EMBO/soda-nel huggingface dataset:
m6A-mediated alternative splicing coupled with nonsense-mediated mRNA decay regulates SAM synthetase homeostasis (2021)
Figure 6

Figure 6. Endogenous unproductive sams mRNAs have m6A modification at the AG dinucleotide of the distal 3'SS.

(A, B) m6A-IP specifically enriched sams mRNA isoforms that retain the AG dinucleotide at the distal/productive 3'SS. (A) Representative gel-like images (n=3) of semi-quantitative RT-PCR analysis of sams-3, sams-4 and sams-5 in input and immunoprecipitated (IP) RNAs from the smg-2 (yb979) mutant. The splicing patterns were analyzed and presented as in Figure 2B. (B) Relative enrichment of total and each of mRNA isoforms compared to eef-1A.1 mRNA by RT-qPCR. Error bars indicate standard error of mean (n=3).

(C) Examples of normalized Nanopore currents for unmodified and m6A-modified in-vitro transcribed sams-3/sams-4 RNAs at nucleotide positions -4 through +4 relevant to the m6A site. One hundred reads are plotted for each. Color codes are indicated.

(D) Distribution of mean (top) and standard deviation (bottom) of normalized Nanopore currents at nucleotide positions -4 through +4 relevant to the m6A site for 5,000 of the unmodified and 2,680 of the m6A-modified sams-3/sams-4 RNAs and 85 of an endogenous sams-3 mRNA isoform E2/E3L. Color codes are indicated. Central bands represent the median, boxes represent the 25th and 75th percentiles, and whiskers represent the lowest and highest values. A red line at position 0 in the top panel indicates a cut-off line (mean current = 1.7557) that discriminates between the unmodified and m6A-modified sams-3/sams-4 RNAs with accuracies of 64.58% for both.

(E) Machine learning-based classification of sams RNA reads in Nanopore direct RNA sequencing data. The statistics in (D) as well as duration time of the Nanopore current at nucleotide positions -50 through +50 of up to 80% of mapped reads from the unmodified and methylated in-vitro transcribed sams-3/sams-4 and sams-5 RNAs were pooled and used for training with a machine learning algorithm Gradient Boosting and accuracy of the classifier was tested with 20% of the mapped reads. The classifier was also applied to reads identified as unproductive sams-3, -4 or -5 mRNA isoforms in the analysis of endogenous mRNAs shown in Figure 1. Numbers of reads used in the tests are indicated at the top; percentages of reads classified as "m6A-modified" are shown with color code.

(F) Relative importance of the statistics at each position for Gradient Boosting.
Phosphoproteomics Reveals a Distinctive Mec1/ATR Signaling Response upon DNA End Hyper-Resection (2021)
Figure 1

Figure 1. A Distinctive Mec1/ATR Signaling Response in rad9∆ Cells.

A. Model showing the role of Rad9 in activating the Rad53-dependent checkpoint and preventing DNA end resection. For simplicity, the ssDNA-binding heterotrimer RPA and the obligatory Mec1 cofactor Ddc2 have been omitted from the diagram.

B. rad9Δ cells fail to activate the Rad53 checkpoint in the presence of either the DNA alkylating drug MMS (0.02%) or the radiomimetic drug phleomycin (40μg/mL).

C. Quantitative phosphoproteomics dataset showing phosphorylation sites enriched in cells lacking RAD9. Among the most highly enriched sites are Sgs1 T1269, Uls1 T540, Mec1 S1964, and 2 S-N sites in Rfa2. This response is similar in both the presence of 40μg/mL phleomycin (x axis) and 0.02% MMS (y axis). Note downregulation of Rad53 signaling (red dots) due to absence of RAD9.

D. Pie chart showing that S/T-Q phosphorylation comprises a large fraction of rad9Δ-induced phosphorylation (71%), though only accounting for a small fraction (5%) of the entire dataset.

E. Quantitative phosphoproteomics dataset showing that most phosphorylation events induced in rad9Δ cells are dependent on Mec1. These phosphoproteomic analyses were conducted in the presence of 0.02% MMS for 2 hours.

F. Model depicting Mec1-dependent phosphorylation of Sgs1, Uls1, and Rfa2 in response to hyper-resection in rad9Δ cells.
Phosphoproteomics Reveals a Distinctive Mec1/ATR Signaling Response upon DNA End Hyper-Resection (2021)
Figure 3

Figure 3. The STR Complex is Phosphorylated at Multiple Sites in Response to Hyper-Resection.

