EMBO/soda-nel huggingface dataset:
A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast (2022)
Figure 2

Figure 2: Finding stable and transient Emc6 interactors by a multi-faceted LC-MS/MS approach.

(A) Schematic of the workflow used for LC-MS/MS sample preparation of the BioID2-HA-Emc6, TurboID-HA-Emc6, and TurboID-HA-Emc6/ABOLISH strains and their control counterparts (tagged-Sbh1, depicted as grey yeast). All strains were prepared in biological triplicate.

(B) Volcano plots for each of the four distinct LC-MS/MS experiments highlighted in Figure 2A showing -log(p-value) against log2(fold-change) for all proteins identified in the appropriate Emc6/Sbh1 samples. Proteins enriched in the Emc6 samples are colour/pattern-coded as follows: dotted outline for HA-IP of BioID2-HA-Emc6; blue fill for streptavidin-AP of BioID2-HA-Emc6; black, solid outline for streptavidin-AP of TurboID-HA-Emc6; and yellow fill for streptavidin-AP of TurboID-HA-Emc6/ABOLISH. Grey dots represent proteins that passed the p-value and fold-change criteria, but were only identified by one peptide. Only EMC complex components or proteins with EMC client features are named.

(C) High-confidence protein identifications of Emc6 determined by: HA-IP of BioID2-HA-Emc6; streptavidin-AP of BioID2-HA-Emc6; streptavidin-AP of TurboID-HA-Emc6; and streptavidin-AP of TurboID-HA-Emc6/ABOLISH. Colour/pattern-coding is as described in (B). EMC complex members and proteins with classical features of EMC substrates are marked in bold and with asterisks, respectively.

(D) Overlap between the proteins highlighted with asterisks in (C) and yeast EMC clients found by two independent studies (Shurtleff et al, 2018; Bai et al, 2020).
A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast (2022)
Figure 3

Figure 3: Verifying new EMC clients using microscopy and BirA-AviTag combined with ABOLISH

(A) Fluorescence microscopy images of control or Δemc3 strains containing either Alg1 N' tagged with GFP, Gnp1 C' tagged with GFP, Pdr12 N' tagged with GFP or Pho84 C' tagged with GFP. For the Gnp1-GFP strain, the quantified signal depletion was ~10% (p < 0.0001). Topological schematics feature beneath each respective image, with predicted TMDs highlighted in blue (Weill et al, 2019). All images are from two independent biological repeats and are digitally coloured using the 'fire' lookup table (LUT) on Fiji. The same contrast settings were used for each image pair. Scale bar = 5μm.

(B) Depiction of the BirA-AviTag system where BirA-tagged Emc6 can biotinylate stably-interacting AviTag-EMC components (such as Emc2) and transient AviTag-substrates.

(C) Streptavidin blots of diploid strains expressing either AviTag-Fks1, -Gnp1, -Pdr5, -Pdr12 or -Stv1 (as negative control) together with BirA-Emc6, grown in the conditions described in Figure 1E. For each strain, blots showing both the 'lower' (L) and 'higher' (H) contrast settings are shown side-by-side. The expected molecular weight in kDa for each tested protein including AviTag is written in parentheses after the protein name.
p53 Pulse Modulation Differentially Regulates Target Gene Promoters to Regulate Cell Fate Decisions (2019)
Figure 1

