Ludzkie geny
kontrolujące metabolizm
mitochondrialnego RNA
Piotr P. Stępień
Department of Genetics, Warsaw University,
IBB PAN
Mitochondria
Molecular probes
Viable bovine pulmonary artery endothelial cells
Molecular probes
Potential-dependent staining of mitochondria
in CCL64 fibroblasts
Funkcje

Cykl Krebsa

Synteza ATP

Bufor Ca2+

Apoptoza
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Utlenianie
kwasów
tłuszczowych,
cykl mocznikowy
Około 1000 białek, z czego 13 kodowane w mtDNA
Gdy mitochondria są niesprawne
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Różne tkanki mają różne wymagania energetyczne
mięśnie szkieletowe
mięsień sercowy
neurony
komórki β trzustki
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Choroby mitochondrialne
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Starzenie się
Dlaczego się starzejemy?
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4 główne teorie :
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Program ewolucyjny : Geny
Wolne rodniki
Glikacja białek
Defekty w mitochondriach
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Mitochondria a starzenie
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Wolnorodnikowa teoria starzenia:

wolne rodniki, powstające głównie w
mitochondriach mogą prowadzić do powstawania
mutacji w mtDNA, co z kolei może upośledzać
funkcję mitochondriów i przyczyniać się do wzrostu
produkcji ROS (sprzężenie zwrotne)

długowieczność może zależeć od sprawności
łańcucha oddechowego i enzymów usuwających
wolne rodniki
Modele doświadczalne w badaniach
nad starzeniem:

Progerie u ludzi

Myszy transgeniczne

Mutanty Drosophila, C.elegans, drożdży
Progeria Hutchinsona-Gilforda:
lamina A
Zespół Wernera: mutacja w helikazie
WRN
Eksperymentalnie wydłużone życie

Zwierzęta na diecie :Calorie restriction
 Myszy:
Obniżone wytwarzanie wolnych rodników
mniej uszkodzeń oksydacyjnych
– w wyniku słabszego działania
kompleksu I
Indukcja sirtuiny
Eksperyment łączący funkcje
mitochondrialne z apoptozą i
starzeniem (Kujoth et al., Science
2005):
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

Transgeniczne myszy z uszkodzonym
genem mitochondrialnej polimerazy DNA
(brak akt. korektorskiej)
Średnio jedna mutacja na mt genom
Przedwczesne starzenie w wyniku apoptozy,
ale bez zmian ROS
Starzejące się myszy
Dlaczego badamy metabolizm RNA
w mitochondriach?

Niewiele na ten temat wiadomo

Degradacja mtRNA gra podstawowa rolę w
regulacji ekspresji genów w mitochondriach

Nasze najnowsze badania sugerują, że
białka kontrolujące przemiany mRNA w
mitochondriach regulują cykl komórkowy i
apoptozę
M. Gadaleta ( Bari University)

What happens after 50 in human cells?

Strong 13x induction of mt transcription
factors
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Balance between synthesis and degradation
of mt RNA
Our research:



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Yeast model
Mammalian cells
Functions of human mitochondrial proteins
outside mitochondria
Speculations and research plans
Kompleksy degradujace RNA
Prime object of our research
Yeast mitochondrial degradosome
Genes coding for yeast degradosome
subunits :
SUV3
• DExH-box RNA helicase, 84 kDa
•Very ancient gene, orthologs found in purple bacteria,
plants, Drosophila and Homo sapiens ( Stepien et al..,
PNAS 1992)
DSS1
• RNase, homologous to bacterial RNase II
•Isolated as a multicopy suppressor of the SUV3 deletion
•110 kDa
( Dmochowska et al., Curr.Genet., 1995; Dziembowski et al.,
Mol.Gen.Genet, 1998, JBC 2003)
The working model: less is more
wild-type
RNA
Reduced levels of
mostly correctly
processed transcripts
suv3Δ
Secondary degradation routes
Degradation
(Suv3p+ Dss1p)
Reduced transcription
(Rpo41psupor Mtf1psup)
Secondary degradation routes
RNA
Degradation
(Suv3p+ Dss1p)
Transcription
(Rpo41p+ Mtf1p)
Secondary degradation routes
Degradation
(Suv3p+ Dss1p)
Transcription
(Rpo41p+ Mtf1p)
RNA
Normal levels of
correctly processed
transcripts
Accumulation of mis-processed
RNAs and high molecular weight
precursors
suv3Δ, su
Reduced transcription rescues the balance lost due to
disrupted degradation
Experiments in progress:
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Crystalization of the complex

