Mutations of ribosomal protein genes induce overexpression of catalase in Saccharomyces cerevisiae

Abstract Ribosome assembly defects result in ribosomopathies, primarily caused by inadequate protein synthesis and induced oxidative stress. This study aimed to investigate the link between deleting one ribosomal protein gene (RPG) paralog and oxidative stress response. Our results indicated that RPG mutants exhibited higher oxidant sensitivity than the wild type (WT). The concentrations of H2O2 were increased in the RPG mutants. Catalase and superoxide dismutase (SOD) activities were generally higher at the stationary phase, with catalase showing particularly elevated activity in the RPG mutants. While both catalase genes, CTT1 and CTA1, consistently exhibited higher transcription in RPG mutants, Ctt1 primarily contributed to the increased catalase activity. Stress-response transcription factors Msn2, Msn4, and Hog1 played a role in regulating these processes. Previous studies have demonstrated that H2O2 can cleave 25S rRNA via the Fenton reaction, enhancing ribosomes’ ability to translate mRNAs associated with oxidative stress-related genes. The cleavage of 25S rRNA was consistently more pronounced, and the translation efficiency of CTT1 and CTA1 mRNAs was altered in RPG mutants. Our results provide evidence that the mutations in RPGs increase H2O2 levels in vivo and elevate catalase expression through both transcriptional and translational controls.


Introduction
The ribosome is a large complex composed of ribosomal RNAs and ribosomal pr oteins r esponsible for cellular tr anslation.The entir e process of ribosome synthesis, spanning from the nucleolus to the c ytoplasm, inv olves the concerted efforts of hundreds of transacting factors and small nucleolar RNAs (snoRNAs).These factors play crucial roles in assisting with rRNA processing, the assembly of ribosomal proteins, and the ov er all r eor ganization of the ribosomal complex (Pena et al. 2017, Bassler and Hurt 2019, Klinge and Woolford 2019 ).Ribosome synthesis is a highly energy-intensive process that demands precise regulation, especially in response to variations in nutrient availability and environmental stresses (Matos-P er domo and Machin 2019 ).
Defects in ribosome biogenesis or mutations in ribosomal proteins have been associated with a range of human congenital diseases, collectiv el y r eferr ed to as ribosomopathies (Kang et al. 2021 ).Diamond-Blackfan anemia (DBA), for instance, is an inherited bone marrow failure syndrome characterized by symptoms such as anemia, congenital malformations, h ypotroph y at birth, and growth retardation.In DBA, the involvement of 20 ribosomal protein genes (RPGs) has been identified (Da Costa et al. 2020 ).Another ribosomopathy is the Shwachman-Diamond syndr ome (SDS), whic h is caused by m utations in the Shwac hman-Bodian-Diamond syndrome (SBDS) gene.In yeast, the homolog of SBDS, Sdo1, has been identified as a critical release factor of the biogenesis factor Tif6 (Menne et al. 2007 ).This yeast model is consistent with observations in SDS, where ribosome assembly is defective (Wong et al. 2011 ).Additionally, a connection be-tween ribosomal defects and cancer has been r e v ealed.Mutations in ribosomal proteins and variations in ribosomal RN A (rRN A) copy numbers have been found in various types of tumors (Kampen et al. 2020, Pecor ar o et al. 2021, Elhamamsy et al. 2022 ).Beyond impairing translation, these ribosomal mutations may alter the translation efficiency of oncoproteins, disrupt translationindependent functions of ribosomal proteins (Zhou et al. 2015 ), affect energy metabolism, and generate significant o xidati ve stress (Kampen et al. 2020 ).Consequentl y, tar geting ribosome biogenesis has emerged as a potential therapeutic strategy (Jiao et al. 2023 ).
Proper ribosomal functioning is closely associated with maintaining cellular redox homeostasis (Kampen et al. 2020 ).Cells with ribosomal defects often exhibit increased levels of reactive oxygen species (ROS) (Ambekar et al. 2010, Turi et al. 2019 ).In pancreatic cancer cells, ribosomal protein Rpl10 (uL16) has been identified as a regulator of mitochondrial ROS levels (Yang et al. 2018 ).The RPL10-R98S m utation, whic h is associated with leukemia, enhances peroxisome activity, leading to ele v ated ROS le v els (Kampen et al. 2019 ).ROS encompass byproducts generated during the incomplete reduction of oxygen, including superoxide anion (O 2 − ), hydr ogen per oxide (H 2 O 2 ), and hydr oxyl r adical (HO • ).These unpaired ROS can potentially damage cellular macromolecules such as proteins , lipids , and DNA (Imla y 2003 ).ROS can act as signaling molecules at low concentrations that control intracellular ROS homeostasis (D'Autreaux and Toledano 2007 ).Howe v er, at higher concentrations, cells acti vate antio xidant mechanisms or may e v en initiate cell death via a poptosis (Perr one et al. 2008 ).
Mitoc hondria, primaril y r esponsible for po w ering cells, are a major intracellular source of ROS (Zorov et al. 2014 ).
In response to stress, several transcription factors play a crucial role in reprogramming gene expression in yeast (Jamieson 1998 ).Y ap1, Y ap2, and Gcn4 are bZip-type transcription factors with DNA-binding and dimerization domains (Fernandes et al. 1997 ).Ya p1 m utants exhibit r educed activities of se v er al antioxidant enzymes, including super oxide dism utase (SOD), glucose-6phosphate dehydrogenase, and glutathione reductase (Schnell et al. 1992 ). Msn2 and Msn4, which share a 66% sequence homology and contain zinc finger binding motifs at the C-terminus (Estruch and Carlson 1993 ), are STRE (stress-responsive element)-binding pr oteins r equir ed for activ ating CTT1 , DDR2 , and HSP12 (Sc hmitt and McEntee 1996 ).Mutants of Msn2 and Msn4 display heightened sensitivity to v arious str esses, including carbon source starv ation, heat shoc k, osmotic str ess, and o xidati v e str ess (Estruc h and Carlson 1993 ).Hog1 is a mitogen-activated protein kinase that responds to hyperosmolality (Brewster et al. 1993 ).In the presence of ele v ated le v els of ROS, cleav a ge of 25S rRNA occurs at the ES7 r egion, r esulting in pr efer ential tr anslation of mRNAs r equired for the o xidati ve stress response (Shedlovskiy et al. 2017 ).It is worth noting that this specific cleav a ge is an iron-dependent c hemical r eaction r ather than an enzymatic mechanism (Zinskie et al. 2018 ).
In our study, we observed a significant increase in catalase activity among the RPG mutants during the stationary phase.Inter estingl y, this ele v ated catalase activity could be reduced by the addition of vitamin E but not by vitamin C. We identified the transcription factors Msn2, Msn4, and Hog1 as k e y regulators of this pr ocess.Furthermor e, our anal ysis r e v ealed a higher pr oportion of 25S rRNA cleav a ge in the RPG mutant strains, indicating an increase in o xidati ve stress.Ad ditionally, we observed an enhanced translation efficiency of catalase genes, specifically CTT1 and CTA1 .These findings suggest that RPG mutations induce oxidativ e str ess and activ ate the str ess r esponse thr ough both tr anscriptional and translational regulatory mechanisms.

