Ongoing DNA transfer from the mitochondrial
to the nuclear genome
Bioinformatic and Comparative Genome Analysis Course
HKU-Pasteur Research Center, Hong Kong, China
August 17-29, 2009
Miria Ricchetti
(mricch@ pasteur.fr)
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1. The genomes of eukaryote cells
The genomes of eukaryote cells
mitochondria
chloroplasts (in plants)
nucleus
Origin of mitochondria and chloroplasts
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
Animals
Fungi
Plants
Slime moulds
Protozoa
Algae
Chloroplasts
Sulfolobus
EUCARYOTES
Thermoplasma
Extreme
halophiles
Cyanobacteria
Mitochondria
Methanogens
Purple
photosynthetic
bacteria
Thermoacidophiles
Gram-positive
bacteria
Spirochetes
Green
photosynthetic
bacteria
EUBACTERIA
ARCHAEBACTERIA
UNIVERSAL
ANCESTOR
Mitochondria
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.
View of mitochondria within a cell
McBride HM et al. 2006. Curr. Biol. 16: R551-600;Bossy-Wetzel E, et al 2003. Curr
Opin Cell Biol 15, 706-16..
Chloroplasts
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.
Eberhard S. et al. 2008. Ann. Rev. Geneti. 42 463-515
Puthiyaveetil S, Allen JF. 2009, Proc Biol Sci. 276:2133-45
Genomes size
Saccharomyces cerevisiae
Homo sapiens
Nuclear genome
Mitochondrial genome
1.2 x 107 bp
85,779 bp
2.9 x 109 bp
16,554 bp
Mt genome size
size mt genome
(kbp)
Reclinomonas americana
(protist)
Arabidopsis thaliana
Saccharomyces cerevisiae
69
67
367
@ 45
85.8
Plasmodium falciparum
(parasitic protist)
17
12-24
average
Homo sapiens
and most vertebrates
protein-coding
genes
16.5
13
6
3
Smallest free-living a-proteobacterial genome
Bartonella henselae
< 2000
@1600
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)
Genes coding for mitochondrial
function and propagation
in Saccharomyces cerevisiae mt proteins are coded:
about 400 in the nucleus
(200 are of bacterial origin)
17 in the mt
Mitochondrial genome products (in humans)
Protein Complex
Encoded by
Encoded by
Mitochondrial
Nuclear
Genome
Genome
__________________________________________________________________
Oxidative phosphorylation
NADH dehydrogenase
Succinate CoQ reductase
Cytochrome b-c1 complex
Cytochrome c oxidase complex
ATP synthase complex
7 subunits
0 subunits
1 subunit
3 subunits
2 subunits
>41 subunits
4 subunits
10 subunits
10 subunits
14 subunits
none
none
Ribosomal proteins
2 rRNAs
22 tRNAs
(13 mRNAs)
none
Other mitochondrial proteins
none
All, e.g., mitochondrial
DNA pol, RNA pol,
other enzymes,
structural proteins
Protein synthesis apparatus
(~80 in total)
Mitochondrial Genome
• 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
Genetic Organization of Human Mitochondrial DNA
H strand synthesis
D-loop
H strand transcription
16S rRNA
23S rRNA
CYB
L strand
transcription
H
STRAND
ND1
ND6
16.5 kb
ND5
ND2
L strand
synthesis
L
ND4
STRAND
CO1
ND4L
ND3
CO2
CO3
Genes encoding proteins
rRNA genes
tRNA genes
Genetic organization of S. cerevisiae mitochondrial DNA
Exons
21S rRNA
CO2
var
Noncoding
ATPase 9
Cytochrome b
Introns
CO3
85.7 kb
par
ATPase 6
ATPase 8
15S rRNA
CO1
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, 28:223-31
Trifunovic A et al. 2004 Nature, 429:417-23
Alexander 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, 168-71
Goldin E et al. 2004. Hum Mutat. 24, 460-5.
Willett-Brozick JE et al. 2001. Hum Genet 109, 216-23.
Chloroplast genome size
protein-coding
genes
Porphyra purpurea
(red alga)
253
average
130
group of Apicomplexans
(parasitic protists)
ancestral cyanobacterial genome
30
@ 3000
Genome size reduction in mitochondria
• 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
Successful gene transfer requires:
• 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
Phylogenetic trees constructed by nuclear and mt DNA, suggest that
mt and nuclear genomes have evolved in concert throughout much,
if not most, of the evolutionary history of the domain Eukarya.
from: Gray et al, Genome Biology (2001)
Why do organellar genes and up in the nucleus?