We hope to resolve the structure

Biochemical analysis is on the way
Prime object of our research
Wielki problem biologii molekularnej

Z sekwencji genu nie potrafimy określić
funkcji białka
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Konieczność badań biochemicznych
Our approach:

We identify human orthologues in silico

We clone cDNA of a candidate, attach
fluorescent tag, and check mito localization

We silence the gene by siRNA and watch for
phenotypes
Our research on human genes:
We identified, cloned cDNAs and analyzed 4 human nuclear
genes:
SUV3 helicase ( Dmochowska et al., Acta Biochim. Pol. 1999;
Dmochowska et al., Cytogen. Cell Genet, 1999; Minczuk et al., NAR 2002, Minczuk et al..,
BBA in press)
polynucleotide phosphorylase (Piwowarski et al.,
J.Mol.Biol. 2003)
poly(A) polymerase Tomecki et al.., NAR 2004)
RNase unpublished)
(
Part I : polyadenylation

The role of human mtRNA polyadenylation
is not known :
evolutionary paradox: diverse roles of
poly(A) tails
The paradox

In procaryotes polyA tails are signal for
degradation
In eukaryotic cytosol polA tails stabilize mRNA
In plant mitochondria : polyA tails destabilize
Yeast mitochondria do not polyadenylate at all

Human mitochondria ?????????
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Human mitochondrial poly(A)
polymerase
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We identified the nuclear gene
Cloned the cDNA
Demonstrated mito localization by GFP
fusion
siRNA PAP silencing
siRNA of human mt polyA
polymerase : ND3 mtRNA
Two kinds of mito poly(A) tails:

Poly(A) : 40 – 50 A residues
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Oligo(A) : 5 A residues
Both poly(A) and oligo(A)
mitochondrial mRNAs are stable

Oligo(A) mRNA is translatable
Nagroda PTBioch. im . J. Parnasa
oraz Nagroda PTGen 2005:

„Identification of a novel human nuclearencoded mitochondrial poly(A) polymerase”

Rafał Tomecki, Aleksandra Dmochowska,
Kamil Gewartowski, Andrzej Dziembowski,
Piotr P. Stępień

Nucleic Acid Research 32:6001, 2004
PART II: Our research on human
SUV3 helicase:

Human SUV3 gene is the orthologue of
yeast SUV3
Genetics : a global science
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Craig Venter : EST

Buy yourself a gene
Overexpressed hSuv3myc is localized in
mitochondria in HeLa cells
hSuv3p is localized in the mitochondrial matrix
in HeLa cells
1. crude mitochondrial fraction
2. cytosolic fraction
3. total cell extract
1. soluble submitochondrial fraction
2. membrane submitochondrial fraction
3. total extract of purified mitochondria
1. mitoplasts
2. post-mitoplast supernatant
3. total extract of purified mitochondria
Heterologically expressed hSuv3p has DNA and
RNA unwinding activity in vitro
Comparison of efficacy of RNA and DNA
unwinding reaction mediated by hSuv3p
as function of decreasing enzyme
concentrations. 4.7 pM substrate and the
following enzyme concentrations were
used:
2 & 8 0.66 fM;
3 & 9 66 fM;
4 & 10 6.6 pM;
5 & 11 0.66 nM
6 & 12 66 nM.
The substrate and released strand were
separated in a TBE polycrylamide gel and
visualised by exposition of dried gel onto X-ray
film for 24 h.
TWO QUESTIONS:


what is the SUV3 function in mitochondria,
is there a human mito degradosome?
what is the SUV3 function outside of
mitochondria ?
We employed yeast two-hybrid
system to find human SUV3
interactors
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

our results :
XIP
Hsp60
Human XIP protein (HBXIP)