Strains and media
All Sacc harom yces cerevisiae str ains used in this study are listed in Table S1 (Supporting Information) .The strains derived from the Open Biosystems wer e v alidated by PCR to ensure the module had integrated at the correct positions before use (Longtine et al. 1998 ).To generate the deletion str ains, the PCR pr oducts containing the KanMX (kanamycin resistance gene) (or clon-NAT, nourseothricin sulfate resistance gene) and the flanking regions wer e tr ansformed to the tar get str ain, and the colonies that sho w ed antibiotic resistance were picked up.The correct genome replacement of the gene with KanMX (or cloN AT) w as examined with PCR (Longtine et al. 1998 ).
For the tr ansformation pr ocess, yeast cells in the log growth phase wer e harv ested and subsequentl y washed once with Li/TE (100 mM lithium acetate , 10 mM Tris , and 1 mM EDTA).T he cells were then mixed with carrier DNA (single-strand DNA), purified PCR products, and PEG/Li/TE, follo w ed b y thorough mixing.The mixture was incubated for 30 min at 30 • C and then for an additional 15 min at 42 • C. Finally, the cells were spread onto the selective plates.
Unless otherwise indicated, all strains were grown at 30 • C in a rich medium (yeast extract peptone) or synthetic dropout medium containing 2% glucose .T he o v ernight cultur e was subcultured in the fresh medium and grown until the OD 600 reached 0.3-0.5 (log phase), 1.0 (late log phase), or 4.0-6.0(stationary phase).

Growth test
T he o v ernight cultur es wer e normalized to OD 1 and subjected to 10-fold serial dilution.A volume of 5 μl from each dilution was spotted on the various plates: yeast extract peptone dextrose (YPD) plates, YPD plates with 0.3 mM menadione or 5 mM H 2 O 2 , YPGal (yeast extract peptone galactose) plates or YPGal plates with 5 mM H 2 O 2 .Menadione is a super oxide-gener ating r ea gent that triggers cell o xidati v e str ess (Kim et al. 2011 ).

Western blotting
To detect the tar get pr oteins thr ough western blotting, the proteins separated b y SDS PAGE w ere transferred to a PVDF membrane (Bio-Rad) using a semidry transfer device (Bio-Rad).Membr anes wer e incubated with TBST solution (Tris-buffer ed saline with 0.1% Tween ® 20 detergent) containing primary antibodies.Anti-GFP (Thermo) and anti-TAP (Thermo) were purchased; anti-Act1 was derived from the Taiw an y east resour ce center.anti-uL8 and anti-eS24 were generated in this lab.Protein signals were detected by Clarity TM ECL Substrate (Bio-Rad).Images were acquired with MultiGel-21 (T opBio, T aiwan).

RN A prepar a tion and northern blotting
Total RN A w as extr acted fr om yeast cells using the hot phenol method (Collart and Oliviero 2001 ), and RN A w as resolved in the formaldehyde a gar ose gel and tr ansferr ed to a nitrocellulose membrane .T he probes were labeled with a Biotin 3end labeling kit (Thermo) and continually hybridized and detected with North2South ® Chemiluminescent hybridization and detection kit (T hermo).T he probe sequence is listed in Table S2 (Supporting Information) .

