© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Florida, Florida, Gainesville, Florida, USA (E.L.P.). should Correspondence be addressed to K.K. ( 4 Biological Sciences, The Scripps Research Institute, Jupiter, Florida, USA. 1 mutated commonly most a third the is not Rps26 cancer. to and susceptibility resistance a predict would ribosomes of lack a proteins, in ribosomal insufficiency from expected are perhaps tissues erating cancer of risk increased a with paired greatly defects) developmental with coupled (often cies and deficien growth proliferative phenotype: paradoxical seemingly exhibit These diseases a range symptoms of but tissue-specific share a syndrome 5q– and asplenia, congenital DBA, as such diseases, located is structure. the in which resolved not nearby, was Rps26, of extension C-terminal acid 21–amino a because partly and sequence, consensus Kozak the to conform not did studies these in mRNA the because partly understood, well not in AUG (with 1–3) position positions −3 the at nucleotide conserved highly the eIF2 between contact a identified have studies structural and codon start the of upstream window ten-nucleotide a of tance magnitude of order an by production protein affect sequence this in changes nucleotide Kozak, by described Originally codon. start the of upstream diately imme sequence the is mRNA an of efficiency translation the affect to known is that element one understood, poorly remain differences con ditions cellular different under vary can mRNA given any for ciency efficiencies differing vastly with ribosomes to recruited are mRNAs different that show studies ing homeostasis protein of Translational control of gene expression is integral to the maintenance gain-of-function heretofore specialized ribosomes from recognition pathogenesis with We Max B Ferretti Kozak sequence elements Rps26 directs mRNA-specific translation by recognition of nature structural & molecular biology molecular & structural nature Received 14 October 2016; accepted 27 June 2017; published online 31 July 2017; Present Present addresses: Henry Samueli School of Engineering and Applied Science, University of California, Los Angeles, California, USA (E.A.W.); The University of Department of Integrative Structural and Biology,Computational The Scripps Research Institute, Jupiter, Florida, USA. Haploinsufficiency of ribosomal proteins underlies a number of of number a underlies proteins ribosomal of Haploinsufficiency

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1 analyses mRNAs. α , 3 13– 17– 4 and and Harriet Harriet L. Wilkes Honors College, Florida Atlantic University, Boca Raton, Florida, USA. , Elizabeth L , PottsElizabeth

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These reveal to stress induced by high-salt and high-pH conditions is augmented augmented is conditions high-pH and high-salt by induced stress to they demonstrate that the transcriptional well-characterized response DBA.of Moreover, pathogenesis the in role a play could homeostasis recognition of the Kozak sequence and suggest that proteinperturbed for basis molecular the reveal data These Kozak sequence. of the tion ing for preferential translation of mRNAs with mutations in high the salt −4or high posi pH, cells generated ance to high salt and high pH. Finally, we show that upon exposure to stitutive activation of these pathways and therefore to increased resist conditions, respectively. Correspondingly, Rps26 depletion led to con pathways, which regulate the response to high-salt and high-pH stress to mRNAs of bound ing by translated preferentially were (−4G) −4 position at guanosine a with mRNAs demonstrated that and −4 atfurthermore position an adenosine with that firmed Rps26 was required for preferential translation of mRNAs assays con reporter Luciferase Kozakupstream of tion sequence. the mRNAs enriched in (WT) cells than in those enriched in +Rps26 ribosomes. Furthermore, bound to type of ribosome bound a subset ofspecific mRNAs, and that mRNAs each that show data The pools. ribosome both to mRNAsbound the ( (+Rps26) from ribosomes yeast cells, followed by RNA-seq Rps26-depleted to identify of separation initiation. translation during codon start the data as crosslinking protein in DBA do

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------© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. tion on polysome recruitment of mRNAs enriched in the two ribos two the in enriched mRNAs of recruitment polysome on tion ( ribosomes deficient ences between these samples did not arise from the TAP tag on differ Rps26- the that demonstrated mRNAs Rps3-TAP-bound sequenced we which in experiments control Importantly, 1.5-fold. than more and pool and 15% were the preferentially in bound to +Rps26enriched ribosomes were ( 13% those, Of pools. ribosome the of either in enriched significantly were mRNAs all of one-quarter than reproducibility. and confidence high with ORFs of majority overwhelming the of identification the bled ( experiments between correlation high showed samples purified and grown independently ( proteins or ‘dubious’ as were annotated ‘uncharacterized’ and may not encode regulation gene of appear ance false the reduce to determined experimentally threshold a experiment, per reads more or 128 had 5,784) of total a of out 5,219 (ORFs: frames reading open the of 90% Further, genome. yeast the the from of reads the and 89–95% sample be could pools both from directly. data compared RNA-seq the mRNAs, same the ribosome encountered therefore two and cells, the same the from isolated because were pools Notably, algorithm. analyzed and DESeq2 500, the NexSeq with Illumina an on sequenced pools, ome that 1f Fig. demonstrated Supplementary results Bioanalyzer the and blotting ( northern populations ribosome two the separated of efficiently method this that showed ribosomes) +Rps26 immunoblotting (containing and (containing analysis eluate the SDS-PAGE fractions. ribosome- the bound only collected and gradient sucrose a onto fraction IgG on ( beads purification affinity by ribosomes +Rps26 maintain from to separated then cycloheximide and of interactions, presence mRNA–ribosome the in lysed and vested 1a Fig. ( Rps3 untagged with those to akin polysomes into Rps3 (ref. of Rps3 and Rps26 when Rps3-TAP of are Importantly,repressed. Rps3-TAP complements fully the absence induction separate for ing allow promoter, TET (dox)-repressible doxycycline the the of under control is Rps3-TAP Furthermore, control. galactose-inducible ( which in strain yeast and +Rps26 purified we ribosome, by the of mRNAs translated repertoire the influences Rps26 whether assess sequence Kozak the to adjacent channel, exit mRNA the in located is Rps26 that demonstrate data crosslinking and Structural D RESULTS have roles. physiological ribosomes Rps26-deficient Thus, mRNAs. stress-related of subset a of translation preferential enable to ribosomes of composition the in changes involves that response translational unknown hitherto a by s e l c i t r a  that strain Rps26 We in a dox-repressible Rps26 ome pools. depleted Fig. 1 Fig. Rps26 To validate these data, we determined the effects of Rps26 deple Rps26 of effects the determined we data, these Tovalidate Using cutoff of a conservative RNA-seq analysis showed that 88–95% of the reads from the +Rps26 ∆ upeetr Dt St 1 Set Data Supplementary Rps26 ribosomes contained mature 18S rRNA ( rRNA 18S mature contained ribosomes Rps26 a Fig. 1 Fig. – ). In this strain, Rps26 and Rps3, a distal protein, are under under are protein, distal a Rps3, and Rps26 strain, this In ).

d and upeetr Fg 2c Fig. Supplementary . fe icbto fr h n lcs, el wr har were cells glucose, in h 4 for incubation After ). 2

b +Rps26 5 ). To exclude free mRNAs, we loaded the flowthrough flowthrough the ). Towe mRNAs, loaded free exclude ), and ribosomes containing Rps3-TAP are recruited recruited are Rps3-TAP containing ribosomes and ), Supplementary Fig. 1h Fig. Supplementary

∆ ribosomes Supplementary Fig. 2d Supplementary Rps26 ribosomes are selectively TAP tagged TAPtagged selectively are ribosomes Rps26 , 5 ∆ g . Of the remaining 565 ORFs, 274 (49%) (49%) 274 ORFs, 565 remaining the Of . Rps26 ribosomes) and the flowthrough flowthrough the and ribosomes) Rps26 ). RNAs were isolated from these ribos these from isolated were RNAs ). P

adj ), and all mRNAs were enriched enriched were mRNAs all and ), bind ). Furthermore, data from three three from data Furthermore, ). < that we 0.05, more determined

different ∆ ∆ ∆ Rps26 sample mapped to mapped sample Rps26 Rps26 ribosomes from a from ribosomes Rps26 Fig. 1 Fig. Rps26 ribosomes were were ribosomes Rps26 ). ). ). Thus, RNA-seq ena

mRNAs b Supplementary Supplementary ). Furthermore, Furthermore, ). Fig. 1 Fig. advance online publication online advance 16 ∆ Fig. Rps26 Rps26 , c 2 4 and and . To . 1 d ------

Rps26 depletion should selectively decrease the polysome recruitment ribosomes, with translating interactions functional reflect pulldowns and level of the protein similar to that inaccumulates the parent strain in its absence ( in ( listed also are pool each in represented genes most the with terms GO The 4,084. enrichment, no ∆ ( enriched mRNAs of distribution ( experiments. three of representative are Data (bottom). rRNA 18S and (top) rRNA) (20S ITS1 against probed were Samples Pno1. and Rio2 factors assembly the of TAP-tagging by captured ribosomes ( experiments. five least at of representative are Data control. a loading as used was Rps10 shown. is Rps26 and Rps3 untagged) and (tagged for flowthrough and elution the of blot A western analysis. sequencing for used were complexes mRNA–ribosome containing fractions the only and gradient, a sucrose on loaded was Rps3-TAP.with flowthrough The ( ( mRNAs. 1 Figure c b b a d Rps26 ribosomes. mRNAs in each pool: +Rps26, 865; 865; +Rps26, pool: each in mRNAs ribosomes. Rps26 ) Northern blot analysis of of analysis blot ) Northern ) Separation of of ) Separation Supplementary Data Set 3 Set Data Supplementary GAL1:: GAL1:: TET Supplementary Fig. 3a Fig. Supplementary Rps10 Rps26