XIP is a 9,6 kDa protein interacting with HBx protein
of hepatitis B virus (Melegari et al., 1998)

HBx is responsible for HBV pathogenesis

Similarly to Suv3p, XIP is highly conserved during
evolution
The expression profile of XIP in humans is almost identical as
compared with hSUV3
Xip interacts with carboxy-terminal
fragment of the hSuv3p protein
Pull-down experiment
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XIP-TAPtag fusion was expressed in E.coli,
attached to IgG-agarose
hSUV3-myc fusions were expressed in vitro,
labeled with S-35 methionine
After incubation and elution, samples were
analyzed on SDS_PAGE
Pull-down of in vitro – translated
hSuv3p protein by overexpressed XIP
Do XIP and SUV3 interact in
mitochondria?

Subcellular localisation of XIP was not
known
Xip is localised in nucleus and
cytoplasm of mammalian cells
The site of XIP-SUV3 interaction is
not in mitochondria

Since SUV3 was assumed to be
mitochondrial protein and XIP is not in
mitochondria :
how does it work ???????-
Recent data on XIP

Marusawa et al., EMBO J. 2003
XIP is a cofactor of survivin in apoptosis
suppression
US patent filed
Survivin, 17 kDa:

One of the most tumor-specific human genes
Normally functions in chromosome
segregation
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Survivin supresses apoptosis

Inhibition of survivin induces caspasedependent death in tumor cell lines but not in
normal cells
Eli Lilly Company:

bought siRNA protocoll for survivin
suppression from Isis Co. for 1 Million USD

Ist phase clinical studies on patients with
cancer
Is there a link ????
 SUV3
 XIP
 survivin
Our working model:

Complex SUV3-XIP can regulate apoptosis

In the absence of SUV3 cancer cells
undergo cell death
Patent application:

Warsaw University: „The use of
modulation of hSUV3 expression for
apoptosis induction in cancer” 2005

Published: Minczuk et al., BBA 2005
Minczuk et al.., FEBS J., 2005

Is human SUV3 always in the
nucleus?
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
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Are mitochondria a reservoir of SUV3,
which is released in certain physiological
conditions?
what is the intracellular SUV3 trafficking ?
Is SUV3 really involved in caspase-9
apoptotic pathway?
PART III : Our research on other
human mitochondrial proteins
involved in RNA turnover

PNPase : polinucleotide phosphorylase

Poly(A) polymerase
Podwójna funkcja PNPazy:

W mitochondriach reguluje stabilność
mRNA

Poza mitochondriami wpływa na cykl
komórkowy
Human polynucleotide phosphorylase (hPNPase)
is associated with cellular senescence and
terminal differentiation
Leszczyniecka et al ( PNAS 2002, 99:16636-16641) have
shown that :
hPNPase is up-regulated:

in senescent progeroid fibroblasts,

fibroblasts entering terminal differentiation
after interferon beta treatment
overexpression of hPNPase resulted in growth
inhibition of human melanoma cells, this
suggests the possible use in gene therapy
patent application for the hPNPase was filed
human PNPase is localized in the cytoplasm ( THIS WAS THEIR
SUMMARY

We study human genes involved in mt RNA
turnover: SUV3, Poly(A) polymerase,
PNPase

SUV3 and PNPase seem to play an
additional role in regulating cell cycle
events
Standard approach:
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Biochemical parameters
Interactors ( Flp-in system and TAP-TAG)
Submitochondrial localisation
Influence on RNA turnover
RNA-protein complexes
siRNA studies
Protein models
All four human proteins : SUV3
PNPase
polyA polymerase
are currently overexpressed in their native
conformation, crystallized and analyzed by
X-ray diffraction

(