EMBO/soda-nel huggingface dataset:
Mitotic checkpoint gene expression is tuned by codon usage bias (2022)
Figure 1

Figure 1. Low steady-state mRNA numbers of checkpoint genes mad1+, mad2+, and mad3+.

(A) Overview of the interactions between Mad1, Mad2, and Mad3.

(B) Schematic of marker-less GFP-tagging at the endogenous locus and representative live cell images of Mad1-, Mad2-, and Mad3-GFP strains (average intensity projections).

(C) Representative images of single molecule mRNA FISH (smFISH) staining of S. pombe using probes against GFP (red). DNA was stained with DAPI (blue). The gamma-value was adjusted to make the cytoplasm visible; cell shapes are outlined in blue.

(D) Frequency distribution of mRNA numbers per cell determined by smFISH; combined data from 3, 4, and 5 experiments, respectively, shown separately in Fig EV1C; n = number of cells. Curves show fit to a Poisson distribution.

(E) Frequency distribution of mRNA numbers per cell using FISH probes against the endogenous genes and using either strains expressing the GFP-tagged gene or the endogenous, untagged gene. Curves show fit to a Poisson distribution. The difference for mad1+ is statistically significant, that for mad2+ is not (Fig EV1E). A lower mRNA number for untagged mad1+ was also observed in an independent strain.

(F) Co-staining by smFISH using probes against mad1+ and GFP either in a strain expressing mad1+-GFP as a positive control or in a strain expressing wild-type mad1+ and mad2+-GFP. Cytoplasmic mad1+ (green) or GFP mRNA spots (magenta) were quantified as co-localizing or not with the respective other. For the mad1+-GFP strain, 544 cells and a total of 1,641 mad1 spots and 1,839 GFP spots were analyzed; 48 cells were not considered since they did not contain at least one spot of each type in the cytoplasm. For the mad1+ mad2+-GFP strain, 571 cells and a total of 1,107 mad1 spots and 1,537 GFP spots were analyzed; 158 cells were not considered since they did not contain at least one spot of each type in the cytoplasm.
Mitotic checkpoint gene expression is tuned by codon usage bias (2022)
Figure 2

Figure 2. The checkpoint genes mad1+, mad2+, and mad3+ combine short mRNA and long protein half-lives, explaining low noise.

(A) Cellular protein noise (coefficient of variation, CV = std / mean) in live-cell microscopy images of S. pombe; n = 7 images (Nmt1-GFP), 11 (Mad1-GFP), 19 (Mad2-GFP), 10 (Mad3-GFP); single images had 16-79 GFP-positive and 6-94 GFP-negative (control) cells. Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Mad1, Mad2, and Mad3 all showed significantly lower noise than Nmt1 (Wilcoxon rank sum test; all p < 0.001).

(B) Simulations of stochastic gene expression noise from selected mRNA/protein half-life combinations assuming a constantly active promoter (see Methods). Synthesis rates were set to obtain a mean mRNA number of 4 per cell, and a mean protein number of 6000 per cell. The x-axis of each graph shows time, the y-axis shows mRNA number per cell (blue) or protein number per cell (black).

(C) Theoretical prediction for the coefficient of variation (CV = std / mean) of the protein number per cell, assuming different mRNA and protein half-lives, using the same underlying model as in B. Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3.5, and a mean protein number per cell of 6000 (approx. 100 nM).

(D) mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Lines are regression curves from generalized linear mixed model fits, excluding the measurements at t = 0 in order to accommodate for non-instantaneous labeling by 4tU. Act1+ and ecm33+ were used as long and short half-life controls, respectively; qPCR was performed for the endogenous mRNAs. Half-lives (95 % confidence interval): mad1+ 5.6 min (4.3 - 8.4), mad2+ 7.7 min (6.2 - 10.4), mad3+ 5.2 min (4.3 - 6.9), act1+ 61.8 min (37.2 - 172.3), ecm33+ 5.0 min (4.5 - 5.7).

