2 and its C-terminal tail contributes to stop codon ... · 112 during the translation process...

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The eukaryotic ribosomal protein S15/uS19 is involved in fungal development1

anditsC-terminaltailcontributestostopcodonrecognition2

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Runningtitle:uS19aproteinofthedecodingsiteineukaryotes4

5

Tan-Trung Nguyen,1,†,‡ Guillaume Stahl,2,† Michelle Déquard-Chablat,1 Véronique6

Contamine1andSylvieHermann-LeDenmat1,3,§,*7

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1InstituteforIntegrativeBiologyoftheCell(I2BC),CEA,CNRS,Univ.Paris-Sud,Université10

Paris-Saclay,91198,Gif-sur-Yvettecedex,France11

2Laboratoire deBiologieMoléculaireEucaryote, CBI,Université deToulouse, CNRS,UPS,12

Toulouse,France13

3Ecolenormalesupérieure,PSLResearchUniversity,F-75005Paris,France14

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†Theseauthorscontributedequallytothiswork17

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*For correspondence. E-mail [email protected] or [email protected],19

Tel.(+64)9923463120

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‡Presentaddress:InstitutJean-PierreBourginINRA,AgroParisTech,CNRS,Université22

Paris-Saclay,Versailles,France23

§Presentaddress:SchoolofBiologicalScience,TheUniversityofAuckland,PrivateBag24

92019,Auckland,1142,NewZealand25

26

author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/2020.02.09.940346doi: bioRxiv preprint

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Graphicalabstract27

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AbbreviatedSummary40

S15/uS19isaconservedsmallribosomalproteinthatineukaryotesharborsaflexibleC-41

terminalextensionproposedtointeractwiththeAsitemRNAcodonduringtranslation.42

Here, we describe how C-terminal variants variously affect Podospora anserina43

development and longevity and impact fungal ribosome and polysome formation.We44

reveal that stop codon recognition is significantly altered by the presence of those C-45

terminal variants, which either expand or on the contrary restrict termination46

ambiguity.47

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Summary51

S15/uS19 is one of the fifteen universally conserved ribosomal proteins of the small52

ribosomal subunit.While prokaryotic uS19 is located away from themRNA decoding53

site,cross-linkingstudiesidentifiedeukaryoticuS19C-terminaltailascontactingtheA54

site on the 80S ribosome. Here, we study the effects of uS19 mutations isolated as55

translation accuracy mutations in the filamentous fungus Podospora anserina. All56

mutations alter residues of uS19 C-terminal tail, and cluster to the eukaryote-specific57

decapeptide 138-PGIGATHSSR-147. All mutations modify fungal development and58

cytosolic translation, albeit differently. Two mutations (P138S and S145F) increase59

fungus longevity and display mild effects on translation, while others (G139D and60

G139C) decrease longevity, have stronger effects on translation and confer61

hypersensitivity to paromomycin. By mimicking P. anserina mutations in the yeast62

Saccharomyces cerevisiaeRPS15 gene, we further show that P138S and S145F induce63

hyperaccuraterecognitionofthethreestopcodons,whereasG139DandG139Cimpair64

UAGandUAAcodonrecognition.Noteworthy, inP.anserina,uS19geneticallyinteracts65

with the eRF1 and eRF3 release factors. All together, our data indicate that uS19 C-66

terminal tailcontributes invivo toeukaryotic translationtermination,and identifykey67

amino acids of uS19 that potentially modulate eRF1-eRF3 interaction in the pre-68

terminationcomplex.69

70

Keywords71

S15/uS19 ribosomal protein, A site, stop codon recognition, fungal development,72

Podosporaanserina,Saccharomycescerevisiae73

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Introduction76

Inallorganisms,ribosomesareresponsible forproteinsynthesis.Thesmallsubunit is77

engaged indecoding the informationencoded inmRNA,whilepeptidebondsynthesis78

occurs at the large ribosomal subunit. In eukaryotes, the cytoplasmic large 60S and79

small 40S subunits are together composed of 80 distinct proteins and four ribosomal80

RNAspecies.The60Ssubunitisassembledfrom25S-28SrRNA,5.8SrRNA,5SrRNAand81

47proteins,ofwhichsevenarespecifictoeukaryotes,20toeukaryotesandarchea,and82

20arefoundinallkingdoms(Lecompteetal.,2002).Similarly,the40Ssubunitconsists83

of18SrRNAand33proteins,ofwhichfivearespecifictoeukaryotes,13toeukaryotes84

andarchea,and15arefoundinallkingdoms(Lecompteetal.,2002;Rabletal.,2011;85

Banetal.,2014).86

Ribosomal proteins (r-proteins), in addition to being constituents of ribosomes, play87

major roles in rRNA processing, ribosome assembly, nucleo-cytoplasmic transport of88

ribosomal subunits, and in the functioning of the translational machinery itself89

(Brodersen andNissen, 2005; Ferreira-Cercaetal., 2005;Wilson andNierhaus, 2005;90

Pöll et al., 2009; Woolford and Baserga, 2013; Graifer and Karpova, 2015). The91

assignmentofexactribosomal functionsto individualr-proteins isnoteasytoachieve92

considering the highly cooperative nature of protein-protein and protein-RNA93

interactions intheribosome. Whereastherolesofanumberofbacterialr-proteins in94

translationhavebeenunderstood(BrodersenandNissen,2005;WilsonandNierhaus,95

2005; for reviews), the contributions eukaryotic r-proteins make to translation have96

been less addressed, and the simple idea that eukaryotic r-proteins share the same97

functionalpropertiesinribosomesastheirbacterialcounterpartscanbeerroneousand98

isasubjectofdebate(seeGraiferandKarpova,2015andreferencestherein).99


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Whataddstothedifficultyisthatr-proteinshaveNandC-terminalextensionsthatare100

specific to eukaryotes (Rabl et al., 2011), and these could participate in eukaryote-101

specific functions of r-proteins. One potential example of this is uS19 (also known as102

S15,theorthologofbacterialS19p;(Banetal.,2014)).Inyeast,thisessentialr-protein103

was shown to be involved in the nuclear export of 40S subunit precursors (Léger-104

Silvestreetal.,2004),aroleconservedinmammaliancells(Rouquetteetal.,2005),and105

itwas suggested that theN-terminal extensionof uS19 (seeFig. 1A)maybe involved106

(Léger-Silvestreetal.,2004).107

EukaryoticuS19localizestotheheaddomainofthesmallribosomalsubunit,alocation108

conserved with bacteria (Ferreira-Cerca et al., 2007; Rabl et al., 2011). Interestingly,109

while the bacterial polypeptide is positioned away from thedecoding site (Brodersen110

andNissen,2005)eukaryoticuS19was foundtoneighbor itandtocontact themRNA111

during the translationprocess (Bulyginetal., 2002).Furthermore, site-directedcross-112

linkingstudiesperformedwithdiversemammalianreconstitutedribosomalcomplexes113

showedthatuS19isoneoftheonly,ifnottheonly,eukaryoticr-proteinsthatinteracts114

with theAsitemRNAcodonduring the initiation,elongationand terminationstepsof115

translation(Bulyginetal.,2005;Pisarevetal.,2006;Pisarevetal.,2008).Cross-linkwas116

mappedintheC-terminaltailofhumanuS19(Khaĭrulinaetal.,2008),mostprobablyin117

theeukaryote-specificpeptidePGIGATHSSR(Khairulinaetal.,2010)(seeFig.1AandB).118

However, the lackofstructural resolutionof theC-terminalsequenceofuS19(Rablet119

al.,2011;LomakinandSteitz,2013)preventsvisualizationofuS19interactionwiththe120

mRNA at the decoding site that would confirm the documented in vitro cross-linking121

data. This, in turn, makes establishing the functional role of uS19 in eukaryotic122

translation difficult. Nevertheless, hypothetic roles of the PGIGATHSSR decapeptide123

havebeenproposed,includingselectionoftheinitiationcodonduringinitiation(Pisarev124


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et al., 2008), stabilization of themRNA codon at the decoding site during elongation125

(Khaĭrulina et al., 2008; Khairulina et al., 2010), as well as interaction with the126

polypeptide chain release factor eRF1 during termination (Khairulina et al., 2010;127

GraiferandKarpova,2012;GraiferandKarpova,2015,forreviews).128

To study the ribosomal control of translation fidelity in the filamentous fungus129

Podosporaanserina,MargueritePicard’sgrouphascarriedout inthe70’sveryelegant130

two-stepgeneticscreens that recovered,amongotherribosomalmutations,mutations131

in the AS1 gene that codes for uS19 (Picard, 1973; Picard-Bennoun, 1976; Picard-132

Bennoun,1981;Dequard-ChablatandSellem,1994).Wereporthere that the fourAS1133

mutationsidentifiedinthosestudiesallalterresiduesofthePGIGATHSSRdecapeptide134

that is identicalbetween theP.anserinaandhumanr-proteins (Fig.1B). Interestingly,135

theseAS1mutationsantagonizetheeffectofmissensemutationsinthegenesencoding136

the release factors eRF1 and eRF3 (Picard, 1973; Picard-Bennoun, 1976; Gagny and137

Silar, 1998; This work). By dissecting the phenotypic traits associatedwith eachAS1138

mutation inP.anserina and studying the translationdefects associatedwith eachAS1139

mutationinP.anserinaandinyeast,wedemonstratetheimportanceofuS19tofungal140

developmentandthecriticalroleuS19C-terminaltailplays instopcodonrecognition.141

Ourinvivofindingsconfirmforthefirsttimehypothesesformulatedtoexplaininvitro142

cross-linkingresultsthathadidentifiedcontactsoftheuS19C-terminaltailwiththeA143

sitemRNAcodon(GraiferandKarpova,2015).144

145

146

147

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Results150

AS1anti-suppressormutationsalterresiduesofuS19C-terminaltail151

Mutated alleles of the P. anserina AS1 gene were obtained in the course of two-step152

geneticscreensaimedtostudy,fourdecadesago,theribosomalcontroloftranslational153

ambiguity ineukaryotes(Picard,1973;Picard-Bennoun,1976;Picard-Bennoun,1981).154

First, many su mutations acting as informational suppressors (i.e., they increased155

translationalerror)havebeenobtained,andsecondly,startingfromsomesumutations,156

anti-suppressor mutations (AS) antagonizing the effect of su mutations have been157

isolated (Picard-Bennoun, 1976; Coppin-Raynal, 1981; Coppin-Raynal et al., 1988, for158

reviews). One of these genetic screens was based on an ascospore defective color159

phenotype associatedwith themutation 193 suspected at the time to be a nonsense160

mutation.Indeed,sequencingrevealedmutation193asbeinganUGAstopcodoninthe161

open reading frame encoding a polyketide synthase involved in melanin formation162

