regulation of mitochondrial translation in...

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