Characteristics of the mt genome
• haploid genome
• relatively small size
• maternal inheritance
• normally does not undergo recombination
• relatively rapid sequence evolution
higher risk of genetic drift
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
chloroplasts (in plants)
nucleus
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 mitochondria
and then in the nucleus ?
In plants, a few (nuclear) genes that code for mt
proteins are derived by duplication from nuclear
genes 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
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 genomes
Mt donor unkonwn
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
Mt DNA insertions in the nuclear genome
• genes (ceased in animals, detected in plants)
Ongoing gene transfer from mt to the nucleus
(in plants)
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)
Nucleus
Mitochondria
SDH2
RPS14
RPL5
rice
RPS14
RPL5
wheat
RPS14
RPL5
maize
RPL5 yRPS14
rice
RPL5 yRPS14
wheat
RPL5 yRPS14
maize
see: Sandoval et al. 2004 Gene 324:139
Dual expression (mt and nucleus) of atp9 in Neurospora
The nuclear and the mt copy of atp9 are expressed
at different stages of the life cycle
(germinating spores versus vegetative cells)
Bittner-Eddy et al. 1994 J. Mol. Biol. 235: 881
Dual expression (mt and nucleus) of cox2 in legumes
Adams et al. 1999 PNAS 24:13863
Mt insertions in the nuclear genome
• genes (ceased in animals, detected in plants)
• 620 kbp insertion in Arabidopsis thaliana
Large mt DNA insertion in the centromeric region
of chromosome 2 of Arabidopsis thaliana
A
D
C
B
duplications
D
telomere
A
D
A
D
A
C
B
Mt DNA insertion (@620 kbp)
from Stupar et al. 2001, PNAS 98, 5099. See also Lin et al. 1999, Nature 402, 761.
centro
mere
Mt insertions in the nuclear genome
• genes (ceased in animals, detected in plants)
• 620 kbp insertion in Arabidopsis thaliana
• 7.9 kbp in felines
A 7.9 Kbp insertion of mt origin in the
nuclear 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 amplified
38-76 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)
Mitochondrial, nuclear and NUMT sizes in
some eukaryotic genomes
size
mt DNA (bp)
Homo sapiens
16.571
Mus musc ulus
16.299
Rattus norvegic us
16.3
Fugu rubripes
16.447
Caernorhabditis elegans
13.794
Drosophila melanogaster
19.517
Anopheles gambiae
15.363
Ciona intestinalis
14.788
Plasmodium falc iparum
5.697
Sac c haromyc es c erevisiae 85.779
sc hisac c haromyc es pombe 19.431
Arabidopsis thaliana
366.924
Oryza sativa
490.52
-4
nuc lear (Mbps) NUMTs (BLASTN threshold 10 )
2910
279.17
2500
53.453
2800
6.34
320
5.624
97
126
122.7
534
278.2
0
116.7
11.51
22.9
152
12.5
1.241
12.5
1.614
115.4
198.105
420
409.104
from Richly & Leister 2004 Mol Biol Evol 21, 1081
% 10- 3
9,.6
2.1
0.2
1.8
0.1
0.4
0.0
9.9
0.7
9.9
12.9
171.7
97.4
NUMTs in six hemiascomycetous yeast species
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
from Sacerdot et al. 2008 FEMS Yeast 8, 846-850
Why variable abundance of NUMTs in
different species ?
