Cyclin CLB2 mRNA localization and protein synthesis link cell cycle progression to bud growth

Domain: physics
Published: 2025-11-26
Editorial outcome: major_revision

Abstract
Clb2 is a conserved B-type cyclin essential for mitotic progression in Saccharomyces cerevisiae, with expression tightly regulated at transcriptional and proteolytic levels. However, it remains unclear whether Clb2 protein synthesis is regulated and responsive to cell growth. Here, we show that CLB2 mRNA localizes to the bud via the She2/She3 complex, while Clb2 protein accumulates in the mother nucleus. This mRNA localization enhances translation without affecting protein localization. A structured RNA element, a ZIP-code, is located within the coding sequence and is required, but not sufficient, for both mRNA transport and protein expression. Mutation of this ZIP code disrupts mRNA localization, reduces Clb2 synthesis, increases budded phase duration and daughter cell size. In wild-type cells, Clb2 protein levels scale with bud growth, a coupling lost in ZIP code mutants. These findings reveal a mechanism by which mRNA localization and translation are coordinated to link cell growth with cell cycle progression.

Introduction
Over the past decades, RNA imaging technologies have revealed that hundreds of mRNAs localize to various subcellular compartments, in organisms ranging from bacteria to multicellular eukaryotes1,2, suggesting that mRNA trafficking is a conserved and integral part of gene expression regulation. Current studies suggest that the primary role of mRNA trafficking is to control asymmetric protein distribution to sustain local functions such as cell migration and polarity1. Even in the single-cell organism S. cerevisiae, dozens of mRNAs localize to the endoplasmic reticulum, mitochondria, and the bud3. The best-characterized localized mRNA is ASH1, which is transported to the yeast bud on actin filaments by the She2-She3 complex and the type V myosin motor Myo44–11. The RNA-binding proteins (RBP) Khd1 and Puf6 bind the ASH1 mRNA and inhibit its translation until the bud-localized kinases Yck1 and CK2 phosphorylate Khd1 and Puf6, releasing the inhibition and allowing local translation to occur12–17. The Ash1 protein is subsequently asymmetrically segregated into the daughter nucleus, where it controls mating-type switching3,7. An additional kinase-RBP pair, Cbk1-Ssd1, has been shown to localize to the bud18 and tune the translation of specific mRNAs19–21.

Besides ASH1, multiple mRNAs have been shown to interact with the She2-She3-Myo4 complex and to localize to the bud22. Among these mRNAs is CLB2, which encodes a conserved B-type cyclin, interacting with and controlling the substrate specificity of the cyclin-dependent kinase Cdk123–28. The Clb2 protein contains two nuclear localization signals and two nuclear export signals29. Most of the protein is found in the nucleus and at spindle pole bodies29–31. However, when the protein is overexpressed or in nuclear import mutants, the Clb2 protein accumulates in the cytoplasm or at the bud neck29–31.

Clb2-Cdk1 regulates entry and progression through mitosis in a threshold-dependent manner25,32–36, by phosphorylating transcriptional and post-transcriptional regulators37–39. This triggers a positive feedback loop leading to the transcription of the CLB2 cluster, a set of 35 genes including CLB2, expressed during the G2/M phase transition24,40–42. Aberrant Clb2 expression- depletion or over-expression- results in abnormal mitotic progression and cell size alteration25,27,43. To achieve accurate periodic Clb2 expression, cells combine cell-cycle-dependent mRNA synthesis44,45, controlled mRNA decay46, and proteasome-dependent protein degradation47,48. The molecular events controlling CLB2 transcription and protein degradation, as well as Clb2 function during cell cycle progression, are well characterized. However, it remains unclear whether and how cells modulate CLB2 mRNA translation in response to changes in cell growth, such as bud growth during S. cerevisiae mitosis, and how it may be coupled to cell cycle progression. Previous mathematical modeling suggested that CLB2 mRNA localization and local translation could act as a bud sizer during the G2/M phase checkpoint49, but thus far experimental evidence supporting this hypothesis is lacking.

To study growth phase-dependent CLB2 expression regulation, we performed single-cell measurements of CLB2 mRNA and protein expression throughout the budding yeast cell cycle in fixed and living cells. We combined single-molecule RNA fluorescence in situ hybridization (smFISH)50,51 and immunofluorescence (IF)52,53 to detect CLB2 mRNA and its protein product simultaneously in individual cells. Furthermore, to study dynamic gene expression changes in living cells, we utilized the MS2 system (MBSV6) optimized to endogenously tag unstable mRNAs in S. cerevisiae54–57, and tagged the Clb2 protein with GFP. Our work shows that CLB2 mRNAs are efficiently localized in the bud by the She2-She3 complex during the G2/M phase, while the CLB2 protein is found in the mother nucleus. We show that the CLB2 mRNA has a single ZIP code located in the coding sequence, which is necessary but not sufficient for both mRNA localization and Clb2 protein synthesis. We found that bud localization stimulated CLB2 mRNA translation, as in a CLB2 ZIP code mutant, we observed impaired mRNA bud localization and reduced Clb2 protein synthesis, which were partially rescued in ZIP code rescue strains. Furthermore, we found that the ZIP code mutant showed an increase in bud size at birth and an increase in budded phase duration. Finally, unlike in wild-type (WT) cells, we observed that protein accumulation in the ZIP code mutant showed reduced correlation with bud growth and thus lost the ability to be a consistent predictor of size changes occurring in this compartment. Altogether, we propose that yeast cells have evolved a mechanism that couples the control of CLB2 mRNA bud localization and protein synthesis to regulate Clb2 protein levels in response to bud growth. We suggest that, in coordination with other known mechanisms controlling CLB2 expression, this mechanism helps cells monitoring bud growth and fine-tune cell cycle progression.

Results
CLB2 mRNAs localize in the bud from S phase to Mitosis
To quantify CLB2 mRNA expression throughout the S. cerevisiae cell cycle, we combined smFISH and IF. To monitor cell cycle progression, nuclear localization of the transcription factor Whi5 was used to classify early G1 phase, while G2 and mitotic cells were identified by staining tubulin (Tub1) and monitoring microtubules stretching between the mother and the daughter mitotic spindles (Fig. 1a). CLB2 smFISH revealed that mRNAs are detected from late S phase, when the bud emerges from the mother cell, until the end of anaphase. Quantification of CLB2 mRNAs showed that mRNAs are found in 63% of cells in an unsynchronized population (Fig. 1b, Supplementary Fig. 1a). The expression peak occurred during G2 (average 10.2 ± 5.7 mRNAs/cell) when about 50% of the cells showed an active transcription site (Fig. 1b, c, yellow arrows) with on average 2.9 ± 1.5 nascent RNAs per transcription site, comparable to previous studies46 (Supplementary Fig. 1b). Furthermore, in expressing cells, CLB2 nascent RNA expression showed Poissonian transcription kinetics typical of constitutive genes58, suggesting that this cell-cycle regulated gene is likely transcribed in a single activation event with fixed initiation rate. From late S phase until anaphase, we observed that CLB2 mRNAs localize to the bud from the first stages of bud formation. Throughout the budded phases, we measured up to 65.6% of mRNAs in the bud, as compared to the distribution of the non-localized mRNA MDN1, where only 17.2% of mRNAs are found in this compartment (Fig. 1b, d, Supplementary Fig. 1c, d). CLB2 mRNA bud localization is independent of the S. cerevisiae background since we observed it both in BY4741, used throughout this study, as well as in the W303 background (Supplementary Fig. 1e).

CLB2 mRNAs localize in the bud of living S. cerevisiae cells
To investigate CLB2 mRNA localization dynamics in living cells, we used an MS2 system optimized for yeast mRNA tagging (MS2 binding sites V6, MBSV6)54–56. We inserted 24xMBSV6 in the 3’ UTR (Untranslated region) of the endogenous CLB2 locus (Supplementary Fig. 2a). To confirm that mRNA tagging with MBSV6 did not alter CLB2 mRNA expression, unlike with previous MS2 variants55,59,60, we performed two-colors mFISH with probes targeting either the coding sequence (CDS) or the MBSV6 loops, to compare the expression of the endogenous and the tagged CLB2 mRNA. This confirmed that MS2-tagged mRNAs are full-length and correctly localized in the bud (Supplementary Fig. 2b). Furthermore, comparable mRNA levels were observed whether the mRNA was MS2-tagged, with or without GFP-tagged MS2 coat protein (MCP-GFP), which is used to detect mRNAs in living cells (Supplementary Fig. 2c–e).

To monitor cell cycle progression and bud emergence in living cells, we tagged the bud neck protein Cdc10 endogenously with tdTomato in the CLB2-MS2-tagged strain (Supplementary Fig. 3a). We performed time-lapse imaging every 2 min and measured CLB2 mRNA expression throughout the cell cycle by acquiring z-stacks encompassing the cell volume (Video 1). To reduce perturbations in gene expression due to synchronization protocols61, we quantified CLB2 mRNA expression in unsynchronized cells, using the bud neck marker expression to compare cells. This revealed that up to 62.9% of CLB2 mRNAs localized in the bud (Fig. 1e, f), in agreement with the smFISH quantifications (Fig. 1d). Furthermore, mRNAs were degraded before the end of mitosis with a half-life of 3.8 ± 1.4 min, consistent with previous measurements46, suggesting that the MS2 system does not affect CLB2 mRNA stability (Supplementary Fig. 3b). Interestingly, imaging of mother-daughter pairs for more than one cell cycle showed that the daughter cell initiated CLB2 mRNA expression about 20 min after the mother (Fig. 1g). This observation is in line with previous evidence showing that S. cerevisiae daughter cells are born significantly smaller than mothers and that cell size control occurring during G1 regulates the entry into the next cell cycle62–64.

High framerate imaging every 100 milliseconds (ms) revealed that, as the bud grows, the number of CLB2 mRNAs localized in the bud rapidly increases (Supplementary Fig. 3c, Video 2). The high concentration of mRNAs in the bud and their rapid movement in and outside of the focal plane did not allow tracking single mRNA molecules to investigate CLB2 mRNA bud localization dynamics. We therefore used mathematical modeling to predict the behavior of CLB2 mRNA in the bud of an average G2 cell. The parameters included the bud and mother volumes based on our measurements, mRNA counts and decay rates based on smFISH and live imaging experiments and an apparent mRNA diffusion coefficient based on previous measurements performed in eukaryotic cells65 (“Methods”). A fast coefficient of 0.4 μm2/s was previously measured for non-translating mRNAs, and a slower coefficient of 0.1 μm2/s was measured for translating mRNAs65. Interestingly, our model suggested that if we assumed either a slow or a fast apparent mRNA diffusion coefficient of 0.1 μm2/s or 0.4 μm2/s, respectively, we would not obtain the expected enrichment of the CLB2 mRNA in the bud (Supplementary Fig. 3d, e). To predict the accumulation of about 65% of the mRNA in the bud observed during the G2 phase, the presence of a high-affinity anchoring factor promoting CLB2 mRNA segregation in the bud had to be included in the simulation (Supplementary Fig. 3f). Altogether, these results showed that CLB2 mRNAs were efficiently transported to the bud in a cell cycle dependent manner and the simulation implied that the mRNA might be anchored via a yet uncharacterized mechanism.

