseases, in particular neurodegenerative disorders. This is the result of of the mitochondria's central role in energy production, reactive oxygen species (ROS) biology, and apoptosis. Mitochondrial DNA, whose integrity is strictly related to that of the mitochondrion itself, appears to be involved in pathologies and in the process of ageing. mtDNA mutations are sufficient by themselves to generate major clinical phenotypes

Bossy-Wetzel E et al. 2003. Curr Opin Cell Biol 15, 706-16.Scott SV, et al. 2003. Curr Opin Cell Biol 15, 482-8.Wallace DC & Fan W 2009 Genes Dev. 23:1714-36

Mitochondria and human diseases

Also defects in mtDNA maintenance are associated with an increasing number of human diseases (i.e. optic atrophy)

Spelbrink JN et al. 2001 Nat Genet, 28:223-31Trifunovic A et al. 2004 Nature, 429:417-23Alexander C et al. 2000 Nat Genet, 26:211-5.Delettre C et al. 2000 Nat Genet, 26:207-10

Mitochondria and human diseases

Some diseases are associated with the mutagenic insertion of mt DNA in the nuclear genome.

(it will be discussed later)

Turner C et al. 2003. Hum Genet 112, 303-9.Borensztajn K et al. 2002. Br J Haematol 117, 168-71Goldin E et al. 2004. Hum Mutat. 24, 460-5.Willett-Brozick JE et al. 2001. Hum Genet 109, 216-23.

protein-coding genes

average 130

ancestral cyanobacterial genome 3000

Chloroplast genome size

group of Apicomplexans 30

253Porphyra purpurea(red alga)

(parasitic protists)

• transfer to the nucleus

• function substituted by unrelated nuclear coded proteins (multi subunit RNA pol single subunit RNA pol)

• loss of function: ex. complex I (nad) in S. cerevisiae

Genome size reduction in mitochondria

• movement of nucleic acid from the mt to the nucleus• integration of the DNA in the nuclear genome• expression of the transfered gene (different codon usage)• protein must acquire a transit peptide to allow access to the organelle• coordination of the expression to different energy needs

Successful gene transfer requires:

from: Gray et al, Genome Biology (2001)

Phylogenetic trees constructed by nuclear and mt DNA, suggest thatmt and nuclear genomes have evolved in concert throughout much,if not most, of the evolutionary history of the domain Eukarya.

Why do organellar genes and up in the nucleus?

• haploid genome• relatively small size• maternal inheritance• normally does not undergo recombination• relatively rapid sequence evolution

higher risk of genetic drift

Characteristics of the mt genome

Some references and reviews on the endosymbiotic theory and on the origin of organelles

Margulis, Lynn, 1970, Origin of Eukaryotic Cells, Yale University Press.

Raven JA, Allen JF. 2003. Genomics and chloroplast evolution: What did cyanobacteria do for plants? Genome Biol. 4:209

Andersson SG, Karlberg O, Canback B, Kurland CG. 2003. On the origin of mitochondria: a genomics perspective. Philos. Trans. R. Soc. London Ser. B Biol. Sci. 358:165–7–9

Keeling PJ, Palmer JD. 2008. Horizontal gene transfer in eukaryotic evolution. Nat. Rev. Genet. 9:605–18

2. Intercompartemental DNA transfer

Theoretical DNA transfer in eukaryote cells

mitochondria

nucleus

chloroplasts (in plants)

Chloroplast DNA transfer in the nucleus

Chloroplast DNA transfer in the nucleus

• entire genes (during evolution)• infA in angiosperms• 33 kb of cpDNA on chromosome 10L of rice• transfer of chloroplast DNA into the nuclear genome of Nicotiana tabacum (preintegrated neomycine phosphotransferase gene) frequency: 1:16,000 • chloroplast DNA fragments (NUPT), ongoing

Millen et al, 2001 Plant Cell 13: 645; Yuan et al, 2002 Mol. Gen. Genet. 267: 713 Huang et al, 2003 Nature. 422: 472

Nuclear DNA transfer in mitochondria

Nuclear DNA transfer in mitochondria

• 1 possible case (intra or interspecies ?): gene homologue of bacterial MutS in the mt of the coral Sarcophyton glaucum

