Pr

1
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_at_ pasteur.fr)
2
1. The genomes of eukaryote cells
3
The genomes of eukaryote cells
mitochondria
chloroplasts (in plants)
nucleus
4
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
5
Origin of mitochondria and chloroplasts
Animals
Fungi
Plants
Algae
Protozoa
Slime moulds
Chloroplasts
Sulfolobus
EUCARYOTES
Cyanobacteria
Thermoplasma
Gram-positive bacteria
Extreme halophiles
Mitochondria
Spirochetes
Methanogens
Purple photosynthetic bacteria
Green photosynthetic bacteria
Thermoacidophiles
EUBACTERIA
ARCHAEBACTERIA
UNIVERSAL ANCESTOR
6
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
R551-600Bossy-Wetzel E, et al 2003. Curr Opin
Cell Biol 15, 706-16..
7
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. 2762133-45
8
Genomes size
Nuclear genome
Mitochondrial genome
Saccharomyces cerevisiae
85,779 bp
1.2 x 107 bp
Homo sapiens
16,554 bp
2.9 x 109 bp
9
Mt genome size
protein-coding genes
size mt genome (kbp)
Reclinomonas americana
67
69
(protist)
Arabidopsis thaliana
367
_at_ 45
85.8
17
Saccharomyces cerevisiae
12-24
average
Homo sapiens
13
16.5
and most vertebrates
3
6
Plasmodium falciparum
(parasitic protist)
Smallest free-living a-proteobacterial genome
Bartonella henselae
_at_1600
lt 2000
10
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)

11
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
12
Mitochondrial genome products (in humans)
Protein Complex Encoded
by Encoded by Mitochondrial
Nuclear Genome
Genome ____________________________________
______________________________ Oxidative
phosphorylation NADH dehydrogenase 7
subunits gt41 subunits Succinate CoQ
reductase 0 subunits 4
subunits Cytochrome b-c1 complex 1
subunit 10 subunits Cytochrome c oxidase
complex 3 subunits 10 subunits ATP
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

13
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

14
Genetic Organization of Human Mitochondrial DNA
H strand synthesis
H strand transcription
D-loop
16S rRNA
23S rRNA
CYB
L strand transcription
ND1
H STRAND
ND6
16.5 kb
ND5
ND2
L strand synthesis
L STRAND
CO1
ND4
ND4L
CO2
ND3
ATPase 8
ATPase 6
CO3
Genes encoding proteins
tRNA genes
rRNA genes

15
Genetic organization of S. cerevisiae
mitochondrial DNA
Exons
21S rRNA
Introns
CO2
var
Noncoding
ATPase 9
CO3
85.7 kb
Cytochrome b
par
ATPase 6
ATPase 8
15S rRNA
CO1

16
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. 231714-36
17
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,
28223-31 Trifunovic A et al. 2004 Nature,
429417-23 Alexander C et al. 2000 Nat Genet,
26211-5. Delettre C et al. 2000 Nat Genet,
26207-10
18
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.
19
Chloroplast genome size
protein-coding genes
253
Porphyra purpurea
(red alga)
130
average
group of Apicomplexans
30
(parasitic protists)
_at_ 3000
ancestral cyanobacterial genome
20
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

21
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

22
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)
23
Why do organellar genes and up in the nucleus?
24
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
25
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.
4209 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. 35816579 Keeling PJ,
Palmer JD. 2008. Horizontal gene transfer in
eukaryotic evolution. Nat. Rev. Genet. 960518
26
2. Intercompartemental DNA transfer
27
Theoretical DNA transfer in eukaryote cells
mitochondria
chloroplasts (in plants)
nucleus
28
Chloroplast DNA transfer in the nucleus
29
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 116,000
• 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
30
Nuclear DNA transfer in mitochondria
31
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
32
Chloroplast DNA transfer in mitochondria
33
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 29967879 Cummings et
al, 2003 Curr. Genet 43 131 Stern Lonsdale,
1982 Nature 299 698 Notsu et al, 2002 Mol.
Genet. Genomics 268 434)
34
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
35
Nuclear DNA transfer in chloroplasts
36
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 9290
37
Mitochondrial DNA transfer in chloroplasts
not found until now
38
Mitochondrial DNA transfer in the nucleus
39
Mitochondrial DNA transfer in nucleus

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

40
Mt DNA insertions in the nuclear genome

• genes (ceased in animals, detected in plants)

41
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
42
Ongoing gene transfer from mt to the nucleus (in
plants)
SDH2
Nucleus
rice
RPS14
RPL5
wheat
RPL5
RPS14
maize
RPL5
RPS14
Mitochondria
RPL5
yRPS14
rice
RPL5
yRPS14
wheat
RPL5
yRPS14
maize
see Sandoval et al. 2004 Gene 324139
43
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 2413863
44
Mt insertions in the nuclear genome

• genes (ceased in animals, detected in plants)
• 620 kbp insertion in Arabidopsis thaliana

45
Large mt DNA insertion in the centromeric
region of chromosome 2 of Arabidopsis thaliana
A
D
C
B
duplications
centro mere
telomere
Mt DNA insertion (_at_620 kbp)
from Stupar et al. 2001, PNAS 98, 5099. See also
Lin et al. 1999, Nature 402, 761.
46
Mt insertions in the nuclear genome

