Journal: Nature Communications

Article Title: Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth

doi: 10.1038/s41467-021-21963-1

Figure Lengend Snippet: a , b HEK293 cells transfected with control siRNA ( siCTL ) or siRNA against PRMT7 ( siPRMT7 ) were analyzed by immunoblotting. MMA mono-methyl-arginine, sDMA symmetric di-methyl-arginine, aDMA asymmetric di-methyl-arginine. ACTIN was served as a loading control. c HEK293 cells transfected with siCTL or siPRMT7 together with or without Flag-tagged, wild-type (WT) or enzymatic dead mutant (MT) PRMT7 were analyzed by immunoblotting. d Experimental flowchart for identification of arginine methylation sites regulated by PRMT7 or responsive to PRMT7 inhibitor SGC3027 in HEK293 cells (see detail in “Methods”). e The number of mono-methyl arginine (Rme1) sites detected (column 1), Rme1 sites could be quantified (column 2), Rme1 sites with methylation signals decreased at least two-fold (column 3) or abolished (column 4) when knocking down of PRMT7 was shown. The number of proteins encompass all these methylation sites was also shown (bottom lane). f The overlap between PRMT7 methylome and proteins of which abundance was decreased at least two-fold when PRMT7 was knocked down is shown. g In vitro methylation assay was performed by mixing purified PRMT7 with CCT7, TFG, YBX1, CKMT1B, hnRNPK, hnRNPA2B1 or △Np63α, followed by immunoblotting with anti-MMA antibody (top panel). Methylation was indicated by white asterisk. The expression of proteins was examined by coomassie blue staining (C.B.S) and indicated by black asterisk (bottom panel). h The expression of purified PRMT7 was examined by C.B.S and indicated by black asterisk. i In vitro methylation assay was performed by mixing purified PRMT7 with synthetic short peptides from CCT7, TFG, YBX1, CKMT1B, hnRNPK, hnRNPA2B1, and hnRNPA1 proteins. Amino (N)-terminal of histone H3 and H4 were also included. The reactions were subjected to dot blotting. aa, amino acid. j The expression of purified PRMT7 was examined by immunoblotting. k In vitro methylation assay was performed by mixing purified PRMT7 with hnRNPA1 wild-type (WT) or mutants including R/K (7) (all five arginine (R) residues methylated by PRMT7 were replaced by lysine (K)), R194K, R206K, R218K, R225K, and R232K. The reactions were subjected to immunoblotting. Source data are provided as a .

Article Snippet: Antibodies used in this study are listed as following: Rabbit monoclonal anti-mono-methyl arginine [mme-R] (Cell Signaling Technology, 8015S, 1:2000); PTMScan® mono-methyl arginine Motif [mme-RG] Kit (Cell Signaling Technology, 12235, for immunoenrichment); Rabbit monoclonal anti-symmetric di-methyl arginine motif [sdme-R] (Cell Signaling Technology, 13222S, 1:2000); PTMScan symmetric di-methyl arginine motif [sdme-RG] (Cell Signaling Technology, 13563, for immunoenrichment); Rabbit monoclonal anti-asymmetric di-methyl arginine motif [adme-RG] (Cell Signaling Technology, 13522S, 1:2000); PTMScan asymmetric di-methyl arginine motif [adme-RG] (Cell Signaling Technology, 13474, for immunoenrichment); mouse monoclonal anti-actin (Proteintech, 66009-1-Ig, 1:2000); Mouse monoclonal anti-CARM1(3H2) (Cell Signaling Technology, 12495S, 1:2000); Rabbit monoclonal anti-PRMT5 (EPR5772) (Abcam, ab109451, 1:2000); Rabbit monoclonal anti-PRMT7 (D1K6R) (Cell Signaling Technology, 14762S, 1:2000); Rabbit monoclonal anti-hnRNPA1 (Proteintech, 11176-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPM (Proteintech, 26897-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPA2B1 (Proteintech, 14813-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPK (Proteintech, 11426-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPU (Proteintech, 14599-1-AP, 1:2000); Mouse monoclonal anti-FLAG M2 (Sigma, F1804, 1:5000); Anti-FLAG M2 Affinity Gel (Sigma, A2220, for immunoenrichment); Monoclonal Anti-HA-Agarose, clone HA-7 (Sigma, A2095, for immunoenrichment).