• 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
from Richly & Leister 2004 Mol Biol Evol 21, 1081
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, 402:96-100
Yu & Gabriel 1999 Mol. Cell 4: 873-881
Mt DNA insertion in the nuclear genome
via Non-Homologous End-Joining (NHEJ)
intact chromosome
DSB
+
Ricchetti et al.1999, cited
mt DNA
Analysis of mt insertions during the repair
of DSBs in yeast
DSB
NHEJ
mt DNA insertion
Mt insertions found at DSB sites in yeast
Digested I-SceI sites:
ATTACCCTGTTAT3'
TAATGGGAC
+
CAGGGTAAT
3'TATTGTCCCATTA
34pAT9
ATTACCCTGTTATattattattttttattattaataataataatttatagggtttattctgttttatcataa
atacgtaaatatctaacttagctctcaaattatattacTAACAGGGTAAT
(A)
34pAS16
ATTACCCTGTTATttagaatatttttaattaaataatataattaaatgaataccaaacttatattatattta
tatttatatttatatttcTAACAGGGTAAT
(A)
34pAS15
ATTACCCTGTTATctttattatatttaagaatattattataattattattattattattatttttaataatt
aaaaatattaataataagtaaatattaattattgttcatttaatcattccaaaaatttaggtaatgatactg
cttcgatcttaattggcatatttgcatgacctgtcccacacaactcagaacatgctccggccacgggagccg
gaaccccgaaaggaggaataagataaatatatagCAGGGTAAT
(ATAA)
(T)
622pBS8
ATTACCCTGTTAagtttccatagaagtaataataataataaatatattaaatattaatataattattaatta
aaataactaatttagatcaatctaaaaaatctaagtgtttagatgataataaagaatatttattaaagtatt
ctattactttaatattttTAACAGGGTAAT
(A)
Ricchetti et al.1999, cited
NUMTs in the yeast nuclear genome
Saccharomyces cerevisiae
Nuclear genome
Mitochondrial genome
1.2 x 107 bp
85,779 bp
by DSB repair:
6 NUMTs
size: single 47-97 bp
multiple 166-382 bp
homology :100%
size: 22-230 bp
homology :86-100%
by BLAST search:
30 NUMTs
Ricchetti et al.1999, cited
Yeast mitochondrial genome and origin of NUMTs
NUMTS
at DSBs
in the yeast genome
found > once
4. Impact of NUMTs insertion on the eukaryotic genomes
211 NUMTs detected in humans
• Sequence analysis of the genome of Homo sapiens
• PCR sampling of humans from different ethnic backgrounds
Ricchetti et al.2004 PLoS Biology, 2:96-1313
PCR amplification of NUMTs
in the human genome
NUMT
amplified DNA fragment
NUMT
NUMT present
NUMT absent
Insertion polymorphism of three NUMTs
Insertion polymorphism of NUMTs in humans
1
individual
2
sex
3
NUMT 1-74
4
NUMT 2-132
5
NUMT 2-53
6
NUMT 12-89
7
NUMT 13-75
8
NUMT 18-192
9
NUMT 11-541
Caucasian-1
Caucasian-2
Caucasian-3
Caucasian-4
Caucasian-5
Caucasian-6
Caucasian-7
Caucasian-8
Caucasian-9
Caucasian-10
African-1
African-2
African-3
Pygmy-1 (Biaka)
Pygmy-2 (Mbuti)
Pygmy-3 (Mbuti)
Pygmy-4 (Biaka)
Chinese-1
Chinese-2
Japanese-1
Japanese-2
Female
Male
Male
Male
Female
Male
Female
Male
Male
Female
Male
Female
Male
Male
Male
Female
Female
Male
Male
Male
Female
-/-/-/+
-/-/+
-/-/-/-/-/+
+/+
-/+
-/+/+
-/-/-/-/+
-/-/-/-
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
-/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
-/+/+
-/+
-/+
-/+
-/+
-/+/+
-/+
-/+
-/+
-/+/+
-/+
+/+
-/-/+
-/-/+
-/+
-/+
-/+
-/-/-/-/-/-/-/+
-/+
+/+
-/-/-/+
-/-/+
-/+
-/-/-/+
-/-/-
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
-/+
-/+
+/+
-/-/-/+/+
-/-/-/-/+/+
-/+
+/+
-/+
-/+
-/-/-/+
-/-/+
-/-/+
-/-
+/+
-/+/+
+/+
-/+
-/+
-/+
-/+/+
-/+
-/-/+
-/+
-/-/-/-/-/-/+
-/+
-/+
*
*/*
*
*
Ricchetti et al.2004, cited
*
*
*
*
*
Frequency of alleles carrying the NUMT
NUMT
%
2-132
13-75
2-53
18-192
12-89
1-74
98
95
48
29
21
21
Insertion polymorphism of NUMTs in humans
1
individual
2
sex
3
NUMT 1-74
4
NUMT 2-132
5
NUMT 2-53
6
NUMT 12-89
7
NUMT 13-75
8
NUMT 18-192
9
NUMT 11-541
Caucasian-1
Caucasian-2
Caucasian-3
Caucasian-4
Caucasian-5
Caucasian-6
Caucasian-7
Caucasian-8
Caucasian-9
Caucasian-10
African-1
African-2
African-3
Pygmy-1 (Biaka)
Pygmy-2 (Mbuti)
Pygmy-3 (Mbuti)
Pygmy-4 (Biaka)
Chinese-1
Chinese-2
Japanese-1
Japanese-2
Female
Male
Male
Male
Female
Male
Female
Male
Male
Female
Male
Female
Male
Male
Male
Female
Female
Male
Male
Male
Female
-/-/-/+
-/-/+
-/-/-/-/-/+
+/+
-/+
-/+/+
-/-/-/-/+
-/-/-/-
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
-/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
-/+/+
-/+
-/+
-/+
-/+
-/+/+
-/+
-/+
-/+
-/+/+
-/+
+/+
-/-/+
-/-/+
-/+
-/+
-/+
-/-/-/-/-/-/-/+
-/+
+/+
-/-/-/+
-/-/+
-/+
-/-/-/+
-/-/-
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
+/+
-/+
-/+
+/+
-/-/-/+/+
-/-/-/-/+/+
-/+
+/+
-/+
-/+
-/-/-/+
-/-/+
-/-/+
-/-
+/+
-/+/+
+/+
-/+
-/+
-/+
-/+/+
-/+
-/-/+
-/+
-/-/-/-/-/-/+
-/+
-/+
*
*/*
*
*
Ricchetti et al.2004, cited
*
*
*
*
*
NUMTs as genetic markers
NUMTS as genetic tools to follow the geographic distribution of
species or populations and to resolve phylogenetic ambiguities
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.
