Ms023

Cells were seeded on coverslips (Corning) and either transduced with Sm proteins (as above) or allowed to grow in the presence of 0.01% DMSO, 1 µM GSK591 (Cayman), or 1 µM MS023 (Cayman) for 2 days. Cells were then washed with 37°C PBS (Hyclone) and fixed with 4% paraformaldehyde at 20–25°C for 10 min followed by washing with 4°C PBS. Residual aldehyde was quenched with 0.1 M glycine in 20–25°C PBS for 15 min. Permeabilization was performed using 0.1% Triton X-100 at 20–25°C with gentle rotation for 30 min. Cover slips were then washed with PBS and blocked for one hour using 0.1% Fish Skin Gelatin in PBS. Primary antibody for FLAG (Sigma-Aldrich, F1804; 1:50) or SNRPB (ProteinTech 16807-1-AP; 1:125) was added to the cover slip and incubated overnight at 4°C in blocking buffer. coverslips were washed with PBS and incubated with secondary antibody (Goat anti-Rabbit Dylight 488, Thermo Fisher Scientific 35552 or Goat anti-Mouse Alexa Fluor 555, Thermo Fisher Scientific A28180; 1:1000) with gentle rotation while protected from light for 1 hr at 20–25°C followed by PBS wash and mounting with DAPI prolong gold anti-fade (Thermo Fisher Scientific). Imaging was performed with an Olympus IX-70 inverted microscope with a 60× objective.

HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) already including 1% glutamine and supplemented with 10% fetal bovine serum (FBS; Life Technologies), penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were cultured at 37°C in a 5% CO2 humidified atmosphere. The cells were tested free of mycoplasma contamination. MS023 was purchased from Cayman chemicals; GSK591 was purchased from Sigma Aldrich. Both MS023 (10 µM) and GSK591 (5 µM) were used for 48 h treatment, together with DMSO as control. For triple SILAC, HeLa were grown in ‘‘Light’’, ‘‘Medium’’ and ‘‘Heavy’’ SILAC DMEM (Thermo Fisher Scientific), supplemented with either L-Arginine, L-Lysine or their medium (Arg6: Sigma-Aldrich; Lys4: Sigma-Aldrich) or heavy (Arg10: Sigma-Aldrich; Lys8: Sigma-Aldrich) isotope-counterparts. Arginine and Lysine were added at a concentration of 84 mg/L and 146 mg/L, respectively. SILAC media were supplemented with 10% dialyzed FBS (GIBCO, Life Technologies), 100 U/ml Penicillin and 100 mg/ml Streptomycin. HeLa cells were grown in the respective heavy-isotopes containing media for at least 9 replication cycles, to ensure full incorporation of isotope-encoded amino acids, with a careful monitoring of growth rate, viability and overall morphology, to guarantee that normal cell physiology was preserved.

A549 cells were grown with 0.01% DMSO, 1 µM GSK591 (Cayman), or 1 µM MS023 (Cayman) for 2 days. Cells were harvested using trypsin (Corning) and washed once with 4°C PBS supplemented with PRMT inhibitors. Cells were then resuspended in RIPA buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8 at 4°C, 0.25% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) supplemented with 40 U/mL RNaseOUT (Thermo Fisher Scientific) and protease inhibitor (Thermo Fisher Scientific). Lysates were incubated on ice for 10 min followed by sonication for 5 s at 20% amplitude with a probe-tip sonicator using a 1/8 inch tip. Lysates were then spun at 10,000×g for 10 min at 4°C. Supernatants were transferred to new low-adhesion RNase-free microcentrifuge tubes and normalized to the same protein concentration using bicinchoninic acid (Pierce). Primary antibody targeting CHTOP (LSBio, LS-C193506; 5 μg) or SNRPB (ProteinTech, 16807-1-AP; 5 μg) was added followed by incubation overnight at 4°C with gentle rotation. The next morning, Protein G agarose (Millipore-Sigma) was equilibrated in lysis buffer and added to the lysates at 4°C with gentle rotation. The beads were washed three times with lysis buffer followed by resuspension in 1× Laemmli buffer for western blotting with CHTOP (as above), SNRPB (as above), Rme2s (CST, 13222), and Rme2a (CST, 13522) antibodies.

A549 cells were grown in the presence of 0.01% DMSO, 1 µM GSK591 (Cayman), or 1 µM MS023 (Cayman) for 2 days. Following a 2-day incubation, the media were removed and replaced with media containing 5 µg/ml actinomycin D (Sigma-Aldrich) with 0.01% DMSO, 1 µM GSK591, or 1 µM MS023 for 60 min. RNA was isolated using RNeasy Mini Kit (QIAGEN) and poly(A)-RNA sequencing was performed as above.