Techniques: Transfection, Western Blot, Mutagenesis, Methylation, In Vitro, Purification, Expressing, Staining

Journal: Nature Communications

Article Title: Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth

doi: 10.1038/s41467-021-21963-1

Figure Lengend Snippet: a The number of arginine methylation sites detected (the sum of mono- and di-methylation sites after removing duplicates) (column 2), arginine methylation sites could be quantified (column 3), arginine methylation sites with methylation signals decreased at least two-fold (column 4) or abolished (column 5) in PRMT4-, PRMT5- or PRMT7-knockdown experiments (lane 2, 3, and 4) following mass spectrometry analysis as described in Fig. . The number of proteins encompass all these methylation sites was also shown (bottom three lanes). For comparison, data shown for PRMT7 in Fig. was also included here. b , c Motif analysis was performed for PRMT4- ( b ) and PRMT5- ( c ) regulated arginine methylation sites using iceLogo . d , f Rates of somatic mutations at all arginine sites in the proteome and PRMT4- ( d ) or PRMT5- ( f ) regulated methylation sites in human cancers ( p = 6.54e−08 ( d ) and p = 0.1684 ( f ) by the Fisher’s exact test). e , g Rates of somatic mutations in the vicinity of all arginine sites in the proteome or PRMT4- ( e ) or PRMT5- ( g ) regulated methylation sites in human cancers (±5 nucleotides) ( p = 3.89e−108 ( e ) and p = 2.57e−45 ( g ) by the Fisher’s exact test). h , i KEGG pathway analysis using Metascape for PRMT4 ( h ) and PRMT5 ( i ) methylome. Representative terms from the top 20 enriched GO term clusters were shown.

Article Snippet: Antibodies used in this study are listed as following: Rabbit monoclonal anti-mono-methyl arginine [mme-R] (Cell Signaling Technology, 8015S, 1:2000); PTMScan® mono-methyl arginine Motif [mme-RG] Kit (Cell Signaling Technology, 12235, for immunoenrichment); Rabbit monoclonal anti-symmetric di-methyl arginine motif [sdme-R] (Cell Signaling Technology, 13222S, 1:2000); PTMScan symmetric di-methyl arginine motif [sdme-RG] (Cell Signaling Technology, 13563, for immunoenrichment); Rabbit monoclonal anti-asymmetric di-methyl arginine motif [adme-RG] (Cell Signaling Technology, 13522S, 1:2000); PTMScan asymmetric di-methyl arginine motif [adme-RG] (Cell Signaling Technology, 13474, for immunoenrichment); mouse monoclonal anti-actin (Proteintech, 66009-1-Ig, 1:2000); Mouse monoclonal anti-CARM1(3H2) (Cell Signaling Technology, 12495S, 1:2000); Rabbit monoclonal anti-PRMT5 (EPR5772) (Abcam, ab109451, 1:2000); Rabbit monoclonal anti-PRMT7 (D1K6R) (Cell Signaling Technology, 14762S, 1:2000); Rabbit monoclonal anti-hnRNPA1 (Proteintech, 11176-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPM (Proteintech, 26897-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPA2B1 (Proteintech, 14813-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPK (Proteintech, 11426-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPU (Proteintech, 14599-1-AP, 1:2000); Mouse monoclonal anti-FLAG M2 (Sigma, F1804, 1:5000); Anti-FLAG M2 Affinity Gel (Sigma, A2220, for immunoenrichment); Monoclonal Anti-HA-Agarose, clone HA-7 (Sigma, A2095, for immunoenrichment).

