自噬与核糖体的关系

KellyandBedwell

EffectofUbp3andautophagyontranslationalefficiency

Translationisseverelyreducedduringnitrogenstarvation(SchmelzleandHall2000).ThisisaconsequenceofTOR1inactivation,whichgreatlyreducestranslationinitiationonmostmRNAsandallowstheexpressionpatternofspecificmRNAstoincreaseinresponsetonutrientlimitation.However,selectiveautophagyofproteins,includingeIF4GandeRF3,couldpossiblyfine-tunetranslationduringnitro-genstarvation.Toaddressthispoint,wefirstaskedwhetherautophagyhadanyinfluenceontheoveralltranslationaldown-regulationthatoccursuponnitrogenstarvationbycar-ryingoutpolysomeanalysisinwild-typeandatg7Δstrains.Weobservedarobustlevelofpolysomesduringnutrient-richgrowthinbothstrains(Fig.7A)(polysome/monosome

FIGURE7.EffectsofautophagyandUbp3pontranslationfollowingtheonsetofnitrogenstarvation.(A)Polysomeanalysisonwild-typeandatg7Δstrainsunderconditionsofnitrogenexcess(left)andnitrogenstarvation(right).(P/M)Polysome-to-monosomeratio(wheremono-someincludesthesumofthe40S,60S,and80Speaks).(B)Schematicofthedualluciferasereadthroughreporters.Eachreportercontainsei-theranin-framenonsenseorsensecodonbetweentheRenillaandfireflyORFs.(C)Effectoftheubp3ΔmutationontherelativeefficiencyoftranslationterminationandeRF3abundanceduringgrowthinnitrogenexcess.(D)Effectofnitrogenstarvation( N)ontherelativeefficiencyoftranslationterminationinwild-typeandatg7Δstrains.Allexperi-mentswerecarriedouttwoormoretimeswithsimilarresults.Bargraphsareplottedasmean±standarddeviation.

904RNA,Vol.21,No.5

ratiosof2.0).Followingnitrogenstarvationfor2h,weob-servedaseverereductioninthepolysomepoolsinbothstrains(polysome/monosomeratiosof<0.2).TheseresultsdemonstratethattheTOR1-mediateddown-regulationoftranslationinitiationduringnitrogenstarvationistheprima-rymeansofcontrollingbulktranslationduringnitrogenstar-vation,andthismechanismwasnotadverselyaffectedbythelossofautophagyintheatg7Δstrain.However,wecannotex-cludethepossibilitythatthereductioninribosomecontentorthedepletionofspecifictranslationfactorsmayalterthetranslationofspecificmRNAs.

WenextaskedwhetherthedeubiquitinaseUbp3pinflu-encestranslationterminationduringgrowthinnutrient-richconditionsusingdualluciferasereadthroughreporters.Thesereporterconstructscontainanonsensecodon(orasensecodonascontrol)insertedin-framebetweentheRenillaandfireflyreadingframes(Fig.7B;Grentzmannetal.1998;Keelingetal.2004).Thisconfigurationallowsustousethefirefly/Renillaratioasanindicatoroftheeffi-ciencyoftranslationtermination.Wefoundthattheubp3Δstrainexhibited1.5to2.5-foldmorereadthroughatnonsensecodonsthanthewild-typestrain,althoughthesteady-statelevelofeRF3didnotchange(Fig.7C).Thissuppressorphe-notypesuggeststhatUbp3p(andbyinference,theubiquitin–proteasomesystem)influencestheefficiencyoftranslationterminationduringnormalgrowthconditions.

Finally,weexploredtheeffectofautophagyontranslationterminationduringnitrogenstarvation.Theefficiencyoftranslationterminationisdeterminedbytherelativeabun-danceofreleasefactors,aminoacyltRNAs,andterminatingribosomes(Betneyetal.2010).Duringnitrogenstarvation,thesteady-statelevelsofbothribosomesandeRF3arere-duced.Accordingly,thereductionineRF3maymodulatetheefficiencyoftranslationterminationduringthesecon-ditions.Totestthispossibility,weuseddualluciferaseread-throughreporterstocomparetherelativestopcodonreadthroughlevelsinwild-typeandatg7Δstrainsinnutri-ent-richandnitrogen-starvedconditions.Inthewild-typestrain,thereadthroughobservedwasmodestlyreduced(10%–25%)atthethreestopcodonsduringnitrogenstarva-tion(Fig.7D),indicatingthattheefficiencyoftranslationter-minationhadmarginallyincreased.Thisanti-suppressoreffectwasnotobservedintheatg7Δstrain,indicatingthatautophagy(andpossiblythereducedlevelofeRF3)mayfine-tunetheefficiencyoftranslationterminationduringni-trogenstarvation.DISCUSSION

Inthisstudy,wefoundthatthetranslationfactorseIF4GIandeRF3undergorapidandselectiveUbp3p-dependentdegradationbyautophagyfollowingtheonsetofnitrogenstarvation(Figs.1D,E,2).eIF4GIwaspreviouslyshowntobedegradedduringeithernitrogenstarvationorrapamycinexposure,althoughthemechanismofdegradationwas

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