• interspecies (horizontal transfer): ribosomal (rps2 and rps11) and respiratory (atp1) proteins between distantly related flowering plants

Pont-Kingdon et al, 1998 J.Mol.Evol. 46: 419; Bergthorsson et al, 2003 Nature 424: 197

Chloroplast DNA transfer in mitochondria

Chloroplast DNA transfer in mitochondria

• tRNA set in higher plants

• 12 Kbp of DNA (originated from the inverted repeated region of the chloroplast genome) in maize • 5 independent transfers of the chloroplast rbcL to the mt genome in angiosperms (rbcL :ribulose-1,5-biposphate carboxylase/oxygenase)

• 17 stretches of plastid-like sequences found in the mt DNA of Oryza sativa L. (rice) 17-6653 bp; 6.3% of mt genome; 61-100% identity

Ellis J. 1982. Nature 299:678–79 ; Cummings et al, 2003 Curr. Genet 43: 131; Stern & Lonsdale, 1982 Nature 299: 698; Notsu et al, 2002 Mol. Genet. Genomics 268: 434)

Chloroplast DNA transfer in mitochondriaand then in the nucleus ?

In plants, a few (nuclear) genes that code for mt proteins are derived by duplication from nucleargenes of ancestrally chloroplast origin

(rps13 in some angiosperms, ribosomal protein S13 in Arabidopsis thaliana)

Adams et al, 2002 Plant Cell 14: 931; Mollier et al, 2002 Curr. Genet. 40: 405

Nuclear DNA transfer in chloroplasts

• two open reading frames, int and dpoB, in the large inverted repeat of the chloroplast genome of the green alga Oedogonium cardiacum no sequence similarity with genes in chloroplast genomesMt donor unkonwn

Nuclear DNA transfer in chloroplasts

Brouard JS et al. 2008. BMC Genomics 9:290

Mitochondrial DNA transfer in chloroplasts

not found… until now

Mitochondrial DNA transfer in the nucleus

Mitochondrial DNA transfer in nucleus

• entire genes (during evolution)• mt DNA frangments (NUMT), ongoing

• genes (ceased in animals, detected in plants)

Mt DNA insertions in the nuclear genome

ex. rsp10 gene (protein of the mt ribosome) in angiosperms

from: Knoop et al. 1995, Curr. Genet; Adams et al. 2000, Nature

Ongoing gene transfer from mt to the nucleus(in plants)

see: Sandoval et al. 2004 Gene 324:139

Ongoing gene transfer from mt to the nucleus (in plants)

SDH2

RPS14

RPL5 RPS14

Nucleus

Mitochondria rice

RPL5 RPS14 wheat

RPL5 RPS14 maize

RPL5 rice

RPL5 wheat

RPL5 maize

RPS14

RPS14

Bittner-Eddy et al. 1994 J. Mol. Biol. 235: 881

Dual expression (mt and nucleus) of atp9 in Neurospora

The nuclear and the mt copy of atp9 are expressedat different stages of the life cycle

(germinating spores versus vegetative cells)

Adams et al. 1999 PNAS 24:13863

Dual expression (mt and nucleus) of cox2 in legumes

• genes (ceased in animals, detected in plants)

• 620 kbp insertion in Arabidopsis thaliana

Mt insertions in the nuclear genome

from Stupar et al. 2001, PNAS 98, 5099. See also Lin et al. 1999, Nature 402, 761.

Mt DNA insertion (620 kbp)centromere

telomere

D D D BA AA C

A

D

B

C

duplications

Large mt DNA insertion in the centromeric regionof chromosome 2 of Arabidopsis thaliana

• genes (ceased in animals, detected in plants)

• 620 kbp insertion in Arabidopsis thaliana

• 7.9 kbp in felines

Mt insertions in the nuclear genome

A 7.9 Kbp insertion of mt origin in thenuclear genome of domestic cat

7.9 kb of a typically 17.0-kb mitochondrial genome inserted to a specific nuclear chromosomal position in the domestic cat.

the intergrated segment has subsequently become amplified38-76 times and now occurs as a tandem repeat macrosatellite

