Novel determinants of spontaneous readthrough of nonsense mutations [Elektronische Ressource] = Beschreibung neuer Faktoren mit Einfluss auf das spontane Überlesen von Stopcodon-Mutationen / vorgelegt von Frederic Pacho

Novel determinants of spontaneous readthroughof nonsense mutationsBeschreibung neuer Faktoren mit Einfluss auf das spontane Überlesen von Stopcodon-MutationenDer Naturwissenschaftlichen Fakultätder Friedrich-Alexander-Universität Erlangen-NürnbergzurErlangung des Doktorgradesvorgelegt vonFrederic Pachoaus Bad SchwalbachAls Dissertation genehmigt von der Naturwissenschaftlichen Fakultätder Universität Erlangen-NürnbergTag der mündlichen Prüfung: 31. August 2010Vorsitzender der Promotionskommission: Prof. Dr. Eberhard BänschErstberichterstatter: Prof. Dr. Klaus von der MarkZweitberichterstatter: Prof. Dr. Thomas WinklerTable of contents 1 Summary / Zusammenfassung.......................................... 1 ................................ 2 Introduction.......................................................................... 5 ............................. 2.1 Epidermolysis bullosa .....................................5.................................................... 2.2 A Herlitz patient with an unexplained mild phenotype .................8...................... 2.3 Anticodon–codon recognition .................................9............................................ 2.4 Termination of translation ..................................1.1.............................................. 2.5 Readthrough of termination codons ............................12...................................... 2.5.1 Discovery of leaky termination c ..................
Publié le : vendredi 1 janvier 2010
Lecture(s) : 17
Source : D-NB.INFO/1006940413/34
Nombre de pages : 85
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Novel determinants of spontaneous readthrough of nonsense mutations
Beschreibung neuer Faktoren mit Einfluss auf das spontane Überlesen von Stopcodon-Mutationen
Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur
Erlangung des Doktorgrades
vorgelegt von Frederic Pacho
aus Bad Schwalbach
Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät
der Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung:
Vorsitzender der Promotionskommission:
Erstberichterstatter:
Zweitberichterstatter:
31. August 2010
Prof. Dr. Eberhard Bänsch
Prof. Dr. Klaus von der Mark
Prof. Dr. Thomas Winkler
Table of contents
1 Summary / Zusammenfassung..........................................................................1
2 Introduction.......................................................................................................5
2.1 Epidermolysis bullosa.........................................................................................5
2.2 A Herlitz patient with an unexplained mild phenotype.......................................8
2.3 Anticodon–codon recognition.............................................................................9
2.4 Termination of translation.................................................................................11
2.5 Readthrough of termination codons..................................................................12
2.5.1 Discovery of leaky termination codons...................................................................12
2.5.2 Natural modifications of tRNAs..............................................................................13
2.5.3 Determinants of stop codon readthrough in bacteria...............................................13
2.5.4 Determinants of readthrough of termination codons in yeast.................................14
2.5.5 Determinants of readthrough of termination codons in mammals..........................15
2.6 Enhancement of readthrough by treatment with aminoglycoside antibiotics....16
2.6.1 Mechanism of aminoglycoside action on ribosomes...............................................16
2.6.2 Determinants of aminoglycoside effects on readthrough........................................18
 3 Working hypothesis / Strategy.........................................................................19
4 Materials..........................................................................................................20
4.1 Primer............................................................................................................... 20
4.2 Reagents........................................................................................................... 21
4.3 Enzymes........................................................................................................... 21
4.4 Plastic............................................................................................................... 21
4.5 Buffers.............................................................................................................. 21
4.6 Media................................................................................................................ 22
4.7 Eukaryotic cell lines.........................................................................................22
4.8 Bacterial strains................................................................................................ 22
4.9 Plasmids............................................................................................................ 23
5 Methods...........................................................................................................25
5.1 DNA................................................................................................................. 25
5.2 RNA.................................................................................................................. 28
5.3 Cell culture....................................................................................................... 28