(Coppin and Silar, 2007). Phenotypically, while the stop-codonmutation 193affected163

ascosporepigmentation,ina193sudoublemutantcontext,pigmentationwasrestored164

(partiallyor totally),and ina193suAS triplemutantcontext,ascosporepigmentation165

was anew defective (see Table 1). Some of the su and AS genes have been cloned166

(DebuchyandBrygoo,1985;SilarandPicard,1994;Silaretal.,1997;GagnyandSilar,167

1998; Dequard-Chablat and Silar, 2006), including the AS1 gene that codes for the168

cytosolicr-proteinuS19(Dequard-ChablatandSellem,1994; thereinuS19wasnamed169

S12 according to electrophoretic nomenclature). In all cases, only actors of the170

translation machinery were identified as initially predicted by the elegant pioneer171

genetic approaches carried out by Marguerite Picard (Picard, 1973; Picard-Bennoun,172

1976;Picard-Bennoun,1981).173


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The genetic screen leading to isolationofmutated alleles in theAS1 gene is hereafter174

briefly redrawn. Four alleles (AS1-1, AS1-2, AS1-3 and AS1-4) were selected as175

antagonizing the effect of su1mutations in the gene encoding translation termination176

factor eRF3. A fifth allele (AS1-5) was obtained as alleviating the growth defect177

associatedwith a su2-5 mutation in the gene encoding translation termination factor178

eRF1 (Table 1 and Discussion). While AS1-2, AS1-3 and AS1-4 were obtained after179

nitrosoguanidine mutagenesis, AS1-1 and AS1-5 were obtained by chance. The AS1-1180

alleleappearedspontaneouslyina193su1-42background(Table1)andtheAS1-5allele181

arosefromthesinglesu2-5mutant(Picard-Bennoun,1981).182

AllAS1mutationsaremissensemutations(Table1).Aspreviouslyreported(Dequard-183

ChablatandSellem,1994),theAS1-4andAS1-5mutationsaffectthesameGlycinecodon184

substituted for an Aspartate and a Cysteine, respectively (position 139 P. anserina185

numbering; Fig. 1B).We report here that themissenseAS1-1mutation gives rise to a186

Proline to Serine exchange (position 138), while the AS1-2 and AS1-3 mutations are187

indeedidenticalandchangeaSerinetoaPhenylalanine(position145;Table1andFig.188

1B).Hereafter,onlytheAS1-2allelewillbeconsidered.189

Alignmentoftheaminoacidsequencesof14representativeeukaryoticr-proteinsuS19190

and three prokaryotic counterparts is presented Fig. 1A. AS1 mutations modify very191

close residues that all fall in the C-terminal part of uS19 (Fig. 1A), precisely in the192

PGIGATHSSR decapeptide mapped to neighbor the A site mRNA codon (Fig. 1B)193

(Khairulinaetal.,2010).ChangesinducedbytheAS1mutationsaffectresiduesstrictly194

conserved in human,P. anserina, andmost eukaryotes. There is an exception for the195

phylogeneticSaccharomycotinagroup(exemplifiedherebytheuS19sequencesfromS.196

cerevisiae,Candidaglabrata andKluyveromyces lactis; Fig. 1A). In this group, uS19 C-197

terminaltailisdistinguishablewithadeficitofthreeinternalresiduesthatcreatesgaps198


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in the alignment (Fig. 1A and B). Outside these gaps, the very end of all eukaryotic199

sequences(lasteightresidues)retainedahighlevelofconservation.200

201

PhenotypictraitsassociatedwiththeAS1mutationsdefinetwoclassesofmutants202

ThefourAS1mutationswerestudiedforthephenotypestheyproducedonP.anserina203

organism. The present study supplements reports about the phenotypic traits204

associated with AS1 mutations and especially with the AS1-4 allele (Picard-Bennoun,205

1976;Picard-Bennoun,1981;Kieu-NgocandCoppin-Raynal,1988;Belcouretal.,1991;206

Dequard-Chablat and Sellem, 1994; Contamine et al., 1996). Phenotypic features that207

characterize the development of the filamentous fungus andpoint out the differences208

betweenAS1mutantstrainsaresummarizedinTables2and3.209

First, none of the AS1 mutations affected ascospore germination that triggers the P.210

anserina life cycle (e.g., germination efficiency was equivalent to that of wild type).211

Nevertheless,theAS1-4andAS1-5germinatingascosporesdisplayedastrongphenotype212

withanalteredgrowthofthethalliandtheproductionofspindlymycelia(Table2).On213

the contrary, AS1-1 and AS1-2 germinating ascospores produced a wild-type aerial214

myceliumwithonlyaslightlyalteredgrowthphenotype.215

Life span is another phenotypic trait that characterizes the filamentous fungus P.216

anserina.At27°Constandardgrowthmedium(Experimentalprocedures),alloftheAS1217

mutantsshowedamodifiedlifespancomparedtowildtypereferencestrains(Table2).218

Inthematingtypemat+context,allAS1mutantsdisplayedahighlyextendedlifespan219

(10to50timeslongerthanwildtype),andduringthecourseofthework,themajority220

ofthemat+subcultureskeptgrowing(withtheexceptionoftheAS1-2mutant). Inthe221

matingtypemat-context,AS1mutantsdifferbetweenthem.Ononehand,theAS1-1and222

AS1-2mutantsstilldisplayedanextendedlifespan(3to7timeslongerthanwildtype),223


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whereasontheotherhand,AS1-4andAS1-5showedashortenedlifespan(5to8times224

shorterthanwildtype).LifespandiscrepancybetweentheAS1-4mat+andAS1-4mat-225

strainshasbeenalreadydescribed(Contamineetal.,1996)andwasshowntostemfrom226

thermp1gene(Contamineetal.,2004).Thatfilamentousfungusspecificgeneistightly227

linked to themating type locuswhere it exists under twonatural alleles: rmp1-2 and228

rmp1-1(withinthemat+andmat-locus,respectively).Theinfluenceofrmp1onfungus229

deathtimingwasreportedforotherP.anserinamutantsbutnofunctionalexplanation230

has been established yet (Sellem et al., 2005; El-Khoury and Sainsard-Chanet, 2009;231

Adametal.,2012;seeDiscussion).232

Finally,vegetativegrowthofeachAS1mutantwasexaminedat27°Caswellasat low233

(11°C) and high (35°C) temperature (Experimental procedures). Starting from 2 day234

germinatingascospores,fiveindependentsubculturesofeachAS1mutantwereusedto235

calculate an averagegrowth rate expressed in cmper day (Table3). In all conditions,236

vegetativegrowthoftheAS1-1andAS1-2mutantsbarelydifferedfromthewildtype.In237

particular, the wild-type, AS1-1 and AS1-2 mat+ strains all exhibited an equivalent238

reduced growth rate at 35°C when compared to their mat- counterparts (Growth239

reductionwasof84%,86%and83%,respectively,seeTable3).Differencebetweenthe240

mat+andmat-contextisanewduetothermp1genewhosermp1-2alleleisknowntobe241

responsible for the temperature sensitive growth of wild-type mat+ strain at 35°C242

(Contamineetal.,2004).Noteworthy,themat+vs.mat-differenceingrowthat35°Cwas243

not detected for theAS1-4 andAS1-5mutants (Table 3). This pointwill be discussed244

further.245

246

Altogether,detailedphenotypicanalysesrevealedtwoclassesofAS1mutants.First,the247

AS1-1 (P138S) andAS1-2 (S145F)mutants that differ very little from each other and248


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from thewild-type strain for their germination phenotype and vegetative growth but249

arehoweververydifferentfromwildtypewhenexaminedforlongevity,bothmutants250

displaying an extended life span. In addition, life span analysis reveals a difference251

betweentheAS1-1andAS1-2mutants,theformergrowinglonger(comparisonismade252

for the same mating type context). Secondly, AS1-4 (G139D) and AS1-5 (G139C)253

developmentisalwaysdistinguishablefromthewildtypeandclearlydifferentfromthe254

oneofAS1-1andAS1-2mutants.TheAS1-4andAS1-5strainssharesimilarphenotypic255

alterations but defects are exacerbated in AS1-4 (especially in the mat- context),256

indicatingthatG139DaminoacidexchangeismoredeleteriousthanG139Csubstitution257

tofungaldevelopment.258

259

DefectsinribosomefunctionfurtherpinpointthedichotomousnatureoftheAS1mutations260

Considering the role of yeast uS19 in small subunit assembly (Ferreira-Cerca et al.,261

2007), we first examined whether AS1 mutant alleles affected the production of 40S262

ribosomalsubunit(r-subunit)inP.anserina.DuetotheshortenedlifespanoftheAS1-4263

mat- andAS1-5mat-mutants (Table2), experimentswereall carriedout in themat+264

context. Equivalent amounts of cell extracts fromwild-type andmutantmat+ strains265

grownat27°Constandardgrowthmediumwere fractionatedonsucrosegradients in266

the absence ofMg2+, to dissociate ribosomes andpolysomes into free40S and60S r-267

subunits (Experimental procedures). AS1 mutations led to either modest (AS1-1 and268

AS1-2) or clear (AS1-4 and AS1-5) reduction in the amounts of free 40S r-subunits269

relativeto60Sr-subunits(Fig.2bottompanels).Decreasein40Sr-subunitswasfurther270

estimated by calculating 40S-to-60S ratios for at least three independent sucrose271

gradients using two independent extractions for each strain (Fig. 2 histograms). In272

agreementwith the phenotype traits associatedwithAS1mutants,AS1-4 showed the273


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strongestdeficitin40Sr-subunitswitha40-to-60Sratioof41%incomparisonto69%274

forthewild-typestrain.AS1-1andAS1-2displayedequivalentlyslightlyreduced40S-to-275