Rps3 Isolation of of Isolation a ) The strain used to purify ribosomes lacking Rps26 ( Rps26 lacking ribosomes purify to used strain ) The Elution ∆ Rps3-TAP ∆ Rps26A Rps26 ribosomes in the presence of dox but maintains a Cytoplasmic translation Rps26B Rps3

∆ ∆ Rps26 Rps26 and +Rps26 ribosomes by affinity purification purification affinity by ribosomes +Rps26 and Rps26

∆ IgG column Rps26 ribosomes and characterization of bound bound of characterization and ribosomes Rps26 nature structural & molecular biology molecular & structural nature +Rps26 Supplementary Fig. 1 Fig. Supplementary Untagged TAP tagged Rps3 ∆ . Source data for for data . Source Rps26 ribosomes compared to immature immature to compared ribosomes Rps26 enriched +Rps26 – YP-Gal +dox 15% d No enrichment P ). We hypothesized that if ribosome ribosome if that Wehypothesized ). adj < 0.05 by DESeq2) in +Rps26 and and +Rps26 in DESeq2) by < 0.05 72% 60 18 20 20 c S Flowthrough enriched ∆Rps26 DNA repair Phosphorylation Cell cycle Regulation oftranscription 80 S S S 13% Rps26 S d ). Original blot images are are images blot Original ). are available online. available are

Rio2-TAP Polysomes Rps3-TAP

TAP-Pno1 ∆ Rps26, 741; 741; Rps26, ∆Rps26 Rps3-TAP YPD d ) The ) The ∆ Rps26). Rps26). Fig. 2 a

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. efficient translation. efficient highly imparts which sequence, Kozak the of enriched recognition in Rps26 ( ribosomes mRNAs +Rps26 translated contrast, highly In the and transcriptome. ribosomes +Rps26 entire on enriched mRNAs than translated on translation enriched mRNAs efficient less with associated be to 5 long is unclear, observations for these ( half-lives 3 the of lengths average the in differences icant ( excluded were short, be to tend which proteins, ribosomal encoding mRNAs tran the or mRNAs ( whole a as scriptome +Rps26-enriched than abundant less were on 5 enriched longer mRNAs Specifically, tics. and data sequencing the of mRNAs on enriched +Rps26 ( ribosomes mRNAs by bound preferentially that ofon had Rps26 a recruitment depletion effect polysome smaller ( versus the +Rps26 in to bound mRNAs enriched and of number ribosomes distribution we coupled sucrose-gradient fractionation with RT-qPCR to track the described previously As pool. +Rps26 the in enriched mRNAs of online. available are figure this in graphs for data ( respectively percentiles, 90th and 10th the to extend 838; +Rps26, 763; +Rps26, length: ORF 4,363. all, 575; sets data existing using ( units. arbitrary AU, mean. the indicates midline the range; the represent plots Box others. all for run were duplicates and Rox1, and Eap1 Abf1, for run were experiments independent ( * mean. the represent Bars cultures. separate three from parallel in purified ribosomes of (right) quantitation and (left) blot a western are Shown Rps3. to relative occupancy Rps26 for 2 Figure nature structural & molecular biology molecular & structural nature b Supplementary Fig. 3e Supplementary ) Translational downregulation (measured by bound ribosomes) of mRNAs enriched in +Rps26 or or +Rps26 in enriched mRNAs of ribosomes) bound by (measured downregulation ) Translational Further analysis revealed that the mRNAs enriched on +Rps26 +Rps26 on enriched mRNAs the that revealed analysis Further Rps26 a Rps3 ∆ p2 rbsms ifrd n hi pyia characteris physical their in differed ribosomes Rps26

∆ ′ UTRs (untranslated regions) and longer ORFs, and and ORFs, longer and regions) (untranslated UTRs Rps26 ribosomes bind a distinct set of poorly translated mRNAs. ( mRNAs. translated poorly of set a distinct bind ribosomes Rps26 Supplementary Fig. 2e Fig. Supplementary Supplementary Fig. 2f Fig. Supplementary d

5 UTR (nt) +Rps26

∆ ′ – Dox Rps26, 736; all = 5476. Box edges represent the interquartile range, midlines indicate the median, and lower and upper whiskers whiskers upper and lower and median, the indicate midlines range, interquartile the represent edges Box = 5476. all 736; Rps26, 250 100 150 200 50 0 +Rps26 ∆ Rps26 pools when Rps26 was replete or depleted or Rps26 was replete when depleted Rps26 pools in vivo in 58– ∆ Enrichment by Fig. 2d Fig. ribosome pool 5 **** Rps26 ribosomes were much less efficiently efficiently less much were ribosomes Rps26 ′ – 6 UTRlength ∆ 1 h Rps26 . . **** ). Quantification of ). showed analyses Quantification these . –Rps26 c + Dox i. 2 Fig. ) Analysis of all mRNAs in in mRNAs all of ) Analysis – f P ∆ ). This finding held true even when when even true held finding This ). < 0.0001, Kolmogorov–Smirnov test. Number of mRNAs analyzed for 5 for analyzed mRNAs of Number test. Kolmogorov–Smirnov < 0.0001, All P Rps26 ribosomes compared to that Rps26 ribosomes ). In contrast, there were ). there no In signif contrast, < 0.05, ). Although the molecular basis basis molecular the Although ). g ), consistent with a role for for role a with consistent ), kDa 25 37 15 ′ UTRs and ORFs are known are known ORFs and UTRs ∆ e Rps26, 575; all, 5,690. Abundance: +Rps26, 804; 804; +Rps26, Abundance: 5,690. all, 575; Rps26, t = 3.795, Degrees of freedom (DF) = 4 (Student’s = 4 (Student’s (DF) freedom of Degrees = 3.795, Fig. Fig. 2b ORF length (nt) ∆ 1,000 2,000 3,000 4,000 5,000 Rps26 ribosomes had had ribosomes Rps26 Rps26-Rps3 0.0 0.5 1.0 1.5

′ 0 5 UTRs or transcript transcript or UTRs advance online publication online advance , +Rps26 27 , Supplementary Figs. 2 Figs. Supplementary c (+Rps26) , ), ), thus validating – Dox 2 8 . Importantly, Importantly, . b **** Enrichment by ribosome pool ; data are mean mean are ; data ORF length ∆ Rps26 * (–Rps26) + Dox All 2 6 - - - ,

b

± Translation units (AU) a ∆ s.d. ** s.d. i. 4a Fig. containing each sequences, mutations at upstream different positions various of by the Kozak (A sequence preceded ( adenosine ferase ribosomes the +Rps26 by in preceded enriched sequence luciferase firefly with encoding yeast transformed Weplasmids assay. devel reporter we luciferase Rps26, dual requires a oped codon start the of upstream sequence Rps26. on relies −10 and −8 −7, −4, −2, −1, positions ( transcriptome yeast entire the reflected and −3 than other position contrast, In positions. these at adenosines containing mRNAs translate entially prefer ribosomes +Rps26 that notion the with consistent is finding and structural and recapitulating the Kozak consensus sequence for yeast for sequence consensus Kozak the recapitulating ( positions −10 and −8 −7, −4, −2, −1, the in dues Kozak by discovered as −3, residue at adenosine an contain to likely were ribosomes +Rps26 on enriched mRNAs that showed analysis This we on focused the sequence immediately upstream of the start codon. tive to Thus, the codon). in start our of enrichment analysis sequence channel, potentially contacting residues −4, and −7 through −10 (rela to bound mRNAs We of Weblogoenrichment used to the sequence interrogate analysis elements Rps26-based and ) Ribosomes isolated from TET-Rps26 cells exposed to dox were probed probed were dox to exposed cells TET-Rps26 from isolated ) Ribosomes –20 –10 Fig. 3b Fig. 10 20 To test whether translation of mRNAs with a Kozak consensus consensus Kozak a with mRNAs of translation whether test To Sam3 0 3 Transcripts (copies/cell) f ). Original blot images are in in are images blot Original ). 3 10 20 30 P Pgk1 0 0 2 4 6 8 ); thus, the ratio of of ratio the thus, ); = 0.0016, Kolmogorov–Smirnov test. ( test. Kolmogorov–Smirnov = 0.0016, , . In addition, adenosines were moderately enriched at resi at enriched moderately were adenosines addition, In . c +Rps26 Stm1 +Rps26

). Thus, the selection of mRNAs containing adenosines at adenosines containing mRNAs of selection the Thus, ).