(E) Protein abundances after translation shut-off with cycloheximide (CHX); n = 3 experiments, error bars = std. Lines indicate fit to a one-phase exponential decay. Cdc2 and Cdc13 were used as long and short half-life controls, respectively. Immunoblots for the endogenous proteins (no tag). A representative experiment shown in Fig S2E.
Mitotic checkpoint gene expression is tuned by codon usage bias (2022)
Figure 3

Figure 3. Codon optimization increases the steady-state mRNA numbers of mad2 and mad3.

(A) The mean CSC value for each S. pombe gene (CSCg) relative to protein number per cell by mass spectrometry (Carpy et al., 2014). CSC was determined using the mRNA half-life data by Eser et al. (Eser et al., 2016) as described in Methods. Colored dots highlight proteins of interest. For Mad2 and Bub1, no protein abundance data was available.

(B) Cumulative frequency distribution of the CSCg values for protein-coding S. pombe genes. The position of spindle assembly checkpoint genes is highlighted.

(C) Schematic of the mad2+ and mad3+ genes. Regions coding for important structural features are highlighted. Black lines in the bottom graph indicate synonymous codon changes in the codon-optimized version.

(D) Scatter plots of whole cell RNA counts versus cell length. Solid lines are regression curves from generalized linear mixed model fits (gray: wild type, black: codon-optimized or ste13∆). Dashed lines: 95 % bootstrap confidence bands for the regression curves. Model estimates of the ratio relative to wild-type mRNA are included with bootstrap 95 % confidence interval in brackets. Two to five replicates per genotype.

(E) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Solid lines: regression curves from generalized linear mixed model fits (dark = ste13+, light = ste13∆), excluding t = 0 to accommodate for non-instantaneous labeling by 4tU. Shaded area: 95 % bootstrap confidence band for ste13+; dashed lines: 95 % bootstrap confidence band for ste13∆. Half-life estimates are included with 95 % bootstrap confidence intervals in brackets. See Fig EV2C for additional statistics. The ste13+ data are the same as in Fig 2.
Mitotic checkpoint gene expression is tuned by codon usage bias (2022)
Figure 4

Figure 4. Codon optimization increases the protein concentrations of Mad2 and Mad3.

(A) Immunoblot of S. pombe protein extracts from cells expressing wild-type (WT) or codon-optimized (co) Mad2-GFP or Mad3-GFP probed with antibodies against GFP and Cdc2 (loading control). Lanes 3-5 are a 1:1 dilution series of the extract from cells expressing the codon-optimized version.

(B) Immunoblot of protein extracts from wild-type (WT) or ste13∆ strains probed with antibodies against Mad2, Mad3, and tubulin (loading control). A 1:1 dilution series was loaded for quantification.

(C) Estimates of the protein concentration relative to wild-type conditions from experiments such as in (A) and (B). Bars are experimental replicates, dots are technical replicates. Two-sided t-tests: p = 0.03 (Mad2-co), 0.004 (Mad3-co), 0.82 (Mad2 ste13∆), 0.15 (Mad3 ste13∆).

(D) Whole-cell GFP concentration from individual live cell fluorescence microscopy experiments (a.u. = arbitrary units). Boxes show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Codon-optimized concentration significantly higher than wild type for both genes (GLMM). Mad2-GFP: n = 468 and 413; Mad2-co-GFP: n = 206 and 366; Mad3-GFP: n = 224 and 127; Mad3-co-GFP: n = 160, 450 and 212 cells.

(E) Representative images from one of the experiments in (D). A single Z-slice is shown. Cells are outlined in gray.
Mitotic checkpoint gene expression is tuned by codon usage bias (2022)
Figure 5

Figure 5. Codon optimization and ste13+ deletion do not significantly affect the steady-state mRNA number of mad1+.