60Sratios(60%)whenintheAS1-5mutantthemeanvalueof40S-to-60Sratiois49%.276

Wenextexaminedtheeffectof40S/60Ssubunitimbalanceonpolysomeprofileswhose277

analysis is a sensitive method for detecting defects in translation. Polysome profiles278

were compared for equivalent amounts of extracts fromwild type andmutantmat+279

strains grown also at 27°C on standard growth medium (Fig. 3A) (Experimental280

procedures). Overall,AS1-1 andAS1-2 strains gave rise to gradient profiles similar to281

that of wild type with equivalent peaks of free 40S, free 60S, monosomes (80S) and282

polysomes(thegridpatterninFig.3Ahelpsthecomparison).Polysomescontentofthe283

AS1-5mutantwasalsocomparable to thatofwild typewhereasmuch less translating284

ribosomes(polysomes)weredetectedfortheAS1-4strain.Consistentwiththereduced285

amountof40SproducedinAS1-4(Fig.2),nopeakoffree40Sr-subunitwasobservable286

onAS1-4gradientprofilesalongwithaconcomitant largeexcessof free60Sr-subunit287

(Fig3A). Similarly, but to a lesser extent,theAS1-5 gradientprofileshardly showeda288

peakoffree40Swhilethepeakoffree60Sincreasedincomparisontowildtype.289

Defects in translation can be also detected by testing sensitivity to paromomycin, a290

translationerror-inducingaminoglycosideantibiotic.Growthrateof thewild typeand291

mutantmat+strainswerecomparedduringninedaysofvegetativegrowthat27°Con292

standard medium with or without 500 μg ml-1 paromomycin (see Experimental293

procedures).Foreachstrain,growthrateofthemyceliumondrug-containingmedium294

wasexpressedasthepercentageofitsgrowthrateondrug-freemedium(Fig.3B).Inthe295

presence of paromomycin, growth rate of thewild-type strainwas 81% compared to296

plain media. Consistent with previous independent report (Kieu-Ngoc and Coppin-297

Raynal, 1988), theAS1-2mutantdisplayedaparomomycin-sensitivitybarelydifferent298


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fromthewildtype(76%ofresidualgrowth)whiletheAS1-1mutantexhibitedaslight299

hypersensitivity (68% of residual growth). In comparison, theAS1-4 (this work and300

Contamineetal.,1996)andAS1-5(thiswork)mutantswerebothfoundhypersensitive301

toparomomycin,displayinggrowthratesalmosthalfreduced.302

Just like ribosome defects, comparison of paromomycin sensitivity differentiates P.303

anserinaAS1mutants into twoclasses:AS1-1 andAS1-2 ononeahandandAS1-4 and304

AS1-5 on the other hand. The AS1-4 (G139D) mutant displays the strongest fungal305

development defect and the strongest ribosomal alterations. In accordance with306

phenotypicanalyses,theAS1.1(P138S)andAS1.2(S145F)mutantsshowlightalteration307

in40SproductionwithnodetectableconsequencesonpolysomeswhileAS1-5(G139C)308

exhibitsintermediaryribosomedefects.309

310

ScAS1-4(G139D)mimeticmutation inducesgrowthandribosomaldefects intheyeastS.311

cerevisiae312

To further evaluate the translation defects associatedwithmutations affecting the C-313

terminaltailofuS19r-protein,thefourP.anserinaAS1mutantsweremimickedinyeast314

S.cerevisiae.Asmentionedbefore,primarysequenceofS.cerevisiaeuS19C-terminaltail315

is quite different from that of P. anserina especially in the decapeptide motif 138-316

PGIGATHSSR-147(Fig.1AandB).BecausethedifferencebetweenyeastandP.anserina317

sequencespreventscopycattingoftheAS1mutationsinthenaturalS.cerevisiaeRPS15318

gene,AS1mutationswereintroducedinaRPS15chimericgenedesignedtocodeforan319

yeast r-protein uS19,whose last 25 amino acids have been exchanged for the last 28320

amino acids of theP. anserinapolypeptide (Fig. 1B; see Experimental procedures for321

details).EachchimericRPS15genewasputunderthecontroloftheendogenousyeast322

RPS15promoterandexpressedfromalowcopyvectorthatwasintroducedinanrps15323


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deletedyeaststrainusingplasmidshuffling.ResultingS.cerevisiaestrainsharboringthe324

AS1-1, AS1-2, AS1-4 or AS1-5 mutations were named ScAS1-1, ScAS1-2, ScAS1-4 and325

ScAS1-5, respectively. In addition, the ScAS1 strain refers to the same rps15 deleted326

strainthatexpressesthewild-typeversionofthechimericuS19r-protein,whileScWTis327

the rps15deleted strain expressing the natural yeastRPS15 gene from the same low328

copyvector.329

The resulting six yeast strainswere compared for their vegetative growth on glucose330

rich medium (Fig. 4A). At all tested temperatures, growth of the ScAS1 strain was331

equivalenttothatoftheScWTreferencestrainindicatingthatthechimericuS19protein332

canfunctionallyreplacetheendogenousyeastsmallr-proteinandthatalbeitdivergent333

insizeandsequence,theC-terminaltailofP.anserinauS19cansubstituteforthatofthe334

S.cerevisiaeprotein.Ofnote, inthecourseofchimeraconstructions(seeExperimental335

procedures),wefoundthatthedeletionoftheverylast15aasofthechimericuS19r-336

proteinwaslethaltoyeast,whichfurtherhighlightstheimportantroleplayedbytheC-337

terminaltailofthiseukaryoticr-protein.338

Allmimeticyeastmutantssupportedwild-typegrowthonsolidglucoserichmediumat339

alltestedtemperatures(Fig.4A).Whentestedforgrowthat30°Cinliquidglucoserich340

medium, theScAS1-4mutant stoodout from theothers anddisplayeda clear slowing341

downofgrowthafter30hoursofculture(Fig.4B).Onsolidmediumat30°C,analtered342

growth of the ScAS1-4 strain could be also observed one day after the incubation of343

serialdilutions(seeFig.4A)butthisdifferencewasnomoredetectableaftertwodaysof344

incubation (Fig. S1). We never observed such a growth delay at low (20°C) or high345

(37°C)temperaturefortheScAS1-4strainortheothermutantstrains(Fig.4AandFig.346

S1).347


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Wenextexaminedthepolysomeprofilesforequivalentamountsofyeastextractsfrom348

allsixstrainsgrownat30°C inglucoserichmedium(Fig.4C).Apart fromtheScAS1-4349

mutationwhichgaverisetoanexcessoffree60Ssubunitandnodetectablepeakoffree350

40S subunit as observed for theP.anserinaAS1-4 strain, thepolysomeprofiles of the351

ScAS1 strain and of the ScAS1-1, ScAS1-2 and ScAS1-5 mimetic mutants did not352

significantlydifferfromthatoftheScWTreferencestrain(Fig.4C).353

ComparedtothecomplexphenotypesassociatedwiththepresenceoftheAS1mutations354

in themulticellular filamentous fungusP.anserina, themimetic ScAS1mutations gave355

risetoverymodesteffectsintheunicellularyeastS.cerevisiae(withinthelimitsofthe356

phenotypes tested), suggesting that ribosomal proteins are not equally important for357

fungidevelopment(Romanietal.,2012).358

359

DependingonuS19tailalterations,stopcodonrecognitionisdifferentlyaffectedinyeast360

mimeticmutants361

Considering that P. anserina AS1 mutations were selected as translational control362

mutations, we next investigatedwhether stop codon recognition could be affected in363

yeastmimeticmutants, despite the absence of strong growth phenotype. Tomonitor364

potentialstopcodonreadthrough,weemployedawidelyuseddicistronicLacZ-lucdual-365

genereportersystemwhereonestopcodon(UAG,UAA,orUGA)isinsertedbetweenthe366

E.coliLacZ and firely luciferase (luc)openreading framesso that firely luciferasecan367

only be produced as a result of nonsense suppression (Fig. 5A). Plasmids bearing the368

dual-genereportersystemwereintroducedinthereferenceandmimeticmutantstrains369

andreadthroughassayswereperformedusingcrudeextractofexponentiallygrowing370

culturesinglucosemediumat30°C(Experimentalprocedures).371


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Thestopcodoncontaining-sequenceoftheLacZ-lucdual-genereportersystem(Fig.5A)372

waspreviouslyshowntopromotehighlevelofreadthroughinyeast(Stahletal.,1995;373

Bidouetal.,2000).Accordingly,wemeasuredareadthroughlevelof7%(UGA)to15%374

(UAG) in theScWTreference strain expressing thenatural yeastRPS15 gene (Fig. 5B;375

see Experimental procedures). In the ScAS1 strain, whatever the stop codon the376

readthrough level was close to that of the ScWT strain, indicating that wild-type377

chimericuS19r-proteindidnotmodifystopcodonrecognitioninyeast.Onthecontrary,378

all four mimetic mutants exhibited altered stop codon recognition (Fig. 5B). When379

compared to ScAS1, the ScAS1-1 and ScAS1-2mutant strains displayed hyperaccurate380

recognitionofthethreestopcodonsand,whateverthestopcodon,anapproximately1.5381

and3-foldenhancementwasfoundintheScAS1-1andScAS1-2strains,respectively(Fig.382

5B). On the opposite, the ScAS1-4 and ScAS1-5 mutant strains both exhibited higher383

readthroughattheUAGandUAAcodonswhereasnosignificantchangewasdetectedin384

the recognition of the UGA codon. In the ScAS1-4 strain, readthrough level increased385

from1.6-(fortheUAAcodon)to1.8-fold(fortheUAGcodon)whencomparedtothatin386

the ScAS1 strain (Fig. 5B). In the ScAS1-5 strain, UAG and UAA readthroughs are387

increasedapproximately1.4-and1.5-fold,respectively.388

OurreadthroughassaysstronglysupportthattheC-terminaltailofuS19contributesin389

vivo to the recognition of the different stop codons. These resultsmoreover revealed390

that depending on residues modified in the decapeptide PGIGATHSSR, translation391

termination ambiguity could be either expanded or on the contrary restricted,which392

further emphasizes the key role playedby this small set of amino acidswithin theC-393

terminalextensionofeukaryoticuS19r-protein.394

395

396


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Discussion397

S15/uS19 is an essential r-protein and one of the fifteen universally conserved398

ribosomal proteins of the eukaryotic small ribosomal subunit. In vitro cross-linking399

studiesusingmammalian40Sand80ScomplexeshaveidentifieduS19asther-protein400

that intimately contacts -via its C-terminal tail- themRNA at the A site codon during401

translation,makinguS19akeycomponentoftheeukaryoticdecodingsite(Graiferetal.,402

2004; Bulygin et al., 2005; Molotkov et al., 2006; Pisarev et al., 2006; Pisarev et al.,403