She2 and She3 are required for ZIP-code-dependent CLB2 mRNA localization
To elucidate the function of CLB2 mRNA localization, we first characterized the mechanism of CLB2 mRNA transport. Previous work showed that CLB2 mRNA is associated with the She2-She3-Myo4 complex and that localization of CLB2 to the bud likely requires She222. To expand this observation and quantify this phenotype, we performed smFISH-IF throughout the cell cycle for the CLB2 mRNA in SHE2 or SHE3 gene deletion strains to test whether the She2-She3 complex, required for ASH1 mRNA transport4–11, was also involved in CLB2 mRNA localization. This revealed that in Δshe2 and Δshe3 strains, localization was strongly impaired (Fig. 2a, Supplementary Fig. 4a) and that during mitosis, only up to 24.5% and 23.6% of CLB2 mRNAs were found in the bud of the Δshe2 and Δshe3 strains, respectively (Supplementary Fig. 4b, c).

As the She2-She3 complex was required for CLB2 mRNA localization, we hypothesized that the CLB2 mRNA might possess a ZIP code akin to the ASH1 ZIP code. Previous work defined the sequence and structure of the (E3) ASH1 mRNA ZIP code bound by She28,66–69. Based on sequence and structure similarity, a pattern search was performed to predict occurrences within the CLB2 mRNA (“Methods”). We identified one high-confidence She2-binding site in the CDS at position 1111–1145 (Fig. 2b, c). To test the predicted site, we generated a CLB2 synonymized mutant whereby the CDS was mutagenized at nine bases to destroy the ZIP code structure, while preserving the protein sequence and the codon usage index (ZIP mut, Fig. 2d, Supplementary Fig. 4d, e). A pattern search confirmed that the ZIP code was destroyed upon synonymization. smFISH revealed that, in the CLB2 ZIP code mutant, the CLB2 mRNA was no longer bud localized (Fig. 2e, f), demonstrating that the identified ZIP code is necessary to control bud mRNA localization, possibly by recruiting the She proteins. Furthermore, the loss of CLB2 mRNA localization was not due to the specific mutations introduced, as other mutants with disrupted ZIP code structure also showed CLB2 mRNA mis-localization (Supplementary Fig. 4d, f). To measure CLB2 mRNA localization, we quantified the mRNA peripheral distribution index (PDI) in budded cells using the RNA Distribution Index Calculator70 (“Methods”). The PDI measures the location of the mRNA relative to the nucleus and allows for the comparison of the localization of distinct mRNA species. An index value equals 1 for diffusely distributed mRNAs or >1 if the mRNA has a polarized pattern, and <1 if the mRNA is distributed closer to the nucleus70 (Fig. 2g). This analysis revealed a PDI of 1.9 ± 0.42 for the CLB2 mRNA, similar to the index value of the control mRNA ASH1 (PDI = 2.2 ± 0.43, Fig. 2h). The PDI value was significantly reduced for CLB2 in the Δshe2 (PDI = 0.5 ± 0.2), Δshe3 (PDI = 0.4 ± 0.13), and CLB2 ZIP code mutant strain (PDI = 0.5 ± 0.16) (ANOVA statistical test: F(4, 185) = 15.74, p < 0.0001), with PDI values similar to the non-localized mRNA MDN1 (PDI = 0.6 ± 0.18, Fig. 2h, Supplementary Fig. 1c, d). Thus, the She2–3 complex is required to transport CLB2 mRNAs to the bud via a conserved ZIP code sequence.

CLB2 mRNA ZIP code and bud localization regulate Clb2 protein levels
To elucidate whether CLB2 mRNA localization influences gene expression, we measured CLB2 mRNA and protein levels in the localization mutants. Using smFISH, we found comparable mature and nascent RNAs counts in the Δshe2 (5.3 ± 5.1 mRNA/cell and 2.7 ± 1.1 nascent RNA/transcription site (TS)) or Δshe3 mutants (5.2 ± 6.2 mRNA/cell and 2.7 ± 1.3 nascent RNA/TS) compared to WT cells (5.4 ± 5.1 mRNA/cell and 2.6 ± 1.3 nascent RNA/TS). While we observed a small but significant increase in the CLB2 ZIP code mutant strain (6.9 ± 6.3 mRNA/cell and 3.4 ± 1.3 nascent RNA/TS) compared to WT cells (Fig. 3a, b). Next, a western blot of the endogenously myc-tagged Clb2 protein showed that the protein expression in Δshe2 or Δshe3 and in the CLB2 ZIP code mutants was significantly reduced compared to WT cells (Fig. 3c, d). Because the ZIP code mutant showed a stronger decrease in Clb2 expression compared to the Δshe mutants, we tested whether the ZIP2 and ZIP3 mutants showed a similar phenotype (Supplementary Fig. 4d, e, Supplementary Fig. 5a, b). As all the mutants showed the same reduction in Clb2 protein expression, this indicated that this region of the CLB2 mRNA is important to control mRNA localization and protein expression. In view of the similarity in the mRNA localization and protein expression phenotypes of the three ZIP mutant strains, we continued the characterization of ZIP code mutant 1 (ZIP1), henceforth called ‘ZIP-mutant’. Furthermore, to test whether the effect of the She proteins and the ZIP code on protein expression was independent of each other, we generated Δshe2-ZIP or Δshe3-ZIP double mutants (Supplementary Fig. 5c–e). Protein expression analysis by western blot showed no significant decrease in protein expression in the double mutants compared to the ZIP mutant, and smFISH showed a lack of mRNA localization in the bud. Altogether, revealing an epistatic interaction and thus suggesting that the She proteins and the ZIP code of the CLB2 mRNA act in the same pathway.

To test whether the decrease in protein expression was due to a change in protein degradation, we performed stability assays by treating WT and localization mutants with the translation inhibitor cycloheximide and measured the protein abundance over time (Fig. 3e, f, Supplementary Fig. 5f, g). No significant difference was observed in the stability of the localization mutants compared to WT cells, suggesting that CLB2 ZIP code and mRNA localization, rather than mRNA or protein stability, regulated Clb2 protein synthesis. Finally, we investigated whether mRNA localization affected Clb2 protein localization. To this end, CLB2 was endogenously tagged with yeast-optimized GFP (yeGFP) in WT and CLB2 mRNA localization mutants. We observed that Clb2 is predominantly found in the nucleus in all tested strains (Fig. 3g), as previously reported for WT cells29–31. Interestingly, Clb2 was observed in the mother nucleus already during the G2 phase (Fig. 3g, top panels), when the mRNAs were already localized to the bud (Figs. 1b, e, 2a), showing an uncoupling between CLB2 mRNA and protein localization. Altogether, these results suggested that CLB2 mRNA localization did not segregate the Clb2 protein in the daughter cell, unlike Ash13,7,71, but rather influenced protein levels.

CLB2 mRNA and protein co-localization suggest more efficient translation in the bud
To simultaneously measure CLB2 mRNA and protein expression in single cells, we generated a strain where 25 myc tags were inserted at the N-terminus of the endogenous CLB2 gene (Fig. 4a). This N-terminal tagging and amplification strategy increased the Clb2 protein signal without significantly affecting growth or protein stability (Supplementary Fig. 6a–d). Next, smFISH and IF were combined to simultaneously detect CLB2 mRNAs and proteins in fixed cells, and IF against tubulin was used to score the cell cycle phase. As immunofluorescence can be noisy in yeast, we tested the specificity of the anti-myc antibody on WT cells (Supplementary Fig. 6e), which demonstrated low levels of non-specific binding of primary and secondary antibodies. This approach showed that the bulk of Clb2 proteins accumulated in the mother (M) nucleus from G2 to mitosis (Fig. 4b, c), at the time when the mRNA was preferentially found in the bud (B) (Fig. 4b, d). This indicated that Clb2 proteins were efficiently localized in the mother nucleus, as observed by live imaging (Fig. 3g) and as shown previously29,31. In addition, Clb2 protein foci were also found in the bud in close proximity to CLB2 mRNAs from G2 to mitosis, suggesting that these foci may represent sites of mRNA translation (Fig. 4b, yellow arrowheads). Quantification of co-localized single mRNAs and protein foci (within 250nm, the resolution of our system) revealed that in WT cells, more co-localized mRNA-protein foci were found in the bud than in the mother cell, while in the localization mutant Δshe2, mRNA-protein foci were preferentially found in the mother cell where the bulk of mRNAs was distributed (Fig. 4e, f). Furthermore, we found a significant reduction of the percentage of bud-localized mRNAs co-localized with protein foci in Δshe2 cells compared to WT cells (Fig. 4g, non-parametric Mann-Whitney test, P < 0.0001), indicating that in localization mutants, CLB2 mRNA translation efficiency may be reduced. It is interesting to note that even in WT cells, only about 25% of the bud-localized mRNAs were found in close proximity to protein foci, suggesting that CLB2 mRNAs are translated unfrequently, as previously reported72. Altogether, these data suggest that in WT cells, CLB2 mRNAs were more efficiently translated in the bud, where the mRNA was actively transported. Furthermore, in Δshe2 cells, we observed an increase of mRNA-protein foci in the mother cell relative to the bud (Fig. 4f), despite protein levels being decreased under these conditions (Fig. 3c, d, Supplementary Fig. 6f), indicating that translation in the mother cell may be less efficient, resulting in reduced Clb2 protein levels in the localization mutants.