Quantitati v e polymerase chain reaction (qPCR)
The cDN As w er e synthesized with a High-Ca pacity cDNA Re v erse Transcription Kit (Thermo).Real-time qPCR (RT-qPCR) was performed with a Po w er SYBR Green PCR Master Mix (Thermo) in a Real-Time PCR machine (Bio-Rad).ACT1 was used as the internal control.Table S2 (Supporting Information) lists the primer sequences.

Sucrose gradient analysis
The cell extracts were separated through a sucrose gradient to separ ate the tr anslated and nontr anslated mRNAs.Yeast cells were collected at different growth stages .Cycloheximide , a translation inhibitor, was added at the final 50 μg/ml concentration before cell collection to stabilize polysome structures .P olysome lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl 2 , 6 mM β-mercaptoethanol, and 200 μg/ml cycloheximide) was used for the pr epar ation of pr otein extr acts.10.5 OD 260 units of pr otein extr acts wer e loaded onto linear 7%-47% sucr ose gr adients and spun at 40 000 rpm in a r otor (SW40; Bec kman) for 2.5 h.Gradient fractions were collected on a density gradient fraction system (Brandel), continuously measuring absorbance at 254 nm.A total of 10% TCA was added to eac h fr action for pr otein pr ecipitation.The pr otein pellets were dissolved in 1x SDS sample buffer.Samples were r esolv ed by SDS-PAGE and detected by western blotting.A volume of 500 μl from each fraction was extracted with Trizol (Thermo) to analyze the RN A levels.RN A w as precipitated with ethanol and dissolved in the DEPC w ater.The RN A le v els wer e measur ed with qPCR.

ROS measurement
1.5 OD yeast cells of WT or RPG mutants were collected at different gr owth sta ges .T he cells were incubated with 10 μM DCFDA (2 ,7 -dic hlor odihydr ofluor escein diacetate, Sigma) for 30 min in the dark.DCFDA is a cell-permeable dye, which is converted to fluor escent after cleav a ge by intr acellular ester ases and oxidized b y R OS.After w ashing once with PBS (phosphate buffered saline), cells were incubated in 2 M lithium acetate for 2 min and then in 0.01% SDS and 0.05% c hlor oform for 2 min.After centrifugation, the fluorescence intensity of the supernatant was measured with the plate reader (SpectraMax ® iD3, Molecular Device) (James et al. 2015 ).The same amount of cells under the same process without DCFDA staining was used for bac kgr ound fluor escence measur ement.
To measure the H 2 O 2 production rate, 0.5 OD cells were resuspended in the solution containing 50 μl 50 mM sodium phosphate buffer (pH = 7.4), 50 μl 50 μM Amplex red (Thermo), and 0.1 U/ml HRP (Sigma).The fluorescence intensity was measured at excitation/emission 571/585 nm in the plate reader at different time points .T he H 2 O 2 production amount in 1 min was calculated.

Microscopy
Cells at the log and stationary phases were harvested and examined with fluorescence microscopy to image the cellular distribution of recombinant GFP (green fluorescent protein) protein.Fluorescence was visualized on a microscope (AxioScope A1; Zeiss) fitted with a Plan Apoc hr omat 100 × 1.40 NA DIC objective and a digital micr oscopy camer a (AxioCam MRm Re v. 3) contr olled with AxioVision LE module Fluorescence Lite (Zeiss).Images were prepared using Photoshop (version 7.0; Adobe).

Zymography
Zymogr a phy was used to detect the activities of catalase and SOD.Yeast cells at the log or stationary phase were harvested and lysed with glass beads in PBS.To test if antioxidants would quench the induction of catalase, uL11a and uL11b wer e cultur ed in the YPD medium with different concentrations of ascorbic acid (vitamin C) or α-tocopherol (vitamin E) to the stationary phase.To determine which catalase was induced in the RPG mutants, ctt1 and cta1 were included in the study.
After measuring the concentrations of proteins with Bradford assay, a fixed amount of pr oteins wer e loaded in 6% native PAGE gel and run at 80 V for 20 min and then 120 V for 100 min at 4 • C. For catalase detection, the gel was incubated with 10 mM H 2 O 2 for 15 min, and then 1% K 3 [Fe(CN) 6 ] and 1% FeCl 3 for 15 min.For SOD detection, the gel was incubated in 0.1% NBT (nitroblue tetrazolium) for 15 min.After washing with ddH 2 O once, the gel was incubated in the 0.1 M potassium phosphate buffer (pH = 7.8) containing 22 mM TEMED (N ,N ,N ,N -Tetrameth yleth ylenediamine) and 28 μM riboflavin for 15 min under the light.The WT sample was included in e v ery gel as a control, and the relative ratios wer e compar ed to WT on the same gel.The intensity of bands was quantitated with Image J.Each assay was performed at least twice .T he same pr epar ations of samples wer e also r esolv ed in SDS PAGE and detected with Coomassie blue staining as the loading controls.

Flow cytometry
To measur e mitoc hondrial membr ane potentials, cells wer e stained with JC-1.JC-1 is a dye that could be accumulated in the mitochondria via the membrane potentials: cells appear orange or green in color when the membrane potentials accumu-late higher or lo w er JC-1 amounts, r espectiv el y. 0.1 OD cells of WT and RPG mutants were collected at different growth stages and resuspended in 1 ml PBS containing 5 μM JC-1 for 30 min (Smiley et al. 1991 ).After washing twice, the cells wer e anal yzed with flow cytometry (Cytomic FC 5000, Beckman Coulter).A total of 10 000 cells were counted for each test.The analyses were repeated twice independently.

