targeting of rna into mitochondria: mechanistic and ... · ndna >1000 proteins, several ncrnas...
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Targeting of RNA into mitochondria: mechanistic and
therapeutic insights
I. Tarassov Journée de la SFEIM 17 Novenmbre 2014, Paris, France
Energy Metabolism
Apoptosis Aging Biosyntheses
Defects in mitochondrial functions are often associated with myopathies and
neuropathies and play a critical role in many age-related diseases
Ophtalmoplegia
(decreased eye associated
Muscles tone)
Parkinson
Alzheimer
Cancer Vascular and heart diseases
In humans:
mtDNA 13 proteins, 2 rRNAs, 21 tRNAs
nDNA >1000 proteins, several ncRNAs (tRNAs, MRP RNA, Rnase P RNA, 5S rRNA)
Genetic double-dependence
Import of RNA into mitochondria
Yeast
Plants
Protozoans
Mammals
tRNAs
RNase P RNA
RNase MRP RNA
5S rRNA Mammals
Import determinants in yeast tRNA(Lys):
Alternative structure of RNA
Kolesnikova et al. (Science, 2000)
Entelis et al . (J Biol Chem, 2001)
Kolesnikova et al. (Mitochondrion, 2002)
Kolesnikova et al. (RNA, 2010)
Also Imported in an
artificial way in human
mitochondria
Smirnov et al. (RNA, 2008)
5S rRNA import determinants
Diabetes Thyroid
Disease Myopathy
Peripheral
Neuropathy
Deafness
CVA / Seizures /
Developmental delay
Respiratory Failure Optic Atrophy / Retinitis Pigmentosa
Cardiomyopathy
Short Stature
Marrow Failure
Liver
Failure
Neurological Non-Neurological
mtDNA mutations cause mitochondrial dysfunctions
>60
(% mutated mtDNA)
Cyto
ch
rom
e c
o
xid
ase a
cti
vit
y
100%
a b
*
*
*
*
*
*
*
*
*
*
mtDNA heteroplasmy and pathogenic threshold
Expression of site-specific
endonucleases
Inhibition of mutant mtDNA
replication
Miothochondrial diesease:
Possible gene therapy aproaches
« Allotopic » expression of
proteins or RNAs
Modulation of heteroplasmy
levels
Mito-TALENs (2013)
Mito-Zn-finger nucleases (2014)
Towards the model of gene therapy : MERRF
(Myoclonic Epilepsy and Red Ragged Fibers)
Which are the biochemical effects of the mutation ?
Hypomodification of the U34 (Yakuzawa et al., 2000)
Poor recognition of AAG codons on mRNA (Yakuzawa et al., 2001; Kirino et al. 2004, 2005)
Low complexes I (ND) & IV (Cox) activity
Low respirtation levels, low DY
Complementation of the MERRF mutation
Complementation of the MERRF mutation in human cells
tRK3 tRK2-93
Complementation is due to induced tRNA import
Expression and import of tRNAs in cybrid cells
Partial correction:
Respiration
DY
Translation
Complexes
I/IV
Cox-cytochemistry
Kolesnikova et al., Science 2000
Kolesnikova et al., Hum Mol Genet 2004
MELAS:
Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like
episodes
MELAS m.3243A>G mutation:
80% of MELAS cases
Carrier frequency ~1/10 000
Defect of Complex I (42% of patients),
Complex IV (29%), Complex III (23%)
Heteroplasmic
Mean onset ~ 10 years
Mean life span
~ 20 – 40 years
No treatement available
G
Recombinant tRNAs design
H. sapiens
mitochondrial
tRNALeu(UUR)
Kirino et al, 2005
CAA
G1-C72
5’
A
A73 A73
A
G C
tRK3
UAA UAA
CAA CAA
identity elements for hmt
LeuRS
determinants of tRNA
import into mitochondria
mutations introduced in
anticodon
Effect of tRK1CAA import on mitochondrial protein synthesis
1
0.8
0.6
0.4
0.2
0
Mel-pLKO Mel-T1CAA 143B
2.5
2
1.5
1
0.5
0
3
tRK1CAA import is acompanied by a generalized increase of mito-translation
Karicheva et al. (Nucleic Acids Res 2011)
Anti-replicative strategy: effect of antigenomic RNAs on heteroplasmy in cybrid cells
containing a deletion in mtDNA
Comte et al., Nucleic Acids Res 2013
Recombinant RNAs imported into
mitochondria of human cells in culture
caused a decrease of mutant mtDNA level.
Oligonucleotides corresponding to the target sequence were inserted into RNA vector molecules.
Stable expression of 5S rRNA-based recombinant RNA genes
intoduced into genome of cybrid cells harboring 65% of KSS
mtDNA molecules
Clones with decreased heteroplasmy levels
5S rRNA can be used as a vector for delivering RNA sequences into
human mitochondria
Oligonucleotides corresponding to the both strands of
target sequence were used to substitute the Loop C –
Helix III region of 5S rRNA
Comte et al., Nucleic Acids Res 2013
Mutation A13514>G (D393G) in ND5 gene, coding for respiratory Complex I subunit; MELAS like syndrome
and MELAS/Leigh/Leber hereditary optic neuropathy syndrome
Recombinant RNA (R20) caused a stable decrease of mutant
mtDNA level.
Tonin et al., J Biol Chem 2014
Developing the antigenomic strategy for point mutations in mtDNA
Design of RNA-vector molecules importable into human mitochondria:
optimisation of RNA stability
The new set of molecules importable into human mitochondria containing various
modifications.
Tonin et al., Biochimie 2014
Patent US 62/011,755 (2014)
Conclusions
In both allotopic and antigenomic strategies, the approach has a limited effect: levels of expression, aminoacylation (tRNA), import, accessibility to the target (antigenomic), etc. might be improved
Artificially established import of cytosolic-type tRNA into mitochondria can be used to deliver tRNAs with either normal or switched identities in human mitochondria to functionnally replace mutant tRNAs
Mitochondrially imported RNA molecules bearing oligonucleotide sequences complementary to mtDNA can affect in a specific manner mtDNA replication thus provoking induced changes of heteroplasmy
Small non-coding RNA molecules (tRNAs, tRNA-derivatives, 5S rRNA, etc.) can serve as vectors to deliver short oligoribonucleotides into mitochondria
Participants & sponsors
Team « Mito »
A.-M. Heckel N. Entelis B. Masquida A. Smirnov A. Smirnova R. Loutre I. Dovydenko I. Chicherin M. Baleva I. Tarassov
Former members:
R. Martin C. Comte O. Kolesnikova O. Karicheva Y. Tonin T. Schirtz M. Vyssokikh T. Schirtz A. Gowher
Collaborations:
P. Kamensky (Moscow University) A. Lombès (Hôpital Salpétrière, Paris) A. Rötig & A. Munnich (Hôpital Necker, Paris) A. Venyaminova (Inst. Fund. Med, Novossibirsk) O. Elpeleg (Jerusalem Univ.) R. N. Lightowlers (Newcastle Univ.) Funding:
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