regulation of mitochondrial translation in...
TRANSCRIPT
Regulation of mitochondrial translation in yeasts
Nathalie BonnefoyMeetochondrie 2017
• Basic processes are conserved
• Facultative aerobe -> powerful selection tool
Yeasts are useful models for mitochondrial studies
glucose CO2 + H2O
O2fermentation
wild-type respiratorymutant
mtDNA
respirationethanol
• Basicprocessesareconserved
• Facultativeaerobe->powerfulselectiontool
• Geneticallyversatile
• MitochondrialtransformationisavailableinbuddingyeasttomanipulatethemtDNA ->reportergenefortranslation
• Manyfeaturesareclosertomammaliancells(mRNAstructure,factors)infissionyeast
Yeasts are useful models for mitochondrial studies
S.pombeS.cerevisiae H.sapiens
Conservationofgeneraltranslationfactors
+/- + +
Transcriptionunits 11 2 3
tRNA punctuation - + +
5’UTRofmRNA Long Veryshort Generallynone
SizeofmtDNA 75-85kb 19kb 16kb
Introncontent 13atmost 4atmost -
S. pombe is an excellent model to studymitochondrial gene expression
• Dualgeneticorigin,specificcode
• Inter-dependenceandcouplingwithotherstepsofmt geneexpression
• Hydrophobicityofmostmt-encodedproteinsrequiresco-insertion
• Assemblyofmostmt-encodedproteinswithnuclear-encodedsubunitstoformtheRCcomplexes->needofconcertation
• Unconventionalandpoorlyunderstoodstartsiteselection
• mt-mRNAuntranslatedregionsvaryalotamongstorganisms
Mitochondrial translation presents many specific features and constraints
Regulation of mitochondrial translation
Any process that modulates the frequency, rateor extent of the chemical reactions and pathwaysresulting in the formation of proteins by thetranslation of mRNA in a mitochondrion.
• Adjustthestoichiometryofthedifferentsubunits• Adapttoavailabilityofcofactors• Respondtoenergeticneeds• Sub-localizesynthesiswheresubunitsareneeded• Coordinatemt synthesiswiththesupplyofnuclear-encodedsubunits
• Thisoptimizationallowsto:Ø Protectmitochondriafromtheoverabundanceofmt-encodedproteinsØ Preventthetoxicaccumulationofnuclear-encodedproteinsatthe
surfaceofthemembrane
Regulation of mitochondrial translation is necessary
Mt-mRNA areabundant andthere is little regulation atthetranscriptionlevel.Translationregulation is needed to:
• Adjustthestoichiometryofthedifferentsubunits• Adapttoavailabilityofcofactors• Respondtoenergeticneeds• Sub-localizesynthesiswheresubunitsareneeded• Coordinatemt synthesiswiththesupplyofnuclear-encodedsubunits
• Thisoptimizationallowsto:Ø Protectmitochondriafromtheoverabundanceofmt-encodedproteinsØ Preventthetoxicaccumulationofnuclear-encodedproteinsatthe
surfaceofthemembrane
Regulation of mitochondrial translation is necessary
Mt-mRNA areabundant andthere is little regulation atthetranscriptionlevel.Translationregulation is needed to:
• Cis-elements inmRNAØ e.g. Stem-loop structureinCox2
• RibosomeitselfØ e.g. Ribsomal subunit isoforms,post-translational modifications
• GeneraltranslationfactorsØ e.g. RRF,IF3
• mRNA specific translational activatorsØ Bona fideØ Dualfunction ->link with other steps
• Generalrepressors oftranslationØ e.g. Ppr5,PTCD1
• Micro-RNAs?
Actors of mt translation regulation
• Cis-elements inmRNAØ e.g. Stem-loop structureinCox2
• RibosomeitselfØ e.g. Ribsomal subunit isoforms
• GeneraltranslationfactorsØ e.g. RRF,IF3
• mRNA specific translational activatorsØ Bona fideØ Dualfunction ->link with other steps
• Generalrepressors oftranslationØ e.g. Ppr5,PTCD1
• Micro-RNAs?
Actors of mt translation regulation
Aep1Aep2
ATP9
Atp22Nca2?Nca3?
