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Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements

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Abstract

We describe a novel approach to separate two ribosome populations from the same cells and use this method in combination with RNA-seq to identify mRNAs bound to Saccharomyces cerevisiae ribosomes with and without Rps26, a protein linked to the pathogenesis of Diamond–Blackfan anemia (DBA). These analyses reveal that Rps26 contributes to mRNA-specific translation by recognition of the Kozak sequence in well-translated mRNAs and that Rps26-deficient ribosomes preferentially translate mRNA from select stress-response pathways. Surprisingly, exposure of yeast to these stresses leads to the formation of Rps26-deficient ribosomes and to the increased translation of their target mRNAs. These results describe a novel paradigm: the production of specialized ribosomes, which play physiological roles in augmenting the well-characterized transcriptional stress response with a heretofore unknown translational response, thereby creating a feed-forward loop in gene expression. Moreover, the simultaneous gain-of-function and loss-of-function phenotypes from Rps26-deficient ribosomes can explain the pathogenesis of DBA.

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Figure 1: Isolation of ΔRps26 ribosomes and characterization of bound mRNAs.
Figure 2: ΔRps26 ribosomes bind a distinct set of poorly translated mRNAs.
Figure 3: Rps26 promotes translation by recognizing specific residues in the Kozak sequence.
Figure 4: Accumulation of ΔRps26 ribosomes activates specific biological pathways.
Figure 5: Cells generate ΔRps26 ribosomes in response to high-salt and high-pH conditions.
Figure 6: Disruption of protein homeostasis by Rps26 insufficiency.

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Acknowledgements

We thank J. Cleveland, J. Joyce and members of the Karbstein lab for comments. This work was supported by the US National Institutes of Health (grants R01-GM086451 (to K.K.) and F31-GM116406 (to M.B.F.)), PGA National (Women's Cancer Awareness Day fellowship to H.G.), the Richard & Helen DeVos Foundation (graduate fellowship to M.B.F.), and the Howard Hughes Medical Institute (Faculty Scholar grant 55108536 to K.K.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health.

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Contributions

Experiments were designed by M.B.F., H.G. and K.K. M.B.F., H.G. and E.L.P. performed and analyzed the experiments. E.A.W. wrote software scripts to analyze sequencing data. The manuscript was written and edited by M.B.F., H.G. and K.K., and all authors support the conclusions.

Corresponding author

Correspondence to Katrin Karbstein.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Rps3-TAP does not perturb ribosome functionality.

Cells containing genomically TAP-tagged Rps3 were grown in parallel to unmodified cells. Growth was tested by (a) serial dilution and (b-c) by continuous measurement of OD600 in liquid culture. Bars represent mean values. d, Rps3-TAP cells were also treated with cycloheximide, run through a sucrose gradient and analyzed by western blot to demonstrate the presence of TAP-tagged Rps3 in polysomes. The small amount of untagged Rps3 is likely the result of degradation. This experiment was replicated twice and representative results are shown. e, Parallel TAP purifications were performed to compare the ribosomes captured by genomically encoded Rps3-TAP with those captured using an inducible TAP tag combined with Rps26 depletion (as in Figure 1) and immature (pre-40S) ribosomes that should not contain Rps26 which were captured by TAP-tagging the assembly factor Rio2. These data show that Rps3-TAP can effectively pull out ribosomes containing Rps26 (middle panel), but do not do so in our Rps26 depletion system (left). This experiment was repeated over five times and representative results are shown. f, Bioanalyzer traces of purified RNA used for the three sequencing replicates. These traces show that the ΔRps26 ribosomes mostly contain 18S, as opposed to 20S, rRNA. g, Quantification 18S vs 20S rRNA in ΔRps26 ribosomes from f, bars represent mean. h, The elution from the genomically encoded Rps3-TAP pulldown was sequenced and compared to the +Rps26 and ΔRps26 ribosome pools using DEseq2. The distributions of p-values show that the mRNAs bound to wild type ribosomes containing Rps3-TAP are different from those on Rps3-TAP ΔRps26 ribosomes (left) but not significantly different from the untagged Rps26-containing ribosomes from the TAP-flow-through (right).

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Supplementary Figure 2 Rps3-TAP pulldown of ΔRps26 ribosomes generates reliable sequencing results.

a, Rps3-TAP is effectively removed from total cell lysates (IN) resulting in lower TAP signal present in the flow-through (FT). This experiment was repeated six times and representative data are shown. b, On average, 73.4% of TAP signal is depleted from FT as compared to input (n=6). Of the total TAP signal in the input, ~14% is captured in the elution. Bars represent mean, error bars = SEM. c, 90.2% of yeast ORFs were represented by >128 reads in each sequencing experiment. Of those with fewer reads, many are annotated as uncharacterized, and may not exist. d, RNA-seq was performed on three biological replicates, which are well-correlated by RPKM analysis. e, Metagene analysis using existing datasets of enriched transcripts after removal of mRNAs encoding ribosomal proteins. Number of mRNAs analyzed: TE: all=5353, +=724, Δ=736; 5’ UTR: all=4229, +=640, Δ=575; ORF length: all=5553, +=737, Δ=741; Abundance: all=5320, +=679, Δ=739; 3’ UTR: all=4788, +=706, Δ=658; Half-life: all=4057, +=572, Δ=531. f, Metagene analyses that showed no difference when comparing enriched transcripts using existing datasets. Number of mRNAs analyzed: 3’ UTR length: all=4926, +=833, Δ=658; Half-life: all=4189, +=694, Δ=531. For e and f, boxplot whiskers represent the 10-90th percentile. Boxes represent the interquartile range, midline is the median. By Kolmogorov-Smirnov test, (****) p<0.0001 and (**) p=0.0095.