Sta tistical anal ysis
In qPCR analysis, the data were shown as mean ± SD, with three inde pendent re plicates ( n = 3) for each condition.Statistical analysis was performed using analysis of variance (ANOVA), follo w ed by a post hoc least significant difference (LSD) test for multiple comparisons.Significance le v els wer e indicated: * P < .05,* * P < .01,and * * * P < .001.A two-tailed Student's T -test was employed for pairwise comparisons between eac h m utant and the wild type (WT) at the same gr owth sta ge to assess statistical significance in these cases ( * P < .05,* * P < .01,and * * * P < .001).The analyses were performed in the Microsoft Excel 2019.

RPG mutants have a higher sensitivity to oxidati v e stress
Sacc harom yces cerevisiae has a haploid genome, but among the 78 RPGs , 59 ha ve two copies , designated as paralogs , such as uL11a and uL11b.Although these paralogous genes possess nearly identical amino acid sequences, differences in their 5 untranslated r egions (UTRs), intr ons, and 3 UTR sequences lead to distinct beha viors (K omili et al. 2007 ).Se v er al par alogs wer e selected in this study.Mutations in uL6a/6b , uL11a/11b , eL24a/24b , and eS24a/24b hav e pr e viousl y been associated with DBA, wher eas m utations in uL2a/2b , eL8a/8b , uL30a/30b , and eL43a/43b have not been reported (Danilova and Gazda 2015 ).As a result, a comparison of phenotypes between these two groups can offer valuable insights.
To assess the sensitivity of RPG mutants to o xidati v e str ess, we conducted growth experiments on YPD plates containing varying concentrations of menadione and H 2 O 2 (Fig. 1 A).Most RPG mutants exhibited a gr owth r ate similar to or slightly slo w er than the WT on YPD plates; ho w e v er, their gr o wth w as significantly impaired in the presence of oxidants, indicating heightened sensitivity to oxidative stress .T his trend persisted when we substituted the carbon source with another fermentative carbon source, galactose (Fig. 1 B).
For further insight into cellular ROS le v els, WT and RPG m utants were subjected to staining with DCFDA (Fig. 1 C).Notably, the cells in the stationary phase displayed lower ROS, suggesting a correlation between ROS production and growth stages.Compared to WT, the mutants sho w ed lo w er R OS at the log phase but similar le v els at the stationary phase (Fig. 1 C).To quantify H 2 O 2 pr oduction r ates, cells wer e also stained with Amplex r ed (Fig. 1 D).Inter estingl y, man y RPG m utants exhibited a higher r ate of H 2 O 2 generation during the log phase than WT (Fig. 1 D).

RPG mutants present higher catalase activity
Yeast employs nonenzymatic and enzymatic antioxidant defense systems to safeguard cells and maintain cellular redox balance.Among the major enzymatic systems responsible for ROS remov al ar e SOD and catalase.We assessed SOD and catalase activities during the log and stationary growth phases to investigate whether RPG mutants exhibit elevated levels of these antioxidant enzymes.The deletion of each paralog, a or b , was labeled as "a" or "b" on the figure.(A) 100 μg cell lysates were prepared from the log phase, and 30 μg cell l ysates wer e pr epar ed fr om the stationary phase .T he SOD activity was anal yzed with zymogr a phy.(B) 100 μg cell l ysates wer e pr epar ed fr om the log phase, and 50 μg cell lysates were prepared from the stationary phase .T he catalase activity was analyzed with zymography.(C) uL11a and uL11b wer e cultur ed in the YPD medium with differ ent concentr ations of ascorbic acid or α-tocopher ol to the stationary phase .T he catalase activity was measured with zymography.The coomassie blue (CB) staining gels were included as the loading controls.Each assay was done independently at least two times .T he r elativ e r atios compar ed to WT wer e indicated in eac h figur e.
In general, most RPG mutants sho w ed a similar or slightly increased SOD activity to the WT (Fig. 2 A).While neither WT nor the mutants in the log phase exhibited significant catalase activity, the catalase activity in most RPG mutants was 2-5 times higher than that of WT during the stationary growth phase (Fig. 2 B).These results indicate that RPG mutations induce a more pronounced increase in catalase activity than SOD activity, consistent with our earlier observation of higher le v els of H 2 O 2 within the mutants (Fig. 1 D).
Given the strong induction of catalase observed in most strains (Fig. 2 B), we selected uL11a and uL11b for further examination.These mutants exhibited similar growth rates and sensitivity to oxidants (Fig. 1 A and B) but displayed the most substantial differences in ROS and H 2 O 2 le v els (Fig. 1 C and D).To assess whether the addition of antioxidants could suppress the overexpression of catalase, we introduced various concentrations of ascorbic acid (Vit C) and α-tocopherol (Vit E) into the cultures.Vit C, being water-soluble, and Vit E, an efficient lipid-soluble antioxidant that terminates lipid peroxidation (Nimse and Pal 2015 ), were chosen for this pur pose.Notabl y, the addition of V it E, but not V it C , led to decr eased catalase le v els (Fig. 2 C), suggesting that the incr ease in catalase expression was partially induced by ROS and lipid oxidation.