(A) Schematic of the mad1+ gene. Regions coding for important structural features are highlighted. Black lines in the bottom graph indicate synonymous codon changes in the codon-optimized version.

(B) Scatter plots of whole cell mRNA counts versus cell length. Solid lines are regression curves from generalized linear mixed model fits (gray: wild type, black: codon-optimized or ste13∆). Dashed lines: 95 % bootstrap confidence bands for the regression curves. Model estimates of the ratio relative to wild-type mRNA are included with bootstrap 95 % confidence interval in brackets.

Two to three replicates per genotype.

(C) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Solid lines: regression curves from generalized linear mixed model fits (dark = ste13+, light = ste13∆), excluding t = 0 to accommodate for non-instantaneous labeling by 4tU. Shaded area: 95 % bootstrap confidence band for ste13+; dashed lines: 95 % bootstrap confidence band for ste13∆. Half-life estimates are included with 95 % bootstrap confidence intervals in brackets. See Fig EV4E for additional statistics. The ste13+ data are the same as in Fig 2.

(D) Comparison between mean CSC values for selected genes (CSCg) and mRNA half-life measured with or without deletion of ste13+. mRNA half-life estimates from Figs 3E, 5C, and EV4D.
Mitotic checkpoint gene expression is tuned by codon usage bias (2022)
Figure 6

Figure 6. Codon identity in mad1+ is important for proper protein concentration.

(A) Immunoblot of S. pombe protein extracts from cells expressing wild-type (WT) or codon-optimized (co) Mad1-GFP probed with antibodies against GFP and Cdc2 (loading control). Lanes 3-5 are a dilution series of the extract from wild-type cells.

(B) Immunoblot of protein extracts from wild-type (WT) or ste13∆ strains probed with antibodies against Mad1 and tubulin (loading control). A 1:1 dilution series was loaded for quantification. Tubulin blot is the same as in Fig 4B.

(C) Estimates of the protein concentration relative to wild-type conditions from experiments such as in (A) and (B). Bars are experimental replicates, dots are technical replicates. Blue lines indicate the mean of all experiments. Two-sided t-tests: p = 0.005 (Mad1-co, n = 4 experimental replicates); p = 0.16 (Mad1 ste13∆, n = 2).

(D) Whole-cell GFP concentration from individual live cell fluorescence microscopy experiments (a.u. = arbitrary units). Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Codon-optimized concentration significantly lower than wild type (generalized linear mixed model). Mad1-GFP: n = 197 and 224; Mad1-co-GFP: n = 80 and 377 cells.

(E) Representative images from one of the experiments in (D). An average projection of three Z-slices is shown; cells are outlined in gray.

(F) Live-cell imaging for time spent in mitosis. The alp7+ gene was deleted to increase the likelihood of spindle assembly checkpoint activation. Localization of Plo1-tdTomato to spindle pole bodies was used to judge entry into and exit from mitosis (also see Figure S4). Exp1: n = 73 (WT) and 94 cells (co); Exp2: n = 126 (WT) and 152 cells (co). Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Measurements for individual cells are shown in addition (gray circles if measurement was exact, red triangles if end of mitosis was not captured because imaging ended). Difference between WT and co: p = 0.14 (Exp1) and 0.15 (Exp2) by Kolmogorov-Smirnov test.
RNA polymerase II contributes to preventing transcription‐mediated replication fork stalls (2014)
Figure 1

Genetic interactions of RNAPII mutations with DNA repair mutations

Viability of single and double mutants of RNAPII and rad52Δ, mre11Δ and cdc44‐8. Tenfold serial dilutions of double mutants obtained by genetic crosses were tested for growth in plain SC medium or supplemented with 10 mM HU or 0.005% MMS.

Tetrad analysis of rpb1‐S751F, rad52Δ and mre11Δ crosses. Δ indicates double mutants that fail to grow.