Lopez et al. 1994, J. Mol. Evol. 39, 554

Mt insertions in the nuclear genome

• genes (ceased in animals, detected in plants)

• 620 kbp insertion in Arabidopsis thaliana

• 7.9 kbp in felines

• DNA fragments of about 300 bp (NUMTs) till > 14 kbp (in most studied eukaryotes)

size

mt DNA (bp) nuclear (Mbps) NUMTs (BLASTN threshold 10-4

) % 10-3

Homo sapiens 16.571 2910 279.17 9,.6

Mus musculus 16.299 2500 53.453 2.1Rattus norvegicus 16.3 2800 6.34 0.2

Fugu rubripes 16.447 320 5.624 1.8Caernorhabdit is elegans 13.794 97 126 0.1

Drosophila melanogaster 19.517 122.7 534 0.4Anopheles gambiae 15.363 278.2 0 0.0

Ciona intestinalis 14.788 116.7 11.51 9.9Plasmodium falc iparum 5.697 22.9 152 0.7

Saccharomyces cerevisiae 85.779 12.5 1.241 9.9schisaccharomyces pombe 19.431 12.5 1.614 12.9

Arabidopsis thaliana 366.924 115.4 198.105 171.7Oryza sativa 490.52 420 409.104 97.4

Mitochondrial, nuclear and NUMT sizes in some eukaryotic genomes

from Richly & Leister 2004 Mol Biol Evol 21, 1081

Yeast species S. cerevisiae C. glabrata K. thermotolerans K. lactis D. hansenii Y. lipolytica

Nuclear genome size (Mb) 12.1 12.3 10.4 10.6 12.2 20.5

Mitochondrial genome size (kb) 85.7 20 23.5 40.2 29.4 47.9

NUMT number 32 14 1 8 145 47

Total transferred mitochondrial DNA (bp) 2356 1423 25 403 9377 2005

Transferred mitochondrial DNA (%) 2.7 7.1 0.1 1 31.8 4.2

NUMTs in six hemiascomycetous yeast species

from Sacerdot et al. 2008 FEMS Yeast 8, 846-850

Why variable abundance of NUMTs in different species ?

from Richly & Leister 2004 Mol Biol Evol 21, 1081

• frequency of DNA transfer ?• vulnerability of mt to stress & other factors?• n° of mt/cell ? (i.e. Plasmodium)• n° of somatic cell divisions from zygotes to meiosis• efficiency of nuclear import of mt DNA and/or its integrattion into the nuclear genome

• rate of loss of NUMTs ?• rates of DNA loss varies from fragments and among species• however, no NUMT loss has been show till now

Types of NUMTs and NUPTs

from Leister. 2006 Trends in Genetics 21, 655-663

3. How do NUMTs integrate in the nuclear genome?

Mitochondrial DNA integrate in the nuclear genome during double-strand break (DSB) repair in yeast

Ricchetti et al.1999 Nature, 402:96-100Yu & Gabriel 1999 Mol. Cell 4: 873-881

DSB

intact chromosome

mt DNA+

Ricchetti et al.1999, cited

Mt DNA insertion in the nuclear genomevia Non-Homologous End-Joining (NHEJ)

mt DNA insertionNHEJ

Analysis of mt insertions during the repair of DSBs in yeast

DSB

(A)

ATTACCCTGTTAT attattattttttattattaataataataatttatagggtttattctgttttatcataaatacgtaaatatctaacttagctctcaaattatattacT A A CAGGGTAAT

(ATAA)

ATTACCCTGTT A T ctttattatatttaagaatattattataattattattattattattatttttaataattaaaaatattaataataagtaaatattaattattgttcatttaatcattccaaaaatttaggtaatgatactgcttcgatcttaattggcatatttgcatgacctgtcccacacaactcagaacatgctc c ggccacgggagccggaaccccgaaaggaggaataagataaatatatagCAGGGTAAT

(T)

(A)

ATTACCCTGTTA agtttccatagaagtaataataataataaatatattaaatattaatata a t tattaattaaaataactaatttagatcaatctaaaaaatctaagtgtttagatgataataaagaatatttattaaagtattctattactttaatattttT A ACAGGGTAAT