2008). Further investigations into the role of uS19 during the translation process404

howeversufferedfromtheabsenceoffunctionalanalysesandthenon-resolutionofthe405

structureoftheC-terminaltail.406

Inthisstudy,welookedbackongeneticscreensthatfourdecadesagohadidentifiedthe407

uS19encoding-geneAS1asanactorofthetranslationfidelityinthefilamentousfungus408

P. anserina (Picard, 1973; Picard-Bennoun, 1976; Picard-Bennoun, 1981). All AS1409

mutations isolated then are now identified and described hereinbefore as altering410

conserved residues of uS19 C-terminal tail and more precisely three residues of the411

eukaryotic specific peptide 138-PGIGATHSSR-147 (modified residues in bold, P.412

anserina numbering), which was shown in vitro to be the contact site of the mRNA413

duringtheelongationandterminationstepsoftranslation(Khairulinaetal.,2010).414

To invivo investigate the translational role of the PGIGATHSSR decapeptide,we have415

considered a widely usedmethod based on a dicistronic LacZ-luc dual-gene reporter416

systemandmeasure translational error rates inyeastS.cerevisiae.AllP.anserinaAS1417

mutationswere recreatedwithin the yeast counterpart uS19 encoding-gene and stop418

codonreadthroughmonitoringrevealedthatallresultingsubstitutions(P138S,G139D,419

G139CandS145F)significantlyaltertheterminationprocessinyeast.Regardlessofthe420

nature of the stop codon, P138S and S145F changes both significantly reduce421


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readthrough level (e.g., increase translational accuracy) whereas substitutions of the422

G139residuedisplayanoppositeeffectandslackenstopcodonrecognitionatUAGand423

UAA(e.g., increase translationalerror).NosignificantmodificationofUGArecognition424

wasdetectableforG139DorG139Cchangessuggestingthatthisglycineresidueplaysa425

role indiscriminatingbetweenthepresenceofaGuanineoranAdenineat thesecond426

position of the stop codon (i.e., the secondnucleotidewithin theA site). Consistently,427

cross-linking studies have shown that in mammalian ribosome complexes the C-428

terminal tail of uS19 mainly contacts the first and second nucleotide of the A site429

(Khairulina et al., 2010; Graifer and Karpova, 2012; Sharifulin et al., 2015). In the430

absenceofasolvedstructureforeukaryoticuS19C-terminal tail,amodelinghasbeen431

proposedthatplacedthelastfourresiduesofthedecapeptide138–PGIGATHSSR-147in432

close contactwith the first and secondpositionof theA site (Khairulinaetal., 2010).433

Even though translational effect associatedwith theS145Pchange suggests an invivo434

proximityofthisresiduewiththeAsite,theproposedpositioningofthedecapeptidein435

thismodeldoesnotplaceG139norP138closetoitwhereasourinvivoresultsclearly436

demonstrate an important, while opposite, implication of these two residues in stop437

codon recognition. A short distance (intra-peptide) influence, either structural or438

functional,cannotbeexcluded.439

440

Considering the genetic screen initially designed to isolate AS (anti-suppressor)441

mutations in P. anserina we could have expected all AS1 mutations to increase442

translationalaccuracy.Howeverinyeast,onlytwoAS1mutations(ScAS1-1(P138S)and443

ScAS1-2(S145F))gaverisetoanhyperaccuraterecognitionofthestopcodonswhereas444

alterations of theG139 residue displayed clearly an opposite effect. Such discrepancy445

couldresultfromthepresenceinP.anserinaofsu(suppressor)mutationsatthetimeof446


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AS genetic isolation(Picard-Bennoun,1976;Belcouretal.,1991;Dequard-Chablatand447

Sellem,1994).Alternatively,wecannotexclude that inyeast themimickingmutations448

had different effects during the termination process all the more because the449

PGIGATHSSRdecapeptide is not conserved in theS.cerevisiae uS19 r-protein (Fig. 1).450

Nevertheless, with the C-terminal tail from P. anserina, S. cerevisiae uS19 chimeric r-451

protein turned out to be functional in yeast anddid not alter stop codon recognition.452

Noteworthy, phenotypic traits associatedwithP.anserinaAS1mutations suggest that453

cleared off the presence of the su mutations, the AS1-4 and AS1-5mutations might454

promote in vivo readthrough on UAG codon as observed in yeast. To get to this455

conclusion,wepaidattentiontotheveryloworabsenceoftemperaturesensitivityofP.456

anserinaAS1-4 andAS1-5mat+ strains compared towild-type,AS1-1 andAS1-2mat+457

strains(Table3).Aspreviouslymentioned,thermp1-2alleleofthemating-type-linked458

rmp1 gene is responsible for the temperature sensitive growth of mat+ strains459

compared to mat- strains (Contamine et al., 2004). Compared to the rmp1-1 allele460

(present inmat- strain,dominantoverrmp1-2 andconsideredas fully functional), the461

sequence of the rmp1-2 allele (present in mat+ strain) contains two nucleotide462

modifications:afunctionallyneutralmissensemutationandanonsensemutation(UAG)463

that truncates the very last 19 amino acids of RMP1 polypeptide (Contamine et al.,464

2004). By promoting readthrough on UAG, the AS1-4 (G139D) and AS1-5 (G139C)465

mutations might restore synthesis of some full length RMP1 polypeptide thereby466

accounting for the relief of the temperature sensitivity observed for both P. anserina467

AS1-4 andAS1-5mat+ strains (Table 3).While indirect that observation nevertheless468

suggests that in P. anserina, substitutions of the G139 residue could increase469

translationalerrorasshowninS.cerevisiae.470

471


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ImportanceoftheC-terminaltailofuS19r-proteintostopcodonrecognitionisfurther472

supportedby the fact thatP.anserinaAS1mutationswere isolatedas interactingwith473

mutations in the genes coding for the release factors eRF1 and eRF3, whose474

interdependentinteractionmediateseukaryotictranslationtermination.eRF3proteinis475

aGTPasethatformsaternarycomplexwithGTPandeRF1,deliveringeRF1totheAsite.476

IntheAsite,eRF1isresponsibleforrecognitionofallthreestopcodonswhereaseRF3’s477

GTPaseactivity enhancespolypeptide release (Hellen,2018, for review).Asdescribed478

hereinbefore, theAS1-1(P138S),AS1-2(S145F) andAS1-4(G139D)mutationsdisplay479

thepropertytodiminishthenon-sensesuppressormutationefficiencyofaP.anserina480

eRF3mutant that contains a unique S380N substitution in the GTPase domain of the481

protein(Picard,1973;Picard-Bennoun,1976;Belcouretal.,1991;Thiswork,seeTable482

1). Serine 380 residue (P. anserina numbering; Serine 319 in Schizosaccharomyces483

pombe;Threonine293 inhumaneRF3A) is locatedbetweentheswitchIandswitchII484

elementsthatarepresentinallGTPasesandessentialforbindingandhydrolysisofGTP485

(Kongetal.,2004;Chengetal.,2009).Intranslationpre-terminationcomplex,eRF1was486

shown to contactboth the switch I and switch II regionsof eRF3and that interaction487

was proposed to tighten the contact between eRF1, eRF3 and the small ribosomal488

subunitduring thedecodingof thestopcodon(Preisetal.,2014).Takingall together,489

wecouldassumethat thesmallr-proteinuS19contributesto that interactionthrough490

itsC-terminal tail.WhileP.anserinaS380NeRF3mutant isassociatedwith loosestop491

codon recognition, presence of P138S, G139D or S145F substitutions in uS19 can492

antagonizetheeffectofS380Nchange.Thus,asanr-proteinneighboringtheAdecoding493

site (Bulygin et al., 2002; Bulygin et al., 2005; Pisarev et al., 2006; Khaĭrulina et al.,494

2008; Pisarev et al., 2008; Khairulina et al., 2010), uS19 could modulate eRF3-eRF1495

interaction in the pre-termination complex, thereby participating in stop codon496


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recognition.497

The fourth P. anserina uS19 mutant (AS1-5, G139C) was isolated as alleviating the498

growthdefectofaneRF1mutantthathasaF407SsubstitutionintheC-terminaldomain499

oftheprotein(Dequard-ChablatandSellem,1994;M.Picard,unpubl.work;Thiswork).500

eRF1 C-terminal domain ismainly responsible for the eRF1-eRF3 interaction (Hellen,501

2018) and the highly conserved Phenylalanine 407 residue (P. anserina numbering;502

residue405inS.pombe;406inhumaneRF1)belongstoahydrophobicpatchthatwas503

shown to interactwithhydrophobic residuesof theC-terminaldomainof eRF3 in the504

crystal structures of human and S. pombe eRF1-eRF3 complexes (Cheng et al., 2009).505

Interestingly, ithasbeenshown that substitutionofF405 toalanine inS.pombe eRF1506

affected cell growth and strongly reduced eRF1 binding to eRF3 (Cheng etal., 2009).507

How G139C uS19 mutant alleviates growth defect of an F407S eRF1 mutant in the508

filamentous fungusP. anserina remainsan opened question but it could be proposed509

thatinthepre-terminationcomplex,theG139Cchangere-establishesafunctionaleRF1-510

eRF3interactionthathasbeenweakenedbytheF407Schange.Althoughthishypothesis511

deserves additional experiments, it anew indicates that uS19 C-terminal tail is an512

importantcomponentoftheeukaryoticdecodingsite.513

514

In theend,onemightargue that translationaccuracy is anexquisitebalancebetween515

numerous,potentiallycompeting,chemicalinteractions(e.g.cognate,near-cognate,non516

cognatecodon-anticodonsinteractions,competingwitheRFsbindingtotheAsite),and517

mimickingmutationsoutside theirnaturalhostmightbeadifficultway todecipher a518

precise role for an individual residue. Nevertheless, we clearly identified a519

translation/terminationdefect inyeastmutants, showingaconservedrole foruS19 in520

controllingAsiteinteractions,tonotablythenucleotidelevel,asG139discriminatesthe521


