constructs pcdna3 1 sc35 cmyc  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

doi: 10.1016/j.jbc.2021.101306

Figure Lengend Snippet: Phosphorylation regulates arginine-rich RNA-binding protein solubility. A , sample preparation and proteomic workflow. Nucleoplasm extracts of HEK293 cells expressing recombinant SRSF2-myc protein were incubated with either calf intestinal phosphatase (+CIP) or distilled water (−CIP) at 37 °C for 1 h. Following this, samples were ultracentrifuged at 100,000 g for 1 h. The soluble and insoluble pellet fractions were desalted and run by either western blot or liquid chromatography coupled with tandem mass spectrometry. B , prespin input (total, T ), supernatant (soluble, S ), and insoluble pellets ( P ) were run by SDS-PAGE and western blotted for myc. The average percent soluble ( sol./(sol. + insol.) ] and insoluble (in sol./(sol. + insol.) ] values were calculated for five biological replicates and displayed below the representative western blot. C , band densitometry of soluble and pellet fraction log 2 -transformed SRSF2-myc band intensities normalized to the total signal in −CIP and +CIP conditions (five biological replicates; Soluble p value = 0.0123; Pellet p value = 0.0372; two-tailed paired t test). D , differential abundance of proteins in the soluble fractions. Fold-change, displayed on the x-axis, was the log 2 value for fraction of signal that was insoluble [insoluble/(insoluble + soluble)] for the pairwise comparison +CIP/−CIP. The t-statistic (−log 10 ( p -Value)) was calculated for all proteins and displayed on the y-axis. Insoluble-enriched proteins were highlighted in red (log 2 (fold change) ≥1, p value < 0.05) and proteins depleted from the insoluble fractions upon dephosphorylation were highlighted in blue (log 2 (fold change) ≤ −1, p -Value < 0.05) squares , respectively. E , an S-graph ranking each protein by the NCPR value. Proteins with NCPR values two standard deviations (2 S.D.) below or above the mean NCPR value of all proteins (0.0076) split the proteome into three groups: highly negative ( yellow , < −0.083, n = 69), remainder ( gray , −0.083 < × < +0.099, n = 3796) and highly positive ( purple , n = 133, > +0.099). RNA-binding proteins in each group are highlighted, including SRSF2 and other SR proteins, which rank among the highest NCPR value proteins in the proteome. F , grouped scatter plot of the log 2 -transformation of the difference of fraction insolubility values of +CIP and mock conditions values for high negative proteins ( yellow ), highly positive proteins ( purple ) and remaining proteins ( gray ). Fraction Insolubility values were compared between groups (unpaired t test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001).

Article Snippet: The pcDNA3.1-SC35-cMyc SRSF2 plasmid was a gift from Kathleen Scotto ( ) (Addgene plasmid #44721).

Techniques: Phospho-proteomics, RNA Binding Assay, Solubility, Sample Prep, Expressing, Recombinant, Incubation, Western Blot, Liquid Chromatography, Mass Spectrometry, SDS Page, Transformation Assay, Two Tailed Test, Comparison, De-Phosphorylation Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

doi: 10.1016/j.jbc.2021.101306

Figure Lengend Snippet: RNA-binding proteins have variable abundance patterns in soluble and pellet fractions following dephosphorylation. A , box and whisker plots of mass spectrometry abundance measurements ( n = 4) of RNA-binding proteins in soluble and pellet fractions in −CIP and +CIP conditions. (two-tailed paired t test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). Module number and color are indicated next to each gene symbol. Whiskers range from min to max values. SRSF2 was significantly depleted from the soluble fraction while nearly significantly enriched to the pellet fraction (Soluble p value = 0.0063; Pellet p value = 0.0518). B , Western blot validation of solubility changes of well-described RNA-binding proteins. Modules, colored accordingly, are paired with the protein name.

Article Snippet: The pcDNA3.1-SC35-cMyc SRSF2 plasmid was a gift from Kathleen Scotto ( ) (Addgene plasmid #44721).