mRNA abundance Rpl3 ∆ Enrichment by ribosome pool

**** mRNA 1 Rps26-enriched mRNAs lacked conservation at any at conservation lacked mRNAs Rps26-enriched Asc1 6 ∆ data indicate that Rps26 is bound in the mRNA exit mRNA exit the in bound is Rps26 that indicate data Rps26

t Abf1 test). Data are representative of four experiments. experiments. four of representative are Data test). ∆ ∆ ∆ Rps26 and +Rps26 ribosomes +Rps26 and Rps26 Rps26, 739; all, 5,459. Translational efficiency: efficiency: Translational 5,459. all, 739; Rps26, Gpr1 Rps26 ribosomes after Rps26 depletion. Three Three depletion. Rps26 after ribosomes Rps26

preference Bop2 All

Mtl1 ∆ ′ Rps26 UTR length: +Rps26, 763; 763; +Rps26, length: UTR Sda1 Renilla Mec1 Supplementary Data Set 3 Set Data Supplementary g

is Translational efficiency Eap1

to firefly luciferase quantified quantified luciferase firefly to mediated 0.0 0.5 1.0 1.5 2.0 2.5 Rox1 +Rps26 Translational efficiency d c Enrichment by ribosome pool **** – ∆ Translational units (AU) g ∆

–20 –15 –10 by 10 ) Metagene analysis analysis ) Metagene Rps26 –5 0 ) and and )

Kozak s e l c i t r a 2 +Rps26 Fig. 3 Fig. Gene enrichment 9 . Crosslinking . Supplementary ∆ All Rps26, Rps26, Renilla 13 . Source . Source **

sequence a , ∆ 31 ), broadly broadly ), Rps26 , 3 2 . This This . luci 2 

4 - - - - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. sistent with findings from Kozak’s findings cells with sistent in mammalian studies con is which translation, impact codon start the of upstream dues ( translation reduced together, −10 to −7 positions as well as positions, −4 and −3 −2, −1, A the of the of effects a given upstream on sequence translation relative to that PDB ( s.e.m. and mean −4G, and −(7–10)G cultures: independent of numbers following the used experiments *** −(7–10)G: with starting right, to left from +Rps26 versus −Rps26 mutations. point individual of recognition the on depletion t **** −(7–10)G: with starting right to left from −Rps26, For −(7–10)G: with starting right to left from +Rps26, For Rps26. (−) depleted or (+) replete with mutations Kozak-sequence from effects test to dox, of presence or absence the in grown TET-Rps26 cells from assays ( ribosomes +Rps26 3 Figure s e l c i t r a  factor Ltv1 ( Ltv1 factor in and WT yeast the 40S assembly in reporter lacking yeast luciferase in defect observed recognition of the the −4 residue. Wefor therefore analyzed translation responsible of the was Rps26 of depletion the accumula rRNA 20S tion moderate to leads depletion Rps26 Because ( such reporter −4G no the of had translation on Rps17 effect or Rps3 of depletion as Rps26, to specific was G ( with at those −4 the position depleted and C or A with mRNAs for enriched ribosomes +Rps26 an with A or C at ( that position with a G at the −4 position. This preference came at the loss of mRNAs scriptome as a whole, sistent data, show which with the sequencing that relative to the tran Rps26-deficient versus Rps26-replete yeast ( tions. Further, translation of mRNAs containing −4G was increased in at −4 and −2 posi the of mRNAs adenosine with translation erential that of the A from indistinguishable was mutants −2G and −4G the of translation and more recent work in yeast = 2.179; ** = 2.179;

d a In Rps26-replete yeast, mutation of individual adenosines at the the at adenosines individual of mutation yeast, Rps26-replete In

P Bits 0.0 0.2 0.4 0.6 0.8 3

= 0.0002, = 0.0002, Relative 3J8 4

, we wondered whether the accumulation of 20S rRNA and not accumulation the , whether we wondered Renilla:firefly ratio (AU) 5 ′

0.00 0.25 0.50 0.75 1.00 1.25 10 1 –10 Rps26 promotes translation by recognizing specific residues in the Kozak sequence. ( sequence. Kozak the in residues specific recognizing by translation promotes Rps26 , ref. , ref. sequence. 10 P –9 ∆ –(7–10)GA **** = 0.0064, = 0.0064, mRNA ( 10 Ltv1). Similar to the Rps26-depleted strain, the the strain, Rps26-depleted the to Similar Ltv1). 1 **** Position upstreamofstartcodon t **** 6 = 4.235; = 4.235; –8 +Rps26 Supplementary Fig. 4 Fig. Supplementary

). The C-terminal 21 amino acids of Rps26 are not resolved in this structure. Source data for for data Source structure. this in resolved not are Rps26 of acids amino 21 C-terminal The ). –4G ** **** a –3C ; ; –7 n Fig. 3 Fig. ∆ –2G = 865) and and = 865) Fig. 3 Rps26 ribosomes specifically enriched mRNAs

–1G +Rps26 –6 t = 3.116. Statistical values determined by two-way analysis of variance (ANOVA). DF = 184. ( = DF 184. (ANOVA). variance of analysis two-way by determined values Statistical = 3.116. P = 0.9712, = 0.9712, d –(7–10)GA –5 **** d 10 , left). These data demonstrate that resi that demonstrate data These left). , , right). Thus, Rps26 is required for pref 1 **** 3 –Rps26 –4

Supplementary Fig. 4b Supplementary –4G . In contrast, in Rps26-depleted cells, cells, . In in Rps26-depleted contrast, Supplementary Fig. 4b Fig. Supplementary **

∆ –3C Rps26 ribosomes ( ribosomes Rps26 –2G –3 ). ( ). t

= 0.2146; = 0.2146; –1G –2 f ) Rps26 (blue) binds mRNA near the −4 position when the start codon (purple) is in the P site (adapted from from (adapted P site the in is (purple) codon start the when position −4 the near mRNA binds (blue) ) Rps26 –1 Fig. 3 Supplementary Fig. 4d Fig. Supplementary 3 ′ b e e

P Relative Bits ). This result is con 0.8 0.0 0.2 0.4 0.6 = 0.7879, = 0.7879, Renilla:firefly ratio (AU) b 5 0.00 0.25 0.50 0.75 1.00 1.25 ; ; ′ –10 P n ). ). In contrast, < 0.0001, = 741) versus all mRNAs in our data set ( set data our in mRNAs all versus = 741) ). This effect effect ). This

t A

= 10.24, 6.229, 7.594, 3.193, or 4.84 versus A versus 4.84 or 3.193, 7.594, 6.229, = 10.24, –(7–10)G

10 –9 advance online publication online advance –Rps26 +Rps26 Position upstreamofstartcodon t 10– = 0.8371; = 0.8371; –8 ∆

Ltv1 Ltv1 –4G 12 *** –7 t , 3 = 8.869; = 8.869; ). ). 3 ------–3C

∆ –6

–2G Rps26 phorylation, cell cycle, DNA repair and, notably, well-characterized well-characterized notably, and, repair DNA cycle, cell phorylation, phos control, transcriptional including processes, regulated highly contrast,translated best the among are proteins ribosomal encoding factors. This is consistent with previous findings showing that mRNAs mRNAs enriched proteins mainly encoded ribosomal and translation and on GeneCodis analysis enrichment for GO-term a as basis specific produce biological outcomes, could we used mRNAs they enriched in each ribosome pool which in pathways biological specific in enriched mRNAs whether Toassess Rim Accumulation −4G. mRNAs containing for preference slight a show that position. for necessary is preferentialthis translation ofthat mRNAs and with a Kozak consensus sequence, of A or Kozak C at the of positions −2 and −4 the recognizes Rps26 that demonstrate all studies structural residue ( −2 the of interactor an neighboring directly residue rRNA residue. −4 the of recognition in defect observed the for responsible that it is depletion of Rps26 and not of accumulation 20S rRNA that is in position −4 translation of the luciferase reporter was sensitive to the residue at the moderate amounts strain accumulates of 20S rRNA P Fig. 3 Fig. –5 = 0.5621, = 0.5621, Importantly, Rps26 directly contacts the −4 position, as well as an an as well as position, −4 the contacts directly Rps26 Importantly, –1G 1 Supplementary Data Set 2 Set Data Supplementary P –4 0 = 0.2228, = 0.2228, f 1 and ref. ref. and n f = 15; −3C, −3C, = 15; pathways ∆ –3 Rps26-ribosome-enriched transcripts Rps26-ribosome-enriched were associated with t –2 = 1.438 (two-way ANOVA). DF = 184. For For = DF 184. ANOVA). (two-way = 1.438 ∆ ∆

Rps26 ribosomes Rps26 have ribosomes lost this preference and instead a of Ltv1 yeast ( yeast Ltv1 – 1 t –1 = 1.223; **** = 1.223; c

6 D ) Weblogo conservation analysis of mRNAs enriched in enriched mRNAs of analysis conservation ) Weblogo

3 ). Thus, the sequencing data, reporter assays and and assays reporter data, sequencing the Thus, ). ′ Rps26 n nature structural & molecular biology molecular & structural nature = 7; −2G, = −2G, 7; c c Bits ; ; 0.0 0.2 0.4 0.6 0.8 n = 5,696). ( = 5,696).