Translation regulators Puf6, Ssd1, and Khd1 do not affect Clb2 protein expression
To investigate if CLB2 translation efficiency was higher in the bud as a result of translation repression prior to localization, we tested if the deletion of the RNA-binding proteins Puf612,13,17, Khd114–16 and Ssd119–21, previously shown to inhibit translation of bud-localized mRNAs, influenced Clb2 protein levels. A western blot of the endogenously myc-tagged Clb2 protein in Δssd1, Δkhd1, and Δpuf6 strains (Supplementary Fig. 6g–i) did not reveal a significant increase in Clb2 protein levels when comparing the mutant strains to WT (ANOVA statistical test: F(3, 8) = 0.7677, p = 0.3), which we would have expected if these proteins were inhibiting CLB2 mRNA translation. Additionally, because Khd1 and Puf6 were shown to be important for ASH1 mRNA localization13,14, we tested CLB2 mRNA localization in the mutants. smFISH for the CLB2 mRNA in the Δssd1, Δkhd1, and Δpuf6 strains showed that the mRNA was still localized in the bud at a similar level as WT cells. Altogether, these results indicated that the factors that control ASH1 mRNA expression were not required to control CLB2 mRNA translation or localization. While other yet unidentified factors may exist, our data suggested the possibility that CLB2 mRNA might also be translated outside of the bud, albeit at a reduced rate.

CLB2 mRNA ZIP-mutant displays increased budded phase duration and cell size at birth
The results so far showed that the absence of CLB2 mRNA bud localization correlated with reduced protein expression, likely due to decreased mRNA translation. To corroborate this finding, we performed time-lapse microscopy. In all strains, the Clb2 protein was tagged with yeGFP while Cdc10 was fused to mCherry to label the bud neck and monitor division time and parent-offspring relationships. We confirmed nuclear accumulation of the Clb2 protein during the budded phase of single cells (Fig. 5a). Nuclear fluorescence peaked ca. 20 min before division in all strains (Fig. 5b, Supplementary Fig. 7a). Peak nuclear fluorescence intensity (Fig. 5b) as well as cumulative nuclear fluorescence over the entire budded phase (Fig. 5c, Supplementary Fig. 7b) was lower in all mutant strains investigated, but for the Δshe strains this reduction was not significant. Given the role of Clb2 protein in entry into and progression of mitosis25,32–34,73 we hypothesized that mutants with lower Clb2 protein levels would show delayed mitotic progression. To allow accurate estimates of the length of the budded phase, we acquired images every minute. This led to slow growth phenotypes when green fluorescence was collected, prompting us to only collect brightfield and red fluorescence for these experiments. The mean values for the duration of the budded phase in the ZIP-mutant strain and the wild type differed by 5.27 min. Even though there was overlap in the distributions (Fig. 5d), bootstrap estimates (10000 runs, sample size 250) of the means showed almost no overlap (Fig. 5e), and the difference in means was overall highly significant (p = 9.95*10−9, two-sided t-test based on the data in Fig. 5d). A similar but smaller increase in budded phase duration was also present in the Δshe strains (Fig. 5e). It seems therefore, that the duration of the budded phase is influenced by Clb2 protein concentration, which in turn is dependent on correct localization and efficient translation of the CLB2 mRNA.

Since Clb2 protein concentration influenced budded phase duration, we hypothesized that bud size would likewise be affected. To relate Clb2 protein concentration to bud size, we collected snapshot images of growing yeast cells and correlated nuclear fluorescence of Clb2-yeGFP against bud size in all cells that were in the budded phase. We found a clear relationship between nuclear fluorescence and bud size in the wild type (Fig. 5f Pearson correlation coefficient 0.43, slope of linear regression 0.25), and a weaker one in the ZIP mutant strain (Fig. 5g Pearson correlation coefficient 0.45, slope of linear regression 0.13). Intriguingly, owing to targeted degradation of the Clb2 protein after mitosis47,48, this correlation between nuclear fluorescence and bud size only held prior to mitosis. In post-mitotic wild-type cells, the correlation was weaker in both strains (Supplementary Fig. 7c, Pearson correlation coefficient 0.32 and 0.14 for WT and Zip mutant, respectively).

A ZIP-code rescue strain shows improved protein expression and cell cycle progression
We have shown that the ZIP code sequence located in the coding region of the CLB2 mRNA is necessary for mRNA localization to the bud via the She2-3 complex. To test if the ZIP code needs to be in the CLB2 coding sequence to promote efficient localization and protein synthesis, we introduced the minimal ZIP code sequence (36 bp) as well as an extended ZIP code region (99 bp) into the 3’ UTR of the ZIP-mutant strain (Fig. 6a, Supplementary Fig. 8a). smFISH uncovered that adding both versions of the ZIP code to the 3’ UTR resulted in a complete rescue of CLB2 mRNA localization in the bud, comparable to WT levels (Fig. 6b, Supplementary Fig. 8b), demonstrating that the WT ZIP code can drive mRNA localization also from a distant location within the CLB2 mRNA. Next, we tested whether the Clb2 protein levels were rescued in the CLB2 ZIP rescue strain. Interestingly, only a partial rescue was observed (Fig. 6c, d, Supplementary Fig. 8c, d), suggesting that CLB2 bud mRNA localization promoted Clb2 synthesis and contributed to controlling about 10–20% of Clb2 protein expression. Next, we tested whether the extended ZIP code would be sufficient to control bud localization and increase the expression of a non-localized mRNA. To this end, we generated a strain where the endogenous RPT2 gene, a constitutive non-localized mRNA, was modified with the CLB2 extended ZIP code (Supplementary Fig. 8e). As the RPT2 coding sequence has a length (1314bp) comparable to the CLB2 coding sequence (1476bp), we inserted the CLB2 ZIP code in frame and at a similar position in the gene. Two-colors smFISH performed on the RPT2-CLB2-ZIP strain or on the control strain using RTP2 probes or CLB2 probes showed that the RPT2-CLB2-ZIP mRNA is not bud localized (Supplementary Fig. 8f). Altogether our data showed that the short CLB2 ZIP code is necessary but not sufficient to drive bud mRNA localization, implying that other yet uncharacterized sequences in the CLB2 mRNA are involved in regulating mRNA localization and translation.

Finally, we investigated the growth and cell cycle distribution of the localization mutants and the ZIP code rescue strain. To measure the distribution of the three major phases of the cell cycle (G1, S, G2/M), DNA was labeled with propidium iodide and measured by flow cytometry, followed by Gaussian mixture modeling to estimate the three cell cycle subpopulations (Fig. 6e, f, Supplementary Fig. 9a). This revealed that, while growth rates of the localization mutants were not significantly affected (Supplementary Fig. 9b), we observed a modest but significant (two-way Anova, Dunnett’s multiple comparison test) increase in the G2/M fraction in the ZIP-mutant but not in the Δshe mutants nor the ZIP-mutant rescue strain (Fig. 6e, f). Altogether, our experiments point to a dual function of the CLB2 mRNA ZIP code motif, promoting both efficient bud localization and translation. For the latter function to take effect, the ZIP motif needs to be in the coding sequence. Consistent with this observation, in the Δshe mutants where the ZIP code is still intact, higher levels of Clb2 protein were observed. In the ZIP-mutant, where both the localization is impaired and the ZIP code is absent, Clb2 protein levels were lowest in bulk populations (Fig. 3c, d) and in individuals across the cell cycle (Fig. 5b). We also observed an increased G2/M fraction in this mutant, suggesting that the duration of these stages is increased at the expense of G1 (Fig. 6e, f). Altogether, the higher G2/M fraction (Fig. 6e, f) and the larger size at budding and division (Fig. 5h) indicated that control of CLB2 mRNA localization and translation play a role in regulating Clb2 protein levels, which in turn report translational maturity as well as size of the bud compartment, providing a mechanism to coordinate growth and cell cycle progression during the G2/M phase transition.

Methods
Yeast cell cultures
Yeast cells were grown in the appropriate media depending on the experiment. For yeast transformation, cells were grown in YEPD (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose). Liquid cultures for smFISH, protein extractions, flow cytometry, and live imaging were done in Synthetic Complete medium (6.7 g/L yeast nitrogen base, 20 g/L glucose and synthetic complete amino acid and vitamins mix) or in Drop-out medium (6.7 g/L yeast nitrogen base, 20 g/L glucose and drop-out mixture lacking leucine or uracil). Drop-out medium lacking leucine was used for the experiments shown in Fig. 1e–g, Supplementary Fig. 3b, c, to maintain the expression of the MCP-2xyeGFP plasmid (pET296). Drop-out media lacking uracil were used for transformation selection during strain construction.

Agar plates 2% (w/v) were made with YEPD or drop-out medium lacking leucine or uracil. For selection of strains expressing the hygromycin or kanamycin markers, cells were plated on YEPD plates with 300 μg/mL of hygromycin or kanamycin antibiotics. Cells were grown at the indicated temperature using constant shaking at 210 rpm. For smFISH, live imaging and flow cytometry, the details of the cell cultures are described below.

Plasmids construction
The plasmids encoding the CLB2 gene N-terminally tagged with myc tags were built as follows. The CLB2 gene was cloned, including its 5’ UTR, CDS, and 3’ UTR (−788 bp before the ATG, +346 bp after the STOP codon) in a YIplac211 vector. To obtain a strain where different N-terminal tags could be inserted in the coding sequence, a gene fragment was synthesized with a BamHI site after the CLB2 ATG start codon. This fragment was sub-cloned into the YipLac211 CLB2-containing plasmid by using restriction enzymes SalI and BrGI. To generate the 5-myc and the 25-myc tagged CLB2 variants, a gene fragment was synthesized encoding 5 synonymized myc tags flanked by BamHI and BglII restriction sites. The myc tags (5 or 25) were cloned into the CLB2 gene at the BamHI site, giving rise to plasmids pET479 and pET491, respectively. The plasmids were cut with restriction enzymes AatII and BglII to generate a cassette including the modifications as well as the sequence for the homologous recombination.