Source data

Supplementary Figure 3 Characterization of Rps26-depleted yeast.

a, Strain construction to accumulate ΔRps26 ribosomes. b, TET:Rps26 cells are deficient in Rps26 when exposed to dox as seen by western blot of total cell lysates from three biological replicates. This experiment was repeated 5 times and representative data are shown. c, Quantification of panel b with Rcl1 as a loading control, bars represent mean values. d, Untreated TET:Rps26 cells have a slightly increased doubling time compared to the parental yeast cell. Boxes indicate interquartile range, the midline is the median and whiskers represent the range.. e-f, Polysome profiles of TET:Rps26 cells -dox (e) or +200ng/ml dox (f) showing fractions used for qPCR analysis. Rps26-depleted cells have reduced polysomes. g, Pgk1 and Rpl3 are genes enriched on +Rps26 ribosomes and the distribution of both transcripts shifts towards lighter polysomes after depletion of Rps26. h, Sda1 and Rox1 mRNAs are enriched on ΔRps26 ribosomes and show a small increase in the middle of the gradient. Data is from three independent experiments for Rox1 and two for all others, error bars = range. For comparison, we attempted to use genes with similar length and TE values. However, all mRNAs in the ΔRps26 sample that we used appeared to be less well translated than expected based on published results, which could affect the outcome of the data, as there are fewer translational units to lose. Nevertheless, in these cases, increases in ribosome recruitment in the middle of the gradient are visible, indicating that the observed effect is not simply an artifact. i, Among ribosome assembly factors required for production of the 40S, 60S, or both subunits, the mRNA encoding Fap7 is specifically enriched in Rps26-containing ribosomes. j, Relative to Rcl1, Fap7 is depleted from cells upon Rps26 depletion. Quantification on the bottom is from two independent experiments, and representative data are shown; error bars = range.

Source data

Supplementary Figure 4 Additional information related to dual-luciferase assays.

a, Cells were transformed with plasmids encoding firefly luciferase and a Renilla luciferase reporter constructs containing a change in the upstream sequence. The signal from each Renilla luciferase variant is normalized to the signal from firefly luciferase and these ratios were then normalized to the ratio observed in A10 cells. b, Nucleotide distribution at the -4 position for mRNAs enriched in each ribosome pool and for the genome as a whole. Sequences analyzed: +:865 Δ:741 All:5790. c, DNA was extracted from cells used for the luciferase assay, RNase-treated and analyzed by qPCR to quantify relative plasmid copy number. Treatment causes no change in the relative copy number of either plasmid, error bars = SD, p=0.7087 t=0.3919 DF=6 by t-test. d, Cells with Rps3 or Rps17 under the control of a dox-repressible promoter translate less Renilla luciferase when it is preceded by a sequence with a -4G compared to the A10. Unlike Rps26, this effect is not mitigated by depletion of either protein. For TET:Rps3, n=13 for all samples and DF=48; -dox (*) p=0.0168 t=2.747 and +dox (*) p=0.0294 t=2.245. For TET:Rps17, n=16 for A10 and n=15 for -4G, DF=48; -dox (****) p<0.0001 t=7.637 and +dox (****) p<0.0001 t=9.284. e, Wild type (BY4741) or ΔLtv1 cells translate less Renilla luciferase when it is preceded by a sequence with a -4G compared to the A10. For WT, n=7 for both constructs, (****) p<0.0001 t=5.488. For ΔLtv1, n=12 for both constructs, (****) p<0.0001 t=9.605. DF=31. For d-e, data was analyzed by two-way ANOVA, bars represent the mean and error bars = SEM.

Source data

Supplementary Figure 5 Salt and pH resistance is not a general response to ribosomal protein depletion or 20S accumulation.

a-g, Cells with Rps3, Rps17 or Rps26 under the control of the dox-repressible promoter were grown to mid-log phase in YPD supplemented with the indicated amount of dox and then exposed to salt, high pH or caffeine stress. The doubling times in stress media were compared to the doubling times without stress. Boxes indicate interquartile range, the midline is the median and whiskers represent the range. h-i Stress sensitivity of WT (BY4741) and ΔLtv1 cells.

Source data

Supplementary Figure 6 Transcriptional regulation of Rps26

a-c, Wild type cells grown to mid-log phase and exposed to stress do not show a specific downregulation of Rps26 mRNA relative to other ribosomal proteins when analyzed by RT-qPCR, n = 2 independent experiments, error bars = range. d, WT cells were transformed with a GAL::Rps26 plasmid or an empty vector (VO), grown in galactose-containing media, and then exposed to salt, high pH or caffeine stress. Error bars = SD. Overexpression of Rps26 did not cause a change in stress sensitivity, even though overexpression of Rps26 under the GAL1 promoter results in a ~11-fold increase (average DDCt = 3.45) in Rps26 mRNA relative to mRNAs encoding Asc1, Pgk1, or Rpl3 (e) as measured by RT-qPCR. Error bars = SEM, n=4.

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Supplementary Figures 1–6 and Supplementary Tables 1–3 (PDF 2287 kb)

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Supplementary Data Set 1

Rps26 dependent enrichment of mRNAs (XLSX 935 kb)

Supplementary Data Set 2

GO terms associated with Rps26 enrichment (XLSX 19 kb)

Supplementary Data Set 3

Uncropped gels and blots (PDF 19188 kb)

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Ferretti, M., Ghalei, H., Ward, E. et al. Rps26 directs mRNA-specific translation by recognition of Kozak sequence elements. Nat Struct Mol Biol 24, 700–707 (2017). https://doi.org/10.1038/nsmb.3442

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