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presenceofandAdenineoraGuanineinthesecondpositionofastopcodon.Tothebest522

ofourknowledgethisisanun-describedfinetuningroleforaribosomalprotein.523

524

Finally, humanuS19-encoding genewas recently reported as aputative cancerdriver525

whose low frequency mutations are associated with poor outcome for patients with526

relapsed chronic lymphocytic leukemia (CLL; Ljungström et al., 2016; Bretones et al.,527

2018).SomerecurrentmutationsclusterattheC-terminaltailofthehumanproteinand528

twoof them induce theexact samechangesasP.anserinaAS1-1 andAS1-2mutations529

(P131S and S138F;H. sapiens numbering). What is the causative effect of ribosomal530

uS19 mutations on CLL pathobiology and how uS19 mutations provoke pleiotropic531

effectsongrowthanddevelopmentofthefilamentousfungusP.anserinaremainlargely532

unknownbutalterationofcellproteomescouldprobablybeapartof (Bretonesetal.,533

2018).534

535

536

537

538

539

540

541

542

543

544

545

546


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ExperimentalProcedures547

548

P.anserinastrainsandcultureconditions549

AllPodosporaanserinastrainsusedinthisstudyderivedfromtheS(UppercaseS)wild-550

typestrain(Rizet,1952;Picard-Bennoun,1976)TheselectionmodesoftheAS1mutants551

wereoriginallydescribed in(Picard-Bennoun,1976)orunpublished(forAS1-5).Since552

selectionmodesare important to thepurposeof thepresentwork theywere recalled553

anddetailedinthetext(seeResultsandTable1).Selectionofsumutantswasoriginally554

describedin(Picard,1973)andthesu1andsu2genesfurtheridentifiedasencodingthe555

eRF3 and eRF1 translation termination factors, respectively (Gagny and Silar, 1998).556

Mutant 193 was described initially in (Picard, 1971) and molecularly analysed in557

(CoppinandSilar,2007).558

Standard culture conditions, media and genetic methods can be accessed at559

http://podospora.i2bc.paris-saclay.fr. The germination phenotype of germinating560

ascopores(Table2)wasobservedongermination(G)mediumaftertwodaysat27°C.561

Vegetative growth and life span measurements were carried out on M2 standard562

medium containing mainly dextrin as a carbon source. To do so, small pieces of563

mycelium developed onto G medium were transferred to M2. For growth rate564

measurements(Table3),dependingonthetemperatureofgrowth,four(11°C)toseven565

(27°C, 35°C) measurements were done over time following the growth of 4-to-6566

independentculturesforeachstrainduringseven(27°C,35°C)tofourteendays(11°C).567

Foreachstrain,alinearcurvewasthenestablishedusingaverageddaily-measurements568

(27°C, 35°C) or averagedmeasurements equally distributed over 14 days (11°C). The569

slopeofeachcurve(incmperday)represents thevegetativegrowthofeachstrain in570

theindicatedcultureconditions.571


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Paromomycinsensitivitywasevaluatedat27°Cbyfollowingoverninedaysthegrowth572

offoursubculturesofwild-typeandmutantmat+strainsonM2mediumsupplemented573

(ornot)with500μgml-1ofparomomycin(Sigma).Foreachstrain,eachsubcultureand574

condition,alinearcurvewasestablishedandtheslopeofeachcurvedisplaysvegetative575

growthincm/day.Theaverageandstandarddeviationofeachsetofdatawereplotted576

ontoFig.3B.577

578

Molecularanalysisofthesu1-1,su1-42andsu2-5mutants579

The genomicDNA from su1-1, su1-42 and su2-5mutantswas extracted from cultures580

grown on cellophane disks placed on M2 medium (27°C, two days). After mycelium581

crushing in a FastPrep-24 machine, extractions used the ZR Fungal/Bacterial DNA582

miniprepKit(Proteigene).PCRamplificationwithextractedDNAwascarriedoutwith583

primerssu1-5’andsu1-3’revforthesu1allelesandprimerssu2-5’andsu2-3’revforthe584

su2-5 allele (for all primers used in this study, see Table S1). PCR fragments were585

purifiedbyPEGprecipitationanddirectlysequencedonbothstrands(BeckmanCoulter586

GenomicsFrance)usingoligonucleotides su1-5’, su1-3’rev, su1-int, su1-int-rev, su2-5’,587

su2-3’rev, su2-int and su2-int-rev. DNA extraction, PCR amplification and DNA588

sequencingwerecarriedoutfromtwoindependentsubculturesforeachmutantstrain.589

The su1-1 and su1-42 alleles were found to be molecularly identical and carry one590

nucleotide substitution in codon380 (P.anserina numbering for eRF3) that induces a591

Serine (AGT) to Asparagine change (AAT). The su2-5 allele carries one nucleotide592

substitutionincodon407(P.anserinanumberingforeRF1)thatcausesaPhenylalanine593

(TTT)toSerinechange(TCT).594

595

S.cerevisiaestrains,ScAS1mimeticmutantsandgrowthconditions596


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Saccharomyces cerevisiae strain ME14-a9 (MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0MET15597

rps15::kanMX4 [pFL38-GAL::RPS15]) (see Léger-Silvestre etal., 2004)was used as the598

recipient strain formimicking the P. anserinaAS1 mutants. ME14-a9 is derived from599

EuroscarfstrainY21731(BY4743background). InME14-a9, thechromosomalcopyof600

RPS15isinactivatedbyakanMX4cassetteandstrainviabilityissupportedbyaplasmid-601

borne copy of RPS15, which is under the control of the conditional GAL1/10-CYC1602

promoter(pFL38-GAL::RPS15,URA3,CEN).603

Strainswere grownon either solid or liquid formof glucose rich or glucose selective604

medium at indicated temperatures. Where required, 5-fluoroacetic acid (5-FOA) and605

G418wereusedatfinalconcentrationsof200μgml-1and100μgml-1,respectively.606

PlasmidspFL36 (LEU2) andpFL38 (URA3)used in thisworkare two lowcopy (CEN)607

vectors (Bonneaud et al., 1991). ScAS1 chimeric genes were initially constructed in608

plasmid pFL38-RPS15wt (Bellemer et al., 2010), which contains the promoter and609

coding sequence (CDS) of wild-type RPS15 gene followed by a PGK1 terminator610

sequence.First,wetookbenefitofthepresenceoftwonearbyEcoRIrestrictionsitesto611

manipulate the 5’ end of RPS15 CDS in order to construct S. cerevisiae/P. anserina612

chimeric alleles.YeastRPS15 CDS containsonenaturalEcoRI site atposition352-356613

encompassing codons 118 and 119 (S. cerevisiae numbering; codon 125 and 126, P.614

anserina numbering; see Fig. 1B). The second EcoRI site used is located in pFL38-615

RPS15wtsequence,12bpdownstreamtheRPS15stopcodon.Aninternaldeletionofthe616

EcoRI-EcoRI fragment of pFL38-RPS15wt generated plasmid yEPU-RPS15-ΔC. A wild-617

typeS.cerevisiae/P.anserina chimericgene(ScAS1)was first constructedbyreplacing618

the EcoRI-EcoRI fragment of pFL38-RPS15wt by an EcoRI-EcoRI fragment amplified619

from pLF61-7D using primers oAS1Eco-Fwd and oAS1-Eco-Rev. Plasmid pFL61-7D620

originatesfromaP.anserinacDNAlibraryconstructedinthepFL61vector((Espagneet621


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26

al.,2008)).ResultingplasmidyEPU-RPS15-AS1expressesthewild-type(WT)versionof622

chimeric uS19 r-protein in which the last 25 amino acids (aas) of S. cerevisiae623

polypeptidehavebeenswitchedforthelast28aasofP.anserinar-protein(Fig.1B).To624

construct mimetic mutants, the EcoRI-EcoRI fragment amplified from pLF61-7D was625

cloned into theEcoRI site of vector Litmus39 (NEBiolabs) yielding L39-AS1-C, which626

servedasquickchangemutationtemplatetoengineerallfourP.anserinaAS1mutations627

usingprimerslistedinTableS1.MutatedEcoRI-EcoRIfragmentswerethenclonedinto628

theuniqueEcoRIsiteofplasmidyEPU-rps15-ΔC.Resultingplasmids(URA3,CEN)were629

calledyEPU-rps15-AS1-1,yEPU-rps15-AS1-2,yEPU-rps15-AS1-4andyEPU-rps15-AS1-5.630

A C-terminal truncated version of wild-type ScAS1chimeric genewas constructed by631

cloningannealedoligonucleotidesoS15-ΔC-FandoS15-ΔC-RintotheEcoRIsiteofyEPU-632

rps15-ΔC.OligonucleotidesweredesignedtoinducethereplacementofProline138(P.633

anserinanumbering)byanochre(TAA)stopcodonsothattheresultingplasmidyEPU-634

rps15-ΔCPro expresses a ScAS1ΔC chimera truncated for the last 15 aasofP.anserina635

partoftheWTversionofchimericuS19r-protein.636

A set of (LEU2, CEN) plasmids was then constructed by substituting in each yEPU637

plasmid theBglII-BglIIURA3 auxotrophic cassetteby theBglII-BglIILEU2 auxotrophic638

cassette PCR amplified fromplasmid pFL36 using the primer pair FLK7w and FLK7c.639

ResultingplasmidswerecalledyEPL-RPS15-AS1,yEPL-rps15-ΔCPro,yEPL-rps15-AS1-1,640

yEPL-rps15-AS1-2, yEPL-rps15-AS1-4 and yEPL-rps15-AS1-5. They were used to641

transformyeaststrainME14-a9.AllbutyEPL-rps15-ΔCProplasmidsustainedgrowthon642

glucose,mediainwhichtheendogenousplasmidpFL38-GAL::RPS15willnotexpressthe643

essential RPS15 copy. Strains were finally cured from this pFL38-GAL::RPS15 (URA3,644