Techniques: RNA Binding Assay, De-Phosphorylation Assay, Whisker Assay, Mass Spectrometry, Two Tailed Test, Western Blot, Biomarker Discovery, Solubility

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

doi: 10.1016/j.jbc.2021.101306

Figure Lengend Snippet: SRSF2 net charge and oligomerization , respectively, increase substantially with dephosphorylation. A , SRSF2 net charge per residue (NCPR) calculated according to phosphorylation state, i.e. , no phosphorylation ( hypo SRSF2), phosphorylation sites previously observed by middle-down mass spectrometry (pSRSF2†, Kundinger & Bishof et al. , 2020) or full phosphorylation ( hyper pSRSF2). Phosphorylation sites represented by turquoise ball and stick . B , line plots of average charge density (window = 21 residues) from C-terminus to N-terminus of SRSF2 that is either nonphosphorylated ( black ), observed phosphorylation by MS ( gray , Kundinger & Bishof et al. , 2020) or fully phosphorylated ( turquoise ) in the RS domain. The SRSF2 protein map is included below the line plot, with the RNA-recognition motif (RRM) domain ( green box ) and RS domain ( black box ) annotated. C , nucleoplasm extracts of HEK293 cells expressing recombinant SRSF2-myc protein were incubated with either calf intestinal alkaline phosphatase (+CIP) or distilled water (−CIP) at 37 °C for 1 h. Following this, samples were split and analyzed by both denaturing and nondenaturing native PAGE and western blotted for myc ( n = 3). D , by denaturing SDS-PAGE ( left ), CIP-treated SRSF2-myc has increased electrophoretic mobility. Equal loading is demonstrated by Histone H3 labeling. Immunoblotting for SRSF2-myc after nondenaturing Blue native PAGE ( right ) identifies various dephosphorylated SRSF2 species not observed in mock-treated samples, including monomer (∼37 kDa, asterisk ) and high-molecular-weight (HMW) species. E , diagram of in vitro kinase reaction using kinase SRPK2 ( black ) and substrate SRSF2 ( green ). Both SRPK2 and SRSF2 were expressed and purified from E. coli and were mixed in the presence (+) or lack thereof (−) ATP and SRPK inhibitor (SRPIN340) and incubated at 30 °C for 30 min. Phosphorylated SRSF2 was represented by a turquoise ring . F , the in vitro reaction was separated by nondenaturing native PAGE, which was transferred and western blotted for SRSF2. Monomeric ( asterisk ) and high-molecular-weight (HMW) oligomeric ( line ) species were unequally observed in the − and + ATP and different SRPIN340 concentration conditions by nondenaturing native PAGE.

Article Snippet: The pcDNA3.1-SC35-cMyc SRSF2 plasmid was a gift from Kathleen Scotto ( ) (Addgene plasmid #44721).

Techniques: De-Phosphorylation Assay, Residue, Phospho-proteomics, Mass Spectrometry, Expressing, Recombinant, Incubation, Clear Native PAGE, Western Blot, SDS Page, Labeling, Blue Native PAGE, High Molecular Weight, In Vitro, Purification, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