ATP8/6
Pet54Pet122Pet494
COX3
Pet111COX2
Mam33Pet309Mss51
COX1
Cbp1Cbs1Cbs2Cbp3Cbp6
CYTb
FactorTargetmRNA
Budding yeast translational activators
VAR1 Sov1
• Allmt-mRNA areconcerned• mRNA stableinmutant,protein absent• Low abundance• Bona fide activators act onthe5’UTRonly• Some areRNAbindingproteins• Poorly conserved inevolution• Mediate several typesofinteractions:with
themRNA,themembrane,theribosome,other activators orcofactors
Startsiteselection
Cox2
Cox1
Cox3
Cit1
Cox4
Cox3/Atp6
Var1Cox1
Cox2Cytb
Role in limiting translation: overexpression of Pet111, the COX2 translational activator,
inhibits Cox1 synthesis
Fiorietal.,Mol Microb 2005
Mtproteinsynthesis Mtproteinsteadystate
Role in adaptation to respiratory switch: Mam33 functions to provide enough Cox1 in
glucose for a rapid adaptation to non-fermentable carbon sources
Roloff andHenry,Mol.Biol.Cell,2015
Mtproteinsynthesis
Role in coupling translation and assembly: In yeast, optimal assembly of the three
complexes is regulated in a different way by translation activators
Cytb incomplexIII:Cbp3/Cbp6
Atp6incomplexV:Atp22
Cox1incomplexIV:Mss51
Cbp1Cbs1Cbs2 Cbp3Cbp6
CYTb
FactorTarget mRNA
Efficient synthesis of Cytb requiresribosome-bound Cbp3-Cbp6 complex
CYTbmRNACYTbmRNA
Cbp3
Cytb
Cbp3Cbp6 Cbp6
complex III
Gruschke etal.J.CellBiol.2010
CYTbmRNA InteractionTuckeretal.PLOSGenet2013
Human UQCC1 and UQCC2 form a complex thatinteracts with CYTb and is essential for translation
and/or immediate stability
Mtproteinsynthesis
Atp22Nca2?Nca3?
ATP8/6
FactorTargetmRNA
Synthesis of Atp6 is dependent upon free F1
Rak etal.,Mol Biol Cell2016
b a F1
F0
In human the lack of F1 regulates Atp6 stabilityrather than translation
Rak etal.FEBSlett 2011
Hela Hela–sh F1b
Mtproteinsynthesis
xx
x
x
Mss51 interacts with the nascent Cox1 protein to mediate its assembly and
feedback regulation
AfterOtt andHerrmann,2010
Mam33Pet309Mss51
COX1
FactorTargetmRNAMam33
xx
x
x
Mss51 interacts with the nascent Cox1 protein to mediate its assembly and
feedback regulation
Mam33 wt
∆cox14
∆pet191∆cox14
Cox1
A
T
Mss51balance Strain TranslationofCOX1
Assembly ofcomplex IV
The equilibrium between the Translation and Assembly forms of Mss51 modifies synthesis of Cox1
wt
Assemblydefect
T A
AT
TA
++ ++
+/- -
+/-++∆cox14
xx
x
x
Mss51 interacts with the nascent Cox1 protein to mediate its assembly and
feedback regulation
AfterOtt andHerrmann,2010
Mam33
Heme b Heme aT
A
Mss51balance Strain TranslationofCOX1
Assembly ofcomplex IV
The equilibrium between the Translation and Assembly forms of Mss51 modifies synthesis of Cox1
wt
Assemblydefect
T A
AT
TA
++ ++
+/- -
+/-++∆cox14Lowheme bMss51hememutant
+/- +/-
An increase of Cox1 and Cytb synthesis in ∆rrf1 cells depends on the specific
translational activators
Cox1--Var1
mtEF-G2
mtRRF1
Ostojic etal.,Nuc.AcidsRes.2017
Mtproteinsynthesis
kDa
Mss51A
Mss51T
- 440
- 232
- 140
wt ∆rrf1
BN-PAGE-Digitonin
Tom40
Deletion of rrf1 does not modify Mss51 accumulation but increases the proportion of the Mss51T complex
Ostojic etal.,Nuc.AcidsRes.2017
Mss51balance Strain TranslationofCOX1
Assembly ofcomplex IV
The equilibrium between the Translation and Assembly forms of Mss51 modifies synthesis of Cox1
wt
Assemblydefect
T A
AT
TA
++ ++
+/- -
+/-++∆cox14lowheme b +/- +/-
+++ +/-∆rrf1
Deletion of the 15 last residues of Cox1 abolishes the feedback regulation
Maybealsoaroleinassemblyandsuper-complexformation…
Garcia-Villegasetal.,J.Biol.Chem.2017
Cox1
Mtproteinsynthesis
Mss51balance Strain TranslationofCOX1
Assembly ofcomplex IV
The equilibrium between the Translation and Assembly forms of Mss51 modifies synthesis of Cox1
wt
Assemblydefect
T A
AT
TA
++ ++
+/- -
+/-++∆cox14lowheme b +/- +/-