CEN)plasmidusing5-FOAandidentityoftheremainingplasmid(oneoftheyEPLset)645

wascheckedbyPCRoncolonyandrestrictionenzymediagnosis.Resultingstrainswere646


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27

namedaccording to theS.cerevisiae/P.anserina chimera theyexpressed:ScAS1(wild-647

type chimera), ScAS1-1 (chimera mimicking the AS1-1 change), ScAS1-2 (chimera648

mimicking AS1-2 change), ScAS1-4 (chimera mimicking AS1-4 change) and ScAS1-5649

(chimera mimicking AS1-5 change). All along DNA constructions, sequencing was650

performed on both strands to check cloned fragments and surrounding regions of651

cloning.652

653

RibosomesandPolysomespurification654

For S. cerevisiae strains, polysomes extractions were carried out starting from655

exponentiallygrowingculturesinglucoserichmediumat30°C.Cellsweretreatedwith656

cycloheximideatafinalconcentrationof100μgml-1 immediatelybeforeharvestingto657

trap elongating ribosomes. Polysomes extractions were performed according to658

(Gregoryetal.,2007).659

ForP.anserina strains,ribosomesandpolysomeswere isolated frommyceliumgrown660

oncellophanedisksplacedonM2medium(27°C,twodays).Myceliumwasdried,frozen661

andplacedintoanitrogencooledTeflonshakingflaskwithfourgrindingstainlesssteel662

beads.Myceliumwas crushed into finepowderusing aMikro-Dismembratormachine663

(Sartorius)andshakingat a frequencyof2600permin for90 seconds.Forpolysome664

extraction, collected mycelium was mixed with cycloheximide (100 μg ml-1) for ten665

minutesat4°Cpriordrying,freezingandcrushing.Ribosomesorpolysomeswerethen666

cold-extractedinavolumeof1mlfor150mgofdriedmycelium.Myceliumpowderwas667

re-suspendedinextractionbuffer[20mMTris-HCl(pH8.0),140mMKCl,1.5mMMgCl2,668

1%TritonX-100,0.5mMDTT,3μlofRNasinribonucleaseinhibitor(Promega)perml669

ofextractionbuffer].Forpolysomeextractionsextracycloheximidewasaddedatafinal670

concentrationof500μgml-1.Suspensionswerefirstclarifiedbycentrifugationat4000g671


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for5minand thesupernatantwas furtherclearedat12000g for10min.Extractsof672

equivalentof10OD260units(approximately400μgofnucleicacids)wereloadedona673

11ml 7–50% sucrose gradient prepared in buffer [50mMTris HCl (pH 7.4), 50mM674

NaCl,1mMDTT]forribosomeextractsandinbuffer[50mMTrisAcetate(pH7.5),50675

mMNH4Cl,12mMMgCl2,1mMDTT]forpolysomeextracts.Gradientswerecentrifuged676

at38000rpmfor2h30(polysomes)to3h30(ribosomes)at4°CinaSW41rotor.677

678

Nonsensecodonreadthroughassays679

To quantify readthrough of nonsense codons, yeast strains ScAS1, ScAS1-1, ScAS1-2,680

ScAS1-4andScAS1-5weretransformedwiththecontrol(pACU-TQ),UAG(pACU-UAG),681

UAA (pACU-UAA) or UGA (pACU-UGA) LacZ-luc reporter plasmid. Plasmid pACU-TQ682

carries the in-frame control, which allows production of 100% of β-galactosidase–683

luciferase fusionprotein.AllreporterplasmidsarepACderivativevectors(Stahletal.,684

1995;Bidouetal.,2000)inwhichtheBglII-BglIILEU2markerwasreplacedbytheBglII-685

BglIIURA3cassettebyamplificationfromplasmidpFL38usingtheprimerpairFLK7w686

andFLK7corbycloningoftheBglII-BglIIURA3cassettefromplasmidpFL38.The51bp687

nonsense-containing sequence (GGG GAT CCC GCT AGC TGG CCA GCA GGA ACA CAA688

STOPCAATTACAGTGGCCA)isborderedbythenatural1024thcodonofthelacZCDS689

andthenatural6thcodonoflucCDS(Fig.5A).Foreachstrain, theβ-galactosidaseand690

fireflyluciferaseactivitieswerequantifiedinthesamecrudeextractsasdescribed(Stahl691

etal.,1995).Theratioofluciferaseactivitytoβ-galactosidaseactivityfromthein-frame692

control construct (pACU-TQ) was taken as reference and termination codon693

readthrough (expressed in percent)was then calculated by dividing the luciferase/β-694

galactosidaseratioobtained fromeachnonsenseconstructby thesameratioobtained695

withthein-framecontrolconstruct.696


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29

697

Acknowledgements698

Thanks are due to Guillaume Canal, Raynad Cossard, Emmanuel Dassa, Agnés699

Delahodde,MichelaEsposito,LarasPitayu,CelineRoudier,CaroleSellemandChristelle700

Vasnier for theirconstructivereviewof theworkandsupport.Tan-TrungNguyenhas701

beenfundedbya3-yearPhDStudentshipfromParis-SudUniversity(Orsay,France).We702

alsogratefullyacknowledgeadditionalsupportfromtheFranco-Vietnameseassociation703

fromParis-SudUniversity.ThisworkwassupportedfromtheFrenchCentreNationalde704

laRechercheScientifique(CNRS)andtheFrenchFoundationFRM(INE20071110914to705

SylvieHermann-LeDenmat).706

707

Authorcontributions708

GS,VCandSHLDconceivedanddesignedthestudy.TTN,VC,andMDCperformedtheP.709

anserinaexperiments.GSandSHLDperformedtheS.cerevisiaeexperiments. TTN,GS,710

MDC,VCandSHLDanalyzedthedata.VCandSHLDsupervisedthework.SHLDwrote711

the original draft. GS and SHLD made major contribution to the writing. All authors712

reviewed,editedandapprovedthefinalmanuscript.713

714

715

716

717

718

719

720

721


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887

888


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38

Table1.SelectionmodeoftheAS1mutantallelesAS1allele Geneticcontext

oftheselectionaCriteriaofselection Missensemutation

inAS1bCodonsubstitution

Reference

AS1-1 193su1-42 Ascoporecolor CCA->TCA P138S Picard-Bennoun(1976);Belcouretal.(1991)

AS1-2/AS1-3c 193su1-1 Ascoporecolor TCT->TTT S145F Picard-Bennoun(1976)AS1-4 193su1-1 Ascoporecolor GGT->GAT G139D Belcouretal.(1991);Déquard-

ChablatandSellem(1994)AS1-5 su2-5 Improvedgrowth GGT->TGT G139C Déquard-ChablatandSellem,

(1994);M.Picard,unpubl.work

a.The193mutationisanonsensemutationinthepolyketidesynthaseencodinggene(CoppinandSilar,2007);su1-1andsu1-42

aremutant alleles of the su1 gene encoding translation release factor eRF3; su2-5 is amutant allele of the su2 gene encoding

translationreleasefactoreRF1(GagnyandSilar,1998);seetextforfurtherdetailsaboutsu1andsu2missensemutations).

b.Mutatednucleotidesareindicatedinboldinthecontextofthemodifiedcodon

c.TheAS1-3allelewas independently isolatedduringthegeneticselectionthatproducedtheAS1-2andAS1-4allelesandafter

foundtobemolecularlyidenticaltoAS1-2


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Table2.PhenotypictraitsoftheAS1mutantstrains

Strain Germinationphenotypea Lifespan(cm)b mat+ mat-

WT aerialmycelium++

10±0.4(15)

9.7±0.7(15)

AS1-1(P138S) aerialmycelium+

>516(26)c

67±29.5(27)

AS1-2(S145F) aerialmycelium+

98.3±60.4(21)

27.5±7.2(24)

AS1-4(G139D) veryspindlymycelium+/--

>421(15)d

1.2±0.4(20)

AS1-5(G139C) spindlymycelium+/-

>483(18)e

2.1±0.2(24)

a.Phenotypeofgerminatingascospore(mat-ormat+)observedafter48hon

germination medium at 27°C. Germination phenotype refers to mycelium

morphology and growth phenotype (thallus appearance). Growth

phenotypes are symbolized by ++ (wild-type,WT), + (slightly altered), +/-

(altered)and+/--(veryaltered).

b. Life span in centimeters (cm ± standard deviation) for indicated strains

measuredonstandardmediumat27°C.Numberofindependentsubcultures

analyzedisindicatedinbrackets.Standarddeviationisgivenwhenalltested

subculturesstoppedgrowingduringthecourseofthework.

c.Allsubcultureskeptgrowingduringthecourseoftheworkexceptonethat

stoppedgrowingat411cm.

d.Sixsubcultureskeptgrowingduringthecourseoftheworkwhereasnine

stoppedgrowingbetween244and388cm.

e.Allsubcultureskeptgrowingduringthecourseoftheworkexcepttwothat

stoppedgrowingat320and433cm.

39


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40

Table3.VegetativegrowthoftheAS1mutantstrainsStrain Vegetativegrowth(cm/day)a

27°C 11°C 35°Cmat- mat+ mat- mat+ mat- mat+

WT 0.72 0.72 0.15 0.15 0.84 0.75c

AS1-1(P138S) 0.75 0.70 0.16 0.16 0.84 0.72c

AS1-2(S145F) 0.75 0.72 0.15 0.16 0.88 0.73c

AS1-4(G139D) NMb 0.74 0.17 0.19 0.80 0.79

AS1-5(G139C) 0.76 0.74 0.20 0.20 0.84 0.80

a.Vegetativegrowthmeasuredonstandardmediumforeachindicatedtemperature

and strain. Mycelium of P. anserina elongates linearly over time and growth

expressed in centimeters (cm)per day corresponds to the slopeof the linear curve

obtained by following growth of at least four independent cultures per strain and

averagingthedata(seeExperimentalproceduresfordetails).

b.HighlyshortenedlifespanoftheAS1-4mat-strain(seeTable2)preventsgrowth

ratemeasurementat27°C(NM=notmeasurable).

c. At 35°C, wild-type (WT), AS1-1 andAS1-2mat+ strains show a clear vegetative

growthslowingdownwhencomparedtotheirmat-counterparts.Growthreduction

(expressed as the (cmper daymat+/cmper daymat-) ratio in percent) is of 84%,

86% and 83%, respectively. The AS1-5 and AS1-4mat+ strains do not show such

slowingdownwithratiosof95%and99%,respectively.


