Journal: bioRxiv

Article Title: Ubiquitin-based pathway acts inside chloroplasts to regulate photosynthesis

doi: 10.1101/2022.06.06.494369

Figure Lengend Snippet: ( A and B ) Chloroplasts isolated from transgenic plants expressing Myc-tagged ubiquitin (6Myc-Ub), and from wild type, were analysed by immunoblotting (A). Similar chloroplasts were treated with thermolysin protease (Ther), or buffer lacking protease (Mock), before immunoblotting analysis (B). The asterisk in B indicates a non-specific band. ( C ) Chloroplasts isolated from 6Myc-Ub plants were separated into membrane pellet (P18) and soluble supernatant (S18; predominantly stroma) fractions by centrifugation at 18,000 × g , and then analysed by immunoblotting. Analysis of control proteins confirmed the efficacy of the protease treatment and fractionation steps. Positions of molecular weight markers (sizes in kD) are shown to the right of the images.

Article Snippet: For analysis of di-Gly modification-enriched peptides, an immunoprecipitation step was performed using a PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit (Cell Signal Technology) according to the manufacturer’s instructions.

Techniques: Isolation, Transgenic Assay, Expressing, Ubiquitin Proteomics, Western Blot, Membrane, Centrifugation, Control, Fractionation, Molecular Weight

Journal: bioRxiv

Article Title: Ubiquitin-based pathway acts inside chloroplasts to regulate photosynthesis

doi: 10.1101/2022.06.06.494369

Figure Lengend Snippet: ( A ) Detection of ubiquitinated proteins in chloroplasts purified from plants expressing Myc-tagged ubiquitin by immunoprecipitation and immunoblotting. Transgenic seedlings expressing ubiquitin with an N-terminal 6×Myc tag (6Myc-Ub) were used to isolate chloroplasts. The 6Myc-Ub chloroplasts were subjected to lysis and membrane solubilization prior to immunoprecipitation (IP) using anti-c-Myc affinity gel. Total lysate (TL; before immunoprecipitation was initiated), IP flow-through (FT), and IP eluate samples were analysed by immunoblotting using anti-Myc and the other indicated antibodies. Ubiquitinated proteins were enriched in the eluted samples, as indicated by the high molecular weight smears, compared with the input sample. ( B and C ) Identification of ubiquitinated chloroplast proteins by immunoprecipitation and mass spectrometry. The 6Myc-Ub elution sample shown in A was analysed by LC-MS/MS, and the data were analysed by MaxQuant. This identified six peptides for ubiquitin (covering 78.4% of the whole ubiquitin protein sequence), as well as the chloroplast proteins listed ( B ) (OEM, outer envelope membrane; IEM, inner envelope membrane). After trypsin digestion, a di-glycine remnant of ubiquitin remains covalently linked to target lysine residues of modified proteins (K-ε-GG), and this is identified by an increased mass of 114 kDa of the lysine residue. A ubiquitinated peptide was detected for the stromal protein PrfB3 (B,C), and the associated fragmentation spectrum is shown (C). The y and b ions are shown in red and blue, respectively, while ions with H 2 O or NH 3 shift are shown in orange. The b ions appear to extend from the N-terminus, and the y ions appear to extend from the C-terminus. The corresponding peptide sequence is presented, with the ubiquitinated K residue is marked in red. The m/z value of the corresponding fragment is indicated in black.

Article Snippet: For analysis of di-Gly modification-enriched peptides, an immunoprecipitation step was performed using a PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit (Cell Signal Technology) according to the manufacturer’s instructions.