doi: 10.1016/j.jbc.2021.101306

Figure Lengend Snippet: Inhibiting SRPKs decreases SR protein phosphorylation and increases cells harboring cytoplasmic SRSF2 granule and tubule structures in HEK293 cells. A , HEK293 cells were incubated with either DMSO (vehicle, VEH) or increasing concentrations of SRPK inhibitor SRPIN340 for a length of 12 h. Immediately following treatment, cells were harvested, run by SDS-PAGE, and immunoblotted for phosphoSR (pSR) proteins using the pan-pSR antibody mAb104. A p-TDP-43 antibody (pTDP-43 band marked by asterisk ) and a Histone H3 antibody were used to confirm specificity of SRPIN340 and equal protein loading, respectively. B , protein band intensities for SRSF4 ( purple ), SRSF6 ( blue ), SRSF10 ( red ), SRSF2/SRSF7 ( gold ), SRSF1/9 ( turquoise ), and separately, pTDP-43 ( green ) were quantified and normalized to the VEH condition (artificially set to value = 1) (multiple t tests, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). Error bars indicate maximum and minimum ranges of band values. The pSRSF2 signal was significantly decreased at 50 μM SRPIN340 concentration, whereas fellow RBP TDP-43 was not. C , validation of HEK293 cell extract fractionation procedure, yielding total (T), cytosolic (C), and nuclear (N) samples. The fractions were immunoblotted for the cytosolic marker GAPDH ( green ) and nuclear marker Histone H3 ( red ). D , immunoblot of nuclear-cytoplasmic fractions for total, endogenous SRSF2 in VEH and SRPIN340 conditions. E , quantification of percent nuclear SRSF2 (nuclear signal divided by sum of cytoplasmic and nuclear signals) in VEH and SRPIN340 conditions (paired t test, ∗ p value = 0.0130). F and G , immunocytochemical (ICC) staining of HEK293 cells for phosphoSRSF2 (pSRSF2)-positive nuclear speckles ( green ) and DAPI+ nuclei ( blue ) in vehicle-treated cells ( F ) and 50 μM SRPIN340-treated cells ( G ). H , the number of cytoplasmic granules observed was divided by the number of cells counted and averaged for 14 independent images in three independent replicates (two-tailed paired t test, ∗ p value = 0.0191). A minimum of 120 cells were counted in each condition per replicate. The error bars represent the range of standard deviation. I and J , ICC staining of HEK293 cells for hypophosphorylated SRSF2 (hypoSRSF2) ( green ) and DAPI+ nuclei ( blue ) in vehicle-treated cells ( I ) and 50 μM SRPIN340-treated cells ( J ). K , the fraction of cells harboring cytoplasmic SRSF2 tubule structures was quantified in four biological replicates and compared (two-tailed paired t test, ∗ p value = 0.0178). L , vehicle- and ( M ) 50 μM SRPIN340-treated HEK293 cells were stained by ICC for TDP-43 ( green ) and DAPI-stained.

Article Snippet: The pcDNA3.1-SC35-cMyc SRSF2 plasmid was a gift from Kathleen Scotto ( ) (Addgene plasmid #44721).

Techniques: Phospho-proteomics, Incubation, SDS Page, Concentration Assay, Biomarker Discovery, Fractionation, Marker, Western Blot, Staining, Two Tailed Test, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

doi: 10.1016/j.jbc.2021.101306

Figure Lengend Snippet: Association of SRSF2 with microtubule proteins. A , I-graph of module 16 (M16) representing hub proteins and corresponding gene symbols as nodes. Node size and edges ( gray ) are reflective of the degree of intramodular connectivity in WGCNA. SRSF2 ( green ) and cytoskeletal-associated proteins ( red ) are highlighted. B , representative western blot of an immunoprecipitation (IP) of recombinant SRSF2-myc in cytoplasm and nucleoplasm extracts isolated from HEK293 cells ( n = 3 replicates per fraction). Co-IP complexes were blotted for α-tubulin (TUBA1A) and β-tubulin (TUBB8). C and D , ICC staining of HEK293 cells for hypophosphorylated SRSF2 (hypoSRSF2) ( green ) and both TUBA1A ( red ; C ) and TUBB8 ( red ; D ) in vehicle-cells or 50 μM SRPIN340-treated cells.

Article Snippet: The pcDNA3.1-SC35-cMyc SRSF2 plasmid was a gift from Kathleen Scotto ( ) (Addgene plasmid #44721).

Techniques: Western Blot, Immunoprecipitation, Recombinant, Isolation, Co-Immunoprecipitation Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

doi: 10.1016/j.jbc.2021.101306

Figure Lengend Snippet: Proposed model of SRSF2 solubility and oligomerization changes regulated by phosphorylation. Equilibrium of structural states (monomer, oligomer, aggregate) of SRSF2 ( green ) in varying states of phosphorylation ( dark turquoise border ). The magnitude of positive ( purple triangle ) SRSF2 net charge is illustrated, inverse to the degree of phosphorylation ( turquoise triangle ). Phosphorylation regulates higher-order SRSF2 solubility, oligomerization, and structure formation.

Article Snippet: The pcDNA3.1-SC35-cMyc SRSF2 plasmid was a gift from Kathleen Scotto ( ) (Addgene plasmid #44721).

Techniques: Solubility, Phospho-proteomics