The synonymized CLB2 ZIP code plasmids were generated by Gibson cloning using the NEB Gibson Assembly Cloning kit (#E5510) using oligos (OAM32, 35, 43, 44, 70, 71, 74, 65) and plasmids (pAM90-97) indicated in Supplementary Data 1. The plasmids were cut using AatII and EcoRI-HF restriction enzymes, and the modified CLB2 inserts were transformed into BY4741 for homologous recombination. The ZIP-code rescue plasmids (pAM097 36-nt ZIP or pET586 99-nt ZIP) were generated by inserting the predicted WT She2 binding site sequence (AGCAGATGACTACGATATACAGTCTCGAACTCTTGCC) at the ClaI restriction site 43 nucleotides downstream of the CLB2 stop codon. The plasmids used to tag the RPT2 gene (pET576 and pET578) were generated by inserting the predicted WT She2 binding site sequence (AGCAGATGACTACGATATACAGTCTCGAACTCTTGCC) at the ClaI restriction site 43 nucleotides downstream of the RPT2 stop codon.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Maekiniemi and colleagues report a novel role for CLB2 mRNA trafficking and localized translation in coordinating cell cycle progression and bud growth in budding yeast. The authors used single-molecule RNA FISH and live-cell imaging to show that the CLB2 mRNA accumulates at the bud of yeast cells in a cell-cycle dependent manner. While Clb2 protein is synthesized in the bud, it does not accumulate in the daughter nucleus. The authors identified a zipcode element within the coding sequence of the CLB2 mRNA and show that mutations that disrupt the She2/She3 binding motif in the zipcode inhibit CLB2 mRNA localization and decrease Clb2 protein expression. Interestingly, deletion of either She2 or She3, or moving the zipcode to the 3’UTR of a zip-mutated CLB2 mRNA, only partially rescue optimal expression of the Clb2 protein, suggesting that the zipcode has yet another undefined function in regulating Clb2 protein synthesis. Finally, expression of a zipcode-mutated CLB2 results in larger daughter cells and loss of synchronization between growth and cell cycle.\nAlthough a role for CLB2 mRNA bud-localization as a sizer of bud growth has already been suggested from mathematical modeling (Spiesser et al., PLOS Comp. Biol, 2015), this manuscript provides the first experimental evidence supporting a mechanism that coordinates growth and cell cycle progression during G2/M phase transition. As such, this work is an important contribution to the field. The results provided are of high quality, especially those from the various single-cell and single-molecule imaging approaches, and their quantitative image analyses.\nFurthermore, the authors provide convincing evidence that the CLB2 zipcode fulfills two roles: promote mRNA localization via the She2-She3-Myo4 pathway; and promote CLB2 expression. However, the most important role of the zipcode seems to be linked to its regulation of CLB2 expression, since the she2/she3 mutants have a minor effect on cell size. While the mechanism of CLB2 mRNA localization has been well characterized in the manuscript, how this zipcode promotes Clb2 expression remains unknown. The authors argue that the zipcode must be in the coding sequence to promote optimal translation, but another possibility is that the zipcode is part of a larger cis-acting element that promotes mRNA translation. This would explain why insertion of the zipcode alone in the 3’UTR of the CLB2 ZIP mutant mRNA or deletion of She2/3 only partially rescue protein expression (Figure 6c-d). Since such dual role for a zipcode is unusual, it would have been nice to have a better characterization of the mechanism of zipcode-mediated regulation of Clb2 expression (i.e polysome gradient, identification of separation of function mutations,..).\nMajor comments:\n- Figure 4C: It is surprising that there’s so many Clb2 protein foci in G1 mother cells knowing that this protein is actively degraded in G1. Since there’s a destruction box element at the N-terminus of Clb2, is it possible that the N-terminal 25 myc-tags may affect the stability of this protein? The authors should look at the degradation of the 25myc-Clb2 protein, as it may be disrupted.\n- This raises the question why the authors inserted their myc tags at the 5’end of CLB2 ORF, which is not commonly used in other publications on Clb2. Since the effect of the she2/3 mutants or zipcode mutation of Clb2 expression is small (25% to 50% reduction), a validation of Figure 3D using an anti-Clb2 antibody to detect the untagged protein would help strengthen their conclusions.\n- The authors should validate that the CLB2 zipcode is sufficient to control the localization and translation of a reporter mRNA, independently of the context of the CLB2 mRNA.\n- Figure 6D: Is it possible to rescue Clb2 protein expression using the zipcode from another bud-localized transcript, like ASH1 for instance? What happen if the ASH1 E3 zipcode is inserted in the 3’UTR of the CLB2 ZIP mut mRNA? Is it a unique role for the CLB2 zipcode to promote protein expression?\nMinor comments:\nThere are a few mistakes in the text, such as “allows” instead of “arrows” (page 5, line 16).\nPage 15, line 9: “The She2-She3 complex localizes the ASH1 mRNA to the bud, with the She2-mRNA interaction mediated via the E3 ASH1 ZIP code located in the ASH1 coding region” ASH1 has 4 zipcodes, and not only one. The E3 zipcode is in the 3’UTR.\nPage 15, line 17: “Puf1” should be replaced by “Puf6”.\nPage 53, line 9, Legend video 2: “One single Z-stack was streamed every 50 ms”. This is not clear. Is that mean a single plane was captured every 50 ms or a Z-stack was captured every 50ms? Also, at page 7, line 4, the Video 2 is described in the main text as “High frame-rate imaging every 100 ms…” Please clarify.", "reviewer_2": "Localization of mRNAs can regulate translation and protein localization. The authors here confirmed that CLB2 mRNA localizes to the bud of yeast cells and seek to understand how this may regulate Clb2 protein levels and localization. They find that CLB2 mRNA localization is dependent on She2 and She3, like the well-studied, bud-localized ASH1 mRNA. The authors identify a ZIP code in the coding sequence of CLB2 mRNA that is necessary for mRNA bud localization. RNA binding proteins that inhibit ASH1 mRNA translation were not found to be important for CLB2 mRNA regulation, overall demonstrating key similarities and differences in the mRNA level regulation of CLB2 and ASH1.\nTo address the function of CLB2 mRNA localization to the bud, the authors suggest that 1) CLB2 mRNA translation is more efficient in the bud and 2) CLB2 mRNA localization to the bud contributes to cell size regulation. While these ideas are very compelling, more evidence is needed to support these conclusions.\nMajor comments\n1. To provide evidence for CLB2 mRNA translation being more efficient in the bud, the authors demonstrate a slight but significant reduction in Clb2 protein expression observed via Western Blot in she2Δ and she3Δ cells and a further reduction in a ZIP code mutant. However, localization to the bud is not sufficient for efficient CLB2 translation since bud localization was rescued with a ZIP code in the 3’UTR but protein levels hardly increased. The authors suggest that the ZIP code in the coding region may be important for efficient translation through some other mechanism, but, alternatively, it could be that localization to the bud does not regulate CLB2 translation very much at all. To further test whether the ZIP code has an effect on translation efficiency separate from localization, the authors could put the ZIP code mutant (with and without 3’UTR Zip rescue) in she2Δ cells. The prediction would be that Clb2 protein levels are further reduced than in cells with she2Δ alone.\n2. The authors provide a secondary line of evidence that bud-localized CLB2 mRNA is more efficiently translated in the bud based on the co-localization of CLB2 mRNA and Clb2 protein foci based on immunofluorescence staining (IF). IF in budding yeast is notoriously tricky, and indeed the pattern of Clb2 protein in live cell images (Fig 3g) is very different from the IF images (Fig 4b and 4e). In particular, the foci that are used for measurements of co-localization with mRNA in Fig 4 are not present in Fig 3. To strengthen this data, the authors need to show that the Clb2 protein foci in the cytoplasm are not artifacts of IF.\n3. Based on the extensive literature of Clb2-CDK driving mitotic entry and progression, there is a clear prediction that lower Clb2 protein will slow G2/M progression resulting in larger cells at the time of cell division. This is supported by the data in Fig 5 where the ZIP code mutants with lower Clb2 protein levels show larger cells at division. However, this does not explain why mother cells would be larger at the time of budding (Fig 5C), which is controlled by the G1 size control mechanism and at a time when no Clb2 is present. This also doesn’t explain why more bud volume is added in G2/M without an increase in budded timing. Is it possible these cells are larger at all cell stages for other reasons?\n4. The authors try to link CLB2 mRNA localization and translation efficiency to cell cycle progression and a potential bud sizer mechanism. The crucial measurement is described in the text as “While nuclear fluorescence tightly predicted bud size in WT and the Δshe mutants’ (Fig. 5k and Extended Data Fig. 7m) this effect was significantly more disperse in the ZIP-mutant strain, indicating a loss of communication between the two compartments.” A scatter plot of nuclear fluorescence vs bud size for the two populations would be more appropriate here. The authors also note that the ZIP code mutant added more material during the budded phase than WT (Fig 5g) and spent longer median time in budded phase (Fig 5h, albeit the authors recognize this result is non-significant). The authors conclude that “control of CLB2 mRNA translation in the bud compartment reports bud size”. These two statements (and the overall description of Fig 5) are difficult to parse, and therefore the intended conclusions and interpretation from the data in Fig 5 are unclear. The results need to be rewritten for clarity, and further experiments are needed that more directly test associations between CLB2 mRNA localization and bud growth. If the intended conclusion is that Clb2 protein levels are regulated by bud size, it would be much more convincing to include other experiments manipulating bud size (latrunculin, or mutants in polarity, vesicle delivery, or septins) and determining the effect on Clb2 protein levels.\nMinor comments\n5. In Fig 2 e, f, explicitly state which stage of the cell cycle is being imaged.\n6. The authors assume the ZIP code mutant abrogates She2 binding, but it would be much stronger to show this directly by co-IP.\n7. A more careful description of strain construction is needed. Are the 5-myc Clb2 WT and ZIP code mutants constructed at the endogenous locus and in the same way?\n8. According to the morphogenesis checkpoint model, the presence of a bud is read out by a change in septin organization, which then leads to inactivation of Swe1, the Clb2-CDK inhibitor (Howell and Lew, 2012). A discussion of how the proposed regulation of Clb2 translation relates to the regulation of Clb2 protein activity by the morphogenesis checkpoint would be very useful for readers to synthesize the conclusions here.\n9. In an effort to make scientific language more inclusive, the authors could consider changing references of mother and daughter cells to parent (or originator) and progeny cells (White, et al, 2021, doi: 10.1038/d41586-021-03490-7).", "reviewer_3": "This manuscript uses a combination of fixed cell and live cell imaging to explore how localization of the CLB2 mRNA affects protein expression, protein localization, cell size, and cell cycle progression. Overall, the experiments are well done, well described, and the conclusions are supported by the data presented. Much of the paper is a beautiful example of how rigorous quantification can provide new insights into biology. The paper doesn’t have all the answers and, in some respects, raises more questions than it answers. It reveals that there must be multiple mechanisms that regulate where translation takes place because translation regulation for CLB2 is very different than ASH1, another well-studied mRNA. The work is exciting in identifying a zip code in the mRNA that is important for localization and perhaps translation.