RPG mutants show higher transcription and protein levels of the catalase
Ther e ar e two types of catalase in yeast: catalase T (Ctt1) in the cytosol and catalase A (Cta1) in peroxisomal and mitochondrial matrices (Cohen et al. 1985, Petr ov a et al. 2004 ).To determine which catalase was induced in the RPG mutants, the catalase zymogr a phy was examined in ctt1 , cta1 , and the combining mutants of ctt1 uL11a and cta1 uL11a .Remarkabl y, onl y the ctt1 strain displayed a significant loss of catalase activity (Fig. 3 A).Subsequentl y, the pr otein quantities of Ctt1 and Cta1 wer e also c hec ked in WT, uL11a , and uL11b strains.In the mutant strains, both proteins demonstrated elevated levels, with Ctt1 displaying a mor e pr onounced incr ease during the stationary phase, wher eas Cta1 exhibited consistent trends in both the log and stationary growth phases (Fig. 3 B).The transcription levels of CTT1 and CTA1 were also checked: the mutants sho w ed higher expressions than WT, and the increase of CTT1 was mor e pr onounced than CTA1 at the log phase (Fig. 3 C) but not at the stationary phase ( Figure S2, Supporting Information ).
The protein levels of two SODs were also examined.Sod1 is a Cu-Zn SOD localized in the cytosol and mitochondrial intermembrane space (Bermingham-McDonogh et al. 1988 ), and Sod2 is a mitochondrial MnSOD (van Loon et al. 1986 ). Sod1 levels remained constant in both WT and mutant strains at different growth phases, while Sod2 exhibited higher expression levels at the stationary phase, with slightly elevated levels in the mutants (Fig. 3 D).These findings align with the insignificant increase in SOD activity observed in the RPG mutants.
The western blotting results of catalase or SOD were consistent with the zymogr a phy data (Fig. 2 ).Consequentl y, the RPG m utants displayed upregulated protein levels and activity of catalase, particularl y e vident during the stationary phase, with Ctt1 showing the most substantial changes among these enzymes.

T he tr anscription factors Msn2, Msn4, and Hog1 are responsible for the upregulation of catalase
Se v er al tr anscription factors involv ed in str ess r esponse wer e deleted to anal yze whic h tr anscription factors ar e the major str ess sensors of the RPG mutants, and the catalase activity was observed at the stationary phase.msn2 , msn4 , hog1 , and zap1 strains sho w ed much lo w er catalase activity (Fig. 4 A).The tran-scription le v els of CTT1 and CTA1 wer e further c hec ked in the msn2 , msn4 , hog1 , and zap1 mutants, as well as the double m utants cr ossed with uL11a , to demonstr ate the r elationship.The RNA le v els of CTT1 wer e decr eased in msn2 , msn4 , and hog1 but not in zap1 .In contrast, the CTA1 RNA levels bar el y c hanged in these mutants (Fig. 4 B).The catalase le v els wer e also examined in these stains, and it was found that the absence of MSN2 , MSN4 , and HOG1 decreased catalase activity in uL11a (Fig. 4 C).
Man y tr anscription factors involv ed in str ess r esponse ar e induced by str ess, r esulting in an incr ease of the pr otein le v els or r elocalizing to the nucleus (Gorner et al. 1998, Westfall et al. 2004 ).The protein levels and cellular distributions of Msn2 and Hog1 wer e tr ac ked in the WT and m utants at differ ent gr owth sta ges.The Msn2-GFP (Rajvanshi et al. 2017 , Mizuno andIrie 2021 ) and Hog1-GFP (Reiser et al. 1999 ) are regularly used to monitor protein status in response to stress or regulation.T herefore , Msn2-GFP and Hog1-GFP were examined in the WT, uL11a , and uL11b mutants.
The pr otein le v els of Msn2 in the m utant str ains wer e similar to those in the WT at the log phase and slightly decreased at the stationary phase (Fig. 4 D).Msn2 was distributed in both the cytoplasm and nucleus in the WT and became more concentrated in the nucleus in the uL11a and uL11b mutants at the log and stationary phases (Fig. 4 E).Hog1 le v els r emained unc hanged in both the WT and mutants at the log phase but increased in the mutants at the stationary phase (Fig. 4 F).Hog1 exhibited a stronger cytoplasmic intensity in the WT, and its nuclear intensity was higher in the mutant strains at the stationary phase (Fig. 4 G).T hus , Msn2 and Hog1 adjusted their cellular localizations, and Hog1 e v en tuned up its pr otein abundances in r esponse to the stress caused by the RPG mutants.The cellular localizations of Msn2-GFP and Hog1-GFP were examined in the cells at different growth stages .T he N/C ratio was calculated from the intensity between the nucleus and cytoplasm from 20 cells.uL11a or uL11b was compared to WT at the same growth stage using the Student's T -test.Ave ± SD. * P < .05 and * * P < .01.