5 ribosomes ′ Supplementary Fig. 4e Fig. Supplementary –10 d AUG P n and and 10 ). We found that +Rps26-ribosome- that We ). found < 0.0001, < 0.0001, = 12; −1G, −1G, = 12; –9 ; ; **** Position upstreamofstartcodon –1 e –8 d are available online. available are –2 ) Results of luciferase reporter reporter luciferase of ) Results

activates e P ∆ ) The effect of Rps26 Rps26 of effect ) The –7 < 0.0001, ** < 0.0001, –3 Rps26 ribosomes cluster in in cluster ribosomes Rps26 P t –4 All ORFs = 8.328; = 8.328; –6 n = 0.6562, = 0.6562, = 13. Data shown are are shown Data = 13. –5 –5

–6 the –8 –7 35 ), demonstrating demonstrating ), –4 –9

, Hog 3 d P P 6 –10 and and = 0.0603, = 0.0603, = 0.0017. = 0.0017. . . Importantly, t –3 = 1.193; = 1.193; Rps26 1 3 e –2 7 and , ( Fig. 1 Fig. 5 , –1 2

6 . In .

3 ′ c -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. test this, we grew WT yeast in rich media containing high salt con salt high containing media rich in yeast WT grew we this, test To of stresses. to these response of cellular the part be formation might ribosomes in the that observed reasoned we phenotypes yeast, stress Rps26-deficient high-pH and high-salt the Given high-pH Yeast pre- 20S ( cells of these in rRNA accumulation the from arise not does resistance stress ∆ Finally, stress. pH and salt to resistance in deficiency Rps26 of role ( Rps17 and Rps3 proteins, ribosomal late-binding other two of depleted strains yeast in defect growth a produced conditions high-pH and high-salt stress to cell-wall produce cascade MAPK tinct dis a activates which caffeine, to resistant not were yeast deficient Rps26- as selective, pathway was phenotype This depleted. is Rps26 when induced are pathways Rim101 the and MAPK Hog1 the both ( conditions acidic mildly in even pathway, high-pH- response the of hallmark a protein, Rim101 the of cleavage minal ( NaCl of the concentrations lower of at MAPK Hog1 phosphorylation increased displayed yeast ( pH Rps26-deficient and salt both to sensitive were cells Rps26-replete whereas media, rich in than conditions high-pH and or high pH. Surprisingly, Rps26-deficient cells grew faster in high-salt salt high without and with media rich in cells yeast Rps26-depleted and for rates Rps26-replete growth we measured Toprediction, this test stresses. these to resistant more are Rps26 of depleted cells that in mRNAs enriched pathway Rim101 the and stress, high-salt to responds that cascade MAPK a pathway Hog1 the including pathways signaling stress-responsive for data Source shown. are data representative in are images blot Original hemagglutinin. HA, cells. ( mM). (0–500 NaCl of concentrations increasing n (W): **** = 1). change (fold conditions no-stress to compared ( ( white. in shown are ribosome of type either in enriched not mRNAs key; the to according colored rectangles, in are ribosomes +Rps26 in enriched mRNAs ( MAPK 4 Figure nature structural & molecular biology molecular & structural nature from ribosomes purified Wethen caffeine. or pH high centrations, f independently grown cultures, as indicated. ( indicated. as cultures, grown independently ). Box edges indicate the interquartile range, midlines indicate the median, and whiskers extend to the minimum and maximum values. Values were were Values values. maximum and minimum the to extend whiskers and median, the indicate midlines range, interquartile the indicate edges Box ). Ltv1 cells also did not show stress resistance, demonstrating that that demonstrating resistance, stress show not did also cells Ltv1 a Because Rps26 depletion increased the relative translation of of translation relative the increased depletion Rps26 Because Hot1 Ssk2 d Ypd1 Ssk1 Sln1

, 378, −120, 120; 120; −120, , 378, form Ssk22 Smp1 d a

– ), Rim101 ( Rim101 ), 3 Accumulation of of Accumulation

f 9 stress Hog1 Pbs2 ) Changes in doubling time in cells containing Rps26 (−dox) or deficient in Rps26 (+dox) after exposure to salt ( salt to exposure after (+dox) Rps26 in deficient or (−dox) Rps26 containing cells in time doubling in ) Changes , which responds to pH stress ( stress pH to responds which , Sho1

Sko1 D Ste11 Supplementary Fig. 5a Fig. Supplementary Rps26 Ste20 Ste50 Msn2/4 Cdc42 1.1–1.4 1.4–2 b enrichmen >2 ∆ ), and cell-wall integrity ( integrity cell-wall and ),

∆ Supplementary Fig. 5h Fig. Supplementary ribosomes Rps26 Rps26 ribosomes ( ribosomes Rps26 e 0.5–0.7 0.7–0.9 <0.5 ∆ , 300, −78, −45; and and −45; −78, , 300, Rps26 ribosomes activates specific biological pathways. ( pathways. biological specific activates ribosomes Rps26 t b Rim101 Vps20

in

response Snf7 Smp1 Rim20 – g Rim101 ), demonstrating the specific specific the demonstrating ), Rim21 Rim Rim13 Fig. 2b Fig. Nrg1 Fig. Fig. 8 d c g Fig. 4d Fig. Dfg16 Rim ) pathways in in ) pathways ) Hog1 phosphorylation (Hog1-p) in Rps26-containing and Rps26-deficient cells after exposure to exposure after cells Rps26-deficient and Rps26-containing in (Hog1-p) phosphorylation ) Hog1 –

, f to h i f , 465, 105, 120, for cells exposed to 0, 100, and 200 ng/ml of dox, respectively. Data are from from are Data respectively. dox, of ng/ml 200 and 100, 0, to exposed cells for 120, 105, , 465, 4 ). 9 are available online. available are ) Proteolytic activation of Rim101 at different pH values in Rps26-containing and Rps26-deficient Rps26-deficient and Rps26-containing in values pH different at Rim101 of activation ) Proteolytic 4 ).

Snf1 0 osmotic , c ( Fig. 4 Fig. Supplementary Data Set 3 Set Data Supplementary advance online publication online advance c ), we hypothesized hypothesized we ), Fig. 4c Fig. , e Wsc2 P ). Additionally, Additionally, ). Fig. 4 Fig. < 0.0001, *** < 0.0001, Slg1 g Wsc3 Rlm1 ) and C-ter and ) Mkk1

Rom1 , and ∆ f Rho1 ). ). Further, Rps26 or +Rps26 ribosomes. mRNAs enriched in in enriched mRNAs ribosomes. +Rps26 or Rps26 Bck1 Pkc1 Swi4 Slt2 h Rom2 Mkk2 ). Thus, Thus, ). ∆ Mid2

Swi6 Rps26 Rps26 Bag7 Mtl1 Bem2 Sac7 3 Lrg1 8 - - - P , = 0.0005, ** = 0.0005, g d Hog1-p The data herein demonstrate key roles for Rps26 in recognizing recognizing in to Rps26 leading sequence, Kozak the of −4 for and −2 at positions adenosines roles key demonstrate herein data The Kozak Rps26 DISCUSSION mRNAs from stress-responsethese pathways by transcriptional response to these stresses with preferential translation of duce pro cells stress, high-pH and mRNAs.high-salt to response Thus,in −4G-containing translate pH high or salt high to exposed are yeast These data strongly suggest that the to salt or high pH stress but not when to exposed caffeine ( A the of that of level the to concentrations translation pH, recovered reporter or of−4G high the from the accumulation of −4G A the with transformed cells WT Weexposed stress. under as a readout for the formation and offunctionality ( yeast, whereas it is indistinguishable from that of the A translation of the −4G depletion of Rps26 using the dual luciferase assay. We have shown that conditions. high-pH and high-salt to response the of part is ribosomes of Rps26-deficient formation the that indicate data These ( cells control non-stressed in those to similar were feine caf to exposed cells in Rps26 of levels the whereas cells, from unstressed ribosomes than Rps26 less contained conditions high-pH or high-salt under grown cells from ribosomes proteins, to any of these normalized when Notably, controls. as Asc1) and Rps8 Rps5, Rps3, (Rps0, subunit ribosomal small the from proteins additional five of levels the assessed We also blotting. western by levels protein Rps26 and control analyzed cells, and cells from non-stressed stressed these Rps26 [NaCl] Fig. 3 Fold change in doubling . Western blots are representative of four independent experiments, and and experiments, independent four of representative are blots . Western Rcl1 We next validated the relative to no stress 0.0 0.5 1.0 1.5 2.0 2.5 Renilla ∆ d

Rps26 ribosomes, thereby augmenting the well-characterized well-characterized the augmenting thereby ribosomes, Rps26 Doxycycline (ng/ml a sequence enforces n **** , right). We used this functional signature of – =27 0 c +Rps26

) Enrichment of mRNAs encoding components of the Hog Hog the of components encoding mRNAs of ) Enrichment P 1 MNaCl = 0.0039 (Wilcoxon signed rank test). Sum of signed ranks ranks signed of Sum test). rank signed (Wilcoxon = 0.0039 n **** reporters to salt, high pH or caffeine stress. As expected expected As stress. caffeine or pH high salt, to reporters 100 =15 ****

n 20 the =15 0 )

translational Renilla –Rps26 e in vivo Fold change in doubling ∆

Rps26 ribosomes in the presence of high salt relative to no stress 10 0. 0. 1. 1. 2. 2. 0 5 0 5 0 5 construct when WT cells were exposed exposed were cells WT when construct reporter is increased in Rps26-deficient translational effects of this physiological Doxycycline (ng/ml n **** =24 100 mMTAPS 1 0 kDa 37 15 50 ∆ Rps26 ribosomes are shown in ovals, ovals, in shown are ribosomes Rps26

pH 8.2 ∆ program n *** =12 Rps26 ribosomes that form when 00 h Rim101 n 20 ** = Rps2 d 0 (HA) Rcl1 9 ), high pH ( pH high ), pH )