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41

Figurelegends892

Fig. 1. P. anserina AS1 mutations affect nearby residues of the highly conserved C-893

terminalpartoftheeukaryoticr-proteinuS19.894

A.Multiplesequencealignmentoffull-lengthuS19proteinfromfourteeneukaryoticand895

three prokaryotic organisms. Alignment was carried out using ClustalW2 program896

(Larkinetal.,2007).Conservedaminoacids(aas)areboxedinblack(identical)andgray897

(similar).Numbering refers to full-lengthprotein sequence and amino acid sequences898

wereretrievedfromtheNCBIdatabase(http://www.ncbi.nlm.nih.gov).NCBIreferences899

are(orderisasinthealignment):PodosporaanserinaXP_001907110,Neurosporacrassa900

XP_965164, Aspergillus niger XP_001397802, Schizosaccharomyces pombe NP_594357,901

Dictyostelium discoideum XP_638126, Zea mays NP_001147395, Oryza sativa902

NP_001051572,Arabidopsisthaliana(isoform1,whichonesharesthehighestsequence903

identity with P. anserina uS19 polypeptide), NP_171923, Drosophila melanogaster904

NP_611136, Xenopus laevis NP_001089043, Homo sapiens NP_001009, Saccharomyces905

cerevisiaeNP_014602,CandidaglabrataXP_446019.1,KluyveromyceslactisXP_455435,906

Bacillus subtilis NP_388001, Escherichia coli NP_289877 and Staphylococcus aureus907

YP_005747034.908

B.C-terminalpartofP.anserina,H.sapiensandS.cerevisiaeuS19r-proteinsasindicated909

in A. Amino acid changes induced by P. anserinaAS1 mutations are indicated. Match910

betweentheAS1allelesandresiduesubstitutionisshownintheinsertedtable.Inyeast911

mimeticmutants(Fig.4and5),the25aasC-terminalendofS.cerevisiaeuS19hasbeen912

exchangedforthe28aasC-terminalendofP.anserinar-protein.Thedecapeptide131-913

PGIGATHSSR-140(H.sapiensnumbering)mappedtoneighbortheAsitemRNAcodonin914

humantranslatingribosome(Khairulinaetal.,2010)ispointedoutbyaline.915


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42

Fig. 2. Production of 40S subunits ismodestly to strongly reduced inP.anserinaAS1916

mutants.917

Equivalent amounts of cell extracts (10 A260nm units) obtained from wild-type and918

mutantmat+strainsgrownat27°Constandardgrowthmediumwere fractionatedon919

7%-50%sucrosegradients.Representativeprofilesofgradientsanalyzedbycontinuous920

monitoringatA254nmareshown(bottom).Atleastthreeindependentsucrosegradients921

and two independent extractions for each strainwere used to quantify the 40-to-60S922

ratios(Top).Ratiosweredeterminedbymeasuringtheareaunderthe40Speakrelated923

totheareaunderthe60Speak.Foreachstrain,the40S-to-60Sratiosareexpressedin924

percentageanderrorbarsrepresentthestandarddeviationofthemean.Significanceof925

thedifferencesbetweenmutantsandwildtypewascalculatedusingaparametrict-test926

(XLSTAT) P-values of 0.0004 (very highly significant) and 0.027 (significant) were927

obtained forAS1-4 andAS1-5, respectivelywhile forAS1-1 andAS1-2 less confidence928

differencewasmeasured(P=0.104andP=0.07,respectively).929

930

Fig.3.PolysomeprofilesandparomomycinsensitivityrevealtwoclassesofP.anserina931

AS1mutants.932

A. Representative polysome profiles obtained for wild-type and mutantmat+ strains933

grown at 27°C on standard growth medium. Equivalent amount (10 A260nm units) of934

cycloheximide-treated mycelium extracts were fractionated on 7%-50% sucrose935

gradientscontainingMg2+.GradientswereanalyzedbycontinuousmonitoringatA254nm936

fromtoptothebottom.Positionsoffree40S,free60S,80Smonosomesandpolysomes937

(translatingribosomes)areindicated.Arrowspointtothe40Sand60Speaksortotheir938

expectedpositions in the gradient.Dotted gridpattern guidesprofile comparison and939

highlightsAS1-5andAS1-4alterations.940


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43

B.Paromomycineffectonvegetativegrowthofwild-typeandAS1mutantmat+strains.941

Four subcultures of each strain were grown at 27°C on standard growth medium942

containing(+)ornot(-)500μgml-1ofdrug.Myceliumgrowthwasfollowedduringnine943

days. For each strain and condition, vegetative growth was measured in cm/day944

(Experimentalprocedures)anddataareexpressedasmeanvalues±standarddeviation.945

Averagepercentageofresidualgrowthinthepresenceofdrug(+)relativetogrowthin946

its absence (-) is indicated above the histogram for each strain. Significance of the947

differencesbetweenmutantsandwildtypewascalculatedusingaparametrict-test(P=948

0.06(AS1-2),P≤0.003(AS1-1;AS1-4;AS1-5)).949

950

Fig. 4. Growth and polysome profiles of the S. cerevisiae ScAS1 mimetic mutants951

distinguishtheScAS1-4strain.952

A. Growth of equivalent 10-fold serial dilutions (indicating OD600) of the indicated953

strainsafter2days(20°C)or1day(30°C,37°C)onglucoserichmedium(seeFigS1).954

ScWT,referencestrainexpressingthewild-typeS.cerevisiaeRPS15gene;ScAS1,strain955

expressing a wild-type RPS15 chimeric gene in which last 75 nucleotides have been956

replacedbythelast84nucleotidesofP.anserinaAS1gene(Fig.1B);ScAS1-1toScAS1-4,957

yeaststrainsmimickingtheAS1-1toAS1-4P.anserinamutations,respectively.958

B.Growthcurvesofthesamestrainsgrownat30°Cinglucoserichmedium.Apartfrom959

ScAS1-4 (diamond), ScAS1 and othermimetic strains show growth curves thatmerge960

withthatoftheScWTreferencestrain(star).961

C.Representativepolysomeprofilesobtainedfortheindicatedstrainsgrownat30°Cin962

glucoserichmedium.Equivalentamountof cycloheximide-treatedyeastextractswere963

fractionated on 7 to 50% sucrose gradients. Gradients were analyzed by continuous964

monitoring atA254nm from top to thebottom.Thepositions of thedifferent ribosomal965


https://doi.org/10.1101/2020.02.09.940346

44

speciesareindicated.ApartfromtheScAS1-4strain,whichdisplaysno40Speakandan966

excessof free60S (as itsP.anserina counterpart, Fig.3A),polysomeprofilesof ScAS1967

andothermimeticstrainsareclosetothatoftheScWTreferencestrain.968

969

Fig. 5. Termination codon readthrough is differently altered in theS.cerevisiaeScAS1970

mimeticmutants.971

A.Schematicrepresentationof theβ-galactosidase(lacZ)-luciferase(luc)dual-reporter972

system used. Positioning of the nonsense-containing region within the lacZ-luc dual-973

gene is indicated by the black box and bordering sense codons of lacZ and lucopen974

reading frames. Only a section of the nonsense-containing sequence (21 nucleotides975

among51;Experimentalprocedures)isgiven.976

B.Quantificationofnonsensecodonreadthroughineachindicatedstraingrownat30°C977

inglucoseselectivemedium(bottom).Strainsweretransformedwithcontrol,UAG,UAA978

and UGA LacZ-luc reporter plasmids and the percent readthrough in each strainwas979

expressedastheratioofluciferaseactivity(measuredfortheindicatednonsensecodon)980

totheonemeasuredforaninframeLacZ-lucconstruct(control),afterβ-galactosidase981

normalization (Experimental procedures). Bars represent mean ± standard deviation982