A. Schematics representing data of all Sgs1 phosphorylation sites detected in a large scale, high coverage, phosphoproteomic dataset (Lanz et al, 2021). Bolded sites indicate those subsequently detected in the IP-MS phospho-mapping experiment in Figure 3C. TIM, Top3 Interacting Motif; HRDC, Helicase and RnaseD C-terminal domain.

B. Immunoblot analysis of Sgs1 N-terminus (amino acids 1-647) from cells treated with increasing doses of the DNA alkylating drug MMS in the absence of RAD9 and/or MEC1. This truncated Sgs1 protein was expressed from its native promoter in this and all subsequent experiments.

C. Quantitative mass spectrometry analysis of phosphopeptides from affinity purified Sgs1 N-terminal fragment expressed in rad9∆ or wild-type 0.02% MMS-treated cells. Error bars represent standard deviation of two or more independent peptide-spectrum matches (PSMs) corresponding to the indicated sites.

D. Rmi1, a component of the Sgs1-Top3-Rmi1 (STR) complex, is hyper-phosphorylated on S/T-Q motifs in rad9Δ cells.

E. Model for Mec1-dependent phosphorylation of the STR complex. For simplicity, the ssDNA-binding heterotrimer RPA and the obligatory Mec1 cofactor Ddc2 have been omitted from the diagram.
Phosphoproteomics Reveals a Distinctive Mec1/ATR Signaling Response upon DNA End Hyper-Resection (2021)
Figure 4

Figure 4. Sgs1 Interacts with the Dpb11 scaffold in rad9∆ cells.

A. Workflow of the SILAC-based quantitative mass spectrometry method for identifying Dpb11 interactions.

B. Identification of Dpb11 interacting proteins in wild-type cells using workflow shown in (A). DPB11-3xHA was overexpressed using the ADH1 promoter. P value calculated using Mann-Whitney u-test.

C. Identification of Dpb11 interacting proteins in rad9∆ cells using workflow shown in a. DPB11-3xHA was overexpressed using the ADH1 promoter. P value calculated using Mann-Whitney u-test.

D. Immunoblot showing co-immunoprecipitation between Dpb11 and Sgs1. DPB11-3xHA was overexpressed from an ADH1 promoter and SGS1-13xMYC was expressed from its endogenous locus.

E. Schematic of the Sgs1 N-terminal truncation used in this study.

F. Immunoblot showing co-immunoprecipitation of Dpb11 with Sgs1. Both DPB11-3xHA and SGS11-647-5xFLAG were expressed at their endogenous loci.

G. Hypothetical depiction of the Dpb11-STR complex engaged at 5' recessed ends. See text for details. For simplicity, the ssDNA-binding heterotrimer RPA and the obligatory Mec1 cofactor Ddc2 have been omitted from the diagram.
Phosphoproteomics Reveals a Distinctive Mec1/ATR Signaling Response upon DNA End Hyper-Resection (2021)
Figure 5

Figure 5. Requirements for Assembly of the Dpb11-STR Complex.

A. Quantitative mass spectrometry analysis of affinity purified Dpb11 complexes, comparing wild-type Dpb11 to the K55A mutant that partially impairs binding through BRCT domain 1. Cells were grown in the presence of 0.04% MMS. Error bars represent SEM of selected proteins for which there were 2 or more peptide-spectrum matches (PSMs).

B. Immunoblots showing co-immunoprecipitation between Dpb11 and Sgs1 in the presence of 0.04% MMS in cells expressing either wild-type or the DPB11K55A mutant. DPB11-3HA or the BRCT mutant was ectopically expressed from its endogenous promoter and co-immunoprecipitated with SGS11-647-FLAG expressed from its endogenous promoter.

C. Quantitative mass spectrometry analysis of affinity purified Dpb11 complexes, comparing Dpb11 purified from rad9∆ cells with or without the 9-1-1 subunit Ddc1. Cells were grown in the presence of 0.04% MMS. Error bars represent SEM of selected proteins for which there were 2 or more peptide-spectrum matches (PSMs).

D. Immunoblots showing co-immunoprecipitation between Dpb11 and Sgs1 in the presence of 0.04% MMS in either wildtype, ddc1Δ, or ddc1-T602A cell lines. DPB11-3HA was tagged at its endogenous locus and co-immunoprecipitated with SGS11-647-FLAG tagged and truncated at its endogenous locus.