Techniques: Purification, Expressing, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Transgenic Assay, Lysis, Membrane, High Molecular Weight, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Sequencing, Modification, Residue

Journal: bioRxiv

Article Title: Ubiquitin-based pathway acts inside chloroplasts to regulate photosynthesis

doi: 10.1101/2022.06.06.494369

Figure Lengend Snippet: ( A ) Detection of the accumulation of ubiquitinated proteins in chloroplasts isolated from CDC48-DN plants by immunoblotting. Chloroplasts were isolated from CDC48-WT and CDC48-DN transgenic seedlings that had been induced with estradiol for two days. Induction of CDC48-DN was used to elevate the levels of ubiquitinated chloroplast proteins by blocking the CHLORAD system, in order to increase the chance of capturing ubiquitination events that would otherwise be below the level of detection, due to low stoichiometry or abundance. The purified chloroplasts were analysed by immunoblotting using anti-ubiquitin and the other indicated antibodies. ( B and C ) Identification of ubiquitinated proteins and peptides in CDC48-DN chloroplasts by di-Gly ubiquitinomics. Proteins extracted from CDC48-DN chloroplasts like those shown in A were digested with trypsin and subjected to immunoprecipitation analysis using an anti-K-ε-GG antibody. The recovered ubiquitinated peptides were submitted to LC-MS/MS analysis, and the data were analysed by MaxQuant. Three independent biological replicates were analysed (Exp1-3), and the overlaps among the identified ubiquitinated peptides (B) and proteins (C) are shown. ( D ) Identification of ubiquitination sites in several chloroplast-encoded proteins by mass spectrometry. Many of the identified ubiquitinated proteins (B,C) were key photosynthetic complex components, and several of these are encoded by the chloroplast genome: PsaA, PsaC (PSI complex), PsbB, PsbC, PsbD (PSII complex), and AtpA (ATP synthase complex). Peptide sequences and representative spectra are shown for these proteins. The y and b ions are shown in red and blue, respectively, while ions with H 2 O or NH 3 shift are shown in orange. The b ions appear to extend from the N-terminus, and the y ions appear to extend from the C-terminus. The ubiquitinated K residues are marked in red in the peptide sequences. The m/z values of the corresponding fragments are indicated in black.

Article Snippet: For analysis of di-Gly modification-enriched peptides, an immunoprecipitation step was performed using a PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit (Cell Signal Technology) according to the manufacturer’s instructions.

Techniques: Isolation, Western Blot, Transgenic Assay, Blocking Assay, Ubiquitin Proteomics, Purification, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Journal: bioRxiv

Article Title: Ubiquitin-based pathway acts inside chloroplasts to regulate photosynthesis

doi: 10.1101/2022.06.06.494369

Figure Lengend Snippet: ( A ) Validation of stromal protein PrfB3 using an in vivo ubiquitination assay. Wild-type protoplasts were cotransformed with constructs encoding HA-tagged PrfB3 (PrfB3-HA) and FLAG-tagged ubiquitin (FLAG-Ub), and the cells were subjected to immunoprecipitation (IP) analysis using anti-HA affinity gel. Total lysate (TL; before IP was initiated) and eluted IP samples were then analysed by immunoblotting using antibodies against: the FLAG tag, to detect polyubiquitinated forms (poly-Ub) of PrfB3-HA or YFP-HA; and the HA tag, to verify that the fusion proteins were present in the samples. Positions of molecular weight markers (sizes in kD) are shown to the left of the images. ( B ) Validation of chloroplast-encoded proteins PsaA and PsbC using an in vivo ubiquitination assay. Protoplasts isolated from CDC48-WT and CDC48-DN transgenic plants were transfected with the FLAG-Ub construct and subjected to estradiol induction. The cells were then analysed by IP using anti-FLAG M2 affinity gel; in this case, ubiquitinated proteins were enriched instead of the target proteins, due to the difficulty of expressing tagged-chloroplast-encoded proteins in plants. Samples (TL, IP) were then analysed by immunoblotting using antibodies against the proteins indicated to the right of the images. Polyubiquitinated forms (poly-Ub) of PsaA and PsbC (and of the known CHLORAD substrate, Toc159) were detected in the IP samples, and these were distinctly larger than their unmodified forms in the TL samples. The poly-Ub forms also increased markedly in abundance in CDC48-DN, suggesting that they are normally subject to CHLORAD degradation. Tic40, which is not a CHLORAD substrate, acted as a negative control. The asterisk indicates a non-specific band. Molecular weight markers are indicated as in A.