Figure 1. A system to modulate p53 expression dynamics and monitor target promoter activation in single cells. (A) Schematic of pulses of p53 expression in response to DNA double strand breaks. p53 dynamics are differentially decoded to induce target genes with diverse downstream functions, including mediating cell cycle arrest and p53 regulation. (B) Schematic of modulation of p53 expression by Nutlin-3, a small molecule inhibitor that prevents p53 degradation by MDM2. Cellular exposure to media with and without Nutlin-3 potentially generates six drug dosing regimens that modulate p53 pulse amplitude, frequency, and duration. (C) Clonal cell lines expressing p53-Venus were engineered to monitor p53 expression dynamics ("input") and two canonically regulated p53 target promoters expressing mCherry ("output") in response to Nutlin-3 treatment. (D) Representative phase contrast and yellow fluorescent (indicating p53-Venus levels) images at the indicated time points for a cell exposed to the "natural dynamics" Nutlin-3 dosing regimen. (E-F) p53-Venus expression in response to the "natural dynamics" Nutlin-3 dosing regimen. Single cell traces (gray; E) and the mean (red; E) are shown for p53-Venus expression in response to each Nutlin-3 regimen. Heat map (F) shows an alternative representation of all traces as shown in (E). N = 51 cells.
p53 Pulse Modulation Differentially Regulates Target Gene Promoters to Regulate Cell Fate Decisions (2019)
Figure 2

Figure 2. Characterization of target promoter activation. (A) Representative phase contrast, yellow fluorescent (indicating p53-Venus levels), and red fluorescent (indicating reporter mCherry levels) images at the indicated time points for MDM2 promoter reporter cells exposed to the "natural dynamics" Nutlin-3 dosing regimen. (B-G) Single cell traces of mCherry expression for the "responding" (light gray) or "not responding" (dark gray) MDM2 promoter or CDKN1A promoter cells exposed to the natural dynamics (B), low amplitude (D), high amplitude (D), high frequency (E), low frequency/short duration (F), or long duration (G) Nutlin-3 dosing regimens. The average trace for responding and not responding cells is shown in red and blue, respectively. Heat maps show alternative representation of all single cell traces below each associated time course plot. N = at least 45 cells per condition.
p53 Pulse Modulation Differentially Regulates Target Gene Promoters to Regulate Cell Fate Decisions (2019)
Figure 3

Figure 3. p53 pulse amplitude generates promoter activation thresholds. Dose-response curves representing the rate of mCherry expression from the MDM2 (pink dots) and CDKN1A (purple dots) promoters as a function of total p53 levels. Values of expression levels were averaged across all cells in the first 15-h response to the long duration Nutlin-3 dosing regimen. Solid black lines represent the best fit to a Hill function model of promoter activation. Dashed lines indicate the half-maximal threshold of p53 levels for promoter activation and the half maximal promoter activity for each promoter.
p53 Pulse Modulation Differentially Regulates Target Gene Promoters to Regulate Cell Fate Decisions (2019)
Figure 4

Figure 4. p53 pulse duration establishes a refractory period. (A) Responding cells were characterized based on the indicated metrics of target promoter activation (timing, magnitude, and rate). (B-G) Effects of p53 duration modulation on target promoter activation in terms of the mean timing (B, E), magnitude (C, F), and rate (D, G) for the MDM2 (B-D) and CDKN1A (E-G) promoters in response to short (light green) and long (dark green) p53 durations. N = at least 45 cells per condition, error bars = SEM. (H) Mean nuclear MDM2-YFP levels in single cells at times corresponding to the first pulse (5.5 h) or second pulse (11 h) of p53 expression in response to natural and long duration Nutlin-3 dosing regimens. N = at least 40 cells per condition, error bars = SEM. * indicates p < 0.05 and ** indicates p < 0.01, two-sample t-test.
p53 Pulse Modulation Differentially Regulates Target Gene Promoters to Regulate Cell Fate Decisions (2019)
Figure 5

Figure 5. p53 pulse frequency is differentially filtered by target promoters. (A) Effects of p53 frequency modulation on target promoter activation in terms of the mean timing (A, D), magnitude (B, E), and rate (C, F) for the MDM2 (A-C) and CDKN1A (D-F) promoters in response to low (light blue), natural (medium blue), high (dark blue) p53 frequencies. N = at least 45 cells per condition, error bars = SEM. (G) Theoretical responses for all-pass, band-pass, and low-pass filters. (H) Percentage of responding cells for the MDM2 and CDKN1A promoters in response to low, natural, and high frequency p53 pulses. Data also shown in Figure S2A. Error bars = SEM. * indicates p < 0.05 and ** indicates p < 0.01, two-sample t-test.
p53 Pulse Modulation Differentially Regulates Target Gene Promoters to Regulate Cell Fate Decisions (2019)
Figure 6