Specie-specific NUMTs
Human-specific NUMTs
Ricchetti et al.2004 PLoS Biology, 2:96-1313
Hazkani-Covo & Graur 2007 Mol. Biol. Evol. 24: 13-18
PCR amplification and sequence analysis of NUMTs
from humans and chimpanzees
NUMT
A
1
NUMT code
74
90
192
53
132
8919-8992
8447-8542
16386-16552
1762-1814
608-739
2
3
3
4
4
4
4
272
76
1323
93
131
152
240
94
96
95
96
96
94
91
6735-7006
4349-4424
1392-2714
14966-15058
958-1088
2895-3046
2221-2460
712
389
487
326
300
286
868
a
984
465
1810
419
431
438
1108
4
342
94
9323-9664
319
a
661
5
949
79
6591-7902
919
5
8781
88
6388-15168
507
*
5
6
7
7
7
8781
527
100
106
164
88
90
98
94
96
6388-15168
2414-2930
1609-1704
12946-13051
2414-2570
709
338
406
270
751
1236
438
512
434
7-505
8-59
8-84
*
*
*
7
8
8
505
59
84
85
94
98
1708-2212
803-861
14846-14929
260
638
472
765
697
556
21
8-1470
11-72
11-163
11-2451
12-68
12-89
13-75
13-123
13-256
14-1023
17-69
17-653
18-156
18-192
20-70
22-47
X-267
*
*
8
11
11
11
12
12
13
13
13
14
17
17
18
18
20
22
X
1470
72
163
2451
68
89
75
123
256
1023
69
653
156
192
70
47
267
96
98
96
93
94
98
94
91
99
93
97
95
94
97
92
100
95
8405-8538
14645-14716
6636-6798
518-2968
4236-4303
3786-3874
9508-9578
5103-5225
978-1233
5578-6600
10128-10195
6812-7464
14366-14521
7969-8160
12947-13016
6176-6222
684-950
406
316
514
2851
419
567
331
448
602
1265
261
1105
530
396
515
385
560
21
244
351
400
351
478
260
325
346
242
192
452
374
204
445
338
293
Y-66
*
Y
66
100
6494-6559
186
Y-71
*
Y
71
100
1268-1338
361
*
*
2-272
3-76
3-1323
4-93
4-131
4-152
4-240
*
*
4-342
*
*
*
*
*
5-949
5-8781 up
d
5-8781 dw
6-527
7-100
7-106
7-164
d
*
*
*
*
*
*
Y-3107
11-541
h
3
size (bp)
1
1
1
2
2
5
mt coord
Y
3107
83
1155-4863
11
541
94
16074-60
6
7
PCR size PCR size
no NUMT
NUMT
344
418
927
1017
825
1017
330
383
365
497
a
8
9
+/+
2
21
21
4
20
10
11
PCR amplification
-/+/sequenced
14
5
1
0
0
1
0
0
2
5
12
2
0
1
1
12
Chimp
+
+
-
21
21
20
21
21
21
21
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
2
0
21
0
0
0
-
0
0
1
+
0
0
3
+
0
0
0
0
0
0
0
0
0
0
2
0
1
0
1
+
+
+
0
0
0
0
0
0
0
0
0
+
+
-
21
21
21
21
1
19
21
21
0
21
21
21
3
21
21
21
0
0
0
0
0
13
0
0
0
21
0
0
0
12
0
0
0
0
0
0
0
0
7
2
0
0
0
0
0
0
6
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
1
5
0
2
0
+
+
+
-
252
0
ND
13
f
1
-
432
0
8
g
13
g
2
-
f
a
21
c
b
21
c
b
21
c
21
21
21
21
e
21
21
b
195
C
B
4
%
identity
100
94
89
100
99
1-74
1-90
1-192
2-53
2-132
2
chr
c
f
f
467
0
ND
13
736
4
8
8
+
+
-
0
+
0
ND
Ricchetti et al.2004,
cited
Human-specific NUMTs in human chromosomes
30
total n° of NUMTs
250
20
200
15
150
10
100
5
50
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22
X
Y
chromosome size (in Mbp)
n° of NUMTs
human-specific NUMTs
25
No strict correlation between chromosome size and nb of NUMTs
Ricchetti et al.2004, cited
Distribution of human-specific NUMTs in chromosomes
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
Ricchetti et al.2004, cited