Article Snippet: For analysis of di-Gly modification-enriched peptides, an immunoprecipitation step was performed using a PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit (Cell Signal Technology) according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, In Vivo, Ubiquitin Proteomics, Construct, Immunoprecipitation, Western Blot, FLAG-tag, Molecular Weight, Isolation, Transgenic Assay, Transfection, Expressing, Negative Control

Journal: bioRxiv

Article Title: Ubiquitin-based pathway acts inside chloroplasts to regulate photosynthesis

doi: 10.1101/2022.06.06.494369

Figure Lengend Snippet: ( A ) Volcano plot representation of the data for all genes identified in quantitative transcriptomic analysis comparing CDC48-DN and CDC48-WT plants, before filtering for chloroplast-related genes . The graph shows -log 10 P values plotted against log2 fold changes; genes deemed to be showing a significant difference in expression in CDC48-DN plants, relative to the CDC48-WT control, are indicated in green (Up) or red (Down). The corresponding dataset is show in table S8. ( B ) Pie chart showing the proportion of mRNAs in the whole transcriptome that are differentially expressed in CDC48-DN plants, as determined by the quantitative transcriptomic analysis. In general, more genes were up-regulated than down-regulated in response to CDC48-DN expression. In contrast, chloroplast-related genes showed the opposite trend, with more genes found to be down-regulated . ( C ) Dot plot showing significantly overrepresented GO terms for mRNAs that are differentially expressed in CDC48-DN plants. Dot size indicates overrepresentation (fold enrichment) compared to the whole genome. Dot colour indicates False Discovery Rate (FDR; -log 10 [ P value]), where higher FDR values indicate more statistically significant enrichment. Dots are not shown for terms lacking statistically significant ( P < 0.05) enrichment. In general, ubiquitin-dependent proteolytic processes are upregulated, whereas photosynthesis-related pathways are down-regulated.

Article Snippet: For analysis of di-Gly modification-enriched peptides, an immunoprecipitation step was performed using a PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit (Cell Signal Technology) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

doi: 10.1101/2020.06.23.167601

Figure Lengend Snippet: a. Schematic of the reactions catalyzed by the three types of protein arginine methyltransferases (Type I PRMT catalyze Rme1 and Rme2a; Type II PRMTs catalyze Rme1 and Rme2s; Type III PRMTs catalyze only Rme1) b. Experimental setup: A549 cells were cultured for 1 week with either DMSO, 1 µM GSK591, or 1 µM MS023. Total protein lysates (8M Urea) were precipitated with TCA followed by complete hydrolysis in 6M HCl and heat. Resulting amino acid products were separated on C18 reversed-phase chromatography and subjected to MS/MS analysis. c. UHPLC trace of the four Arginine amino-acids resolved onto Agilent Zorbax Eclipse Plus C18 RRHD column (2.1×150 mm, 1.8 µm; kept at 40 °C). d. Representation of Arginine amino-acid species detected within the study (R, Rme1, Rme2a and Rme2s). The fragments detected and utilized as quantifiers within the low resolution set-up are depicted in blue; likewise, their matching m/z are annotated e. As an example, the fragmentation pattern (MS/MS trace) of the Rme2s species from its parent peak (insert; m/z = 203.1). f. Calculated abundance of each methylarginine species as a percent of total arginine are shown (DMSO control in gray, GSK591-treated cells in green, and MS023-treated cells are shown in purple).

Article Snippet: Peptides were subjected to successive immunoprecipitations with the Rme1 (CST Kit # 12235), Rme2a (CST Kit # 13474), and Rme2s (CST Kit # 13563) prebound resins.

Techniques: Cell Culture, Reversed-phase Chromatography, Tandem Mass Spectroscopy, Control

Journal: bioRxiv

Article Title: Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

doi: 10.1101/2020.06.23.167601

Figure Lengend Snippet: a. Arginine response was measured, in triplicate, and plotted against known concentration (concentration determined by 1 H-NMR with an adenosine standard). Replicates are shown; 95% confidence intervals are in orange. b. Monomethylarginine (Rme1) response measured in triplicate, as above c. Symmetric dimethylarginine (Rme2s) response measured in triplicate, as above d. Asymmetric dimethylarginine (Rme2a) response measured in triplicate, as above e. Calculated abundance of each methyllysine species as a percent of total lysine are shown