Figure 6. p53 frequency modulation alters cell cycle arrest. (A-B) Representative phase contrast, red fluorescence (indicating geminin-mCherry levels), and yellow fluorescence (indicating p53-Venus levels) images of cells in response to a low (A) or high (B) frequency Nutlin-3 dosing regimen over 40 h. (C) Geminin-mCherry traces in single cells treated with the low, natural, or high frequency Nutlin-3 dosing regimens. N = at least 50 cells per condition. (D-E) Percentage of cells undergoing cell division (D) or positive for geminin-mCherry expression (E) within the 40 h imaging in response to the low, natural, and high frequency Nutlin-3 dosing regimens as shown in (C).
Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD-interaction domain as well as a CTD-mimicking region in Sen1 (2020)
Figure 1

Figure 1: Identification of a Nrd1-Interaction Motif (NIM) in Sen1 that is critical for the integrity of the NNS-complex.

A) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1. Coimmunoprecipitation (CoIP) experiments using TAP-tagged Nrd1 (either wt or ∆CID) as the bait. Representative gel of one out of two independent experiments.

B) Scheme of Sen1 protein. Globular domains are denoted by solid bars whereas intrinsically disordered regions are shown by a line. The disorder prediction was obtained using IUPred (Dosztányi et al., 2005). The sequence of the RNAPII S5P-CTD and Trf4 and Sen1 NIMs is shown on the top. Structural elements that are important for the interaction with Nrd1 CID are indicated. Conserved positions are underlined.

C) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1. CoIP experiments using Nrd1-TAP as the bait in a SEN1 or sen1∆NIM background. Representative gel of one out of two independent experiments.

D) CoIP experiments using HA-tagged Sen1, either wt or ∆NIM, as the bait. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA prior to immunoprecipitation. In these experiment, Sen1 could not be detected in the input extracts.

Data information: Antibodies used for protein detection are listed in Appendix Table S3.
Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD-interaction domain as well as a CTD-mimicking region in Sen1 (2020)
Figure 3

Figure 3: The N-terminal domain of Sen1 plays an essential role in non-coding transcription termination in vivo.

A) Deletion of the N-terminal of Sen1 is lethal. A ∆sen1 strain (YDL2767) covered by an URA3-containing plasmid (pFL38) expressing wt Sen1 was transformed with a TRP1-plasmid (pFL39) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left. After over-night growth in non-selective medium, cells were plated on minimal medium (CSM) containing 5-fluorootic acid (5-FOA) to select those that have lost the URA3 plasmid. The absence of growth in 5-FOA implies that the SEN1 version expressed in the TRP1-plasmid does not support viability.

B) Deletion of the N-terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo. Northern blot analyses of three well-characterized NNS-targets, the CUT NEL025c and the snoRNAs SNR13 and SNR33, in a Sen1-AID (auxin-induced degron) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1. Sen1-AID was depleted for 2h by the addition of 500 μM indole-3-acetic acid (IAA) to monitor the capacity of the plasmid-borne versions of SEN1 to induce transcription termination. The expected RNA species resulting from inefficient termination (RT for readthrough species) are indicated.

C) Overexpression of sen1∆Nter restores viability. Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter (pGAL) in the presence of galactose.

D) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N-terminal domain. Northern blot analysis of a typical NNS-target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose.

Data information: For B) and D) assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control. All results shown were reproduced in at least one additional independent experiment. Probes used for RNA detection are described in Appendix Table S6.
Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD-interaction domain as well as a CTD-mimicking region in Sen1 (2020)
Figure 5

Figure 5: The N-terminal domain of Sen1 can recognize the S5-phosphorylated form of RNAPII CTD and Sen1 C-terminal domain.