Tetrad analysis of rpb1‐S751F crossed by pol32Δ and rad51Δ. Δ indicates triple mutants that fail to grow.

HU and MMS sensitivity of different mutant combinations of rpb1‐1, rpb1‐S751F, pol32Δ and rad51Δ.

Data information: Cells were cultured for 3 days at 30°C.
RNA polymerase II contributes to preventing transcription‐mediated replication fork stalls (2014)
Figure 5

Effect of rpb1‐1 and rpb1‐S751F on replication velocity

Analysis of replication by BrdU incorporation. Cells were synchronized in G1 with α‐factor and released in the presence of BrdU. Samples were taken at the indicated times and processed to measure BrdU incorporation by ChIP analysis. Replication was analyzed in two DNA regions, one covering the early origin ARS508 and the SPF1 gene and the other the late origin ARS603 and the YFL034W gene.

DNA combing analysis of replication. Graphs indicate the distribution of BrdU track length (kb) at 60 and 120 min after α‐factor release in the presence of 40 mM HU, the replication speed (kb/min) determined with the two time points analyzed and the inter‐origin distance (kb) at 60 min after release. The median test was applied.
RNA polymerase II contributes to preventing transcription‐mediated replication fork stalls (2014)
Figure 8

Replication-transcription relationship

Analysis of Rrm3 recruitment in WT and rpb1‐1. Rrm3 IP histogram bars in the y‐axis show the average signal log2 ratio of loci enriched in the immunoprecipitated fraction along the indicated regions in the x‐axis. When the ratio fulfills the P‐value criteria (P 0.01) it is shown in red.

Composite profile of Rrm3 occupancy across the average ORF plotted as Rrm3 percentage of significant ChIP hits per segment. Each gene was divided into 10 equivalent segments from the start and end coordinates (segments 2-10). Two additional segments of the same size were considered upstream (5′) and downstream (3′) of each ORF (segments 1 and 12, respectively). The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted.

Composite profile of Rrm3 occupancy across the average ARS plotted as Rrm3 percentage of significant ChIP hits per segment. Each ARS was divided into 11 equivalent segments from the start and end coordinates. The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted.

Model for a role of RNAPII as a source of genetic instability. Conflicts between transcription and replication machineries affect replication progression causing DSBs at high levels under exogenous replicative stress, which demands the HR machinery for the repair and restart of RFs. RNAPII would have a function facilitating RF progression upon a transcription-replication encounter. This function would be affected in the rpb1 and rpb9 mutants analyzed here, causing RF progression impairment and genome instability.
ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking (2016)
Figure 3

Figure 3. Polarization patterns of BiFC between the Dsl complex, the COPI coat, and COPI cargo

Time-lapse micrographs of agarose-embedded cells were taken at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Representative images of characteristically reoccurring fluorescence localization stages are displayed. In all combinations, fluorescence foci localize to areas of membrane outgrowth in the bud. Often, another prominent fluorescent spot is found in the mother cell on the side of the bud neck. Scale bar 5 µm. Kymographs of all time-lapse datasets shown in this Figure are presented in Fig EV3A.

A-D. Cells expressing complementary BiFC-tagged proteins between subunits of the Dsl complex and COPI were analyzed. Panel (D) shows a BiFC combination that includes a mutated VC-tag. This mutation in the VC part is equivalent to the A206K mutation in GFP, which prevents the formation of YPF dimers (Zacharias et al., 2002).

E, F. The VC-tagged COPI vesicle cargo protein Rer1p was examined in combination with the VN-tagged COPI subunit β’-COP or the VN-tagged Dsl1 protein.

G. BiFC spots in COPI•COPII BiFC combination (β’-COPVN•Sec16pVC) were also polarized.

H. COPI•COPI (α-COPVN•α-COPVC) BiFC signals showed one spot that localized to the bud tip and, at later budding stages, to the bud neck (compare with A-G). Additionally, multiple spots with no apparent polarization tendency were observed, which are reminiscent of a Golgi pattern.