+++ +/-∆rrf1COX1-∆C15 +++ ++
Conservation of Cox1 translational activators in yeasts and human
S.cerevisiae S.pombe H.sapiensName Role Name Role Name Role Disease
Pet309 Cox1synthesis Ppr4 Cox1Synthesis
LRPPRC AllmRNA,stabilization
…
LateonsetLeigh’s
syndromeMam33 Cox1 synthesis
inglucoseMam33 ? P32/
C1QBPNumerous?Chaperone
Cardio-myopathy(mice)
Mss51 Cox1 synthesisFeedback
Heme sensing
Mss51 StabilityofCox1
MSS51 ?Muscleprotein
-
YGR021w ? SPBC8D2.12c
? TACO1 SynthesisofCox1
LateonsetLeigh’s
syndrome
Mt-mtRNAs
cox1
21S
Cox1Cytb
Cox2
Atp6
Rps3
Cox3
Atp9
Kühl etal.Nucl.Acids Res.2011
Ppr4 is the first mRNA-specific translationalactivator in S. pombe mitochondria
Mtproteinsynthesis
Conservation of Cox1 translational activators in yeasts and human
S.cerevisiae S.pombe H.sapiensName Role Name Role Name Role Disease
Pet309 Cox1synthesis Ppr4 Cox1Synthesis
LRPPRC AllmRNA,stabilization
…
LateonsetLeigh’s
syndromeMam33 Cox1 synthesis
inglucoseMam33 ? P32/
C1QBPNumerous?Chaperone
Cardio-myopathy(mice)
Mss51 Cox1 synthesisFeedback
Heme sensing
Mss51 StabilityofCox1
MSS51 ?Muscleprotein
-
YGR021w ? SPBC8D2.12c
? TACO1 SynthesisofCox1
LateonsetLeigh’s
syndrome
TACO1 is the first mRNA-specific translationalactivator in human mitochondria
Mtproteinsynthesis
Weraarpachai etal.Nat.Genet.2009
Mt-mRNA
COX1
TACO1 binds the COX1 mRNA, mostly at the 5’ end
Richmanetal.Nat.Com.2016
COX1
Conservation of Cox1 translational activators in yeasts and human
S.cerevisiae S.pombe H.sapiensName Role Name Role Name Role Disease
Pet309 Cox1synthesis Ppr4 Cox1Synthesis
LRPPRC AllmRNA,stabilization
…
LateonsetLeigh’s
syndromeMam33 Cox1 synthesis
inglucoseMam33 ? P32/
C1QBPNumerous?Chaperone
Cardio-myopathy(mice)
Mss51 Cox1 synthesisFeedback
Heme sensing
Mss51 StabilityofCox1
MSS51 ?Muscleprotein
-
YGR021w ? SPBC8D2.12c
? TACO1 SynthesisofCox1
LateonsetLeigh’s
syndrome
Bourens andBarrientos,EMBOreports2017
MITRAC is a COX1 pre-assembly complex equivalent to the yeast COX1-COA3-COX14 complex
C12ORF62:COX14MITRAC12:COA3MITRAC15:COA1MITRAC7/C12ORF52
Micketal.Cell2012;
For some authors, MITRAC couples optimal complex IV assembly and translation regulation of COX1
Optimalassembly
Feedbackregulation
Adaptationofmt-synthesistoinfluxofnc-encodedproteins
Micketal.Cell2016
Bourens andBarrientos,EMBOreports2017
For others, COX1 synthesis and formation of the CMC1-COX1-COA3-COX14 complex are not impaired
by downstream defects in CIV biogenesis
Mtproteinsynthesis
Mtproteinsteadystate
Mt translation work
• Translational activators: T.D. Fox, A. Tzagoloff
• General translation factors: L. Spremulli
• Feedback regulation: A. Barrientos, T.D. Fox, D. Winge, X. Perez-Martinez
• MITRAC: P. Rehling, A. Barrientos
• PPR proteins: A. Filipovska, X. Perez-Martinez
• Ribosome: J. Herrmann, Martin Ott, Alexey Amunts
• For some of our data: G Dujardin, CJ Herbert, C panozzo, A Bourand-Plantefol, J Ostojic, I Kühl
• Cis elements: Are there some regulating mt translation in human mtRNA? -> Would make sense since untranslated regions are limited
• Ribosome: Role in start site selection? Specificity of ribosomes for translation of RC subunits? Effect of post-translational modifications?
• General factors: Could the phenotype of e.g. RRF and IF3 disease be an assembly defect?
• Translational activators: are they more bona fide translational activators in human? Does MITRAC really play a role in translational control?
• Translation inhibitors: Are Ppr5 and PTCD1 functionning similarly? Are theyother general or specific negative regulators in human, like Smt1?
• Non coding micro-RNA: To what extent nuclear and mitochondria-encodedmiRNA regulate mt translation?
Open questions