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