ATTACCCTGT T A T ttagaatatttttaattaaataatataattaaatgaataccaaacttatattatatttatatttatatttatatttcT AACAGGGTAAT

(A)

34pAT9

34pAS16

34pAS15

622pBS8

ATTACCCTGTTAT3'TAATGGGAC 3'TATTGTCCCATTA

+ CAGGGTAATDigested I-SceI sites:

Mt insertions found at DSB sites in yeast

Ricchetti et al.1999, cited

NUMTs in the yeast nuclear genome

85,779 bp

Mitochondrial genome

Saccharomyces cerevisiae

Nuclear genome

1.2 x 107 bp

by DSB repair:

6 NUMTs size: single 47-97 bpmultiple 166-382 bp

homology :100%

by BLAST search:

30 NUMTs size: 22-230 bp homology :86-100%

Ricchetti et al.1999, cited

Yeast mitochondrial genome and origin of NUMTs

at DSBsin the yeast genomefound > once

NUMTS

4. Impact of NUMTs insertion on the eukaryotic genomes

• Sequence analysis of the genome of Homo sapiens

• PCR sampling of humans from different ethnic backgrounds

211 NUMTs detected in humans

Ricchetti et al.2004 PLoS Biology, 2:96-1313

NUMT

NUMT

amplified DNA fragment

NUMT present

NUMT absent

PCR amplification of NUMTsin the human genome

Insertion polymorphism of three NUMTs

1 2 3 4 5 6 7 8 9individual sex NUMT 1-74 NUMT 2-132 NUMT 2-53 NUMT 12-89 NUMT 13-75 NUMT 18-192 NUMT 11-541Caucasian-1 Female -/- +/+ -/- -/+ +/+ -/- +/+Caucasian-2 Male -/- +/+ +/+ -/- +/+ -/- -/-Caucasian-3 Male -/+ +/+ -/+ -/- +/+ -/- +/+Caucasian-4 Male -/- +/+ -/+ -/- +/+ +/+ * +/+Caucasian-5 Female -/+ +/+ -/+ -/- +/+ -/- -/+Caucasian-6 Male -/- +/+ -/+ -/- +/+ -/- -/+Caucasian-7 Female -/- +/+ -/- -/- +/+ -/- * -/+Caucasian-8 Male -/- +/+ +/+ -/+ +/+ -/- -/-Caucasian-9 Male -/- +/+ -/+ -/+ +/+ +/+ * +/+Caucasian-10 Female -/+ +/+ -/+ +/+ +/+ -/+ -/+

African-1 Male +/+ +/+ -/+ * -/- +/+ +/+ * -/-African-2 Female -/+ -/+ */* -/- -/- +/+ -/+ -/+African-3 Male -/- +/+ +/+ -/+ +/+ -/+ -/+

Pygmy-1 (Biaka) Male +/+ * +/+ -/+ -/- +/+ -/- -/-Pygmy-2 (Mbuti) Male -/- +/+ +/+ -/+ +/+ -/- -/-Pygmy-3 (Mbuti) Female -/- +/+ -/- -/+ +/+ -/+ -/-Pygmy-4 (Biaka) Female -/- +/+ -/+ * -/- +/+ -/- -/-

Chinese-1 Male -/+ +/+ -/- -/- +/+ -/+ -/-Chinese-2 Male -/- +/+ -/+ -/+ -/+ -/- * -/+

Japanese-1 Male -/- +/+ -/+ -/- -/+ -/+ -/+Japanese-2 Female -/- +/+ -/+ -/- +/+ -/- -/+