Article Snippet: Peptides were subjected to successive immunoprecipitations with the Rme1 (CST Kit # 12235), Rme2a (CST Kit # 13474), and Rme2s (CST Kit # 13563) prebound resins.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

doi: 10.1101/2020.06.23.167601

Figure Lengend Snippet: a. Total proteome western blots of all three methylarginine states are shown. Using the CST Rme1 (left), Rme2s (center left panel), and Rme2a (center right panel) antibodies, the changes in methylarginine protein abundance is shown for each of the control (DMSO), GSK591, and MS023 conditions. The right panel shows the Direct Blue 71 (DB71) membrane stain total loaded protein. b. Schematic of PTMScan approach. Purified tryptic peptides, in biological triplicate, were successively immunoprecipitated with the Rme1 PTMScan antibodies, the flowthrough applied to the Rme2a PTMScan antibodies, and that flowthrough applied to Rme2s antibodies. Antibody epitopes are shown. Peptides were eluted and subject to mass spectrometry. A sample of input peptides was reserved for total proteome analysis c. Example ETD spectrum of the C-terminal peptide from small nuclear ribonuclearprotein Sm D1. The peptide fragment from residue 93 to 118 and contains 9x dimethylarginines, all site localized. The region from 350 to 750 m/z of the full mass spectrum (Left/Top) is expanded in the (right/bottom) spectra.

Article Snippet: Peptides were subjected to successive immunoprecipitations with the Rme1 (CST Kit # 12235), Rme2a (CST Kit # 13474), and Rme2s (CST Kit # 13563) prebound resins.

Techniques: Western Blot, Quantitative Proteomics, Control, Membrane, Staining, Purification, Immunoprecipitation, Mass Spectrometry, Residue

Journal: bioRxiv

Article Title: Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

doi: 10.1101/2020.06.23.167601

Figure Lengend Snippet: a. The number of peptides enriched in each of the three replicate sequential immunoprecipitations (Rme1, Rme2a, Rme2s) in each condition (DMSO in gray, GSK591 in green, and MS023 in purple) are shown. Error bars represent standard deviation. b. The percent of enriched peptides containing methylation in each of the three replicate sequential immunoprecipitations (Rme1, Rme2a, Rme2s) in each condition (DMSO in gray, GSK591 in green, and MS023 in purple) are shown. Error bars represent standard deviation c. The fraction of peptides enriched peptides that contain monomethylarginine (Rme1) versus dimethylation (Rme2s) in each of the three replicate sequential immunoprecipitations (Rme1, Rme2a, Rme2s) in each condition (DMSO in gray, GSK591 in green, and MS023 in purple) are shown. Error bars represent standard deviation. d. The stacked histogram shows the abundances of hybrid peptides (containing both Rme1 and Rme2), only demethylated peptides, or only monomethylated peptides enriched in each of the three replicate sequential immunoprecipitations (Rme1, Rme2a, Rme2s) in each condition (DMSO top, GSK591 middle, and MS023 bottom) are shown.

Article Snippet: Peptides were subjected to successive immunoprecipitations with the Rme1 (CST Kit # 12235), Rme2a (CST Kit # 13474), and Rme2s (CST Kit # 13563) prebound resins.

Techniques: Standard Deviation, Methylation

Journal: bioRxiv

Article Title: Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

doi: 10.1101/2020.06.23.167601

Figure Lengend Snippet: a. Volcano plot of monomethylarginine (Rme1) peptide enrichments for GSK591 (left, green) and MS023 (right, purple) treated cells. Log2 fold-change is on the x-axis, while the negative log2 of the p-value is shown on the y-axis (dashed x-axis line represents p ∼ .05). b. Volcano plot of symmetric dimethylarginine (Rme2s) peptide enrichments for GSK591 (left, green) and MS023 (right, purple) treated cells. c. Volcano plot of asymmetric dimethylarginine (Rme2a) peptide enrichments for GSK591 (left, green) and MS023 (right, purple) treated cells. d. 382 proteins found in all three IPs in all three conditions were clustered by row z-score (negative z-score shown in purple, positive z-score shown in orange). e. Each of the three protein clusters of proteins were analyzed for GO term enrichment. Shown is the semantic space REViGO plot for each of the molecular function groupings.