Techniques: Methylation, Mass Spectrometry

Journal: Nature Communications

Article Title: Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth

doi: 10.1038/s41467-021-21963-1

Figure Lengend Snippet: a Overlap among PRMT4, 5, and 7 methylome is shown. Splicing factors among substrates commonly regulated by PRMT4, 5, and 7 are listed. b Metascape CORUM , analysis was performed for substrates commonly regulated by PRMT4, 5, and 7. c Motif analysis was performed for cassette exons commonly regulated by PRMT4, 5, and 7 using CentriMo (v5.0.5) . d HEK293 cells transfected with siCTL or sihnRNPA1 were subjected to alternative splicing analysis as described in Fig. . e Schematic representation of the domain architecture of hnRNPA1. Arginine (R) sites methylated by PRMT4, 5, and/or 7 in the RGG domain were highlighted in red. The PRMTs that methylated each arginine and the methylation status were indicated at the bottom. (RRM: RNA recognition motif; RGG: arginine-glycine-glycine). f HEK293 cells transfected with siCTL or sihnRNPA1 in the presence or absence of wild type (WT) or methylation mutants including R/K (4), R/K (5), R/K (7), and R/K (457) were subjected to alternative splicing analysis as described in Fig. . g HEK293 cells transfected with siCTL or siPRMT4 , 5 , or 7 were subjected to RNA-IP assay using control IgG or anti-hnRNPA1 antibody. n = 3 biological replicates (mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 by unpaired Student t -test, two-tailed). h HEK293 cells transfected with empty vector (CTL) or Flag-tagged, wild-type (WT) or methylation mutants as described in ( f ) were subjected to RNA-IP assay using anti-Flag antibody. n = 3 biological replicates (mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 by unpaired Student t -test, two-tailed). Source data are provided as a .

Article Snippet: Antibodies used in this study are listed as following: Rabbit monoclonal anti-mono-methyl arginine [mme-R] (Cell Signaling Technology, 8015S, 1:2000); PTMScan® mono-methyl arginine Motif [mme-RG] Kit (Cell Signaling Technology, 12235, for immunoenrichment); Rabbit monoclonal anti-symmetric di-methyl arginine motif [sdme-R] (Cell Signaling Technology, 13222S, 1:2000); PTMScan symmetric di-methyl arginine motif [sdme-RG] (Cell Signaling Technology, 13563, for immunoenrichment); Rabbit monoclonal anti-asymmetric di-methyl arginine motif [adme-RG] (Cell Signaling Technology, 13522S, 1:2000); PTMScan asymmetric di-methyl arginine motif [adme-RG] (Cell Signaling Technology, 13474, for immunoenrichment); mouse monoclonal anti-actin (Proteintech, 66009-1-Ig, 1:2000); Mouse monoclonal anti-CARM1(3H2) (Cell Signaling Technology, 12495S, 1:2000); Rabbit monoclonal anti-PRMT5 (EPR5772) (Abcam, ab109451, 1:2000); Rabbit monoclonal anti-PRMT7 (D1K6R) (Cell Signaling Technology, 14762S, 1:2000); Rabbit monoclonal anti-hnRNPA1 (Proteintech, 11176-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPM (Proteintech, 26897-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPA2B1 (Proteintech, 14813-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPK (Proteintech, 11426-1-AP, 1:2000); Rabbit monoclonal anti-hnRNPU (Proteintech, 14599-1-AP, 1:2000); Mouse monoclonal anti-FLAG M2 (Sigma, F1804, 1:5000); Anti-FLAG M2 Affinity Gel (Sigma, A2220, for immunoenrichment); Monoclonal Anti-HA-Agarose, clone HA-7 (Sigma, A2095, for immunoenrichment).

Techniques: Transfection, Methylation, Two Tailed Test, Plasmid Preparation

Journal: Cancer cell

Article Title: Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation

doi: 10.1016/j.ccell.2019.07.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PTMScan Mono-Methyl Arginine 13 Motif [mme-RG] Kit , Cell Signaling Technologies , Kit #12235.

Techniques: Recombinant, Luminescence Assay, MTS Assay, Knock-In, Transgenic Assay, Sequencing, Software