6 encoded 3. f 3 5 +Rps26 ∆ Fold change in doubling 5. Rps26 ribosomes. ∆ 7 5 relative to no stress s e l c i t r a ∆ 0 1 2 3 4 10 Rps26 ribosomes Rps26 ribosomes .5

Doxycycline (ng/ml e Renilla n ****

) or caffeine caffeine ) or =30 by 10 mMcaffeine 0 .5 –Rps26

the Fig. 5 Fig. 5a Fig. 5. n **** 100 =14 5 10 reporter 7.

or the the or 5 n **** 200 =15 d kD 37 15 75 100 – – a ) g c ). ). ). ).  - -

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. indicating that the decreased translation of otherwise well-translated well-translated of translation otherwise that the decreased indicating pathways these were yeast, constitutively in activated Rps26-deficient that showed immunoblotting and assays Growth pathways. Rim101 high-pH-response the and MAPK Hog1 conserved highly and ized but instead cluster pathways, in including the specific well-character tion are not randomly distributed throughout the yeast transcriptome program. translational separate a decode ribosomes Rps26-deficient whereas by Kozak the sequence, encoded ferent structure. Regardless, Rps26 enforces the translational program −4G in WT orribosomes, the −4G-containing mRNAs from might adopt a arises dif that hindrance steric a ameliorate might deficiency Rps26 alternatively, or Additionally, Kozak favored. longer strong no are mRNAs as mRNAs, such of competitiveness increased from simply arise might This −4G. containing mRNAs of translation the Notably, ( cells Rps26-depleted in numbers ome ribos reduced overall the explaining proteins, ribosomal those encoding including mRNAs, translated highly normally the of decreases translation depletion Rps26 translation, efficient sequence, enables Kozak which the of recognition in Rps26 of role critical the of 30 uncovered years the ago Kozak sequence in mutations of effects the for basis molecular a provide data these preferential translation of mRNAs that contain these adenosines. Thus, s e l c i t r a  in mutations with mRNAs of translation increased the and mRNAs t ** t ( condition. YPD-only the to are comparisons All gels. of sets different two on run each and condition each for caffeine) for (four ( shown. are gels these of each from bands RpsX and Rps26 the and Rps26, to relative proteins these of each analyze to run were gels separate size, in identical nearly are Rps26 except proteins ribosomal all Because caffeine. mM 10 with or 8.2, pH 1 M NaCl, with alone, YPD in grown yeast WT from purified ribosomes in Asc1 and Rps26 Rps8, 5 Figure data for for data in are images blot Original s.e.m. and mean are shown Data ANOVA. two-way by determined were values Statistical * YPD: conditions. stress different to exposure a 4-h after cells WT of assays luciferase t caffeine, = 0.4039, DF = 12. Statistical values were determined by Student’s Student’s by determined were values Statistical = 12. DF = 0.4039, caffeine, = 10; DF = 1.708, *** pH, = 8; DF = 2.469, P Remarkably, we that observed mRNAs upregulated by Rps26 deple = 0.0036, = 0.0036, a kDa 15 25 15 25 15 25 15 25 15 25 P = 0.0219, = 0.0219,

d P Cells generate generate Cells – ∆ g = 0.8089, = 0.8089, YPD Rps26 ribosomes were also found to selectively augment augment selectively to found also were ribosomes Rps26 are available online. available are

NaCl t = 3.773, DF = 10; caffeine, caffeine, = 10; DF = 3.773, pH 8.2 t = 2.817. NaCl: NaCl: = 2.817.

t Caffeine = 0.2472, DF = 12. ( = 12. DF = 0.2472, ∆ Rps26 Rps5 Rps26 Rps3 Rps26 Rps0 Rps26 Asc1 Rps26 Rps8 Rps26 ribosomes in response to high-salt and high-pH conditions. ( conditions. high-pH and high-salt to response in ribosomes Rps26 P = 0.0003, = 0.0003, P = 0.5866, = 0.5866, b e Rps26/Rps8 Rps26/Rps0 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 P = 0.8563 (n.s.), (n.s.), = 0.8563 t = 5.967, DF = 8; caffeine, caffeine, = 8; DF = 5.967, YPD YPD b t – = 0.5618, DF = 10. ( = 10. DF = 0.5618, Supplementary Fig. 3e Fig. Supplementary f f P ) Quantification of data in in data of ) Quantification ) NaCl, * ) NaCl, = 0.1343, = 0.1343, 10– NaCl NaCl ** * 12 pH 8.2 pH 8.2 , 3 **** 3 *** ( P Fig. Fig. 6 t = 0.048, = 0.048, Caffeine Caffeine = 0.4983. pH: pH: = 0.4983. t n.s. n.s. = 1.606, DF = 12. ( = 12. DF = 1.606, a ). Because ). Because advance online publication online advance e t ) NaCl, ** ) NaCl, = 2.287, DF = 9; pH, *** pH, = 9; DF = 2.287, f P Rps26/Asc1 t = 0.1018, = 0.1018, c test. n.s., not significant. Data shown are mean and s.e.m. ( s.e.m. and mean are shown Data significant. not n.s., test. , 0.0 0.5 1.0 1.5 f

a Rps26/Rps3 ). ). P . Data are from ribosomes grown and purified in three independent replicates replicates independent three in purified and grown ribosomes from are . Data - - - - 0.0 0.5 1.0 1.5 = 0.8673 (n.s.), (n.s.), = 0.8673 YPD P position position to cancer in observed humans with DBA, whereas the overall for predis the mRNAs account might of specific upregulation tional transla by pathways pro-growth of activation cells, mammalian In How Fap7 depletion. to lead would which (refs. Rps26 of growth the rescues Fap7 of depletion of Fap7 in Rps26-depleted cells explains why overexpression rRNA 18S ( tain cells Rps26-depleted in accumulated moderately is rRNA 20S why explains finding This els were decreased in Rps26-depleted cells ( cells Rps26-depleted in decreased were els +Rps26 ( ribosomes in factors either showed ome-assembly no or enrichment were enriched sequences. Kozak 5 long by characterized mRNAs translated poorly and production, bythe enhancing translation of selectively otherwise ribosome to reduced leading proteins, ribosomal encoding ing those includ mRNAs, essential of translation the disabling by roles genic whereby a model support data the Collectively, depletion. Rps26 upon ostasis home protein cellular in perturbation a to lead sequence Kozak the = 0.008, = 0.008,

n YPD d Interestingly, whereas the large majority of mRNAs encoding ribos ∆ = 7 (YPD) or 6 (all other conditions) independently grown cultures. grown independently conditions) other 6 (all or = 7 (YPD)

) NaCl, ) NaCl, NaCl t Rps26 ribosomes, the mRNA encoding Fap7 was enriched in in enriched was Fap7 encoding mRNA the ribosomes, Rps26 = 1.802, DF = 10. ( = 10. DF = 1.802,

* can NaCl

pH 8.2 ** ∆

*** ribosomal t P Rps26 ribosomes, such as Rps26 found those ribosomes, in DBA, play patho

= 3.299, DF = 10; pH, **** pH, = 10; DF = 3.299, pH 8.2 = 0.5534, = 0.5534, Caffeine ** 42 t = 0.1681. Caffeine: ** Caffeine: = 0.1681. a P n.s. ) Western blots showing the levels of Rps0, Rps3, Rps5, Rps5, Rps3, Rps0, of levels the showing blots ) Western

, Caffeine 4 Fig. 1 Fig. = 0.0004, = 0.0004, 3 n.s. ), ),

Supplementary Fig. 3i Supplementary ∆ 3 nature structural & molecular biology molecular & structural nature 4 protein c Tsr2 cells are expected to be Rps26 deficient, deficient, Rps26 be to expected are cells Tsr2 , even though Rps26-deficient ribosomes con ribosomes Rps26-deficient , though even t and g = 0.6132, DF = 10; pH, pH, = 10; DF = 0.6132, c Relative renilla:firefly ratio ) NaCl, ** ) NaCl, 0.0 0.5 1.0 1.5 t = 5.132, DF = 10; caffeine, caffeine, = 10; DF = 5.132, Supplementary Fig. 1f Fig. Supplementary d

haploinsufficiency Rps26/Rps5 ∆ 0.0 0.5 1.0 1.5

Tsr2 cells Tsr2 YPD * P Supplementary Data Set 3 Set Data Supplementary = 0.0025, = 0.0025, P

= 0.0014, = 0.0014, YPD P < 0.0001, < 0.0001, NaCl NaCl 4 n.s. ). ). Consequently, Fap7 lev 1 Supplementary Fig. 3j Fig. Supplementary ; because Tsr2 stabilizes stabilizes Tsr2 because ; b

t pH 8.2 = 4.015, DF= 10; pH, pH, 10; DF= = 4.015, P ) NaCl, * ) NaCl, n.s. t = 0.1184, = 0.1184, = 3.884. DF = 42. = 42. DF = 3.884. pH 8.2

g t lead = 7.599, DF = 8; DF = 7.599, ) Results of ) Results Caffeine ′ , UTRs and weak weak and UTRs g n.s. ). Furthermore, ). Furthermore,