5.4 qPCR................................................................................................................ 31
5.5 PCR.................................................................................................................. 31
5.6 Sequencing....................................................................................................... 32
5.7 Luciferase assay................................................................................................ 33
5.8 Flow Cytometry................................................................................................ 33
5.9 Immunofluorescence.........................................................................................34
5.10 Western Blot................................................................................................... 34
5.11 Bacteria........................................................................................................... 35
6 Results.............................................................................................................37
6.1 Diagnostic investigations..................................................................................37
6.1.1 Sequence analysis....................................................................................................37
6.1.2 Immunofluorescence detection of laminin-332.......................................................39
6.2 Analysis of mRNA............................................................................................ 41
6.3 Quantification of stop codon readthrough.........................................................45
6.4 Influence of single nucleotides on readthrough................................................48
6.4.1 Influence of the context on readthrough levels in HEK293 cells............................48
6.4.2 Influence of the context on readthrough levels in HeLa cells.................................50
6.4.3 Influence of the context on readthrough levels in NIH3T3 cells............................51
6.4.4 Influence of an expanded context on readthrough levels in HEK293 cells............52
6.5 Influence of context on readthrough.................................................................54
6.6 Aminoglycoside-induced readthrough..............................................................56
7 Discussion........................................................................................................60
7.1 Diversity of genotype-phenotype correlations..................................................60
7.2 Reasons for the unexpected mild phenotype of JEB-NRT................................61
7.3 Stability of premature termination codon containing mRNA............................63
7.4 Translational readthrough.................................................................................66
7.5 Determinants of readthrough in humans...........................................................67
7.6 Aminoglycoside antibiotics...............................................................................69
7.7 Possible mechanisms........................................................................................69
8 Abbreviations...................................................................................................71
9 Literature.........................................................................................................72
10 Acknowledgements........................................................................................79
Summary / Zusammenfassung
1 Summary / Zusammenfassung Origin of this work was a patient with junctional epidermolysis bullosa (JEB). This patient,
JEB-NRT, suffered from generalized blistering of skin and mucosae after birth and had
dystrophic finger- and toenails, symptoms characteristic for JEB. Today, JEB-NRT is six
years old and shows only occasionally localized blistering of the skin, a characteristic
feature of non-Herlitz JEB.
Genotype analysis of the patient JEB-NRT revealed two premature termination codons in
the LAMA3 gene, R943X and R1159X, which are compound heterozygous. However, loss
of both alleles of LAMA3, LAMB3 or LAMC2 is expected to cause the Herlitz form of
JEB, which is characterized by large scale skin blistering, leading to loss of fluid, minerals
and proteins. Herlitz patients normally die in their first year of life, because they are prone
to septicemia and other complications of the disease. Absence of any of the three proteins
(laminin α3, β3 or γ2) composing laminin-332 results in complete loss of extracellular
laminin-332, because only the assembled heterotrimer is secreted. In contrast to the
expected loss of extracellular laminin-332 in JEB-NRT, deposition of laminin-332 in the
basement membrane was detected by immunofluorescence staining in various skin biopsies
obtained from JEB-NRT, although most if not all laminin α3 chains may be truncated.
To elucidate the reason(s) for the difference between the expected and the observed clinical
phenotype of JEB-NRT, his LAMA3 mRNA was analyzed. Premature termination codons
(PTCs) may lead to alternatively spliced mRNA, in which the exon(s) containing the
PTC(s) is(are) skipped; if the reading frame is maintained in the shorter mRNA, a
truncated protein can be produced, that could attenuate the phenotype of JEB-NRT from
Herlitz to non-Herlitz JEB. Conventional PCR revealed that the mRNA was not
alternatively spliced and quantitative PCR showed that the majority (~96%) of LAMA3
mRNA comprises the R943X mutation. This is astonishing, because both PTC mutations
should result in nonsense-mediated mRNA decay (NMD) and degradation of both LAMA3
mRNAs. Even PTCs more distal assign LAMA3 mRNA to NMD[1].