I. β’-COPmGFP fluorescence foci in Dsl1p-depleted GAL-DSL1 cells after incubation in glucose-containing medium (YEPD) for 4 h at 30 °C.

J. Schematic representation of the characteristic BiFC spot localization during different phases of bud outgrowth. BiFC foci are displayed in black, ER is marked dark gray for orientation.
ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking (2016)
Figure 4

Figure 4. Organelle association of β’-COPVN•Dsl3VC BiFC spots

Time-lapse images of agarose-embedded cells were taken at RT with supply of fresh PM Glc+ura medium throughout one budding cycle. Only (F) shows fixed cells immunostained for Sed5p. All cells produced the β’-COPVN•Dsl3pVC BiFC pair. Most strains contained plasmids encoding different organelle markers as indicated below. BiFC signals are pseudocolored green, organelle markers are pseudocolored red. Scale bar 5 µm.

A. RFPHDEL, ER marker

B. Sec13pRFP, ER exit sites marker

C. RFPSed5p, cis-Golgi markers (genomic)

D. CFPGos1p, medial Golgi marker

E. Sec7pCFP, trans-Golgi marker

F. Individual fixed cells carrying the β’-COPVN•Dsl3pVC BiFC pair along with trans-Golgi marker CFPSec7p were used for this panel. The β’-COPVN•Dsl3pVC BiFC signals are pseudocolored white. Immunostaining was used to visualize the cis-Golgi marker Sed5p (red). The trans-Golgi marker CFPSec7p is pseudocolored green.

G. CFPRer1, COPI cargo

H. CFPSec4p, marker for secretory vesicles
ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking (2016)
Figure 5

Figure 5. Effects of the dsl1-5xWA mutation on COPI•COPII BiFC and COPI•Dsl BiFC

A. Fluorescence micrographs of two different COPI•COPII BiFC pairs are shown: Sec16pVN•αCOPVC or Sec16pVN•ε-COPVC. DSL1 cells show dispersed punctate patterns resembling ER exit site patterns. In the dsl1-5xWA mutant, cells exhibit additional bright spots. Scale bar 10 µm.

B. Quantification of fluorescence intensity differences of the strains shown in (A). COPI•COPII BiFC fluorescence is significantly increased in cells carrying the dsl1-5xWA mutant as compared to DSL1 wild type. Mean values + SEM of at least nine independent experiments (n=918) are displayed (two-sample unpaired twotailed ttest).

C. Time-lapse micrographs of cells expressing the β’-COPVN•Dsl3pVC BiFC pair in a dsl1-5xWA background. Cells were imaged embedded in agarose at RT with fresh PM Glc+ura medium supply throughout one budding cycle. Fluorescence signals retain the characteristic polarization pattern as seen DSL1 wild type cells (see Fig 3). Scale bar 10 μm.
ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking (2016)
Figure 6

Figure 6. Dependence of ERAS distribution on cytoskeletal structures

A-B. Involvement of filamentous cytoskeletal structures in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells expressing the Dsl1pVN•ε-COPVC BiFC combination at RT with fresh PM Glc+ura medium supply. (A) Upon disruption of actin filaments with 1,25 μM latrunculin A, COPI•Dsl BiFC signals dispersed. (B) Microtubuli disruption through 24 μg/ml nocodazole did not affect polarization of the COPI•Dsl BiFC signals. Arrowheads: polarized signal, asterisks: unpolarized localization. Fluorescent images are displayed with inverted brightness values. Scale bar 10 μm.

C-E. Involvement of myosin motors in BiFC foci polarization. Time-lapse micrographs of agarose-embedded cells carrying the COPI•Dsl BiFC pairs Dsl1pVN•ε-COPVC or β’COPVN•Dsl3pVC were taken. Either wild type cells (E) or mutants carrying the myosin V mutations myo2-66 (C) or myo4∆ (D) were analyzed. Cells were grown at RT with fresh PM Glc+ura medium supply. In the myo2-66 cells, the BiFC signal polarization was hardly detectable even at permissive temperature, while it was unaffected in myo4∆ compared to the control cells.

