Lecture 23 - Extranuclear inheritance II Flashcards
(21 cards)
What does mtDNA encode?
13 polypeptides
22 tRNAs
2 rRNAs
13 polypeptides that make up components of the electron transfer chain - CytB (encodes a subunit called cytochrome c reductase)
- ND genes (1,2,3,4,5) - encode NADH dehydrogenase
What are features of the Electron transfer chain (ETC)?
Complex 1 - mitochondrial & nuclear encoded subunits
4/5 complexes in electron transfer chain have subunits coded by the mitochondrial DNA - only complex 2 doesn’t (good for control)
Complex 1 - mtDNA-encoded subunits - 7, nDNA-encoded subunits - 49
example of co-assembly of subunits encoded in different genomes (nDNA & mtDNA) work together to perform essential cellular activity.
Complex 1,3,4 - responsible for pumping proteins across mitochondrial inner membrane
Complex 5 - ATP synthase
Describe mtDNA transcription & replication
2 origins of replication, OH & OL, Most genes encoded by Heavy strand.
Transcription commences from HSP & LSP (Here H1,H2 & L)
MtDNA replication is dependent on transcription of a short section from the LSP.
- MtDNA transcription is essential for mtDNA replication
- Transcription factor, co-factors & mtDNA polymerase are all nuclear-encoded
TFAM - essential mitochondrial transcription factor
POLG - only mitochondrial DNA polymerase
Amplification of D-loop (control region) shows binding sites for transcription factors & also DNA polymerase.
OH - origin of heavy strand replication
OL - origin of light strand replication
H1&H2 - transcription start sites for heavy strands
L - transcription start site for light strand
What is TFAM?
transcription factor binds to transcription start sites & is important for transcribing a primer required for replication. mtDNA replication is dependent on a short section from the light strand promoter. TFAM is essential for transcription & replication
What is POLG (enzyme)?
mitochondrial DNA polymerase - fault leads to lack of effectivity of mtDNA.
After RNA primer transcription from the light strand transcription start site, we get transition to DNA, binding of POLG & replication of genome.
How is TFAM a key player?
Tfam +/- Reduced mtDNA copy number, reduced mtDNA transcription & respiratory chain dysfunction in the heart
Tfam -/- Severe mtDNA depletion, abolished oxidative phosphorylation, enlarged mitochondria, growth retardation. Die prior to embryonic day E10.5
Conclusion = TFAM is essential for maintaining mtDNA copy number and for respiratory chain function.
Data showing TFAM is essential for maintaining mtDNA
What is TFAM’s packaging role?
TFAM is required for transcription from the LSP (light strand promoter) & HSP (heavy strand promoter) and is thus essential for mtDNA replication.
It is thought that levels of TFAM may DIRECTLY CONTROL mtDNA COPY NUMBER.
TFAM also has an important packaging role & is the MOST ABUNDANT PROTEIN IN MITOCHONDRIAL NUCLEOIDS. Another is mtSSB (mitochondrial single-stranded DNA binding protein).
The nucleoid proteins regulate the stability, replication, transcription & segregation of mtDNA.
What is TFAM interaction with DNA?
TFAM binding at the LSP & HSP forces the mtDNA into a U-shape.
HMG box domains bind to minor groove of DNA & presence of linker domain pulls the DNA round into U-shape.
This is structurally important for the activation of transcription from these 2 sites.
Describe the interplay between nucleus & mtDNA
- Nuclear-encoded enzymes are required for mtDNA transcription & replication
Co-assembly of nuclear-encoded & mtDNA-encoded subunits of respiratory chain complexes
Majority of proteins that function in the mitochondria are nuclear-encoded; Over 1000 nuclear-encoded products are essential to mitochondrial function.
INTER-GENOMIC COMMUNICATION IS ESSNETIAL
some level of cross-species compatibility.
Variation in sequences or production of mutations, intergenomic communication can break down & lead to disrupted mitochondrial function.
Increasing genetic divergence decreases oxidative phosphorylation.
What are features of mitochondrial?
- Heteroplasmy increases within individuals with age; this has been assoicated with ageing effects & numerous diseases
- Harmless polymorphisms vs disease-causing mutations
- More common than might expect. 1 in 200 carrying known point mutation
- Prevalence: 1 in 4000 children born with an inherited mitochondrial disease in US. 1 in 5000 adults affected.
- Mitochondrial disease includes any disease that results from impaired mitochondrial function.