Article Snippet: Peptides were subjected to successive immunoprecipitations with the Rme1 (CST Kit # 12235), Rme2a (CST Kit # 13474), and Rme2s (CST Kit # 13563) prebound resins.

Techniques:

Journal: bioRxiv

Article Title: Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

doi: 10.1101/2020.06.23.167601

Figure Lengend Snippet: a. The log 2 (fold change) for methylation sites found in both the GSK591 (x-axis) and MS023 (y-axis) for monomethylarginine (Rme1, left), symmetric dimethylarginine (Rme2s, center), or asymmetric dimethylarginine (Rme2a, right) are shown b. Weblogo of Rme1, Rme2s, or Rme2a enriched peptides are shown. Residues shown span -10 to +10 amino acids from the methylated arginine R.

Article Snippet: Peptides were subjected to successive immunoprecipitations with the Rme1 (CST Kit # 12235), Rme2a (CST Kit # 13474), and Rme2s (CST Kit # 13563) prebound resins.

Techniques: Methylation

Journal: bioRxiv

Article Title: Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

doi: 10.1101/2020.06.23.167601

Figure Lengend Snippet: a. 56392 human proteins (Uniprot 2012) were plotted with their RAPID predicted intrinsic disorder percentage of the protein on the x-axis and the log10 of their molecular weight (Da) on the y-axis. The median intrinsic disorder across the proteome (18.1%) is indicated, as is the median molecular weight (31.4 kDa). b. The proteins were plotted with their RAPID predicted intrinsic disorder on the x-axis and their hydrophobicity as calculated by GRAVY on the y-axis. Positive scores are hydrophobic, while negative scores are hydrophilic. The median hydrophobicity across the proteome was -0.37. c. The proteins were plotted with their RAPID predicted intrinsic disorder on the x-axis and their isoelectric point on the y-axis. Positive scores are hydrophobic, while negative scores are hydrophilic. The median isoelectric point across the proteome was 7.03. d. The RAPID percent disorder distribution of the proteome, PDB, Nucleus, RNA-binding, chromatin, and methylarginine (orange) sets are shown as violin plots. The median is indicated with a dark line and the quartiles are shown with a dashed line. e. The molecular weight distribution of the proteome, PDB, Nucleus, RNA-binding, chromatin, and methylarginine (orange) sets are shown as violin plots as in d. f. The GRAVY hydrophobicity distribution of the proteome, PDB, Nucleus, RNA-binding, chromatin, and methylarginine (orange) sets are shown as violin plots as in d. g. The isoelectric point distribution of the proteome, PDB, Nucleus, RNA-binding, chromatin, and methylarginine (orange) sets are shown as violin plots as in d. h. Table showing the number of proteins in each set i. Venn diagram of the intersection between the PTMScan methylarginine containing proteins, RNA binding, Nucleus, and chromatin sets. 153 of the methylarginine containing proteins were not found in these sets. j. Venn diagram showing the intersection human proteins between the PTMScan methylarginine containing proteins and those previously identified to be bound to RNA using RBR-ID. 140 of the methylarginine containing proteins were not found in this set. k. Percent of methylated arginine residues found in intrinsically disordered regions (IDR, light gray) shown as a histogram: Rme1 (91%) in IDRs; Rme2s (95%); Rme2a (88%) l. The PScore distribution, indicating pi-pi mediated liquid-liquid phase separation (LLPS) propensity, is shown for the proteome, PDB, Nucleus, RNA-binding, chromatin, and methylarginine (orange) sets. The median is shown with a dark line. Across the figure, all distributions shown were significant (Kruskal-Wallis test, adjusted p-val < 0.01) as compared to the Uniprot total proteome, except for the pI of the chromatin fraction vs the proteome (n.s.)

Article Snippet: Peptides were subjected to successive immunoprecipitations with the Rme1 (CST Kit # 12235), Rme2a (CST Kit # 13474), and Rme2s (CST Kit # 13563) prebound resins.

Techniques: Molecular Weight, RNA Binding Assay, Methylation