Insertion polymorphism of NUMTs in humans

Ricchetti et al.2004, cited

Frequency of alleles carrying the NUMT

NUMT %

2-132 9813-75 952-53 4818-192 2912-89 211-74 21

1 2 3 4 5 6 7 8 9individual sex NUMT 1-74 NUMT 2-132 NUMT 2-53 NUMT 12-89 NUMT 13-75 NUMT 18-192 NUMT 11-541Caucasian-1 Female -/- +/+ -/- -/+ +/+ -/- +/+Caucasian-2 Male -/- +/+ +/+ -/- +/+ -/- -/-Caucasian-3 Male -/+ +/+ -/+ -/- +/+ -/- +/+Caucasian-4 Male -/- +/+ -/+ -/- +/+ +/+ * +/+Caucasian-5 Female -/+ +/+ -/+ -/- +/+ -/- -/+Caucasian-6 Male -/- +/+ -/+ -/- +/+ -/- -/+Caucasian-7 Female -/- +/+ -/- -/- +/+ -/- * -/+Caucasian-8 Male -/- +/+ +/+ -/+ +/+ -/- -/-Caucasian-9 Male -/- +/+ -/+ -/+ +/+ +/+ * +/+Caucasian-10 Female -/+ +/+ -/+ +/+ +/+ -/+ -/+

African-1 Male +/+ +/+ -/+ * -/- +/+ +/+ * -/-African-2 Female -/+ -/+ */* -/- -/- +/+ -/+ -/+African-3 Male -/- +/+ +/+ -/+ +/+ -/+ -/+

Pygmy-1 (Biaka) Male +/+ * +/+ -/+ -/- +/+ -/- -/-Pygmy-2 (Mbuti) Male -/- +/+ +/+ -/+ +/+ -/- -/-Pygmy-3 (Mbuti) Female -/- +/+ -/- -/+ +/+ -/+ -/-Pygmy-4 (Biaka) Female -/- +/+ -/+ * -/- +/+ -/- -/-

Chinese-1 Male -/+ +/+ -/- -/- +/+ -/+ -/-Chinese-2 Male -/- +/+ -/+ -/+ -/+ -/- * -/+

Japanese-1 Male -/- +/+ -/+ -/- -/+ -/+ -/+Japanese-2 Female -/- +/+ -/+ -/- +/+ -/- -/+

Insertion polymorphism of NUMTs in humans

Ricchetti et al.2004, cited

NUMTs as genetic markers

Podnar et al 2007 J Mol Evol 64: 308-320. Unusual origin of a nulcear pseudogene in the Italian wallizard: intergenomic and interspecific transfer of a large section of the mitochondrial genome in the genus Podarcis (Lacertidae).

Vartanian JP, Wain-Hobson S. 2002 Proc Natl Acad Sci U S A 99: 7566-7569. Analysis of a library of macaque nuclear mitochondrial sequences confirms macaque origin of divergent sequences from old oral polio vaccine samples.

NUMTS as genetic tools to follow the geographic distribution of species or populations and to resolve phylogenetic ambiguities

Human-specific NUMTs

Specie-specific NUMTs

Ricchetti et al.2004 PLoS Biology, 2:96-1313Hazkani-Covo & Graur 2007 Mol. Biol. Evol. 24: 13-18

1 2 3 4 5 6 7 8 9 10 11 12NUMT code chr size (bp) % mt coord PCR size PCR size PCR amplification Chimp

identity no NUMT NUMT +/+ -/- +/- sequenced1-74 * 1 74 100 8919-8992 344 418 2 14 5 1 -1-90 * 1 90 94 8447-8542 927 1017 21 0 0 1 +

1-192 1 192 89 16386-16552 825 1017 21 0 0 2 +2-53 * 2 53 100 1762-1814 330 383 4 5 12 2 -

2-132 * 2 132 99 608-739 365 497 20 0 1 1 -

2-272 * 2 272 94 6735-7006 712a 984a21 0 0 0 -

3-76 * 3 76 96 4349-4424 389 465 21 0 0 1 +3-1323 3 1323 95 1392-2714 487 1810 20 0 0 0 -

4-93 * 4 93 96 14966-15058 326 419 21 0 0 0 -4-131 * 4 131 96 958-1088 300 431 21 0 0 0 -4-152 4 152 94 2895-3046 286 438 21 0 0 2 +4-240 4 240 91 2221-2460 868 1108 21 0 0 0 -