A) Deletion of Sen1 N-terminal domain does not prevent the interaction of Sen1 with RNAPII. CoIP experiments using Rbp3-FLAG as the bait. Assays were performed in a Sen1-AID strain harboring a plasmid expressing either SEN1 or sen1∆Nter upon depletion of Sen1-AID in the presence of IAA for 2h. An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1∆Nter. Nrd1 is detected as a positive control. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.

B) Deletion of the Sen1 N-terminal domain reduces the interaction of Sen1 with the S5P-CTD. CoIP experiments using TAP-Sen1 as the bait. Sen1 proteins were expressed from pGAL in the presence of galactose. Nab3 is detected as a positive control. Representative gel of one out of three independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.

C) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability. Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1-plasmid carrying the SEN1 versions indicated in the scheme on the left. The growth of the strain expressing the Nrd1 CID-sen1∆Nter chimera in 5-FOA implies that this gene can support viability.

D) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1∆Nter mutant. Northern blot assays performed in a Sen1-AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B. Experiments performed in a ∆rrp6 background. Representative gel of one out of two independent experiments.The U4 RNA is used as a loading control. Probes used for RNA detection are described in Appendix Table S6.

E) Sen1 Nter interacts with the C-terminal domain (Cter) of Sen1 both in the presence and in the absence of the NIM in vitro. Pull-down experiments using either a wt or a ∆NIM version of recombinant Sen1 Cter immobilized on glutathione-sepharose beads and a TAP-tagged version of Sen1 Nter expressed in yeast. Representative gel of one out of three independent experiments.

Data information: Antibodies used for protein detection are listed in Appendix Table S3.
Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD-interaction domain as well as a CTD-mimicking region in Sen1 (2020)
Figure 6

Figure 6: High-resolution transcriptome-wide maps of binding sites for different Sen1 variants.

A-C) Comparison of the distribution of Sen1 and RNAPII (Rpb1) at mRNAs and CUTs. Values on the y axis correspond to the median coverage. Note that the scale of Sen1 and Rpb1 datasets have been modified to be able to visualize both in the same plot.

D-F) IGV screenshots of two mRNAs and one CUT exemplifying the trend observed in the metagene plots.

G) Metagene analysis of the distribution of the different Sen1 variants at mRNAs using either the TSS or the PAS as reference point. Values correspond to the median of Sen1 data normalized by the RNAPII signal obtained in the corresponding background. Additional analyses performed using the mean and using quartiles are included in Appendix Figure S5.

H-I) Screenshots of two well-characterized protein-coding genes that are regulated by NNS-dependent premature termination. Values correspond to unnormalized Sen1 occupancy.

J) Profile of Sen1 binding at CUTs calculated as for mRNAs.

K-L) Examples of CUTs exhibiting differences in Sen1 occupancy upon deletion of the NIM or the Nter. Plots correspond to unnormalized data.

M-O) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs.

P) Comparison of the total levels of the different Sen1 variants at mRNAs, CUTs and the termination region of snoRNAs (i.e. a window of 200 nt downstream of the 3' end of the mature snoRNA, snoRNA-ter). Boxes in box-plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR. The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. Datasets for the different Sen1 versions were compared using a t-test (bilateral distributions, unpaired data). p-values≥ 0.05 are indicated as ns (not significant), one asterisk is used when 0.05>p-value≥0.01, two asterisks when 0.01>p-value≥0.001 and three asterisks for p-value <0.001.
Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD-interaction domain as well as a CTD-mimicking region in Sen1 (2020)
Figure 7

Figure 7: Influence of the transcription elongation rate on the requirement of Sen1 Nter for non-coding transcription termination.

A) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter. Northern blot assays performed in a Sen1-AID, ∆rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments. Where indicated, cells were treated with 50 mg/L of 6-azauracile (6AU) for 2h. Representative gel of one out of two independent biological replicates.The U4 RNA is used as a loading control. Probes used for RNA detection are described in Appendix Table S6.