P P to = 0.6934, = 0.6934,

= 0.0388, = 0.0388, Caffeine

cancer? –4G A . Source . Source

** 10 ). ).

------© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ult-oto mcaim drn assembly during mechanisms quality-control ribosomal escape can ribosomes these how explain may ribosomes Rps26-deficient of relevance physiological the Importantly, milieu. extracellular the in programs changes of face the in survival transcriptional cell for allow that and translational coordinates that loop feed-forward a creating stress, during roles physiological play also ( stress high-pH and high-salt to that and Rim101 stress-response pathways. Most important, the data Hog1 show the from proteins encoding those including sequence, Kozak the in position −4 the at mutations with mRNAs of translation the that but also sequence, Kozak a of strong mRNAs containing in translation are deficient ribosomes proteins. ribosomal lated encoding mRNAs, those including trans of highly recruitment for ribosome required is Rps26 contrast, of mRNAs subset Hox from the family.of for a specific translation In specific developmental pathologies. Similar to Rps26, Rpl38 is required to leads Rpl38 protein subunit ribosomal large the of deficiency that In their groundbreaking work, Barna and colleagues Physiological cancer to ribosomal proteins, or rRNA modifications, that other have of also been linked depletion from effects mRNA-specific of will assessment allow subpopulations ribosome of purification for herein method described the Importantly, repertoire. translational the of alteration effects mRNA-specific have which can factors, translation-initiation of binding or features mRNA of specific recognition in involved similarly be might proteins These mRNA channel. the exit near located are also subunit small the from proteins have to other ribosomal linked been also DBA 3e Fig. 1 Sets ( proteins ribosomal of translation in decrease homeostasis. protein in changes to leading thereby sequence, Kozak the in deviations with mRNAs of translation the increasing accumulate, blue) (light Rps26 lacking ribosomes conditions, high-pH or high-salt in ( sequences. Kozak canonical containing mRNAs of translation the enhancing thereby ovals), red (dark sequence ( 6 Figure nature structural & molecular biology molecular & structural nature ribosomes of a b a ) Rps26 (orange ovals) recognizes positions −2 and −4 in the Kozak Kozak the in −4 and −2 positions recognizes ovals) (orange ) Rps26 Nevertheless, the data herein demonstrate not only that that only not demonstrate herein data the Nevertheless, ∆ Rps26 ribosomes are produced by WT yeast upon exposure exposure upon yeast WT by produced are ribosomes Rps26 and , f

), accounts for growth and developmental defects. Of note, note, Of defects. developmental and growth for accounts ), Disruption of protein homeostasis by Rps26 insufficiency. insufficiency. Rps26 by homeostasis protein of Disruption 47 2 , 4 8 ), and the resulting loss of ribosomes ( ribosomes of loss resulting the and ), 53 .

roles , 5 4 .

for

ribosomes ∆ Rps26 ribosomes selectively increase increase selectively ribosomes Rps26 44– Fig. 6 Fig.

Rps26 insufficiency,or lacking 4 6 b , with net effects that lead to to lead that effects net with , salt orpHstress ) When Rps26 is insufficient or insufficient is Rps26 ) When b ). Thus, Thus, ).

ribosomal Supplementary Data Data Supplementary

51 advance online publication online advance ∆ 49 , Rps26 ribosomes ribosomes Rps26 5 2 , Supplementary Supplementary 5 and function function and 0 2 demonstrated

proteins 3 , , and several ∆ Rps26 Rps26 -

6. 5. 4. 3. 2. 1. claims jurisdictional in published maps and affiliations. institutional reprints/index.htm Rps26 and allow for the fully reversible loss and reincorporation of of reincorporation and loss reversible fully the for allow and Rps26 Tsr2store might ribosomes, nascent to Rps26 delivering to addition identified Tsr2, for Rps26, been has chaperone a that specific note to interesting is it regard, that In ribosomes. pre-existing from assembly of formation to lead that conditions stress the that observation the with consistent to the leads levels of Rps26 downregulation that it that is not likely Together,indicate data these ( 11-fold about increased mRNA Rps26 of levels the though even yeast, of sensitivity stress the on effect an have not did Furthermore, promoter less. galactose the via Rps26 of perhaps expression forced proteins, ribosomal other of those to 6a Figure in shown data The ribosomes. Rps26-deficient of production the for allow could which proteins, ribosomal other of investigate to whether levels RT-qPCR of Rps26 mRNA used decrease more under stress we than those questions, these addressing start To Rps26. lack that produced are subunits 40S made newly instead, whether, or ribosomes Rps26-containing mature from formed are ribosomes us these leads to stress ask and whether high-pH high-salt during formed are ribosomes Rps26-deficient that observation The How Reprints and permissions information is available online at at online available is information permissions and Reprints The authors declare no competing interests.financial and all authors support the conclusions. sequencing data. The manuscript was written and edited by M.B.F., H.G. and K.K., performed and analyzed the experiments. E.A.W. wrote software scripts to analyze Experiments were bydesigned M.B.F., H.G. and K.K. M.B.F., H.G. and E.L.P. the US National Institutes of Health. responsibility of the authors and does not necessarily represent ofviews the official Institute (Faculty Scholar grant 55108536 to K.K.). The content is solely the Foundation (graduate fellowship to M.B.F.), and the Howard Hughes Medical Cancer Awareness Day fellowship to H.G.), the Richard & Helen DeVos GM086451 (to K.K.) and (to F31-GM116406 M.B.F.)), PGA National (Women’s This work was supported by the US National Institutes of Health (grants R01- We thank J. Cleveland, J. Joyce and members of the Karbstein lab for comments. version of the pape Note: Any Supplementary Information and Source Data files are available in the pape the the in available are references, and codes accession statements of including Methods, and data availability any associated M protein. the COMPETING FINANCIAL INTERESTS COMPETING AU Ac

ethods k T Ingolia, N.T., Lareau,L.F. &Weissman, J.S.Ribosomeprofilingofmouseembryonic Genome-wide Weissman,J.S. & J.R.S. N.T.,Newman, Ingolia, S., Ghaemmaghami, processes. developmental and P.cellular Lasko, in Translational& control J. Kong, of control Translational L. Veylder, De & D. Inzé, R., Beyaert, K., Kelen, Der Van cancer. in control Translational R.J. Schneider, & S.C. Formenti, D., Silvera, apoptosis. and stress Translationalin N. control Sonenberg, & M. Holcik, 789–802 (2011). 789–802 stem cells reveals the complexity and dynamics of mammalian proteomes. profiling. Science ribosome using resolution nucleotide with translation of vivo in analysis Genet. Rev. Nat. expression. gene eukaryotic Cancer Rev. Biol. Cell Mol. H no

are O wle R R r

CONT 55–

.

Rps26-deficient 324 – d c 5

7 demonstrate that Rps26 levels decreased similarly similarly decreased levels Rps26 that demonstrate 10 , 218–223 (2009). 218–223 , gme . We thus speculate that . that We speculate thus

r l RIBU 6 . . Publisher’s with neutral Nature remains regard to note: Springer , 254–266 (2010). 254–266 , .

, 318–327 (2005). 318–327 , ∆ 13 Rps26 ribosomes do not support active ribosome ribosome active support not do ribosomes Rps26 nts , 383–394 (2012). 383–394 , T I ONS Crit. Rev. Biochem. Mol. Biol. Mol. Biochem. Rev. Crit.

ribosomes de novo de formation of formation ∆

formed? Rps26 ribosomes are formed are formed ribosomes Rps26 Supplementary Fig. 6d Fig. Supplementary ∆ http://www.nature

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3 147 Nat. . . In , e of ). ).  ,