Decrease in tr ansla tion elonga tion triggers ca talase o verexpression
Ribosomal defects lead to an increase in ROS (Ambekar et al. 2010, Turi et al. 2019 ), and mitoc hondria ar e the principal places for ROS pr oduction.The mitoc hondria membr ane potentials wer e measured in each mutant to investigate potential links between RPG m utants and mitoc hondrial status.Among the tested str ains, onl y uL6a , uL6b , and eS24a sho w ed lo w er potential, while others displayed ele v ated potential ( Figur e S1, Supporting Information ).
Ho w e v er, e v ery m utant str ain exhibited higher catalase le v els (Fig. 2 B).Ther efor e, mitoc hondrial status might not be the k e y factor triggering catalase production.
Many important physiological pathways can be affected by ribosomal pr otein defects.Se v er al m utant str ains in the r elated pathw ays w ere selected to assess catalase le v els ( Figur e S3A, Supporting Information ).This information may be correlated with the primary factor triggering catalase production.The TOR complex is the primary sensor of nutrients and upregulates growth-Figur e 5. T he o v er expr ession of catalase in RPG m utants is also r egulated at the tr anslation le v el.(A) The cells wer e cultur ed with differ ent concentrations of CHX to the stationary phase, and the catalase activity was analyzed with zymography.The relative ratios compared to WT were indicated.(B) The growth tests of strains on the plates containing different concentrations of CHX.(C) The position of the probe and the cleavage site at 25S rRNA were indicated.The cleaved 25S rRNAs were analyzed in the cells at log phase, log phase with the treatment of 0.25 mM H 2 O 2 for 1 h, and stationary phase with northern blotting.The cleaved 25S rRNA band was indicated with an asterisk ( * ).(D) The fractions of mRNAs at the translation (the sum of the RNA le v els at the 80S and polysome peaks) were analyzed at different growth stages with qPCR.Each mutant was compared to WT at the same stage using the Student's T -test.n = 2 * P < .05;* * P < .01;and * * * P < .001.related genes, including ribosome biogenesis and protein synthesis (Po w ers and Walter 1999 ).Deletion of Tor1, the component of TORC1, resulted in a slight increase in catalase activity.Gcn4 is the transcription activator that responds to amino acid starvation (Hinnebusch and Natarajan 2002 ), and the catalase activity did not increase in gcn4 .TIF1 and TIF4631 are one of the paralogous genes that code for translation initiation factors, specifically corresponding to eIF4A and eIF4G, respectively; TIF3 is the coding gene of eIF4B (Altmann and Linder 2010 ).The catalase activity of tif1 , tif3 , and tif4631 was e v en lower than that of the WT ( Figure S3A, Supporting Information ).To investigate whether the translation defect caused the decrease in catalase levels, low amounts of cycloheximide (CHX), an inhibitor of translation elongation, were included in the culture and found to trigger catalase activity efficiently.The WT did not sho w gro wth defects at 30 ng/ml CHX (Fig. 5 B), but this dosa ge trigger ed a 2-fold increase in catalase activity.Although the gro wth defects w orsened in a dose-dependent manner, catalase activity plateaued at concen-trations higher than 50 ng/ml CHX (Fig. 5 A).uL11a and uL11b mutants sho w ed higher gro wth defects to w ar d CHX (Fig. 5 B), and CHX e v en induced catalase activity in these mutants (Fig. 5 A).T hus , translation elongation efficiency might be a critical factor triggering catalase ov er expr ession.

Overexpression of catalase in the RPG mutants is also regulated at the translational control level
The pr e vious study sho w ed that higher R OS w ould cleave 25S rRNA and change the mRNA pr efer ence of ribosomes (Shedlovskiy et al. 2017 ).Since H 2 O 2 was higher in the RPG mutants (Fig. 1 D), 25S rRNA might be cleaved in response to stress regulation.RNA was pr epar ed fr om WT, uL11a , and uL11b , and the potential cleav a ge was observed with northern blotting.yap1 , a transcription factor for o xidati v e str ess r esponse, was included as a positiv e contr ol as it has been shown to have 25S rRNA cleav a ge (Shedlo vskiy et al. 2017 ).T he clea v a ge was insignificant at the log phase and enhanced after H 2 O 2 addition.At the stationary phase, compared to the WT, uL11a , and uL11b cells sho w ed lo w er 25S rRNA and higher cleav a ge bands (Fig. 5 C), which suggests the ROS le v els wer e higher in these str ains.
From the data abo ve , disturbance in translation enhanced catalase expression (Fig. 5 A), and the rRN A w as cleaved in the RP mutants (Fig. 5 C).T hus , tr anslational contr ol might be critical in adjusting the protein expressions related to the stress caused by RPG mutations .T he translation profiles were analyzed in the WT and m utant str ains at the log, late log, and stationary phases to dissect the dynamic responses.SOD1 and ACT1 (actin gene) were included for comparison with CTT1 and CTA1 to exclude the potential disturbance of translation from deletion of ribosomal protein.Cell extr acts wer e fr actioned thr ough sucr ose gr adients, and the sedimentation positions of the 40S (fr ac.3), 60S (fr ac.4), 80S (fr ac.5), and polysome (frac.6-11) were checked with western blotting ( Figure S3B, Supporting Information ).RN A w as extracted from eac h fr action and anal yzed with qPCR.The fr actions of nontr anslated (frac.1-4) and translated (frac.5-11) were calculated.The RNA fractions in the polysome were compared between different gr owth sta ges of WT, uL11a , and uL11b str ains (Fig. 5 D).As the contr ols, the fr actions of tr anslated ACT1 and SOD1 wer e lo w er in the RPG mutants at the log phase, and no difference was observed between WT and RPG mutants at the late log or stationary phase .Meanwhile , only 20%-30% of CTT1 and CTA1 mRNAs were translated at the log phase, and this increased to 50%-80% at the late log phase as cells entered the stationary phase, with the RPG mutants exhibiting higher levels than the WT.Activ el y tr anslated CTA1 mRNAs ac hie v ed a plateau and no discrimination among the strains at the stationary phase .T he changes in CTT1 mRNAs wer e differ ent: the tr anslated fr actions wer e incr eased to 80% and 90% in WT and uL11b but decreased in uL11a (Fig. 5 D).
The data above suggest that RPG mutants sho w ed different dynamics to trigger the translation of CTT1 and CTA1 mRNAs: the m utants tr anslated the catalase genes at earlier stages than WT in response to the H 2 O 2 le v els in the cells .T he upregulation of catalase genes in RPG mutants depends on transcriptional and tr anslational contr ols to r egulate o xidati ve homeostasis.