Colonisation rate of the human genome
27 NUMTs are specific to humans and have colonised human
chromosomes in the last 4-6 Myrs
Ricchetti et al 2004, cited
5000 Alu sequences have colonised human chromosomes
in the last 4-6 Myrs
from: Batzer & Deininger et al, 2002 Nature. Genet. Rev. 3, 370
180 Alu seq : 1 NUMT
NUMTs as potential mutagens
Insertion sites of NUMTs in the human genome
exon
25
intron
promoter
intergene
number of NUMTs
20
15
10
5
0
NUMTs common to
humans and chimpanzees
human-specific NUMTs
NUMTs with insertion
polymorphism in humans
Ricchetti et al.2004, cited
NUMTs preferentially insert in genes (in humans)
80% NUMTs inserted in genes
genes (with introns) < 25% of the human genome
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
Disease
NUMT
severe type I factor (F) VII deficiency
(rare bleeding disorder, family history)
251 bp NUMT insertion in
IVS acceptor splice site
Borensztajn et al, 2001 Brit. J. Haemat. 117, 168
sporadic case of Pallister-Hall syndrome
(de novo insertion)
72 bp NUMT insertion into
exon 14 of the GLI3 gene
Turner et al, 2003 Hum. Genet. 112, 303
bipolar affective disorder
(cosegregation in family pedigree)
Willet-Brozick et al, 2001 Hum. Genet 109, 216
41 bp NUMT insertion at the
breakpoint junction of a reciprocal
constitutional translocation
t(9;11)(p24;q23)
Disease related to NUMT insertion:
sporadic case of Pallister-Hall syndrome (GLI3)
ctgcccagcctgctcagcctcacgcccgcccagcagtaccgcctcaaggccaag
wt
ctgcccagcctgctcagcctcacgcccggtctaacaacatggctttctcaactttt
aaaggataacagctatccattggtcttaggccccaaaaattttcccagcagtaccg +NUMT
cctcaaggccaag
LPSLLSLTPAQQYRLKAK
LPSLLSLTPVSNNMAFSTFKG*
from: Turner et al, 2003 Hum. Genet. 112, 303
wt
+NUMT
Impact of new NUMT and Alu insertions on human diseases
17 known disease-related Alu insertions
1,500,000 Alu seq/ human genome
4 known disease-related NUMT insertions
211 NUMTs /human genome
NUMT insertions can modify the exon/intron pattern
Some NUMT insertions in genes
hypothetical protein
a
1
2
b
1
2
3
4
3
NUMT 12-89
hypothetical protein
a
1
2
3
4
5
b
1
2
3
4
5
6
NUMT 17-653
hypothetical protein
a
1
2
3
4
5
6
7
8
b
1
2
3
4
5
6
7
8
10
9
NUMT 5-8781
protein Q8N7L5
a
1
2
3
4
5
6
b
1
2
3
4
5
6
Ricchetti et al.2004, cited
7
7 8
NUMT 1-74
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
5. How do nucleic acids move to the nucleus?
• lysis of mitochondria ?
• capture of cytoplasmic nucleic acids ?
• illegittimate transport ?
• membrane fusion ?
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 normal
growth and stress conditions
Acknowledgements
Laurent Chatre
Benjamin Montagne
Cecile Fairhead
Fredj Tekaia
Bernard Dujon
Unité de Génétique Moléculaire des Levures
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a re ne ed ed to s ee th i s pi c tu re.
QuickT ime ™an d a
TIFF ( Uncomp res sed) deco mpre ssor
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