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Ferreira-Cerca, S., Pöll, G., Gleizes, P.-E., Tschochner, H. & Milkereit, P. Roles of P.Roles Milkereit, & P.-E.,Tschochner,H. Gleizes, G., Pöll, S., Ferreira-Cerca, initiator AUG the of recognition in nucleotides flanking of role Possible M. Kozak, Nakagawa, S., Niimura, Y., Gojobori, T., Tanaka, H. & Miura, K. Diversity of preferred the of comparison and Compilation H.A. Boer, de & C.K. Watanabe, R., Hamilton, translational the from upstream sequences of analysis and Compilation M. Kozak, logo sequence a WebLogo:Brenner, S.E. & J.M. Chandonia, G., Hon, G.E., Crooks, in lengths mRNA of analysis H. Liu, Genome-wide P.O. Brown, & E.H. Hurowitz, Arava, Y. B. Koch, T.V. Pestova, & C.U.T. Hellen, M.M., Yusupov, V.G., Kolupaeva, A.V., Pisarev, I. Boria, the process: ubiquitous a from diseases Diverse B. Triggs-Raine, & J. Armistead, Stumpf, C.R. & Ruggero, D. The cancerous translation apparatus. disorders anemia Blackfan Non-Diamond J.M. Liu, & A. Shimamura, N., Burwick, B.L. Ebert, A. Bolze, J.E. Farrar, Hussain, T. TranslationalC.-C. efficiency Wang, & Yeh,L.-S. K.-J., Chang, G., Lin, S.-J., Chen, Y. Kim, S. enhance Dvir, codon initiator AUG the preceding nucleotides six least At M. Kozak, that codon initiator AUG the flanking sequence a define mutations Point M. Kozak, efficiency the affect codon initiator AUG the to close The mutations Point M. D. Kozak, Ruggero, & B.S. Taylor, A.B., Olshen, M.V., Moreno, C.R., Stumpf, and localization cytoplasmic direct sequences Promoter E.K. O’Shea, & B.M. Zid, G.A. Brar, uaytc iooa poen i mtrto ad rnpr o pe1S RA and rRNA pre-18S of function. ribosome transport and maturation in proteins ribosomal eukaryotic ribosomes. eukaryotic by codon Res. Acids Nucleic genomes. eukaryote in codon initiation translation the around sequences nucleotide Res. Acids Nucleic sequence context around the AUG startcodons in mRNAs. eukaryotic in site start generator. eukaryotes. cerevisiae Saccharomyces cerevisiae Chem. initiation translation eukaryotic in mRNA of complexes. contacts and position Ribosomal update. database paradox. ribosomopathy Dev. Hematol. syndrome. 5q– and syndrome Diamond Shwachman function: ribosome of screen. interference asplenia. congenital Blood complex. initiation translation in context yeast. sequence its by affected significantly is codon initiation non-AUG a of Res. Acids Nucleic codon has a pronounced effect on the translational efficiency in yeast. in expression cells. mammalian in translation ribosomes. eukaryotic by translation modulates preproinsulin rat of translation of cycle. cell (2013). mammalian the of landscape translational yeast. in starvation during mRNAs of translation profiling. ribosome

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. qPCR mRNA shift assay. SDS-PAGE by analyzed and DTT) followed by mM western blotting. 2 heparin, mg/ml 1 KCl, mM 500 Buffer, (Ribo buffer high-salt forin resuspendedr.p.m.were pellets resulting The 100,000 h. 2.5 at rotor 100.1 TLA Beckman a in spun and DTT) mM 2 layered over 100 400 DTT. mM 2 and (Sigma) cocktail KOAc, 2.5 mM Mg(OAc) phase and flash-frozen in ribosome buffer (20 mM Hepes/KOH, pH 7.4, Purification100 of mM ribosomes from TET-Rps26 cells. genome tures file listed above. We obtained upstream bases from determinedthe by Weblogo analysis start codon in transcripts that were associated with Rps26-mediatedwas selectiondetermined was using GENECODIS life measurements were derived from existing data sets pool. Translational efficiency, 5 sets of mRNA characteristics to analyze the transcriptsIdentification considered and enrichedcharacterization in each of enriched transcripts. standard settings expressionanalysis on three biological replicates was done with DESeq2, using seq-count version 0.6.1 was used to generate gene counts, and differential gene the Saccharomyces Genome Database with Flexbar 2.4 and aligned to the sequences adaptorremove to trimmed were 500 NextSeq the from generated Bioinformatic processing. cell at a final concentration of 1.8 pM. libraries were pooled at equimolar ratios and loaded onto the NextSeq 500 flow ments was 200–600 bp with insert sizes in the range of 80–450 bp. The validated DNA was amplified via PCR to generate the final libraries. The final size of frag repaired, 3 dTTP, preserving strand information. The double-stranded cDNAof place in incorporatingdUTP synthesizedwas was strand secondthen cDNA.The of end- strand first the generate to reverse-transcribed and primed random-hexamer Total RNA sample prep kit (Illumina). Briefly, chemically fragmented RNA was TruSeqStranded the with processed then Yeastand Gold (Illumina) KitZero Ribo- the RNAwith ribosomal of depleted DNase-treatedRNAwas Thetotal Biolabs).England (New I DNasewith treated then andassessment quality for Technologies)(Agilent Bioanalyzer 2100 Agilent an on run and (Invitrogen) Illumina sequencing. phenol-chloroform-isoamyl alcohol extraction. the TCA–DOC method and analyzed by western blotting. RNA was isolated by via fractions precipitated these Proteinwas from pooled. were polysomes and ated on a 10–50% sucrose gradient as described fraction and collected were ribosomes, +Rps26 containing flowthrough, the elution steps were performed. The IgG eluate, containing 100 (Sigma Aldrich). TAP purification was performed as described 0.15. After 4 h, cells were harvested after the addition of OD100ataninoculatedintoandmedia prewarmedYPD media with YPGal with 0.2 with pKK3566 (TET–Rps3-TAP). An overnight culture in YPGal supplemented cells for this experiment by transforming strain yKK636 (GAL-Rp26; GAL-Rps3)Isolation of mRNAs bound to blotting. Plasmids used in this work are listed in niques tech recombination standard by created were or Collection YeastKnockout are listed in Yeast strains and plasmids. ONLINE doi: (TET-Rps26) deficiency WepKK3792.transforming withby yKK491 induced 10.1038/nsmb.3442 µ g/ml cycloheximide was included in all steps and only the IgG-binding and 6 2 5 . The identity of generated strains was verified by PCR and western and PCR by verified was strains generated of identity The . µ 8 by using the custom-built program STARTSEQ written in Python. g/ml dox was grown to mid-log phase. Cells were washed three times ′

adenylated and ligated to PCR adaptors. The purified adaptor-ligated METHODS Supplementary Table 1 µ 6 5 L of sucrose cushion (Ribo Buffer, 500 mM KCl, 1 M sucrose, . Purified RNA was quantified in a Qubit 2.0 Fluorometer 2 ) supplemented with 1 mg/ml heparin, protease inhibitor We generated a yeast strain with dox-inducible Rps26 Demultiplexed and quality-filtered raw reads (fastq)

Saccharomyces cerevisiae ′ D 2 and 3 9 Rps26 and wild-type ribosomes. . ORF-length data were taken from the SGD_fea S. cerevisiae 3 and were obtained from the GE Dharmacon 7 ′ . Enrichment of nucleotides upstream of the UTR lengths, mRNA abundance and half- 5 8 with TopHat version 2.0.9 (ref. µ L of each clarified yeast lysate was was lysate yeast clarified each of L genome (S288C, SacCer3) from 5 Supplementary Table 2 1 . Fractions containing the 80S Cells were grown to mid-log 59– strains used in this study 6 ∆ 1 . GO-term enrichment Rps26 ribosomes, and µ S. cerevisiae We used existing data g/ml cycloheximide 51 , 6 We generated 3 , except that reference 6 4 . ). HT 600 of - - - -