and means were determined for at least five independent measurements. The983

significanceofdifferencesinsignalsbetweeneachmimeticmutantandtheScAS1strain984

isindicated:*P<0.02,**P<0.002(parametrict-test).985

986

987

988

989

990


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131 140

P138S

G139D

G139C

S145F

A

P. anserina 1 MADE-YNAEEAAE-LKKKRTFRKFSYRGVDLDALLDLTSDELRDVVHARARRKINRGLKRRPMGLIKKLRKAKQEAK-PN

N. crassa 1 MADE-YNAEEAAE-LKKKRTFRKFSYRGIDLDALLDLGSDELRDVVHARARRRINRGLKRKPMGLIKKLRKAKQEAK-PN

A. niger 1 MADEHYNAEEAAE-IKKRRQFRKFTYRGIDLDQLLDLSSEQLRDVVHARARRRFNRGLKRKPMGLIKKLRKAKQEAR-PN

D. discoideum 1 ---------MSEQ--IKKRTFKKFTYSGVALESLLDLKEEQLISLLRCRARRKLRRETPIKHVNFLKKCRASKAAVTQVG

Z. mays 1 MADVDVEPEVAAG-APKKRTFRKYSYRGVDLDALLDMSTDDLVQLFPARARRRFQRGLKRKPMALIKKLRKAKKDAP-AG

O. sativa 1 MADVEVEAEVAAAGAPKKRTFRKYSYRGVDLDALLDMSTDDLVQLFPARARRRFQRGLKRKPMALIKKLRKAKKDAP-AG

A. thaliana 1 MAD–-VEPEVAAAGVPKKRTFKKFAFKGVDLDALLDMSTDDLVKLFSSRIRRRFSRGLTRKPMALIKKLRKAKREAP-QG

D. melanogaster 1 -----MADQVDEN-LKKKRTFKKFTYRGVDLDQLLDMPNNQLVELMHSRARRRFSRGLKRKPMALIKKLRKAKKEAP-PN

X. laevis 1 ------MAEVEQK---KKRTFKKFTYRGVDLDQLLDMSYEQVMQLYCARQRRRLNRGLRRKQNSLLKRLRKAKKEAP-PM

H. sapiens 1 ------MAEVEQK---KKRTFRKFTYRGVDLDQLLDMSYEQLMQLYSARQRRRLNRGLRRKQHSLLKRLRKAKKEAP-PM

S. pombe 1 MAEENHDEAVRVAELRKKRTFRTFAYRGVELEQLLDLSAEQLVDLFHARARRRMLRGLGPNASRFIRKLRKAKSEAP-LN

S. cerevisiae 1 --------MSQAVNVK-KRVFKTHSYRGVDLEKLLEMSTEDFVKLAPARVRRRFARGMTSKPAGFMKKLRAAKLAAP-EN

C. glabrata 1 --------MSQATGAKSKRVFKTHSYRGVDLEKLLEMSTEDFIKMTPARVRRKFSRGVSGKPNGFMKKLRAAKLACP-EN

K. lactis 1 --------MSEAAAPR-KRSFKTYSYKGVDLEKLLEMPTEDFVKLAPARVRRKFARGLSEKPAGLMKKLRAAKLSAP-EN

B. subtilis 1 -----------------------------------------------------MARSLKKGP—-FVDGHLMTKIEKLNET

E. coli 1 -----------------------------------------------------MPRSLKKGP—-FIDLHLLKKVEKAVES

S. aureus 1 -----------------------------------------------------MARSIKKGP—-FVDEHLMKKVEAQEGS

C-terminal part

P. anserina 78 EKPDLVKTHLRDMIVVPEMIGSVVGIYSGKEFNQVEIKPEMVGHYLGEFSISYKPVKHGRPGIGATHSSRFIPLK 152

N. crassa 78 EKPDLVKTHLRDMIVVPEMIGSVIGIYSGKEFNQVEIKPEMVGHYLAEFSISYKPVKHGRPGIGATHSSRFIPLK 152

A. niger 79 EKPDLVKTHLRDMIVVPEMIGSVIGIYSGKEFNQIEVKPEMVGHYLGEFSISYKPVKHGRPGIGATHSSRFIPLK 153

D. discoideum 70 EKPALVKTHARNILIVPEMIGSVIGIYNGKVFNQVEVKPEMIGHYTGEFSLSYKSVNHGRPGIGATHSSRFIPLK 144

Z. mays 79 EKPEPVKTHLRNMIIVPEMIGSIVGVYNGKTFNQVEIKPEMIGHYLAEFSISYKPVKHGRPGIGATHSSRFIPLK 153

O. sativa 80 EKPEPVRTHLRNMIIVPEMIGSIVGVYNGKTFNQVEIKPEMIGHYLAEFSISYKPVKHGRPGIGATHSSRFIPLK 154

A. thaliana 78 EKPEPVRTHLRNMIIVPEMIGSIIGVYNGKTFNQVEIKPEMIGHYLAEFSISYKPVKHGRPGVGATHSSRFIPLK 152

D. melanogaster 74 EKPEIVKTHLRNMIIVPEMTGSIIGVYNGKDFGQVEVKPEMIGHYLGEFALTYKPVKHGRPGIGATHSSRFIPLK 148

X. laevis 71 EKPEVIKTHLRDMIILPEMVGSMVGVYNGKAFNQVEIKPEMIGHYLGEFSITYKPVKHGRPGIGATHSSRFIPLK 145

H. sapiens 71 EKPEVVKTHLRDMIILPEMVGSMVGVYNGKTFNQVEIKPEMIGHYLGEFSITYKPVKHGRPGIGATHSSRFIPLK 145

S. pombe 80 EKPATVKTHLRNMIILPEMVGSVVGIYNGKLFNQVEIRPEMIGHYLGEFSITYKPTKHGRPGIGATHSSRFIPLK 154

S. cerevisiae 71 EKPAPVRTHMRNMIIVPEMIGSVVGIYNGKAFNQVEIRPEMLGHYLGEFSITYTPVRHGR--AGAT-TSRFIPLK 142

C. glabrata 72 EKPAVVKTHLRNMIIVPEMIGSVVGIYNGKVFNQVEIRPEMLGHYLGEFSITYTPVRHGR--AGAT-TSRFIPLR 143

K. lactis 71 EKPAVVRTHLRNMIIVPEMIGSVVGVYNGKVFNQVEIRPEMVGHYLGEFSITYTPVRHGR--AGAT-TSRFIPLR 142

B. subtilis 26 DKKQVVKTWSRRSTIFPQFIGHTIAVYDGRKHVPVFISEDMVGHKLGEFAPTRTYKGHAS---DDKKTRR----- 92

E. coli 26 GDKKPLRTWSRRSTIFPNMIGLTIAVHNGRQHVPVFVTDEMVGHKLGEFAPTRTYRGHAA---DKKAKKK----- 92

S. aureus 26 EKKQVIKTWSRRSTIFPNFIGHTFAVYDGRKHVPVYVTEDMVGHKLGEFAPTRTFKGHVA---DDKKTRR----- 92

B

P. anserina 125 EFSISYKPVKHGRPGIGATHSSRFIPLK 152

H. sapiens 118 EFSITYKPVKHGRPGIGATHSSRFIPLK 145

S. cerevisiae 118 EFSITYTPVRHGR--AGAT-TSRFIPLK 142

P138S AS1-1

S145F AS1-2

G139D AS1-4

G139C AS1-5


https://doi.org/10.1101/2020.02.09.940346

40S-

to-6

0S ra

tio (%

)

Fig. 2 Tan-Trung Nguyen et al.

40S 60S

40S 60S

40S 60S

40S 60S

40S 60S

0

20

40

60

80

WT AS1-1 AS1-2 AS1-4 AS1-5

69%

60% 59%

41%

49%

WT


https://doi.org/10.1101/2020.02.09.940346

76%

60% 56% 68%

81%

(-) Paromomycin (+) Paromomycin

A25

4nm

WT

60S

80S

Polysomes 40S

Top Bottom

A25

4nm

60S

80S

Polysomes 40S

AS1-1

Top Bottom

A25

4nm

60S

80S

Polysomes 40S

AS1-2

Top Bottom

A

Fig. 3 Tan-Trung Nguyen et al.

A25

4nm

60S 80S

Polysomes 40S

AS1-5

Top Bottom

60S 80S

Polysomes 40S

A25

4nm

AS1-4

Top Bottom WT AS1-1 AS1-2 AS1-4 AS1-5

0.6

0.4

0.2

0

0.8

vege

tativ

e gr

owth

(cm

/day

)

B


https://doi.org/10.1101/2020.02.09.940346

C

Fig.4Tan-TrungNguyenetal.

A

ScWT ScAS1 ScAS1-1 ScAS1-2 ScAS1-4 ScAS1-5

20°C 30°C 37°C 0,1

1

10

100

1000

10000

100000

0 10 20 30 Time in hours

ScAS1-4

★

w 104

103

102

10

1

0.1

105

Arb

itrar

y U

nits

(Opt

ic D

ensi

ty)

B

60S

ScWT

Top Bottom

A25

4nm

80S Polysomes 40S

ScAS1

Top Bottom

A25

4nm

80S Polysomes 40S 60S

ScAS1-1

60S

Top Bottom A

254n

m

80S Polysomes 40S

ScAS1-2

60S

Top Bottom

A25

4nm

80S Polysomes

40S

ScAS1-5

60S

Top Bottom

A25

4nm

80S Polysomes

40S

ScAS1-4 60S

Top Bottom

A25

4nm

80S Polysomes

40S

1 10-1 10-2 1 10-1 10-2 1 10-1 10-2 OD600


https://doi.org/10.1101/2020.02.09.940346

Fig.5Tan-TrungNguyenetal.

% R

eadt

hrou

gh

24

28

20

16

12

8

4

0

B

A β-galactosidase (lacZ)

AUG codon 1024 codon 6 STOP

Luciferase (luc)

UAG UAA…GGA ACA CAA UGA CAA UUA CAG… G T Q * Q L Q

ScWT

ScAS1

ScAS1-1 (P138S)

ScAS1-2 (S145F)

ScAS1-4 (G139D)

ScAS1-5 (G139C)

UAG UAA UGA

*

**

*

**

***

**

**

**

*


https://doi.org/10.1101/2020.02.09.940346

Supportinginformation

TableS1.OligonucleotidesusedinthisstudyName Sequence(5’->3’)

su1-5’ CCAGCGCAGTCCGAATTCGsu1-3’rev GCTAGCCTTGACATAATTCATAGsu1-int GCCAAGGCGGTAGAGAAGACsu1-int-rev GTCGCGAGCAGCAGCCTTG su2-5’ CGCGGCGAGTCTCTTCTTCsu2-3’rev TGGTCGGCATGCAGTGTCTCsu2-int CGCTTGCTGAGCTTCTCGAGsu2-int-rev GTCACGGGTTCCACTCAG oAS1-Eco-Fwd GCGAaTTCTCTATCTCATACAAGCCaGTCAAGCoAS1-Eco-Rev CTGAATTCTTATCCCAGCCCTTACTTcAatGGAATGAAACGoS15-ΔC-F AaTTCTCTATCTCATACAAGCCaGTCAAGCACGGTAGATAAGoS15-ΔC-R AATTCTTATCTACCGTGCTTGACtGGCTTGTATGAGATAGAGoAS1-1-Fwd GTAGATCAGGTATCGGTGCTACCCACTCTTCoAS1-1-Rev CCTGATCTACCGTGCTTGACTGGCTTGoAS1-2-Fwd CCACTTTTCTCGTTTCATTCCATTGAAGTAAGGoAS1-2-Rev CGAGAAAAGTGGGTAGCACCGATACCGGGTCTACCoAS1-4-F3 CAGATATCGGTGCTACCCACTCTTCTCGTTTCoAS1-4-R3 AGCACCGATATCTGGTCTACCGTGCTTGACtGoAS1-5-F3 CAGATATCGGTGCTACCCACTCTTCTCGTTTCoAS1-5-R3 AGACCATGTATCGGTGCTACCCACTCTTCTCG FLK7w TTGGCGTAATCATGGTCATAGCTGTFLK7c TATTGGACGACCGACACGAAAAGACEcoRIcloningsiteornucleotidesthatarepartoftheEcoRIsiteareinbold.Lowercase

letters indicate silentmutations introduced to adaptP. anserinaAS1 codons to the S.

cerevisiae codonusage.Ochre stop codon is in italics. Underlinednucleotides indicate

changes introduced to create the ScAS1-1, ScAS1-2,ScAS1-4 andScAS1-5mutations in

theyeastRPS15chimericgene.


https://doi.org/10.1101/2020.02.09.940346

30°C 37°C

ScWT

ScAS1

ScAS1-1

ScAS1-2

ScAS1-4

ScAS1-5

ScWT

ScAS1

ScAS1-1

ScAS1-2

ScAS1-4

ScAS1-5

Day 1

Day 2

Fig. S1. Growth delay of the ScAS1-4 strain. Growth of equivalent 10-fold dilutions (indicating OD600) of the indicated yeast strains on glucose rich medium. Growth of the same plate was observed after one and two days of growth at the indicated temperature. The growth delay observed for the ScAS1-4 strain at 30°C after one day of growth (*) fades at day 2 albeit individual colonies appear smaller than those of the reference ScAS1 strain.

OD600 1 10-1 10-2 10-3 10-4 1 10-1 10-2 10-3 10-4

*


https://doi.org/10.1101/2020.02.09.940346

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