In order to elucidate the reason for the escape of the mRNA containing the R943X
mutation from NMD, a reporter gene vector was constructed, that allowed analysis of the
efficiency of (premature) termination codons in their context: HEK293 cells were infected
with retroviruses which contain a CMV promoter-driven EGFP reporter gene and selected
for cells with stably integrated reporter genes. Between the ATG of the EGFP reporter gene
page 1 of 81
Summary / Zusammenfassung
and the remaining EGFP coding sequence, oligonucleotides which contain a termination
codon surrounded by six nucleotides on each side were inserted. EGFP fluorescence levels
detected by flow cytometry were used to calculate the percentage of readthrough over the
termination codon inserted.
The R943X TGA termination codon in its context led to low levels (~1.0%) of
readthrough. Termination codons with other contexts derived from LAMB3 PTCs allowed
no detectable readthrough. To determine which positions on the mRNA are involved in
readthrough, the three nucleotides in front of and behind the TGA termination codon were
replaced individually by every other nucleotide.
Analysis of the levels of readthrough allowed deduction of a consensus sequence for
efficient readthrough: (T/A)(A/G)(C/T) TGA CT(A/C). Extension of the context by three
nucleotides in both directions revealed that these further positions have minor impact on
readthrough, with exception of thymidine at positions four (+7) and five (+8) behind the
TGA termination codon, which impairs readthrough.
Readthrough could be enhanced, as expected from literature, by treatment with
aminoglycoside antibiotics. G418 raised readthrough levels nearly sixfold, whereas
gentamicin enhanced readthrough threefold for the R943X context. Other contexts were
less inducible by aminoglycoside antibiotics, even in long term experiments.
page 2 of 81
Summary / Zusammenfassung
Ein Patient mit junktionaler Epidermolysis Bullosa (JEB) war Ursprung dieser Arbeit.
Dieser Patient, JEB-NRT, litt nach der Geburt an Blasenbildung der gesamten Haut und der
Schleimhäute. Zusätzlich hatte er dystrophe Finger- und Zehennägel, typische Symptome
der JEB. Heute ist JEB-NRT sechs Jahre alt und leidet nur noch gelegentlich an örtlich
begrenzter Blasenbildung der Haut, charakteristisch für die nicht-Herlitz Form der JEB.
Die genetische Analyse des Patienten JEB-NRT zeigte vorzeitige Stopcodon-Mutationen in
beiden Allelen des LAMA3 Gen: R943X und R1159X. Der Verlust von beiden Allelen der
Gene LAMA3, LAMB3 oder LAMC2 führt zu der Herlitz-Variante der JEB, die durch
Blasenbildung der gesamten Haut gekennzeichnet ist; dies führt zu Verlust von Wasser,
Mineralien und Proteinen. Patienten mit Herlitz JEB sterben normalerweise innerhalb eines
Jahres nach der Geburt, da sie sehr anfällig für eine Sepsis und andere Komplikationen
sind. Das Fehlen auch nur eines der drei Proteine (Laminin α3, β3 oder γ2), aus denen
Laminin-332 zusammengesetzt ist, führt zu einem Verlust von sämtlichem extrazellulärem
Laminin-332, da nur das zusammengesetzte Heterotrimer sezerniert werden kann.
Immunfluoreszenzfärbungen auf Hautbiopsien von JEB-NRT zeigten, im Gegensatz zu
dem erwarteten Verlust von extrazellulärem Laminin-332, dass alle drei Proteine
extrazellulär vorkommen. Es ist jedoch möglich, dass fast alle Laminin α3-Ketten verkürzt
sind.
Um die Gründe für diesen Unterschied zwischen dem erwarteten und dem beobachteten
klinischen Bild von JEB-NRT aufzuklären, wurde seine LAMA3 mRNA analysiert.