\nOne thing I am trying to understand is: the localization mutants only show a subtle decrease in protein expression. Why is localization of the mRNA to the bud important? The authors suggest that the mRNA may be more efficiently translated in the bud than in the mother based on data in Figure 4 that show there is greater colocalization of mRNA and protein in the bud than the mother. But the difference is quite subtle (a mean of less than 1 colocalization spot in the mother compared to ~ 2 colocalization spots in the bud). While the difference is statistically significant it seems extremely small from a biological perspective. Could the authors comment on whether this observation?\nIn Figure 4, the authors show that protein expression increases in G2/M and protein is more frequently found in the bud (but it is still found in the mother more frequently). mRNA expression increases significantly in the bud in G2/M. In WT cells only 25% of mRNA associated with protein suggesting that mRNA is infrequently translated. In the mRNA localization mutant, the authors found more colocalizing mRNA and protein in the mother and less in bud - was this done as a function of the cell cycle (like in WT)? Did authors quantify mRNA and protein (like in c and d) and do they see a decrease in amount of protein at the single cell level (like in WB)?\nThe data presented in Extended Data Figure 7 are confounding. Up until this point, the data in the manuscript seemed to support the argument that the zip code in the mRNA interacts with the she2-she3 system to localize to the bud and localization to the bud affects protein translation and hence protein expression levels (to some extent). But the extended data in figure 7 suggest that delta-she mutants that prevent localization of the mRNA to the bud don’t exhibit a cell growth defect. In the discussion, the authors rationalize this observation with the explanation that perhaps the zip code serves a dual function – promoting bud localization and efficient translation. If that is the case, wouldn’t we expect to see differences between the delta-she2 mutant and the zip code mutant in the single cell single molecule experiments in Figure 4? As far as I can tell, these experiments weren’t done with the zip code mutant. But doing so, could potentially provide experimental evidence for the authors’ claims.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We would like to thank all the reviewers for the constructive criticism. Following the\nsuggestions, we have significantly revised the manuscript and addressed all the referees’\ncomments in the point-by-point rebuttal in the pages below. A summary of the key\nmodifications is provided here:\nFor the revised manuscript we generated 5 new strains:\n- We generated ZIP mutant strains combined with the deletions of SHE2 or SHE3 (New\nExtended data Figure 5c,d,e). The strains showed an epistatic eIect which\nsuggested that the CLB2 ZIP code and the SHE2/3 complex belong to the same\npathway.\n- We generated a CLB2 ZIP code rescue strain with an extended ZIP code region of 99\nnt to test whether regions adjacent to the ZIP code loop (36 nt) also contributes to\nmRNA localization and translation (New Extended data Figure 8a,b,c,d). This\nrevealed that the extended region also is capable of rescuing CLB2 bud mRNA\nlocalization and induce a small increase in Clb2 protein expression, comparable to\nthe short (36 nt) ZIP code.\n- We generated strains where the CLB2 ZIP code was introduced in the coding\nsequence of a non-bud localized mRNA, RPT2 (New Extended data Figure 8e,f).\nsmFISH revealed that this mRNA is not bud localized, thus demonstrating that the\nCLB2 ZIP code is necessary but not suIicient to drive bud mRNA localization.\nFor the revised manuscript we added the following controls:\n- We tested a commercially available anti-Clb2 antibody to test if we could use it\ninstead of the anti-myc antibody (Rebuttal, page 5). This unfortunately did not work.\n- We tested whether N-terminally tagging the Clb2 protein with 25 myc tags\nsignificantly changed the protein stability compared to the 5 myc-tagged strain (New\nExtended data Figure 6c,d). Our results show no significant change in stability.\n- We added a control smFISH-immunofluorescence where we verified the specificity\nof the anti-myc antibody (New Extended data Figure 6e).\nFor the revised manuscript we added the following new experiment:\n- To more precisely quantify the duration of the budded phase in the WT and in the\nlocalization mutants, we re-shot timelapse imaging experiments with higher\ntemporal resolution (New Figure 5d,e,f,g). This data, combined with an improved bud\ncell segmentation pipeline, revealed a significant increase in the budded phase\nduration both for the ZIP code and the SHE deletion mutants.\nformat requirements. All the text modifications are highlighted in yellow in the re-submitted\ndocument.\n- We wrote a 150 words abstract.\n- We changed the titles of paragraphs to make them shorter.\n- We changed the text describing Figure 5, to improve clarity.\n- We extended the discussion, as per recommendation.\n- We generated a new Zenodo repository version (new doi:\n10.5281/zenodo.16032630) with the updated figures raw data, code and a readme\nfile.\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Maekiniemi and colleagues report a novel role for CLB2 mRNA traIicking\nand localized translation in coordinating cell cycle progression and bud growth in budding\nyeast. The authors used single-molecule RNA FISH and live-cell imaging to show that the\nCLB2 mRNA accumulates at the bud of yeast cells in a cell-cycle dependent manner. While\nClb2 protein is synthesized in the bud, it does not accumulate in the daughter nucleus. The\nauthors identified a zipcode element within the coding sequence of the CLB2 mRNA and\nshow that mutations that disrupt the She2/She3 binding motif in the zipcode inhibit CLB2\nmRNA localization and decrease Clb2 protein expression. Interestingly, deletion of either\nShe2 or She3, or moving the zipcode to the 3’UTR of a zip-mutated CLB2 mRNA, only partially\nrescue optimal expression of the Clb2 protein, suggesting that the zipcode has yet another\nundefined function in regulating Clb2 protein synthesis. Finally, expression of a zipcode-\nmutated CLB2 results in larger daughter cells and loss of synchronization between growth\nand cell cycle.\nAlthough a role for CLB2 mRNA bud-localization as a sizer of bud growth has already been\nsuggested from mathematical modeling (Spiesser et al., PLOS Comp. Biol, 2015), this\nmanuscript provides the first experimental evidence supporting a mechanism that\ncoordinates growth and cell cycle progression during G2/M phase transition. As such, this\nwork is an important contribution to the field. The results provided are of high quality,\nespecially those from the various single-cell and single-molecule imaging approaches, and\ntheir quantitative image analyses.\nFurthermore, the authors provide convincing evidence that the CLB2 zipcode fulfills two\nroles: promote mRNA localization via the She2-She3-Myo4 pathway; and promote CLB2\nexpression. However, the most important role of the zipcode seems to be linked to its\nregulation of CLB2 expression, since the she2/she3 mutants have a minor eIect on cell size.\nWhile the mechanism of CLB2 mRNA localization has been well characterized in the\nmanuscript, how this zipcode promotes Clb2 expression remains unknown. The authors\nargue that the zipcode must be in the coding sequence to promote optimal translation, but\nanother possibility is that the zipcode is part of a larger cis-acting element that promotes\nmRNA translation. This would explain why insertion of the zipcode alone in the 3’UTR of the\nCLB2 ZIP mutant mRNA or deletion of She2/3 only partially rescue protein expression (Figure\n6c-d). Since such dual role for a zipcode is unusual, it would have been nice to have a better\ncharacterization of the mechanism of zipcode-mediated regulation of Clb2 expression (i.e\npolysome gradient, identification of separation of function mutations,..).\nconstructive criticism. As detailed in the point-by-point rebuttal, for the revised manuscript\nwe generated new strains and performed experiments to further characterize the ZIP code-\nmediated regulation of CLB2 expression.\nMajor comments:\n- Figure 4C: It is surprising that there’s so many Clb2 protein foci in G1 mother cells knowing\nthat this protein is actively degraded in G1. Since there’s a destruction box element at the N-\nterminus of Clb2, is it possible that the N-terminal 25 myc-tags may aIect the stability of\nthis protein? The authors should look at the degradation of the 25myc-Clb2 protein, as it may\nbe disrupted.\nTo address the reviewer’s question, we first tested whether we could use a commercially\navailable anti-Clb2 antibody to compare the stability of the myc-tagged Clb2 proteins to the\nendogenous Clb2 protein. As previously commercially available antibodies are no longer\navailable (e.g. Santacruz (sc-9071) used for example, in PMID: 34787675), we tested the\nantibody sold by Cusabio (https://www.cusabio.com/Custom-Antibodies/CG22-Antibody-\n12888599.html#a04).\nUnfortunately, this antibody exhibited high levels of non-specific binding to unknown\nendogenous proteins (see image below). Protein extracts were collected from the following\nstrains: wild-type (WT, no tag), 5-myc Clb2, 25-myc Clb2 and a strain where the CLB2 coding\nsequence was replaced by another coding sequence (eIectively a Dclb2 strain). The extracts\nwere loaded in duplicate on an acrylamide gel and separated by size. After transfer on a\nnitrocellulose membrane, the membrane was cut along the dashed line (see picture below).\nOn the left side (panel a, i), it is shown the membrane hybridized with the anti-Clb2 antibody,\nwhile on the right-side (a, ii), it is shown the membrane hybridized with the anti-myc\nantibody. On the left side, the black asterisk (*) indicates the expected size of the untagged\nClb2 protein, the blue (*) indicates the expected size of the 5-myc Clb2 protein, while the\norange (*) indicates the expected size of the 25-myc protein. Panel b shows the loading\ncontrol, which is a comassie staining of the acrylamide gel after transfer.\nprotein expression, while the anti-myc provided a clean and specific signal.\nAs we could not use the anti-Clb2 antibody, we compared the stability of the 25-myc Clb2\ntagged protein to the 5-myc Clb2 version. We performed protein stability assays to compare\n5\nthe stability of the 5-myc Clb2 and 25-myc Clb2 proteins as we previously did for Figure 3\ne,f). Cells were grown to mid-log phase and incubated with 100 µg/mL cycloheximide to\nblock mRNA translation. Time-points were collected at the indicated times. As shown below,\nthe stability of the 5-myc Clb2 and 25-myc Clb2 proteins is comparable. This new figure is\nnow included as Extended data Figure 6, c and d.\nAltogether, the new data suggests that N-terminally tagging the Clb2 protein with 25-myc\ntags does not significantly aIect Clb2 protein expression compared to 5-myc tags. This is\nconsistent with the growth assays results reported in Extended Figure 6,b, showing no\nsignificant growth defect in the myc-tagged strains compared to each other and to the\ncontrol strain, suggesting that the tagged strains can be used for cell physiology\nexperiments. Furthermore, the observed Clb2 decay pattern is comparable the one\nindependently reported in other publications using either tagged or untagged versions of\nClb2 (Hendrickson et al. Curr. Bio 2001 PMID 11719221, or Bäumer et al. Febs Letters 2000\nPMID 10692575, Schwab et al. Cell 1997 PMID 9288748).