Discussion
Man y par alogs of RPGs wer e included in this study.The slowgr owing RPG m utants showed higher sensitivity to oxidants in the growth test.Howe v er, man y par alogs hav e compar able gr owth rates at normal conditions but behave with different sensitivity to w ar d the oxidants (Fig. 1 A and B).Although most paralogs have an identical amino acid sequence or only one or two amino acid differences , they ma y ha ve unique functions .Mor e and mor e studies have shown that ribosomes are created with heterogeneity.Different ribosomal protein paralogs , rRNA modifications , and posttr anslational modifications gener ate the "specialized ribosomes."They demonstr ate differ ent pr efer ences for specific mRNAs containing r egulator elements, suc h as internal ribosome entry sites or upstream open reading frames, and behave as a regulator for cell homeostasis (Xue andBarna 2012 , Norris et al. 2021 ).
We tried to correlate the catalase activity with the H 2 O 2 gener ation r ates.Ho w e v er, the connections wer e poor.For example, eL43a and eL43b sho w ed similar rates (Fig. 1 D), but eL43a had higher catalase activity; uL30b had higher H 2 O 2 but uL30a had higher catalase (Fig. 2 B).The catalase activity correlated better with gr owth r ates, while eL43a and uL30a had slo w er gro wth rates .T hus , the ROS level or H 2 O 2 production is not the only factor to trigger catalase expression.In our study, we found Vit E but not Vit C could r epr ess catalase ov er expr ession (Fig. 2 C).The dif-ference might be due to their properties: Vit E is lipid soluble, and ViC is water soluble.While Vit E exerts antioxidant effects by scavenging lipid peroxyl radicals, it is not an efficient scavenger of •OH and alk oxyl r adicals ( •OR) in vivo (Nimse and P al 2015 ), whic h suggests that the signaling to induce catalase might be from the lipid sources.
The increase of catalase activity is growth stage-dependent: the activity was bar el y detected at the log phase, but it became obvious at the stationary stage (Fig. 2 B).The previous study shows that pero xisome acti vity is enhanced in the RPL10 m utant, r esulting in ele v ated ROS le v els (Kampen et al. 2019 ).Cta1, the catalase in the peroxisomal matrix, increased in RPG mutants but did not ele v ate fr om the log to the stationary phase (Fig. 3 B).In contr ast, Ctt1 increased at the stationary phase (Fig. 3 B).When CTT1 but not CTA1 was deleted in uL11a , the increase of catalase activity at the stationary phase disa ppear ed (Fig. 3 A right panel).T hus , the growth-dependent catalase activation in RPG mutants is mainly contributed by cytosolic catalase Ctt1.Deletion of CTA1 but not CTT1 induced the catalase le v els, impl ying that the cells may upregulate the CTT1 expression to compensate for the loss of CTA1 , but not vice versa (Fig. 3 A left panel).
The defects in RPG mutants may disturb other pathways besides translation, and ribosome biogenesis and translation are under man y r egulations .T hus , we included the mutants in other pathways in the assay: tor1 , the component of TORC1; gcn4 , the tr anscription activ ator in r esponse to amino acid starv ation; tif1 and tif4631 , one paralog of translation initiation factor eIF4A and eIF4G; tif3 , the coding gene of eIF4B (Altmann and Linder 2010 ).Ho w e v er, no catalase activity was significantly increased as RPG mutants in these strains ( Figure S3A, Supporting Information ).T hus , the ele v ated catalase activity might be unique to RPG mutations.
Out of the transcription factors we inv estigated, onl y the absence of Msn2, Msn4, or Hog1 led to a reduction in both catalase activity and transcription in the RPG mutants .T hese factors are known for recognizing stress-response elements (STRE), like AGGGG or GGGGA, particularly during stress conditions (Stewart-Ornstein et al. 2013 ).Notably, genes such as CTT1 , CTA1 , and SOD1 also contain STRE elements (Rajvanshi et al. 2017 ).Ho w ever, it is important to highlight that only the transcription levels of CTT1 wer e adv ersel y affected.This implies that the r egulatory mec hanisms governing these antioxidant enzymes may differ significantly.
Cells change protein synthesis in response to o xidati ve stress.Besides tr anscriptional contr ol, tr anslational contr ol is also critical to maintaining homeostasis.H 2 O 2 could regulate translation at the initiation, elongation, and termination sta ges (Gr ant 2011 ).In winter rye lea ves , post-transcriptional control regulates the activation of catalase .T he N7-methylation on the cap enhances the translation efficiency of catalase mRNA.Translation activation is induced by blue light and H 2 O 2 (Schmidt et al. 2006 ).A previous study sho w ed that higher ROS le v els cleav ed 25S rRNA and c hanged the mRNA pr efer ence of ribosomes in yeast (Shedlovskiy et al. 2017 ).In our data, uL11b sho w ed higher R OS le v els (Fig. 1 C); indeed, this strain sho w ed a stronger cleavage band than WT and uL11a (Fig. 5 C).Ho w e v er, uL11a sho w ed similar R OS levels to WT (Fig. 1 C) but still sho w ed higher cleav a ge le v els than WT (Fig. 5 C).Another study further indicated that r edox-activ e, ribosome-bound iron potentially promotes the Fenton reaction for rRNA cleav a ge (Zinskie et al. 2018 ).T hus , the mutant ribosome ma y ha v e alter ed ir on le v els or bounding form, triggering the Fenton reaction.Our study found the translation efficiency of the CTT1 and CTA1 genes was enhanced starting from the late log phase .T he translational preference might be from the cleavage of 25S rRNA in RPG m utant str ains.Alternativ el y, the RPG deletion may change the ribosome selection to w ar d certain mRN As (Xue and Barna 2012 ).Or both factors contribute to the translational enhancement.