Vorzeitige Stopcodon-Mutationen können zu alternativ gespleißter mRNA führen, in der
die Exone, die die Mutation enthalten, ausgelassen werden; wird das Leseraster in der
kürzeren mRNA beibehalten, kann eine verkürzte Version des Proteins produziert werden,
welche möglicherweise den Phänotyp von JEB-NRT von Herlitz zu nicht-Herlitz JEB
abmildern kann. Normale PCR zeigte, dass keine alternativ gespleißte mRNA vorlag und
quantitative PCR belegte, dass ein Großteil (~96%) der LAMA3 mRNA die Mutation
R943X enthielt. Dieses Ergebnis war überraschend, da beide vorzeitigen Stopcodon
Mutationen zu nonsense-mediated mRNA decay (NMD) und Abbau beider mRNAs führen
sollten; andere Stopcodon-Mutationen, die weiter distal im LAMA3 Gen liegen, führen
auch zu NMD[1].
Um die Gründe zu klären, warum die mRNA, die die R943X Mutation enthält, dem NMD
entgehen kann, wurde ein Reportergenvektor konstruiert. Dieser ermöglichte die Messung
page 3 of 81
Summary / Zusammenfassung
der Durchlässigkeit von Stopcodon-Mutationen in ihrem genetischen Kontext: HEK293
Zellen wurden mit Retroviren infiziert, die ein EGFP Reportergen enthielten, das von
einem CMV Promoter gesteuert wurde. Zellen mit stabil ins Genom integriertem
Reportergen konnten mit Hilfe von Puromycin selektioniert werden. Zwischen dem ATG
des EGFP Reportergens und der restlichen kodierenden Sequenz des EGFP wurden
Oligonukleotide eingesetzt, die Stopcodons enthielten, die auf jeder Seite von sechs
Nukleotiden (genetischen) Kontextes umgeben waren. EGFP Fluoreszenzlevel wurden mit
Hilfe von Durchflusszytometrie bestimmt und genutzt, um den Prozentsatz des Überlesens
des jeweiligen Stopcodons zu berechnen.
Das R93X TGA Stopcodon in seinem genetischen Kontext führte zu niedrigen
Überleseraten (~1.0%). Stopcodons in anderen Kontexten, abgeleitet von Stopcodon-
Mutationen im LAMB3 Gen, ermöglichten keine messbaren Überleseraten. Um den
Einfluss einzelner Positionen der mRNA auf die Überleseraten zu bestimmen, wurden die
drei Nukleotide vor und hinter dem TGA Stopcodon einzeln gegen jedes andere Nukleotid
ausgetauscht.
Die Messung der Überleseraten ermöglichte die Ableitung einer Konsensussequenz, die
hohe Überleseraten ermöglicht: (T/A)(A/G)(C/T) TGA CT(A/C). Eine Erweiterung des
untersuchten Kontextes um weitere drei Nukleotide in beide Richtungen zeigte, dass diese
Positionen nur einen geringen Einfluss auf die Überleserate haben; einzig Thymidin an den
Positionen vier (+7) und fünf (+8) hinter dem TGA Stopcodon beeinträchtigt das
Überlesen.
Die Überleserate konnte, wie schon in der Literatur beschrieben, durch eine Behandlung
mit Aminoglykosidantibiotika weiter verstärkt werden. Im R943X Kontext erhöhte G418
die Überleserate knapp sechsfach, Gentamicin dreifach. In anderen Kontexten war die
Überleserate durch Behandlung mit Aminoglykosidantibiotika weit weniger induzierbar,
auch wenn die Behandlung über einen längeren Zeitraum erfolgte.
page 4 of 81
Introduction
2 Introduction
2.1 Epidermolysis bullosa Epidermolysis bullosa (EB) is a disease of skin and mucosae with variable phenotypes.
The main reason for this variability is the number of genes that can be affected. On the
other hand, missense or nonsense mutations in the same gene may lead to completely
different phenotypes.