E. Quantitative mass spectrometry analysis of affinity purified Dpb11 complexes, comparing Dpb11 purified from rad9∆ cells with or without Mec1. Cells were grown in the presence of 0.04% MMS. Error bars represent SEM of selected proteins for which there were 2 or more peptide-spectrum matches (PSMs).

F. Immunoblots showing co-immunoprecipitation between Dpb11 and Sgs1 in the presence of 0.04% MMS in either wildtype, mec1Δ, tel1Δ or mec1Δ tel1Δ cell lines. DPB11-3HA was tagged at its endogenous locus and co-immunoprecipitated with SGS11-647-FLAG tagged and truncated at its endogenous locus.
Phosphoproteomics Reveals a Distinctive Mec1/ATR Signaling Response upon DNA End Hyper-Resection (2021)
Figure 6

Figure 6. A BRCT-Sgs1 Fusion Protein Promotes MMS Sensitivity in a Ddc1-dependent Manner.

A, B. Schematics depicting the rationale for designing a BRCT-Sgs1 chimera. The BRCT3/4-Sgs1 fusion protein is expected to engage at DNA lesions via the 9-1-1 clamp. For simplicity, the ssDNA-binding heterotrimer RPA and the obligatory Mec1 cofactor Ddc2 have been omitted from the diagram.

C. Dilution assay of wild-type, ddc1Δ, or ddc1-T602A cells expressing BRCT3/4-SGS1 in the presence of 0.005% MMS.

D. Immunoblot of Rad53 mobility shift assay in cells treated with 0.02% MMS expressing either DPB11BRCT3/4 alone or the BRCT3/4-SGS1 chimera. Dilution assay of wild-type, ddc1Δ, or ddc1-T602A cells expressing BRCT3/4-SGS1 in the presence of 0.005% MMS.

E. Dilution assay of wild-type or rev3Δ cells expressing BRCT3/4-SGS1 in the presence of 0.0025% MMS.
Phosphoproteomics Reveals a Distinctive Mec1/ATR Signaling Response upon DNA End Hyper-Resection (2021)
Figure 7

Figure 7. The B3/4-Sgs1 Fusion Protein Impairs Homologous Recombination.

A. Diagram of the break-induced replication (BIR) assay used in this study. Red line represents galactose-inducible HO endonuclease cut site.

B. Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3/4 fused to wild-type or mutant versions of SGS1. Mutants include a helicase deficient allele of SGS1 (SGS1hd) or an allele lacking the Top3 Interacting Motif (TIM; SGS1TIM). Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.

C. Measurement of BIR efficiency in cells carrying an empty vector or expressing DPB11BRCT3/4 fused to either SGS1, TOP3, or RMI1. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.

D. Measurement of BIR efficiency in cells carrying an empty vector or DPB11BRCT3/4 fused to either SGS1, hBLM, PIF1, or RRM3. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.

E. Diagram of the gene conversion (GC) assay used in this study. Red line represents galactose-inducible HO endonuclease cut site.

F. Measurement of gene conversion efficiency in cells carrying an empty vector or expressing DPB11BRCT3/4 fused to wild-type or mutant versions of SGS1. Mutants include a helicase deficient allele of SGS1 (SGS1hd) or an allele lacking the critical amino acid residues responsible for binding to Top3 (SGS1TIM). Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.

G. Diagram of the single-strand annealing (SSA) assay used in this study. Red line represents galactose-inducible HO endonuclease cut site.

H. Measurement of single strand annealing in cells carrying an empty vector or expressing DPB11BRCT3/4. Error bars represent SEM of at least 3 replicate experiments. AA strain contains perfect homology on either side of a GAL-inducible break. FA strain contains 3% sequence divergence on either side of the break (homeologous product).

I. Ratio AA / FA repair product computed from data in (H). Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.
Phosphoproteomics Reveals a Distinctive Mec1/ATR Signaling Response upon DNA End Hyper-Resection (2021)
Figure 8

Figure 8. Disruption of the Sgs1-Dpb11 Interaction Modulates Recombination Outcomes.

A. Diagram of the Sgs1 acidic patch (AP) truncations used in this study.

B. Histogram showing abundance of Sgs1 peptides in Dpb11-HA co-immunoprecipitation mass spectrometry experiments from cells expressing either SGS1 acidic patch truncation mutants or wild type SGS1.. Each mutant was compared in a SILAC experiment against the wild-type control. Error bars represent SEM of 2 or more Sgs1 peptide-spectrum matches (PSMs).