F. Fluorescence micrographs of β’-COPVN•Dsl3pVC cells carrying the myo2-66 defect. COPI•Dsl BiFC, as well as ER or Golgi markers were analyzed. While the BiFC spots were dispersed in these mutants, they retained their association with ER and Golgi. Scale bar 5 μm.
ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking (2016)
Figure 7

Figure 7. Subcellular localization of COPI•Dsl interactions in immuno-electron microscopy

A-B. Immuno-electron micrographs showing the buds and the neck region of two β’-COPVN•Dsl3pVC cells. Both parts contain an overview image (left) and two zoomed details (right). Slides were decorated with antibody against COPI and visualized with protein A-conjugated 10 nm gold particles. Gold particles mark electron-dense areas in apposition to membranes and/or vesicular structures, which are localized at the side of the bud and the bud neck of the mother cell. Scale bar 100 nm.

C. Fluorescence micrograph of a budding cell carrying β’-COPVN•Dsl3pVC (green) and RFPHDEL (red). Arrowheads mark BiFC signals which may correspond to the localizations found in (A) and (B). Scale bar 2 µm.

D. COPI-positive, electron-dense structures in close apposition to perinuclear ER. 10 nm gold particles mark COPI. Scale bar 100 nm.

E. Accumulation of COPI-positive vesicles. 10 nm gold particles mark COPI. PM: plasma membrane, nuc: nucleus. Scale bar 100 nm.
Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion (2014)
f1

(a) RT–PCR analysis of LC3, IL-8, CHOP and GADD34 in U2OS cells subjected to glutamine deprivation (−Q) or exposed to TPG (1 μM). (b) Immunoblot analysis of ER stress- and autophagy-related proteins in U2OS cells subjected to glutamine deprivation or TPG treatment. Samples are run in triplicate. (c) Cytokine array analysis of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. (d) IL-8 enzyme-linked immunosorbent assay of conditioned media from U2OS and A549 cells grown in the presence (+Q) or absence (−Q) of glutamine. Error bars in all figures represent s.d. of three biological replicates.
Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion (2014)
f2

(a) Autophagosomes were visualized in U2OS cells stably expressing a GFP-LC3 reporter construct. GFP-labelled puncta were examined after 24 h in the presence (+Q) or absence (−Q) of glutamine, with or without co-addition of 100 nM CCI-779 (CCI) and 400 nM BafA1. Scale bar, 10 μm. (b) Graphical summary of experiments performed as described in a. Percentage of cells with >10 puncta per cell from three independent experiments is depicted. Bars represent mean±s.d. from three independent experiments (>50 cells per experiment). The statistical significance (P value) was determined by a two-tailed, paired Student’s t-test. *P0.05. (c) Immunoblot analysis of U2OS cells subjected to glutamine deprivation with or without 400 nM BafA1. Cells were pretreated with BafA1 for 1 h before and during exposure to glutamine-deficient medium. Autophagic activity was monitored by detection of p62 and LC3-II proteins. (d) U2OS mCherry-GFP-LC3 cells were cultured in the presence (+Q) or absence (−Q) of glutamine for 18 h. Red vesicles denote autolysosomes, whereas yellow vesicles represent autophagosomes. Bars indicate numbers of yellow vesicles (autophagosomes) or red vesicles (autolysosomes) per cell±s.d. (e) Images of U2OS mCherry-GFP-LC3 cells cultured for 18 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm. (f) Phagophore formation in mCherry-ULK1. Scale bar, 10 μm. (g) mCherry-ATG5-expressing U2OS cells after 24 h in the presence (+Q) or absence (−Q) of glutamine. Scale bar, 10 μm.