Figure 1 .
Figure 1.RPG mutants are more sensitive to o xidati ve stress.(A)-(C) Normalized cell cultures were serially diluted and spotted on the plates indicated in the figures at 30 • C. (A) YPD and YPD with 0.3 mM menadione or 5 mM H 2 O 2 .(B) YPGal and YPGal with 1 mM H 2 O 2 .The growth test were done independently twice.(C) and (D) WT and the RPG mutants ( a was shown by a white bar and b was shown by a black bar) at the log and stationary phases were collected.Total ROS le v els wer e detected with DCFDA (C), and the H 2 O 2 production rates were detected with Amplex Red (D).Each assay was done independently three times .T he a verages and standard deviations were shown.Each mutant was compared to WT at the same stage using the Student T test.* P < .05,* * P < .01,and * * * P < .001.

Figure 2 .
Figure 2. RPG mutants showed higher SOD and catalase activity, and treatment of α-tocopherol could reduce the catalase activity in the RPG mutant.(A) and (B) Cells in the log phase (top panel) and stationary phase (bottom panel) were collected, and the normalized whole cell lysates were prepared.The deletion of each paralog, a or b , was labeled as "a" or "b" on the figure.(A) 100 μg cell lysates were prepared from the log phase, and 30 μg cell l ysates wer e pr epar ed fr om the stationary phase .T he SOD activity was anal yzed with zymogr a phy.(B) 100 μg cell l ysates wer e pr epar ed fr om the log phase, and 50 μg cell lysates were prepared from the stationary phase .T he catalase activity was analyzed with zymography.(C) uL11a and uL11b wer e cultur ed in the YPD medium with differ ent concentr ations of ascorbic acid or α-tocopher ol to the stationary phase .T he catalase activity was measured with zymography.The coomassie blue (CB) staining gels were included as the loading controls.Each assay was done independently at least two times .T he r elativ e r atios compar ed to WT wer e indicated in eac h figur e.

Figure 3 .
Figure 3. RPG mutants showed higher transcription and protein levels of the antioxidant enzymes.(A) The catalase activities in the mutant strains at the stationary phase were analyzed with zymography.The relative ratios compared to WT were indicated.The results of two replicates were shown in the left panel.(B) The protein levels of Ctt1 and Cta1 were analyzed with western blotting.The experiments were done twice .T he quantification is indicated below the figure.(C) RNA was pr epar ed fr om the WT and RPG m utants ( a w as sho wn b y a white bar and b w as sho wn b y a black bar), and the transcription level of each gene was analyzed with qPCR.ACT1 was used as the internal control.The data were shown as mean ± SD ( n = 3) and analyzed with ANOVA follo w ed b y an LSD test.* P < .05;* * P < .01;and * * * P < .001.(D) The pr otein le v els of Sod1 and Sod2 wer e anal yzed with western blotting.The quantification is indicated below the figure.

Figure 4 .
Figure 4. Msn2, Msn4, and Hog1 are responsible for the upregulation of catalase.(A) and (C) Cell extracts prepared from different strains at the stationary phase were analyzed with catalase zymography.The relative ratios compared to WT were indicated.(B) RNA was pr epar ed fr om the cells at the log phase .T he transcription levels were analyzed with qPCR.ACT1 was used as the internal control.The data were shown as mean ± SD ( n = 3) and analyzed with ANOVA follo w ed b y an LSD test.For each set, eac h stain was compar ed to WT or uL11a .* P < .05,* * P < .01,and * * * P < .001.(D) and (F) The protein levels of Msn2-GFP and Hog1-GFP were analyzed with western blotting.The relative ratios compared to WT were shown.(E) and (G) The cellular localizations of Msn2-GFP and Hog1-GFP were examined in the cells at different growth stages .T he N/C ratio was calculated from the intensity between the nucleus and cytoplasm from 20 cells.uL11a or uL11b was compared to WT at the same growth stage using the Student's T -test.Ave ± SD. * P < .05 and * * P < .01.