each gradient fraction, TU of each gene in Rps26-replete cells from the TU in Rps26-depleted cells. For over all of the gradient fractions. We obtained the values those summingsampleand that in bound ribosomes ofnumber the by lationalbymultiplyingunits(TU) mRNAthe content gradienteachsample in content to represent the fraction of mRNA in that sample. We calculated trans mRNAcontent, normalized RNaseto levels, Pand divided by totalmRNA the for that gradient. This each sample by comparing each sample’s ers listed in master mix per the manufacturer’s instructions on a BioRad IQ2, usingper thethe manufacturer’sprim instructions. qPCR was performed with Excella 2× SYBR reverse transcription was carried out with Protoscript II (New England Biolabs) chloroform extraction from a fixed percentage of each sample’s total to volume,each sample, and which was used for normalization, RNA was isolated by phenol- additionofng0.65of final two samples contained 10–11 and 12–13 ribosomes, respectively. After the omes. We were able to clearly resolve peaks for up to nine bound ribosomes. containing(iv–xiii)eachmonosomes, two,one four,andthree,The80S ribos etc. were collected: (i) unbound RNAs, (ii) free 40S and 60S ribosomal subunits, (iii) 40,000 r.p.m. for 165 min. Gradients were at SW-41Tifractionated, (Beckman) rotoran in spun and gradient sucroseand 25–45% a ontothe following samples described above. 5,000 OD units of clarifiedas lysatefractionation (~50 sucrose-gradient for prepared then were and phase mid-log with TET-Rps26 yeast at an OD depletion of Rps26 by inoculating YPD media (with or without 0.2 Analysis System (Growth Curves USA) or with a Synergy 2 multi-mode multi-mode 2 Synergy a with microplate or reader (BioTek). USA) Curves (Growth System Analysis Curve Growth Microbiology Automated C Bioscreen the in measured were with NaOH. were Cells grown at 30 °C with rapid shaking, and doubling times cells were grown in YPD + stress,100 mM high-pH TAPSFor caffeine. bufferedmM 10 to pH+ 7 YPD (no or stress)NaCl, orM pH 1 8.2 + YPD follows: as cultures) at OD 0.1 to test stress tolerance. The composition of stress media was as indicated. Cells in mid-log phase were transferred to stress media (or control grown and depleted of Rps26 as described above with either 0.2 or 0.1 Stress-response growth curves. turer’s protocol, with assay volumes scaled down to 15%. onPerkinElmera EnVision 2104 Multilabel Readeraccording to themanufac activity was measured with the Promega Dual-Luciferase Reporter Assay System detailed below, and harvested in mid-log phase. Cells were lysed, and luciferase were grown for cells 4 h WT under stressstress, conditions under (or assays in minimalluciferase media Foras controls),transcription. mRNAas on effects ferences did not arise from differential translation, although we cannot exclude addition( demonstrated that the copy numbers of these plasmids did not change upon dox Rps26 as described above and harvested in mid-log phase. Control experiments Supplementary Fig. 4a (listedin sequences six ofprecededonebyferase firefly luciferase preceded by a 10-adenosine upstream sequence and assay.Luciferase pKK3968. Both strains were grown as described for TET-Rps26.with yKK489 transforming by cells TET-Rps17 and pKK4015, with yKK493 Rps15.Rps3andof Depletion fied RNA was reverse-transcribed and analyzed by qPCR as described above. mid-log phase by hot phenol extraction. After ethanol precipitation, 1 fromRNA.totalqPCR cellular Fraction TU mR = NA ∑ of conten (Fractio SupplementaryFig. 4c mR Supplementary Table 3 NA t TET-Rps26 cells were transformed with plasmids encoding plasmids with transformed were TET-Rps26cells no = in f mRNA 2 each ( ∆ C invitro C ). Cells were then grown in selective media, depleted of v t nature structural & molecular biology molecular & structural nature t gradient was then transformed into arbitrary reference units of alue 10 in of –transcribed RNase P RNARNase–transcribedP from 600 each × We generatedTET-Rps3 transformingbycells ggradient For stress-tolerance tests, TET-Rps26 cells were Total RNA isolatedwas fromgrowing cells in of 0.05. Cultures were grown until they reached ( ), ensuring), thatdox-dependent observed dif samp RNas samp . We calculated the percentage of mRNA in le samp C eP le t = value to the smallest ( ) normalizatio × ∑ le ( ) Nu mR − mb Minimu ∆ SupplementaryTable 1 NA TU value by subtracting the er mR conten of µ nc l of lysate) were loaded t m ribo NA oefficien C o t conten so valu C e f me t Bacillussubtilis value obtained eo ntir si Renilla t g f µ t f n ) µ µ eg g/ml dox) radien g of puri g/ml dox ract radien iion) luci and t ) t ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. indicated in the respective figure legends. Unpaired, two-tailed, Student’s analysis. Statistical ribosomal subunits. were tested against yeast lysates antibodies and either recombinantfour protein These or Peptide.purified 40S England New by protein each from peptides against raised wereRps26 and Rps10 to Antibodies LLC.Josman, proteinsby recombinant purified against raised were Rcl1 and Fap7 to Antibodies Iowa. UniversitymaintainedTheandat NIHof the ofNICHD the createdby Bank, DevelopmentalHybridomaStudiesthe fromobtained J.Frankel, wasby oped Tub1,devel against antibody monoclonal The Vanderbilt(Asc1).University di Parma (Rps8) were gifts from M. Seedorf, Universität Heidelberg (Rps3), G. Dieci, Università Scientific. Anti-Rps5 was from ProteinTech (16964-1-AP). Polyclonal antibodies (HA.C5) from Abcam (ab18181). Anti-TAP (CAB1001)Hog1-p detect was to from used wasThermo Fisher Antibodies. purified as previously described into stress media at a starting OD of 0.7 and harvested after 4 h. Ribosomes were intodifferentwere beforephasemid-loginoculatedseeded Cells being media. purificationcells. fromRibosome stressed with pKK3678 (3HA-Rim101). pathway activation was tested as described or 500 mM NaCl for 5 min, collected, and analyzed by western blotting. Rim101 above. Cells were then transferred to YPD containing 0 mM, 100 mM, 300 mM Rps26 cells to mid-log phase at 30 °C in YPD with or without dox, as described Stress pathway activation. nature structural & molecular biology molecular & structural nature The phospho-p38 (D3F9) antibody from Cell Signaling Technologies 6 8 , L. Valášek, Czech Academy of Sciences (Rps0) and A. Link, Various statistical tests were used as appropriate and as and appropriate as used were tests Variousstatistical We tested HOG pathway activation by growing TET- 6 7 . HA-tagged Rim101 was detected with anti-HA with detected was HA-taggedRim101 . 6 6 . 3 9 with TET-Rps26 cells transformed BY4741 yeast cells wereyeastcellsBY4741grown to t tests -

68. 67. 66. 65. 64. 63. 62. will be made available upon request. Figures1 under accession code Data availability statement. reporter construct) were tested. parisons was used for data sets in which two factors (for example, cell type and used. Finally, a two-way ANOVA with Holm–Sidak correction for multiplechange),(no of1 nonparametric the two-tailedWilcoxoncom testrankwas signed hypotheticalForvalueratestestingachanges from growth-curveused. was in not an assumption or normality, the nonparametric Kolmogorov–Smirnov test was there which in those or sets, datalargerFor sets. data small on used were A

Dieci, G., Bottarelli, L. & Ottonello, S. A general procedure for the production of production the for procedure general A S. Ottonello, & L. Bottarelli, G., Dieci, aiaa M, iaa A, eahm, . Tkhr, . Mea T Sphingolipids T. Maeda, & T. Takahara, M., Terashima, A., Kihara, M., Tanigawa, and Reconstitution change J.R. Lorsch, & S.F.,J.S. Mitchell, Nanda, S.E., fold Kolitz, Acker,M.G., of estimation Moderated S. Anders, & W. Huber, M.I., Love, with junctions splice TopHat:discovering S.L. Salzberg, & L. Pachter,Trapnell, C., B.S. Strunk, M.S. Longtine, euae h yat ihomlrt gyeo rsos pathway. response glycerol high-osmolarity yeast the regulate initiation. translation yeast of DESeq2. with data RNA-seq for dispersion RNA-Seq. intermediates. assembly 40S by initiation in modification and deletion gene 555–560 (2005). 555–560 proteins. ribosomal eukaryotic against reagents antibody (2012). 2861–2870 (1998). Life Sciences Reporting Summary – 6 are available with the paper online. Other data and custom scripts Bioinformatics t al. et et al. et GSE8620 iooe seby atr peet rmtr translation premature prevent factors assembly Ribosome Additional modules for versatile and economical PCR-based economical and versatile for modules Additional

25 Sequencing data have been deposited at NCBI-GEO , 1105–1111 (2009). 1105–1111 , 3 . Source data for Methods Enzymol. Methods Saccharomyces cerevisiae Saccharomyces for this article is available. Science Genome Biol. Genome Figures 1

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– 15 rti Pp. Lett. Pept. Protein 5 and . , 550 (2014). 550 , 10.1038/nsmb.3442 Yeast o. el Biol. Cell. Mol. Supplementary

14 , 953–961 ,

12 32 - , , nature research | life sciences reporting summary Corresponding Author: Katrin Karbstein Date: Jun 8, 2017

Life Sciences Reporting Summary Nature Research wishes to improve the reproducibility of the work we publish. This form is published with all life science papers and is intended to promote consistency and transparency in reporting. All life sciences submissions use this form; while some list items might not apply to an individual manuscript, all fields must be completed for clarity. For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Research policies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist.

` Experimental design 1. Sample size Describe how sample size was determined. For sequencing, ribosome purification and mRNA shift experiments, sample size was limited by the cost of assays. For growth curves and other experiments, the n was chosen to be large enough to identify any biologically relevant phenotypes. 2. Data exclusions Describe any data exclusions. For quantification of western blot images, bands were excluded if they were distorted or obscured by a transfer or blotting artifact. All other data was included. 3. Replication Describe whether the experimental findings were reliably reproduced. Experimental findings for pull downs, mRNA shift assays, phenotypic assays, etc were successfully replicated with minor variance between experiments (which is included in the data and accounted for via the indicated statistical tests). 4. Randomization Describe how samples/organisms/participants were allocated into Yeast strains used for this experiment were clonal. When different experimental groups. treatments were applied to a strain, cells always originated from a common culture. 5. Blinding Describe whether the investigators were blinded to group allocation No, data analysis was performed by the same researcher who performed during data collection and/or analysis. the experiment so blinding was not possible. Instead, all data analysis was performed using a standard set of steps that were held consistent between samples. Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used. June 2017

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` Software Policy information about availability of computer code 7. Software Describe the software used to analyze the data in this study. Statistical tests were performed with GraphPad Prism 6, sequencing analysis is described in the methods section, upstream sequences of genes were retrieved using a custom script that is available upon request. For all studies, we encourage code deposition in a community repository (e.g. GitHub). Authors must make computer code available to editors and reviewers upon request. The Nature Methods guidance for providing algorithms and software for publication may be useful for any submission.

` Materials and reagents Policy information about availability of materials 8. Materials availability Indicate whether there are restrictions on availability of unique All strains and plasmids generated for this study are freely available from materials or if these materials are only available for distribution by a our lab. Any commercial reagents are noted in the methods section. for-profit company. 9. Antibodies Describe the antibodies used and how they were validated for use in All antibodies are described (with references or product numbers, as the system under study (i.e. assay and species). appropriate) in the methods section (page 22). 10. Eukaryotic cell lines a. State the source of each eukaryotic cell line used. All yeast strains used are described in supplementary table 3

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