4-342 * 4 342 94 9323-9664 319a 661a21 0 0 0 -

5-949 5 949 79 6591-7902 919 21c0 0 1 +

5-8781 upd5 8781 88 6388-15168 507b 21c

0 0 3 +

5-8781 dwd5 8781 88 6388-15168 751b 21c

0 0 2 +6-527 6 527 90 2414-2930 709 1236 21 0 0 0 +7-100 * 7 100 98 1609-1704 338 438 21 0 0 1 -7-106 7 106 94 12946-13051 406 512 21 0 0 0 -7-164 7 164 96 2414-2570 270 434 21 0 0 1 +

7-505 * 7 505 85 1708-2212 260 765 21e0 0 0 +

8-59 * 8 59 94 803-861 638 697 21 0 0 0 +8-84 * 8 84 98 14846-14929 472 556 21 0 0 0 -

8-1470 * 8 1470 96 8405-8538 406b 21c0 0 2 +

11-72 * 11 72 98 14645-14716 244 316 21 0 0 0 -11-163 11 163 96 6636-6798 351 514 21 0 0 0 -

11-2451 11 2451 93 518-2968 400 2851 21 0 0 0 +12-68 * 12 68 94 4236-4303 351 419 21 0 0 0 +12-89 * 12 89 98 3786-3874 478 567 1 13 7 0 -13-75 * 13 75 94 9508-9578 260 331 19 0 2 0 -13-123 13 123 91 5103-5225 325 448 21 0 0 0 -13-256 13 256 99 978-1233 346 602 21 0 0 0 -

14-1023 14 1023 93 5578-6600 242 1265 0 21 0 0 -17-69 * 17 69 97 10128-10195 192 261 21 0 0 0 -17-653 17 653 95 6812-7464 452 1105 21 0 0 0 -18-156 18 156 94 14366-14521 374 530 21 0 0 1 -18-192 18 192 97 7969-8160 204 396 3 12 6 5 -20-70 * 20 70 92 12947-13016 445 515 21 0 0 0 -22-47 * 22 47 100 6176-6222 338 385 21 0 0 2 -X-267 X 267 95 684-950 293 560 21 0 0 0 -

Y-66 * Y 66 100 6494-6559 186 252 0 NDf 13f1 -

Y-71 * Y 71 100 1268-1338 361 432 0 8g 13g2 -

Y-3107 Y 3107 83 1155-4863 467 0 NDf 13f0 +

11-541 h 11 541 94 16074-60 195 736 4 8 8 0 ND

NUMT

A CB

PCR amplification and sequence analysis of NUMTs from humans and chimpanzees

Ricchetti et al.2004,cited

0

5

10

15

20

25

30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

total n° of NUMTs

human-specific NUMTs

50

100

150

200

250

n°

of N

UM

Ts

chro

mos

ome

size

(in

Mb

p)

Human-specific NUMTs in human chromosomes

No strict correlation between chromosome size and nb of NUMTs

Ricchetti et al.2004, cited

1 2 6 753 4 8 9 10 11

13

12

14 1615 17 18 19 20 21 22 X Y

Distribution of human-specific NUMTs in chromosomes

Ricchetti et al.2004, cited

5000 Alu sequences have colonised human chromosomesin the last 4-6 Myrs

from: Batzer & Deininger et al, 2002 Nature. Genet. Rev. 3, 370

180 Alu seq : 1 NUMT

27 NUMTs are specific to humans and have colonised humanchromosomes in the last 4-6 Myrs

Ricchetti et al 2004, cited

Colonisation rate of the human genome

NUMTs as potential mutagens

0

5

10

15

20

25

number of NUMTs

NUMTs common tohumans and chimpanzees

human-specific NUMTs NUMTs with insertionpolymorphism in humans

exon

intron

promoter

intergene

Insertion sites of NUMTs in the human genome

Ricchetti et al.2004, cited

80% NUMTs inserted in genesgenes (with introns) < 25% of the human genome

NUMTs preferentially insert in genes (in humans)

Diseases associated with the insertion of NUMTs

Diseases associated with the insertion of a NUMT include disorders as diverse as:

• a sporadic case of the Pallister-Hall syndrome, a multiple congenital anomaly characterised by hypothalamic hamartoma and bone deformities