\nNevertheless, our control experiments do not exclude a change of stability compared to the\nnon-tagged Clb2 protein. It is possible that myc-tagging causes an increase in Clb2 stability\nthat could explain some of the Clb2 protein foci observed in G1 cells by FISH-IF, yet without\ncausing a fitness decrease.\n- This raises the question why the authors inserted their myc tags at the 5’end of CLB2 ORF,\nwhich is not commonly used in other publications on Clb2. Since the eIect of the she2/3\nmutants or zipcode mutation of Clb2 expression is small (25% to 50% reduction), a\nvalidation of Figure 3D using an anti-Clb2 antibody to detect the untagged protein would help\nstrengthen their conclusions.\nThe choice of adding the myc tags at the N-terminus of the Clb2 protein was motivated by\nthe goal of simultaneously measuring newly synthesized Clb2 proteins and the encoding\nvisualization of co-localized mRNA and protein foci. This approach was inspired by the\ntranslation reporter systems developed in the Singer lab for mammalian cells (Wu et al.\nScience 2016 PMID 27313041). This system combines nascent protein imaging by N-\nterminally tagging a protein of interest with 24 copies of the Sun-Tag (Tanenbaum et al. Cell\nthe MS2 system. While this reporter was mainly used for translation measurement in living\ncells, it was also used for mRNA-protein colocalization in fixed cells by smFISH-IF (Wu et al.\nFigure 5 and Supplementary Figure 6, PMID 27313041).\nAs detailed in the previous point, the use of a commercially available antibody targeting the\nendogenous Clb2 protein proved to be unsuccessful. Nonetheless, our data shows that the\ntagging the Clb2 protein with 5-myc tags provides a reliable (across tens of biological\nreplicates) and background-free approach for measuring Clb2 protein expression,\nsuggesting that even small Clb2 changes can be detected using an anti-myc antibody.\nImportantly, we independently confirmed the Clb2 protein expression reduction in the DSHE\nand ZIP code mutants by using CLB2-GFP tagged strains and single-cell fluorescence\nmeasurements (Figure 5b,c and Extended data figure 7a,b). In these strains, the GFP tag is\nintroduced at the C-terminus of the Clb2 protein, and the extent of protein reduction is\nconsistent with the western-blot measurements.\n- The authors should validate that the CLB2 zipcode is suIicient to control the localization\nand translation of a reporter mRNA, independently of the context of the CLB2 mRNA.\nTo address this point, we generated new strains where we first tested the ability of an\nextended version of the ZIP code (99 nt), to rescue CLB2 mRNA localization and translation\n(New Extended data Figure 8a,b,c,d). This showed similar results as the minimal 36 nt ZIP\ncode. Next, the endogenous RPT2 gene was modified with the CLB2 Zip code (New\nExtended Figure 8e,f). The RPT2 gene was selected because it has a length comparable to\nthe CLB2 gene, and we knew from our previously unpublished smFISH results that the RPT2\nmRNA is not bud localized in S. cerevisiae. Thus, we inserted in the RPT2 coding region, at a\nposition similar to the CLB2 ZIP code position, a CLB2 ZIP-code variant of 99 bp. We used a\nlonger ZIP code variant to test the importance of the CLB2 ZIP code neighboring sequence\nin controlling bud mRNA localization and possibly translation (the characterization of this\nlonger variant is reported in the New Extended data Figure 8e,f). The results reported below\nshow that the 99 bp Zip code variant is not suIicient to localize the RPT2 mRNA to the bud.\nThus, it is possible that, like the ASH1 mRNA, the CLB2 mRNA harbors additional localization\nelements in the coding sequence or in the UTRs contributing to bud mRNA localization.\nand will be further investigated in future work.\n- Figure 6D: Is it possible to rescue Clb2 protein expression using the zipcode from another\nbud-localized transcript, like ASH1 for instance? What happen if the ASH1 E3 zipcode is\ninserted in the 3’UTR of the CLB2 ZIP mut mRNA? Is it a unique role for the CLB2 zipcode to\npromote protein expression?\nWhile we find that the reviewer suggests an interesting experiment, we think that this request\nfocusses on characterizing ASH1 ZIP code, thus it exceeds the goal of characterizing the\nCLB2 ZIP code properties and function. Furthermore, our data in Extended Figure 6 f-h\nsuggests that the translation regulation of the ASH1 and CLB2 mRNAs is diIerent, thus\nmaking diIicult to interpret a potential rescue of CLB2 mRNA translation with the ASH1 E3\nZIP code. In future studies we will continue investigating the translation regulation of bud-\nlocalized mRNAs.\nThere are a few mistakes in the text, such as “allows” instead of “arrows” (page 5, line 16).\nThank you for spotting this typo. It is now corrected in the revised version of the manuscript.\nPage 15, line 9: “The She2-She3 complex localizes the ASH1 mRNA to the bud, with the She2-\nmRNA interaction mediated via the E3 ASH1 ZIP code located in the ASH1 coding region”\nASH1 has 4 zipcodes, and not only one. The E3 zipcode is in the 3’UTR.\nGood point. This sentence was misleading. We corrected it in the revised manuscript.\nPage 15, line 17: “Puf1” should be replaced by “Puf6”.\nThank you for spotting this typo. It is corrected in the revised manuscript.\nPage 53, line 9, Legend video 2: “One single Z-stack was streamed every 50 ms”. This is not\nclear. Is that mean a single plane was captured every 50 ms or a Z-stack was captured every\n50ms? Also, at page 7, line 4, the Video 2 is described in the main text as “High frame-rate\nimaging every 100 ms…” Please clarify.\nGood point. This description was not clear. We corrected it in the revised manuscript.\nIndeed, we meant a single plane was captured every 50 ms.\nReviewer #2 (Remarks to the Author):\nLocalization of mRNAs can regulate translation and protein localization. The authors here\nconfirmed that CLB2 mRNA localizes to the bud of yeast cells and seek to understand how\nthis may regulate Clb2 protein levels and localization. They find that CLB2 mRNA localization\nis dependent on She2 and She3, like the well-studied, bud-localized ASH1 mRNA. The\nauthors identify a ZIP code in the coding sequence of CLB2 mRNA that is necessary for\nmRNA bud localization. RNA binding proteins that inhibit ASH1 mRNA translation were not\nfound to be important for CLB2 mRNA regulation, overall demonstrating key similarities and\ndiIerences in the mRNA level regulation of CLB2 and ASH1.\nTo address the function of CLB2 mRNA localization to the bud, the authors suggest that 1)\nCLB2 mRNA translation is more eIicient in the bud and 2) CLB2 mRNA localization to the\nbud contributes to cell size regulation. While these ideas are very compelling, more evidence\nis needed to support these conclusions.\nconstructive criticism. To address the reviewer’s concerns, we generated new strains and\nperformed experiments to further characterize the ZIP code-mediated regulation of CLB2\nmRNA localization and protein expression.\nMajor comments\n1. To provide evidence for CLB2 mRNA translation being more eIicient in the bud, the\nauthors demonstrate a slight but significant reduction in Clb2 protein expression observed\nvia Western Blot in she2Δ and she3Δ cells and a further reduction in a ZIP code mutant.\nHowever, localization to the bud is not suIicient for eIicient CLB2 translation since bud\nlocalization was rescued with a ZIP code in the 3’UTR but protein levels hardly increased.\nThe authors suggest that the ZIP code in the coding region may be important for eIicient\ntranslation through some other mechanism, but, alternatively, it could be that localization\nto the bud does not regulate CLB2 translation very much at all. To further test whether the\nZIP code has an eIect on translation eIiciency separate from localization, the authors could\nput the ZIP code mutant (with and without 3’UTR Zip rescue) in she2Δ cells. The prediction\nwould be that Clb2 protein levels are further reduced than in cells with she2Δ alone.\nThe reviewer raises an important point that is central to the message of the paper. What is\nthe contribution of mRNA localization to Clb2 protein expression? Are the ZIP code functions\nin translation and localization separated?\nAs detailed in the paper, the ZIP code rescue strain (Figure 6) shows a small (15-20%) but\nsignificant increase in Clb2 protein expression. We suggest that this 15-20% increase\ncorresponds to the contribution of mRNA localization in controlling Clb2 protein expression.\nWe suggest that the mRNA localization-dependent mechanisms of Clb2 protein expression\nregulation works alongside other well-known mechanisms of Clb2 protein expression\nregulation (including, proteasome-dependent PMID: 8020094, APC-dependent PMID:\n12152084, Hct1-dependent PMID: 9288748). This point was rephrased in the discussion to\nimprove clarity.\nTo address whether the ZIP code functions in translation and localization are separated we\ngenerated two new strains in which we combined the deletion of SHE2 or SHE3 with the ZIP\ncode mutant. As hypothesized by the reviewer, we would expect in the double mutants a\nfurther reduction of Clb2 protein levels if the eIect of the She proteins and the ZIP code on\nClb2 protein expression were independent. However, our data showed an epistatic eIect as\nin the double mutant strains (ZIP mut + Dshe2 and ZIP mut + Dshe3) we did not observe a\nfurther decrease in Clb2 protein expression (Figure below and New Extended data figure 5\nc,d,e).\neven\nthough future studies should confirm and characterize this interaction in in vitro and in vivo.\n2. The authors provide a secondary line of evidence that bud-localized CLB2 mRNA is more\neIiciently translated in the bud based on the co-localization of CLB2 mRNA and Clb2 protein\nfoci based on immunofluorescence staining (IF). IF in budding yeast is notoriously tricky, and\nindeed the pattern of Clb2 protein in live cell images (Fig 3g) is very diIerent from the IF\nimages (Fig 4b and 4e). In particular, the foci that are used for measurements of co-\nlocalization with mRNA in Fig 4 are not present in Fig 3. To strengthen this data, the authors\nneed to show that the Clb2 protein foci in the cytoplasm are not artifacts of IF.\nThe reviewer raises a good point, which gives us the opportunity to discuss the Clb2 protein\nexpression results in more detail.\nFirstly, we only partly agree with the statement that “the pattern of Clb2 protein in live cell\nimages (Fig 3g) is very diIerent from the IF images (Fig 4b and 4e)”. In both experimental\nsetups we see that the Clb2 protein is mainly nuclear localized, suggesting that both tagging\nmethods (N-terminal and C-terminal) preserve the expected Clb2 nuclear localization.\nHowever, we agree that the immunofluorescence data in fixed cells looks spottier than the\nGFP data collected in living cell. We think that this spotty profile is due to the primary and\nsecondary antibodies signal amplification, which is even further amplified by the presence\nof 25-myc tags in the Clb2 protein. This could potentially allow the detection of single Clb2\nproteins.\nTo address the reviewer’s concern, we added the requested control in the new Extended\ndata Figure 6e. We did not add it earlier to keep the manuscript concise, but we agree that\nthis is an important technical control that should be in the paper. As shown below, we\nperformed immunofluorescence using the anti-myc antibody both in wild-type cells and the\n11\n25-Clb2 myc-tagged strain. The experiments shown below were done on the same day and\nthe data were collected using the same imaging conditions. Furthermore, to compare the\ntwo images side-by-side, the same contrast and brightness levels were used. The\nimmunofluorescence shows low levels of non-specific binding in wild-type cells, suggesting\nthat the signal detected in the 25-Clb2 myc tagged strain is specific. Nevertheless, some\nbackground is indeed present, possibly explaining part of the unexpected Clb2 protein signal\ndetected during G1.