C. Serial dilution assay of sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH.

D. BIR assay in rad9Δ sgs1Δ cell lines expressing either SGS1 or sgs1ΔACIDICPATCH. Error bars represent SEM of 3 independent experiments. Error bars represent SEM of at least 3 replicate experiments. P value was calculated with a two-tailed, unpaired t-test. * = p≤0.05; ** = p≤0.01; *** = p ≤0.001.
Specific N-glycans regulate an extracellular adhesion complex during dendrite patterning (2022)
Figure 2

Figure 2 AMAN-2/Golgi alpha-mannosidase II requires enzymatic activity in PVD to form higher order branches

Types of N-linked glycans. The shared pentasaccharide core consists of two N-acetylglucosamines (blue squares) and 3 mannoses (green circles) attached onto an Asparagine residue with an N-X-S/T consensus site. Glycan types vary by identity of additional sugars onto the pentasaccharide core.

Drawing showing the enzymatic activity of AMAN-2 in the medial Golgi. AMAN-2 cleaves two specific α1,3 and α1,6 mannose linked residues boxed in red, allowing for the formation of complex and paucimannose type N-glycans. Arrows denote other enzymes. Swainsonine specifically inhibits enzymatic activity of alpha-mannosidase II. (Man= mannose, Gn= N-acetylglucosamine).

Table showing cell-specific rescue experiments of PVD defects. AMAN-2 cDNA is expressed under the control of PVD, muscle, and epidermal specific promoters in the indicated double mutant backgrounds. Rescue is defined by restoration of the enhanced PVD phenotype back to that of the single hypomorphic mutants alone in aman-2(gk248486) null mutant backgrounds enhanced by lect-2(gk846764) or mnr-1(dz213). 25 transgenic animals and their non-transgenic siblings were scored for each line. Numbers indicate number of biological replicates that showed rescue.

Multiple sequence alignment of human MAN2A1 (Hs; acc# NP_002363.2), mouse MAN2A1 (Mm; acc# NP_032575.2), zebrafish Man2a1 (Dr; acc# NP_001103497.2), fruit fly Alpha-Man-IIa (Dm; acc# NP_650494.2) and C. elegans AMAN-2 (Ce; acc# NP_505995.2) created by COBALT (constrained based multiple sequence alignment tool). Conserved catalytic sites D306 (black arrow) and D443 (white arrow) are boxed in orange.

Fluorescent images and tracings of wild type control (top) and lect-2(gk846764) hypomorphic animals (bottom) fed on plates with 300µm swainsonine vs a DMSO control. PVD is visualized by the wyIs581 transgene. The cell body is denoted with an asterisk. Anterior is to the left and dorsal is up in all panels. Vesicular gut autofluorescence is visible as white circular staining. Scale bars represent 20μm.

Quantification of the number of full tertiaries in denoted genetic backgrounds (aman-2 null is gk248486). Black data points indicate DMSO and orange data points show swainsonine treated animals. Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001; ns= not significant). n = 12 for all genotypes and are biological replicates.

Quantification of the number of quaternary dendrites in wild type control animals fed on plates with and without 300µm swainsonine. Animals treated with swainsonine show a significant decrease in quaternary branch number, akin to the data in Figure 1C. Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001). n = 12 for each experiment and are biological replicates.
Specific N-glycans regulate an extracellular adhesion complex during dendrite patterning (2022)
Figure 4

Figure 4 AMAN-2/Golgi alpha-mannosidase II modulates N-glycans on DMA-1/LRR-TM to regulate PVD morphogenesis

Schematic of DMA-1/LRR-TM with mutated N-glycan attachment sites S1-S4 indicated.

Quantification of the number of quaternary branches in DMA-1::2xFLAG control animals and animals with combinations of DMA-1::2xFLAG N-glycan attachment sites mutated. Note that DMA-1::2xFLAG control animals display a slightly reduced number of quaternary dendrites compared to wild type animals (Fig.EV3C). Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.

Quantification of the number of quaternary branches in DMA-1::2xFLAG control animals alone and in combination with an aman-2(gk248486) null mutant (shaded in red). Control data is identical as in (B) and shown for comparison only. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.