THRESHOLD LEVELS; % of mutated mtDNA genomes above which disease is evident.
Threshold where compensation is no longer possible.
Only relevant to mitochondrial diseases that arises from mutation in mtDNA. It is possible easily for mitochondrial disease to arise due to nDNA. But in that case, heteroplasmy isn’t relevant.
Describe random segregation of mtDNA variants
Mixed load of mitochondrial is germ cell, random segregation during oogenesis means some oocytes have a higher mutant load than others. This means there is more likely there is a diseased phenotype.
This makes prediction about mitochondrial disease inheritance difficult
Describe features of mitochondrial disease.
- diseases associated with all 13 mtDNA encoded ETC (electron transport chain) subunits
- Inheritance follows a maternal rather than Mendelian pattern
- Disorders reflect deficiency in the biogenetic function of the mitochondrial
- Most evident in ‘energy intensive’ tissues that have the highest ATP requirement.
Affects lots of tissues with high energy requirements. Energy-intensive tissues affected most as inactive cells can cope with a lot of mitochondria being defective, as not as much ATP is needed.
What are Cell specific OXPHOS (oxidative phosphorylation) requirements?
The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutants in the mitochondrial translation factor EFG1.
Different tissues have very varying OXPHOS requirements.
Patients have lower levels of mtDNA-encoded OXPHOS proteins compared to controls.
Levels of protein in different complexes in different tissue types.
Patients with mitochondrial translation factor cEFG1 mutation have problems with protein levels of all complexes that have components that reside in mitochondrial genome.
Levels are different (lower) in certain tissue types.
What is inheritance seen in mitochondrial disease?
- Mitochondrial disorders can result from mutations in: mtDNA; nuclear genes encoding OXPHOS subunits; proteins required for their translation & assembly
- Mitochondrial disorders that are caused by nuclear mutations display Mendelian inheritance patterns
- Mitochondrial diseases caused by mtDNA mutations are maternally inherited but follow laws of population dynamics.
What is LHON - Leber’s Hereditary Optic Neuropathy?
- degeneration of the retinal ganglion cells & their axons
- acute onset blindless in young adulthood; more commonly affects males
- mtDNA point mutations in genes encoding NADH dehydrogenase subunits
Affected by many secondary mutations or polymorphic variants; genetically heterogenous disease.
What is MERRF - Myoclonic Epilepsy & Ragged Red Fibre disease?
- symptoms include myoclonus, myopathy, seizures, ataxia, peripheral neuropathy, dementia
- mtDNA gene MT-TK encoding tRNA - mitochondria unable to make proteins - mitochondria unable to make proteins required for functional respiratory chain
- Huge variation in severity between different patients & significant variations in tissue distributions of mutant mtDNA - due to random segregation results in varying mutant load in different tissues; some will not exceed threshold levels.
What is KSS - Kearns Sayre Syndrome?
- Multi-system disorder, particularly CNS & eyes
- large-scale deletions in mtDNA
- generally not maternally inherited; new somatic mutations.
- 100% dead, 20-90% disease; 2-% asymptomatic
- DNA polymerase gamma (POLG) - mutation in nuclear-encoded gene results in defects in mtDNA
- replication process affected
How does Polymerase gamma lead to mitochondria disease?
Results in an array of mtDNA mutations (secondary mutations), deletions or mtDNA depletion.
POLG-related disorders are clinically heterogenous
Lots of different issues can arise from POLG gene that cause problems with mtDNA replication. Some mutations make it more likely that there will be small mutations occurring in mitochondrial genome (secondary mutations).
Is mitochondrial DNA disease inheritance predictable - A1555G mutation?
No - it is unpredictable. Sudden occurrence common in mitochondrial disease.
Conclusion:
Risk for deafness increased with increasing mutant load BUT the level of heteroplasmy did not correlate with disease severity
THEREFORE:
mutant load is a very important determinant of phenotype but it is not the only variable.
% = mutant load (level of heteroplasmy)
What are implications in other disease?
Mutations in the mitochondrial genome has also been associated in more well-known disease - e.g mutation known in 1.5% diabetes, colorectal cancer, chronic fatigue syndrome & ageing
VARIABILITY IN mtDNA DISEASE SEVERITY
What are important points to remember?
- random segregation during oogenesis
- threshold levels of mutant load
- tissue-specific OXPHOS requirements
- random segregation leading to variable mutant load in tissues
- presence of secondary mutations/polymorphisms in mtDNA or nDNA