\n3. Based on the extensive literature of Clb2-CDK driving mitotic entry and progression, there\nis a clear prediction that lower Clb2 protein will slow G2/M progression resulting in larger\ncells at the time of cell division. This is supported by the data in Fig 5 where the ZIP code\nmutants with lower Clb2 protein levels show larger cells at division. However, this does not\nexplain why mother cells would be larger at the time of budding (Fig 5C), which is controlled\nby the G1 size control mechanism and at a time when no Clb2 is present. This also doesn’t\nexplain why more bud volume is added in G2/M without an increase in budded timing. Is it\npossible these cells are larger at all cell stages for other reasons?\nWe agree with the reviewer that some data in the initial submission were confounding.\nWe\nbelieve that this was caused by the fact that the temporal resolution of our timelapse\nimaging was not suIiciently to precisely measure the changes we wanted to capture. While\nthe durations of the budded phase we included in the initial submission did not show\nsignificant diIerences between mutants, those diIerences (ca. 5 min) were based on\ndatasets in which frames were acquired every 5 minutes. We reacquired images with a\nhigher frame rate (every 60 s) to test if in the data submitted initially the acquisition rate\nmasked diIerences in duration. We found that imaging GFP once per minute led to slower\n12\ngrowth even at the very low LED power and exposure used in the experiment. Therefore, we\nrepeated the experiment while only acquiring bright field and red fluorescence, which did\nnot reduce growth rate appreciably, and which allowed us to determine the duration of the\nbudded phase with higher accuracy than in the initial submission. We found a highly\nsignificant increase in G2/M of around 5 min, consistent with the low time resolution data in\nthe initial submission. Given that a 5 min absolute increase in budded phase duration\ncorresponds to a relative increase in duration of around 10%, the diIerence in added bud\nvolume in Fig. 5g in the initial submission can be explained. Nonetheless, in this\nresubmission we have omitted subplots Fig. 5c-g of the initial submission for clarity.\n4. The authors try to link CLB2 mRNA localization and translation eIiciency to cell cycle\nprogression and a potential bud sizer mechanism. The crucial measurement is described in\nthe text as “While nuclear fluorescence tightly predicted bud size in WT and the Δshe\nmutants’ (Fig. 5k and Extended Data Fig. 7m) this eIect was significantly more disperse in\nthe ZIP-mutant strain, indicating a loss of communication between the two compartments.”\nA scatter plot of nuclear fluorescence vs bud size for the two populations would be more\nappropriate here. The authors also note that the ZIP code mutant added more material\nduring the budded phase than WT (Fig 5g) and spent longer median time in budded phase\n(Fig 5h, albeit the authors recognize this result is non-significant). The authors conclude that\n“control of CLB2 mRNA translation in the bud compartment reports bud size”. These two\nstatements (and the overall description of Fig 5) are diIicult to parse, and therefore the\nintended conclusions and interpretation from the data in Fig 5 are unclear. The results need\n13\nto be rewritten for clarity, and further experiments are needed that more directly test\nassociations between CLB2 mRNA localization and bud growth. If the intended conclusion\nis that Clb2 protein levels are regulated by bud size, it would be much more convincing to\ninclude other experiments manipulating bud size (latrunculin, or mutants in polarity, vesicle\ndelivery, or septins) and determining the eIect on Clb2 protein levels.\nAs detailed in the point above, we reacquired timelapse data to determine G2/M duration\nmore precisely. We found small but highly significant increases in budded phase duration in\nall mutants studied (New Figure 5d,e). Additionally, we have reacquired microscopy images\nto determine the correlation of bud compartment size and nuclear fluorescence (New\nFigure 5f,g). To get more reliable signal for both the bud neck marker and the fluorescently\nlabeled Clb2 protein, we acquired images at maximum LED power. To avoid irradiation-\ninduced growth phenotypes, we cultivated agarose pads in the dark and ran a single\nmeasurement per position, reasoning that acquiring many snapshot images would preserve\ncorrelations between bud compartment size and nuclear fluorescence. In WT, we found\nstrong correlation between nuclear fluorescence and bud size before mitosis (Fig. 5f), which\ndisappeared during and after mitosis (Extended data Fig. 7c). No strong correlation was\nevident for the ZIP mutant. We proceeded to restructure the content of Fig. 5 and extended\ndata Fig. 7 based on the new data collected and rewrote the relevant sections in the text.\nMinor comments\n5. In Fig 2 e, f, explicitly state which stage of the cell cycle is being imaged.\nThank you for the suggestion. Cells shown in Figure 2e are in G2 phase. The cells analyzed in\nFigure 2f include all budded cells. We added this information in Figure 2e and in the legend\nof Figure 2.\n14\n6. The authors assume the ZIP code mutant abrogates She2 binding, but it would be much\nstronger to show this directly by co-IP.\nWe agree with the reviewer that with our work we do not test directly the binding of She2 to\nthe CLB2 ZIP code. However, earlier work from Shepard et al. 2003 independently identified\nthe CLB2 mRNA as an interacting partner of She2, She3 and Myo4 (PMID: 13679573). Based\non those observations and on the new double mutant strains generated for the revised\nmanuscript (ZIP mut + Dshe2 and ZIP mut + Dshe3, New Extended data figure 5 c,d), we\nsuggested that a likely hypothesis is that the ZIP code mutant abrogates She2 binding. In the\nfuture, we are planning to continue the biochemical characterization of the ZIP code-She2\nbinding, including by doing co-IP and in-vitro binding experiments.\n7. A more careful description of strain construction is needed. Are the 5-myc Clb2 WT and\nZIP code mutants constructed at the endogenous locus and in the same way?\nThe 5-myc and the ZIP code mutants were all integrated at the CLB2 endogenous locus, and\nthey were generated using the same strategy. Following the reviewer’s suggestion, we now\nextended the Material and methods section describing in more detail plasmids and strains\nconstruction.\n8. According to the morphogenesis checkpoint model, the presence of a bud is read out by\na change in septin organization, which then leads to inactivation of Swe1, the Clb2-CDK\ninhibitor (Howell and Lew, 2012). A discussion of how the proposed regulation of Clb2\ntranslation relates to the regulation of Clb2 protein activity by the morphogenesis\ncheckpoint would be very useful for readers to synthesize the conclusions here.\nWe agree with the reviewer that it is important to try to reconcile our results with previous\nliterature. We briefly discussed this point in the Discussion and pointed the readers to\nrelevant literature.\n9. In an eIort to make scientific language more inclusive, the authors could consider\nchanging references of mother and daughter cells to parent (or originator) and progeny\ncells (White, et al, 2021, doi: 10.1038/d41586-021-03490-7).\nWe thank the reviewer for the suggestion. We will incorporate this change in future\nmanuscripts.\nReviewer #2 (Remarks on code availability):\nREADME files that I could find. I'm not very familiar with zenodo, so maybe I just missed it.\nWe thank the reviewer for noticing that we missed a readme file. We now included a readme\nfile in the new Zenodo repository associated with the revised manuscript.\nReviewer #3 (Remarks to the Author):\nThis manuscript uses a combination of fixed cell and live cell imaging to explore how\nlocalization of the CLB2 mRNA aIects protein expression, protein localization, cell size, and\ncell cycle progression. Overall, the experiments are well done, well described, and the\nconclusions are supported by the data presented. Much of the paper is a beautiful example\nof how rigorous quantification can provide new insights into biology. The paper doesn’t have\nall the answers and, in some respects, raises more questions than it answers. It reveals that\nthere must be multiple mechanisms that regulate where translation takes place because\ntranslation regulation for CLB2 is very diIerent than ASH1, another well-studied mRNA. The\nwork is exciting in identifying a zip code in the mRNA that is important for localization and\nperhaps translation.\nWe would like to thank the reviewer for the positive assessment of our work and for the\nconstructive criticism. We agree with the reviewer that this work (including the new\nexperiments done for the revisions of this manuscript) opens many avenues that we hope\nwe and others will be able to follow up in the future.\nOne thing I am trying to understand is: the localization mutants only show a subtle decrease\nin protein expression. Why is localization of the mRNA to the bud important? The authors\nsuggest that the mRNA may be more eIiciently translated in the bud than in the mother\nbased on data in Figure 4 that show there is greater colocalization of mRNA and protein in\nthe bud than the mother. But the diIerence is quite subtle (a mean of less than 1\ncolocalization spot in the mother compared to ~ 2 colocalization spots in the bud). While the\ndiIerence is statistically significant it seems extremely small from a biological perspective.\nCould the authors comment on this observation?\nThe data in Fig 4c are not corrected for compartment size. Rather than concentrations,\nactual counts are given. The bud compartment is always strictly smaller than the mother\ncompartment. Assuming a bud that has half of the volume of the mother, a 2:1 diIerence in\ntranslation foci becomes a 4:1 diIerence. At the low counts of mRNAs, we are observing\n16\n(<10 at peak expression), 0-1 transcripts located in the mother cell is the smallest amount\nwe can hope for in the presence of localization noise. Biologically, this mechanism is part of\na larger mechanism that finetunes Clb2 expression levels. Clb2CDK is subject to post-\ntranslational modifications via phosphorylation by e.g. SWE1, which titrates activity.\nIn Figure 4, the authors show that protein expression increases in G2/M and protein is more\nfrequently found in the bud (but it is still found in the mother more frequently). mRNA\nexpression increases significantly in the bud in G2/M. In WT cells only 25% of mRNA\nassociated with protein suggesting that mRNA is infrequently translated. In the mRNA\nlocalization mutant, the authors found more colocalizing mRNA and protein in the mother\nand less in bud - was this done as a function of the cell cycle (like in WT)? Did authors\nquantify mRNA and protein (like in c and d) and do they see a decrease in amount of protein\nat the single cell level (like in WB)?\nWe thank the reviewer for this suggestion. We did indeed quantify the CLB2 mRNA and\nprotein in the localization mutant as a function of cell cycle phase, but we did not report the\nanalysis in the first version of the manuscript. Below, on the left, we report Clb2 protein\nexpression measured by immunofluorescence in WT cells and in the Dshe2 mutant. The\nanalysis shows a consistent reduction in Clb2 protein expression across the cell cycle in the\nlocalization mutant. This is also visible when the data from the diIerent cell cycle phases is\ncompiled; Figure below, left panel (not included in the manuscript) and right panel, New\nExtended Data Figure 6f. Conversely, CLB2 mRNA expression does not show diIerences in\nthe localization mutant, regardless of the cell cycle phase (not shown).\nin the manuscript seemed to support the argument that the zip code in the mRNA interacts\nwith the she2-she3 system to localize to the bud and localization to the bud aIects protein\ntranslation and hence protein expression levels (to some extent). But the extended data in\nfigure 7 suggest that delta-she mutants that prevent localization of the mRNA to the bud\ndon’t exhibit a cell growth defect. In the discussion, the authors rationalize this observation\nwith the explanation that perhaps the zip code serves a dual function – promoting bud\nlocalization and eIicient translation. If that is the case, wouldn’t we expect to see\ndiIerences between the delta-she2 mutant and the zip code mutant in the single cell\nsingle molecule experiments in Figure 4? As far as I can tell, these experiments weren’t\ndone with the zip code mutant. But doing so, could potentially provide experimental\nevidence for the authors’ claims.