Turner C et al. 2003. Hum Genet 112, 303-9

Diseases associated with the insertion of NUMTs

Diseases associated with the insertion of a NUMT include disorders as diverse as:

• a familial plasma factor deficiency, a severe type I factor VII deficiency resulting in severe bleeding

Borensztajn K et al. 2002. Br J Haematol 117, 168-71

Diseases associated with the insertion of NUMTs

Diseases associated with the insertion of a NUMT include disorders as diverse as:

• a mucolipidosis Type IV, showing a moderate phenotype of this usually severe neurodegenerative disorder

Goldin E et al. 2004. Hum Mutat. 24, 460-5

Diseases associated with the insertion of NUMTs

Diseases associated with the insertion of a NUMT include disorders as diverse as:

• a familial bipolar affective disorder associated to a constitutional chromosomal translocation with a NUMT at the junction site

Willett-Brozick JE et al. 2001 Hum Genet 109, 216-23

Diseases associated with the insertion of NUMTs

The diversity of these pathologies reflects a mutagenic process that can target a large variety of genes.

Three diseases related to NUMT insertions

NUMT

251 bp NUMT insertion inIVS acceptor splice site

Disease

severe type I factor (F) VII deficiency(rare bleeding disorder, family history)Borensztajn et al, 2001 Brit. J. Haemat. 117, 168

sporadic case of Pallister-Hall syndrome(de novo insertion)Turner et al, 2003 Hum. Genet. 112, 303

72 bp NUMT insertion into exon 14 of the GLI3 gene

41 bp NUMT insertion at thebreakpoint junction of a reciprocal

constitutional translocationt(9;11)(p24;q23)

bipolar affective disorder(cosegregation in family pedigree)

Willet-Brozick et al, 2001 Hum. Genet 109, 216

ctgcccagcctgctcagcctcacgcccgcccagcagtaccgcctcaaggccaag

ctgcccagcctgctcagcctcacgcccggtctaacaacatggctttctcaacttttaaaggataacagctatccattggtcttaggccccaaaaattttcccagcagtaccgcctcaaggccaag

LPSLLSLTPAQQYRLKAKLPSLLSLTPVSNNMAFSTFKG*

from: Turner et al, 2003 Hum. Genet. 112, 303

wt

+NUMT

wt+NUMT

Disease related to NUMT insertion:sporadic case of Pallister-Hall syndrome (GLI3)

17 known disease-related Alu insertions1,500,000 Alu seq/ human genome

4 known disease-related NUMT insertions211 NUMTs /human genome

Impact of new NUMT and Alu insertions on human diseases

NUMT insertions can modify the exon/intron pattern

hypothetical protein

1 32 4

1 32

a

bNUMT 12-89

hypothetical protein

1 2 3 4 5

1 2 3 4 65

a

bNUMT 17-653

hypothetical protein

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8 9 10

a

bNUMT 5-8781

protein Q8N7L5

1 2 3 4 5 6 7

1 2 3 4 5 6 7 8

a

bNUMT 1-74

Some NUMT insertions in genes

Ricchetti et al.2004, cited

Generation of novel nuclear exons by NUMTs

Nuclear insertions of organelle DNA in yeast, H. sapiens, Arabidopsis, and rice:45 insertions contributed sequences to a total of 49 protein-coding exons in 34 genes.

Noutsos et al. 2007 Trends Genet. 23:597–601

NUMTs as regulatory sequences?

Discussion on unpublished data by Chatre and Ricchetti, 2009

• lysis of mitochondria ?• capture of cytoplasmic nucleic acids ? • illegittimate transport ?• membrane fusion ?

5. How do nucleic acids move to the nucleus?

Mitochondria-nuclear interactions

Human HeLa cells. Subpopulation of perinuclear mitochondria under normal growth conditions. By Laurent Chatre, Institut Pasteur

Perspectives

Analysis of the possible regulatory function of NUMTs in other eukaryotes,including humans

Analysis of mt-nuclear interactions and mt DNA release under normalgrowth and stress conditions

Acknowledgements

Laurent ChatreBenjamin Montagne

Cecile FairheadFredj TekaiaBernard Dujon

Unité de Génétique Moléculaire des Levures

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