• genes (ceased in animals, detected in plants)
• 620 kbp insertion in Arabidopsis thaliana
• 7.9 kbp in felines

47
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
48
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 gt 14 kbp (in most studied eukaryotes)

49
Mitochondrial, nuclear and NUMT sizes in some
eukaryotic genomes
from Richly Leister 2004 Mol Biol Evol 21, 1081
50
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
51
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
52
Types of NUMTs and NUPTs
from Leister. 2006 Trends in Genetics 21, 655-663
53
3. How do NUMTs integrate in the nuclear genome?
54
Mitochondrial DNA integrate in the nuclear
genome during double-strand break (DSB) repair
in yeast
Ricchetti et al.1999 Nature, 40296-100 Yu
Gabriel 1999 Mol. Cell 4 873-881
55
Mt DNA insertion in the nuclear genome via
Non-Homologous End-Joining (NHEJ)
intact chromosome
DSB
mt DNA

Ricchetti et al.1999, cited
56
Analysis of mt insertions during the repair of
DSBs in yeast
DSB
NHEJ
mt DNA insertion
57
Mt insertions found at DSB sites in yeast
Digested I-SceI sites
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58
NUMTs in the yeast nuclear genome
Mitochondrial genome
Nuclear genome
Saccharomyces cerevisiae
85,779 bp
1.2 x 107 bp
by DSB repair
size single 47-97 bp multiple 166-382 bp
6 NUMTs
homology 100
by BLAST search
30 NUMTs
size 22-230 bp
homology 86-100
Ricchetti et al.1999, cited
59
Yeast mitochondrial genome and origin of NUMTs
NUMTS
at DSBs
in the yeast genome
found gt once
60
4. Impact of NUMTs insertion on the eukaryotic
genomes
61
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, 296-1313
62
PCR amplification of NUMTs in the human genome
NUMT
amplified DNA fragment
NUMT
NUMT present
NUMT absent
63
Insertion polymorphism of three NUMTs
64
Insertion polymorphism of NUMTs in humans
Ricchetti et al.2004, cited
65
Frequency of alleles carrying the NUMT
NUMT 2-132 98 13-75 95 2-53 48 18-192
29 12-89 21 1-74 21
66
Insertion polymorphism of NUMTs in humans
Ricchetti et al.2004, cited
67
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). Vartan
ian 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.
68
Specie-specific NUMTs
Human-specific NUMTs
Ricchetti et al.2004 PLoS Biology,
296-1313 Hazkani-Covo Graur 2007 Mol. Biol.
Evol. 24 13-18
69
PCR amplification and sequence analysis of NUMTs
from humans and chimpanzees
Ricchetti et al.2004, cited
70
Human-specific NUMTs in human chromosomes
No strict correlation between chromosome size and
nb of NUMTs
Ricchetti et al.2004, cited
71
Distribution of human-specific NUMTs in
chromosomes
Ricchetti et al.2004, cited
72
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
73
NUMTs as potential mutagens
74
Insertion sites of NUMTs in the human genome
Ricchetti et al.2004, cited
75
NUMTs preferentially insert in genes (in humans)
80 NUMTs inserted in genes genes (with introns)
lt 25 of the human genome
76
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
77
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
78
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
79
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
80
Diseases associated with the insertion of NUMTs
The diversity of these pathologies reflects a
mutagenic process that can target a large variety
of genes.
81
Three diseases related to NUMT insertions
Disease severe type I factor (F) VII
deficiency (rare bleeding disorder, family
history) Borensztajn et al, 2001 Brit. J.
Haemat. 117, 168
NUMT 251 bp NUMT insertion in IVS acceptor
splice site
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 the breakpoint junction
of a reciprocal constitutional translocation t(91
1)(p24q23)
bipolar affective disorder (cosegregation in
family pedigree) Willet-Brozick et al, 2001 Hum.
Genet 109, 216
82
Disease related to NUMT insertion sporadic case
of Pallister-Hall syndrome (GLI3)
ctgcccagcctgctcagcctcacgcccgcccagcagtaccgcctcaaggc
caag
wt
ctgcccagcctgctcagcctcacgcccggtctaacaacatggctttctca
actttt aaaggataacagctatccattggtcttaggccccaaaaatttt
cccagcagtaccg cctcaaggccaag
NUMT
LPSLLSLTPAQQYRLKAK
wt
LPSLLSLTPVSNNMAFSTFKG
NUMT
from Turner et al, 2003 Hum. Genet. 112, 303
83
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
84
NUMT insertions can modify the exon/intron pattern
85
Some NUMT insertions in genes
Ricchetti et al.2004, cited
86
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. 23597601
87
NUMTs as regulatory sequences?
88
Discussion on unpublished data by Chatre and
Ricchetti, 2009
89
5. How do nucleic acids move to the nucleus?

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

90
Mitochondria-nuclear interactions
Human HeLa cells. Subpopulation of perinuclear
mitochondria under normal growth conditions. By
Laurent Chatre, Institut Pasteur
91
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
92
Acknowledgements
Laurent Chatre Benjamin Montagne Cecile
Fairhead Fredj Tekaia Bernard Dujon Unité de
Génétique Moléculaire des Levures