B) Model for the role of the N-terminal domain and the NIM in the control of Sen1 action at ncRNAs. Nrd1 and Sen1 are likely independently recruited to the elongation complex. The recruitment of Nrd1 and Nab3 is mediated by the recognition of specific RNA sequences and enhanced by the interaction of Nrd1 CID with the S5P-CTD via the CID. Sen1 would be recruited either by unspecific binding to the nascent RNA or by possibly transient protein-protein interactions with RNAPII. The interaction of Sen1 NIM with Nrd1 CID also enhances Sen1 recruitment. The interaction of Sen1 Nter with the CTD could facilitate the loading of Sen1 to the nascent RNA in the vicinity of paused RNAPII. Sen1 would translocate along the nascent RNA to induce dissociation of the elongation complex. The release of Sen1 from Nrd1 would be required for the CID to be available to recruit TRAMP for subsequent processing/ degradation of the released RNA.

EXPANDED VIEW FIGURE LEGENDS
OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP-mediated microtubule anchoring (2021)
Figure 1

Figure 1 OCRL deficiency leads to perinuclear lysosome positioning

A NHF, Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation, then immunostained with LAMP1 antibody. Dashed lines in images outline cell boundaries. Scale bar, 10 μm.

B Wildtype (Ocrl+/y) and Ocrl-/- MEF cells were grown in complete media or 5 h serum starvation, then immunostained with indicated antibodies. Dashed lines in images outline cell boundaries. Scale bar, 10 μm.

C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A. The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment.

D, E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B. The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment.

F, G Representative images of the trajectory of GFP signals from NHF (n= 4) and Lowe 1676 (n= 4) cells transfected with lysosome-GFP. Trajectories were obtained as described He et al., 2016() and labeled in different colors with the same colored circles showing the start of the track. G. Measured MSD (⟨∆r2 (t)⟩) as a function of delay time t for the mobile lysosome trajectories taken at F.

H Diffusion coefficient of lysosome in the experiments shown in G. Means ± SEM of n = 4 cells per group.

I Snap images of WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs transfected with lysosome-GFP. After transfection, WT, OCRL knockout and IOB MEFs were left in serum starvation for 8 h. Scale bar, 5 μm.

J Quantitative analyses of tubulated lysosomes in the experiments shown in H. n = 3 per group in which 10 cells were counted per condition per experiment.

Data information: In (C,D,E,J), data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.
OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP-mediated microtubule anchoring (2021)
Figure 2

Figure 2 OCRL deficiency impairs lysosome positioning leading to mTOR inactivation and abnormal nutrient sensing

A Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K in NHF, Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments.

B Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR , p-p70 S6K (Thr389) and total p70 S6K in WT (Ocrl+/y) and IOB MEFs grown in complete medium or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments.

C Immunoblot analysis of GFP, p-mTOR (Ser2448), total mTOR , p-p70 S6K (Thr389) and total p70 S6K in Lowe 1676 transfected with or without GFP-OCRL.

D NHF and Lowe 1676 cells were grown in complete media, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunoblotted using antibodies against endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K.

E Quantification of p-mTOR (Ser2448) over total mTOR ratios normalized to NHF cells as shown in D, n = 3.

F Quantification of p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells as shown in D, n = 3.

G WT and IOB MEFs were grown in 1% FBS, 15% FBS media with or without 2 μmol MHY1485 for 4 days. Relative cell number is the ratio of cells on day 4 normalized to day 1, three independent experiments. ****, #### P < 0.0001, calculated by two-way ANOVA.

H NHF and Lowe 1676 cells were grown in 1% FBS, 15% FBS media with or without 2 μmol MHY1485 for 4 days. Relative cell number is the ratio of cells on day 4 normalized to day 1, three independent experiments. ****, #### P < 0.0001, calculated by two-way ANOVA.

Data information: In (A - F), data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.