Based on the level of tissue separation at the dermal-epidermal junction, EB is classified
into EB simplex, dystrophic and junctional EB. In the junctional subtype, three different
forms can be distinguished clinically as well as on the molecular level:
·
·
·
junctional EB with pyloric atresia (MIM #226730)
junctional EB, non-Herlitz type (MIM #226650)
junctional EB, Herlitz type (MIM #226700)
In junctional EB with pyloric atresia, mutations are found in the genes coding for α4- or
β6-integrin, which are components of the hemidesmosomes.
The non-Herlitz form of junctional EB can be caused either by mutations in the gene
COL17A1 or by mutations in any of the three genes encoding laminin-332, LAMA3,
LAMB3 and LAMC2. If LAMA3, LAMB3 or LAMC2 are affected, homozygous or
compound heterozygous nonsense mutations are known to result in the lethal Herlitz type
of disease.
Patients affected by junctional EB suffer from generalized blistering of skin and mucosae,
eventually accompanied by atrophic scarring. Typical symptoms also include dystrophic
finger and toenails. Besides impairment of the skin and its appendages, the patients may
suffer from anemia, growth retardation, tooth abnormalities, gastrointestinal and
genitourinary alterations. In the Herlitz form of junctional EB, both alleles of one of the
three genes encoding the laminin-332 protein chains contain a premature termination
codon caused by frameshift mutations, out-of-frame exon skipping or nonsense mutations.
The mode of inheritance of Herlitz junctional EB is autosomal recessive. The disease
occurs with a frequency of 1-2 per one million births.
page 5 of 81
Introduction
Absence of any of the laminin α3, β3 or γ2 chains excludes laminin-332 deposition in the
basement membrane. All three chains contain a central rod-like domain essential for
heterotrimerization. The assembled laminin-332 has a cruciform shape where the long arm
of the cross is built up by a twisted coiled-coil formed by the central domains of the
laminin α3, β3 and γ2 chains. Only the assembled heterotrimer can be secreted by basal
keratinocytes.
Figure 1: Structure of the laminin-332 molecule Laminin-332 is a heterotrimer composed of laminin α3, β3 and γ2 chains. The central coiled-coil of laminin-332 is formed by the rod-like structure of each chain, whereas the N-termini of the chains form the short arms of the cross shaped molecule. The C-terminus of the α3 chain contains five laminin globular domains, three of which mediate binding of laminin-332 to integrins, syndecan and heparan (LG1 to LG3).
Even premature termination codons C-terminal of the coiled-coil region lead to Herlitz
junctional EB. The reason is degradation of the mutated mRNA by nonsense-mediated
mRNA decay if the mutation is situated in front of the last exon[2,3].
Patients with the Herlitz form of junctional EB suffer from an increasing loss of fluid,
minerals and proteins from their large skin wounds and fail to thrive. They are prone to
septicemia and normally die in their first year of life.
The severe phenotype of Herlitz junctional EB can be explained by the major importance
page 6 of 81
Introduction
of laminin-332 as anchoring protein in the basement membrane. Cellular integrins, mainly
the hemidesmosomal α6β4 integrin, bind to the C-terminal globular domains 1-3 of the
laminin α3 chain (LG1-3)[4,5]N-terminal short arms of all chains mediate laminin-332. The
integration into the basement membrane and binding to the dermal anchoring collagen VII.
Without laminin-332, adhesion of the basal keratinocytes to the basement membrane and
dermis is impossible.
Additionally, laminin-332 influences motility, growth, polarity and differentiation of the
basal keratinocytes via outside-in signaling subsequent to binding to α3β1- and α6β4-
integrins[6-8] .
Figure 2: Organization of the dermal-epidermal junction Laminin-332 is the central anchoring filament at the dermal-epidermal junction . The globular domains of laminin α3 interact with cellular integrins, whereas the N-terminal parts of all three components of laminin-332 mediate its anchorage to the basement membrane and binding to dermal collagen VII (adapted from[9]).
page 7 of 81
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