\nWe agree with the reviewer that the expectation would be that in the ZIP code mutant we\nwould observe reduced co-localization of protein foci with mRNA by FISH-IF. However, we\ndecided not to do this experiment for this rebuttal for the following reasons. First, as the Clb2\nprotein expression in the ZIP mutant is very low (20-30% of WT), we expect that the signal\nwould be too noisy to be reliably detect by IF. Considering the eIort it would have taken to\nmake the strain and to perform and analyze FISH-IF data, we decided to prioritize other type\nof experiments we could do with the current lab personnel. Second, our data already\ndemonstrated, although indirectly, that the ZIP code mutant has a translation defect as the\nprotein reduction is not due to a change in mRNA expression, nor protein stability. We remain\nopen to generate this strain for future studies.\nReviewer #3 (Remarks on code availability):\nThe zenodo link provides all raw data for the paper. It also provides code (in R and Python)\nfor data analysis of FACS data and imaging data. As far as I can tell scripts are provided but\nthere are no readme files. I didn't install and run the code.\nWe thank the reviewer for noticing that a readme file was missing. We now included the file\nin the new Zenodo repository associated with the revised manuscript.\nReviewer #4 (Remarks to the Author):\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank this reviewer for the constructive criticism!\nReviewer #1 (Remarks to the Author):\nThe authors have properly answered the comments of the reviewers. Overall, this is a\nvery interesting work which uses leading-edge quantitative imaging to unveil a new\nmechanism linking mRNA localized translation to cell growth and cell cycle progression.\nI think this manuscript is now acceptable for publication in this journal.\nWe would like to thank this reviewer for the constructive criticism and for taking the time\nto evaluate our work.\nReviewer #2 (Remarks to the Author):\nIn this revision, the authors have greatly improved the experiments and clarity of the\ntext. I am convinced that CLB2 mRNA localization in the bud supports efficient\ntranslation of Clb2 protein, and, consistent with the field, the authors have provided\nconvincing evidence that Clb2 protein levels affect cell division timing. However, I\nremain concerned with the interpretations and conclusions surrounding CLB2 mRNA\nlocalization to the bud reflecting bud growth. This paper is strong and doesn’t require\nany further experiments, but the interpretations in the results and discussion sections\nneed to be more cautious.\nThe data is very convincing that Clb2 protein levels correlate with duration of the\nbudded phase and cell division timing. However, the relationship between Clb2 protein\nlevels and bud size are less straightforward. On several occasions, the authors have\nattempted to explain that Clb2 protein levels are a function of bud size (a couple\nexamples below), an interpretation that is not well supported with the data in this\nmanuscript. An alternative explanation could be that bud size is only a function of time,\ni.e. more time in budded phase results in bigger buds. Since lower Clb2 protein levels\nextend the duration of the budded phase, the bud sizes could simply be larger due to\nlower Clb2 protein levels. While both interpretations are possible, the data presented in\nthe paper does not directly address this issue. As stated in the original review, if the\nintended conclusion is that Clb2 protein levels are regulated by bud size, it would be\nmuch more convincing to include other experiments that manipulate bud size with\nindependent approaches (latrunculin, or mutants in polarity, vesicle delivery, or septins)\nand determine the effect on Clb2 protein levels. These experiments are probably\noutside the scope of the current manuscript, so I recommend just that the causal\nrelationship between Clb2 mRNA localization, protein levels, and bud size be restricted\nto speculation in the Discussion section.\nExample statements to reconsider:\n- 297 “Taken together, this chain of dependences can function as a mechanism that\nmeasures maturity of the bud compartment; active localization of a transcript to the bud\ncompartment may provide preferential access of that transcript to the ribosomes that\nare localized in that compartment, and a larger bud compartment harbors more\nribosomes. The concentration of the resulting protein should then reflect bud size.” –\nThis argument is unsupported and speculative.\n- 310 “If CLB2 mRNA localization were part of a mechanism that measures bud\nmaturity, mutants with non-localized transcript would be expected to be born larger, as a\nresult of the parent cell spending more time in the budded phase. Indeed, ZIP mutant\ncells were on average 10.9% larger at birth than wild type cells (Fig. 5h), but no\nsignificant effect was evident for the Δshe mutants (Extended data Fig. 7d,e).” – While\nthe data supports a correlation between CLB2 mRNA localization and cell size, the text\nhere implies causation where CLB2 mRNA measures bud maturity.\n- 430 “Altogether, our data suggest that by coupling CLB2 mRNA bud localization and\nprotein synthesis, cells coordinate cell growth with cell cycle progression by sensing the\nbud translation capacity via CLB2 mRNA transport and localized translation.” – This\npoint about sensing bud translation capacity is highly speculative and not a direct\nconclusion of the data presented.\nReviewer #2 (Remarks on code availability):\nI was able to access the code and the README file.\nWe would like to thank this reviewer for the significant effort taken to provide valuable\nfeedback and constructive criticism.\nWe understand the criticism and have revised the manuscript, specifically the Results and\nDiscussion sections, to reduce the implied causality between CLB2 mRNA localized\ntranslation and bud size control. In the final paragraph of the Discussion, our model now\nstates that CLB2 mRNA localization and nuclear protein levels may either reflect bud size\nor the duration of budding phase. This clarification acknowledges both possibilities and\nemphasizes that further experimental work is needed to test these hypotheses.\nReviewer #3 (Remarks to the Author):\nI appreciate the authors' due diligence in preparing this revised manuscript. They have\nsatisfactorily addressed all my concerns - either through experiments or explanations. I\nam supportive of publication.\nReviewer #3 (Remarks on code availability):\nThe zenodo link provides all data and code for the paper. It now includes a readme file.\nWe would like to thank this reviewer for the constructive criticism and for taking the time\nto review our work.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe would like to thank this reviewer for the constructive feedback and for dedicating\ntime and effort to evaluate our work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have properly answered the comments of the reviewers. Overall, this is a very interesting work which uses leading-edge quantitative imaging to unveil a new mechanism linking mRNA localized translation to cell growth and cell cycle progression. I think this manuscript is now acceptable for publication in this journal.", "reviewer_2": "In this revision, the authors have greatly improved the experiments and clarity of the text. I am convinced that CLB2 mRNA localization in the bud supports efficient translation of Clb2 protein, and, consistent with the field, the authors have provided convincing evidence that Clb2 protein levels affect cell division timing. However, I remain concerned with the interpretations and conclusions surrounding CLB2 mRNA localization to the bud reflecting bud growth. This paper is strong and doesn’t require any further experiments, but the interpretations in the results and discussion sections need to be more cautious.\nThe data is very convincing that Clb2 protein levels correlate with duration of the budded phase and cell division timing. However, the relationship between Clb2 protein levels and bud size are less straightforward. On several occasions, the authors have attempted to explain that Clb2 protein levels are a function of bud size (a couple examples below), an interpretation that is not well supported with the data in this manuscript. An alternative explanation could be that bud size is only a function of time, i.e. more time in budded phase results in bigger buds. Since lower Clb2 protein levels extend the duration of the budded phase, the bud sizes could simply be larger due to lower Clb2 protein levels. While both interpretations are possible, the data presented in the paper does not directly address this issue. As stated in the original review, if the intended conclusion is that Clb2 protein levels are regulated by bud size, it would be much more convincing to include other experiments that manipulate bud size with independent approaches (latrunculin, or mutants in polarity, vesicle delivery, or septins) and determine the effect on Clb2 protein levels. These experiments are probably outside the scope of the current manuscript, so I recommend just that the causal relationship between Clb2 mRNA localization, protein levels, and bud size be restricted to speculation in the Discussion section.\nExample statements to reconsider:\n- 297 “Taken together, this chain of dependences can function as a mechanism that measures maturity of the bud compartment; active localization of a transcript to the bud compartment may provide preferential access of that transcript to the ribosomes that are localized in that compartment, and a larger bud compartment harbors more ribosomes. The concentration of the resulting protein should then reflect bud size.” – This argument is unsupported and speculative.\n- 310 “If CLB2 mRNA localization were part of a mechanism that measures bud maturity, mutants with non-localized transcript would be expected to be born larger, as a result of the parent cell spending more time in the budded phase. Indeed, ZIP mutant cells were on average 10.9% larger at birth than wild type cells (Fig. 5h), but no significant effect was evident for the Δshe mutants (Extended data Fig. 7d,e).” – While the data supports a correlation between CLB2 mRNA localization and cell size, the text here implies causation where CLB2 mRNA measures bud maturity.\n- 430 “Altogether, our data suggest that by coupling CLB2 mRNA bud localization and protein synthesis, cells coordinate cell growth with cell cycle progression by sensing the bud translation capacity via CLB2 mRNA transport and localized translation.” – This point about sensing bud translation capacity is highly speculative and not a direct conclusion of the data presented.", "reviewer_3": "I appreciate the authors' due diligence in preparing this revised manuscript. They have satisfactorily addressed all my concerns - either through experiments or explanations. I am supportive of publication.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We would like to thank this reviewer for the significant effort taken to provide valuable feedback and constructive criticism.\nWe understand the criticism and have revised the manuscript, specifically the Results and Discussion sections, to reduce the implied causality between CLB2 mRNA localized translation and bud size control. In the final paragraph of the Discussion, our model now states that CLB2 mRNA localization and nuclear protein levels may either reflect bud size or the duration of budding phase. This clarification acknowledges both possibilities and emphasizes that further experimental work is needed to test these hypotheses."}]

Decision letter