Fluorescent images (left) and tracings (center) of PVD in animals of denoted backgrounds (aman-2(null) is gk248486). S4 corresponds to the endogenous alteration of N-glycan attachment site N386 of DMA-1. PVD is visualized by the dzIs117 transgene. The cell body is marked with an asterisk. Scale bars represent 20μm.

The schematics on the right detail the molecular context of the indicated backgrounds. Green indicates a relatively normal PVD structure and red a heavily defective arborization. Black attachments indicate wild type, red indicate blocked sites, green indicates abnormal N-glycans chains, and purple indicates presumably incomplete but present N-glycans chains.

Proposed model of how the function of the Menorin complex could be modulated by complex stability. Note that other mechanisms are possible (see discussion). Yellow circles represent normal N-glycan chains, while red stars represent abnormal N-glycans aman-2 mutant animals. Putative functionality is indicated below.

EXPANDED VIEW FIGURE LEGENDS
Identification of PPM1D as an essential Ulk1 phosphatase for genotoxic stress-induced autophagy (2016)
Figure 1

Figure 1. Involvement of PPM1D in Ulk1 dephosphorylation and autophagy induction.

(A) p53-dependent dephosphorylation of Ulk1 at Ser637. p53+/+ and p53−/− MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells assessed by propidium iodide (PI) staining. α-Tubulin was used as a loading control. A semiquantitative analysis of protein expression is shown in Fig. EV1. (B, C) PPM1D-dependent dephosphorylation of Ulk1 at Ser637 and the induction of autophagy. PPM1D+/+ MEFs and PPM1D−/− MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. Dead cells (%) indicates the population of apoptotic cells. (C) Semiquantitative analysis of protein expression is shown (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant. (D–I) Suppression of etoposide-induced autophagy in PPM1D−/− MEFs. (D, E) The indicated MEFs were treated with etoposide (10 µM) for 6 hr and then immunostained with an anti-LC3 antibody (green). Representative images are shown in (D). LC3 puncta are observed in etoposide-treated PPM1D+/+ MEFs. (E) The proportion of cells with LC3 puncta was calculated (n > 100 cells in each experiment). Data are shown as the mean  ±  SD (n = 3 experiments). *p < 0.05, **p < 0.01. (F, G) The indicated MEFs were treated with etoposide (10 µM) for 6 hr and then analyzed using electron microscopy. Representative images are shown in (F). Many autophagic vacuoles (arrows) can be seen in etoposide-treated PPM1D+/+ cells (upper panel). “N” indicates the nucleus. Bar = 2 µm. A representative autophagosome (AP) and autolysosome (AL) are shown in the insets. (G) The number of autophagosomes and autolysosomes in each cell were counted (n > 8 cells). Red lines indicate the mean value. *p < 0.05. (H, I) Analysis of autophagic flux. The indicated MEFs were treated with etoposide (10 µM) for 6 hr in the presence or absence of bafilomycin A1 (10 nM), and the expression of each protein was examined by western blotting. α-Tubulin was included as a loading control. (I) Semiquantitative analyses of protein expression (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.
Identification of PPM1D as an essential Ulk1 phosphatase for genotoxic stress-induced autophagy (2016)
Figure 2

Figure 2. Confirmation of PPM1D involvement in Ulk1 dephosphorylation and the induction of autophagy.

(A, B) Effect of stable expression of PPM1D in PPM1D−/− MEFs. PPM1D−/− MEFs and PPM1D-transfected PPM1D−/− MEFs were treated with etoposide (10 µM), and the expression of each protein was examined by western blotting. (B) Semiquantitative analyses of protein expression in (A) (n = 3; mean ±  SD). *p < 0.05, **p < 0.01, NS: not significant. (C, D) Effect of a PPM1D inhibitor on etoposide-treated PPM1D+/+ MEFs. PPM1D+/+ MEFs were treated with etoposide (10 µM) in the presence or absence of GSK2830371 (30 µM), and the expression of each protein was examined by western blotting. (D) Semiquantitative analysis of protein expression in (C) (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant. (E, F) Effect of the transient overexpression of PPM1D. PPM1D+/+ MEFs were transfected with a PPM1D-Flag plasmid in the presence or absence of bafilomycin A1 (10 nM), and the expression of each protein was examined by western blotting. (F) Semiquantitative analyses of protein expression in (E) (n = 3; mean  ±  SD). *p < 0.05, **p < 0.01, NS: not significant.