Ja 25

Bacteria were grown either at 37 °C (E. coli BW25113 derivatives) or 18 °C (E. coli ΔbipA CFT073 derivatives) up to OD₆₀₀ of 0.5 in 50 mL of Neidhardt MOPS minimal medium [80] (link) supplemented with 0.1% casamino acids (w/v), 0.5% glycerol (w/v) and 100 μg/mL carbenicillin and l-arabinose was added to a final concentration of 0.2% (w/v). Cultures grown at 37 °C were harvested 10 min after induction by pouring them into precooled centrifuge bottles containing 100 g of crushed ice and centrifuged at 10,000 rpm for 10 min at 4 °C (Beckman JLA16.250 rotor). Cultures grown at 18 °C were harvested 5 h after induction by collecting into precooled centrifuge bottles and pelleting at 10,000 rpm for 10 min at 4 °C (Beckman JA25.50 rotor). Lysates were prepared the same as for polysome profiling of E. coli (see below).

E. coli cells of the BL21 strain, with or without the pET28a-obgE were grown at 37°C to OD600 of ∼0.5. The cell cultures were diluted to a series of concentrations (10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, and 10⁻⁷). 2 µl of each dilution was dropped on LB plate (1 mM IPTG) and incubated at 37°C for 10 h.
E. coli cells of BL21 strain with or without pET28a- obgE were grown at 37°C to OD600 of ∼0.5, and 1 mM IPTG was added to both cultures. After 5 h incubation at 30°C, cells were lysed by sonication, and clarified at 15,000 rpm in a JA 25.50 (Beckman Coulter) for 30 min. Equal amounts of cell extracts were loaded gently onto the top of a12 ml 10% to 40% sucrose gradient in Binding buffer, and the gradients were centrifuged in a SW41 rotor (Beckman Coulter) for 3.5 h at 39,000 rpm and 4°C. Gradients were analyzed using a Teledyne Isco fractionation system (Teledyne Isco).

Yeast cells were grown to a density of 10⁷ cells/ml before harvesting and freezing at −80°C. The cell pellet was then ground in a freezer mill cryogenic grinder. The resulting protein powder was resuspended in SP lysis buffer (20 mM HEPES pH 7.9, 100 mM KAc, 2 mM MgCl₂, 1 mM DTT, 3 mM EDTA, 0.1% igepal, 10% glycerol and cOmplete protease inhibitor (Roche)) and centrifuged at 20 000 × g for 15 min (JA-25.50, Beckman Coulter). The supernatant containing the total protein extract was then either stored at −80°C until use or further processed for enrichment of nuclear proteins. For nuclear protein enrichment, the sample was centrifuged for 90 min at 42 000 rpm (Ti45 rotor, Beckman Coulter). The lipid-rich layer and the soluble fraction were discarded, while the lower murky layer (the chromatin fraction) was washed with SP lysis buffer by centrifugation for 90 min at 38 000 rpm in an SW60 rotor (Beckman Coulter). The pellet was resuspended in SP lysis buffer (2 ml per 10 g of starting material) using a dounce homogenizer before adding NH₄SO₄ (500 mM final concentration). The solution was stirred for 30 min at 4°C, polyethylenemine was added to 0.1% final concentration, the solution was stirred for another 15 min at 4°C, and the solution was centrifuged for 90 min at 38 000 rpm (SW60 rotor). The resulting supernatant (nuclear protein extract) was then stored at −80°C until use.

CtSRα and the deletion variants and CtSRβ were cloned as hexa-histidine-tagged proteins via the pETHis vector26 (link)60 (link). Untagged CtSRβΔTM was cloned into the pET24a vector (Novagen). CtSR His-tagged CtSRα and untagged CtSRβΔTM were expressed individually using the auto-induction method in E. coli BL21 (DE3) cells at 24 °C overnight61 (link). Pellets of cells expressing CtSRα and CtSRβΔTM were lysed together in the lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 20 mM imidazole and 0.02% (v/v) Nonidet-P40 ) using a microfluidizer. After the lysis, the cell debris was removed by centrifugation at 67,000 g using JA25.50 (Beckman) rotor. The supernatant was collected and incubated at 4 °C for 1 h on a rotating wheel. The CtSR complex was then purified as described for mammalian SR47 (link). His-tagged CtSRα, CtSRα deletion variants and CtSRβΔTM were expressed and purified similarly.

Cells were harvested at 3500 rpm in Heraeus Multifuge, and pellets were washed twice with cold H2O, resuspended in 100 mM Tris HCl (pH 9.4), 10 mM DTT, and 10 μg/ml nocodazole, and incubated for 20 min on ice. Cells were washed with ice-cold H2O, resuspended in spheroplasting buffer (1 M sorbitol, 50 mM Tris HCl [pH 7.5], 1 mM CaCl2, 1 mM MgCl2, 10 μg/ml nocodazole, 350 U lyticase L4025-Sigma) and incubated 30 min on an orbital platform at 4°C. Spheroplasts were sedimented in a Beckman Coulter JA25.50 at 6000 rpm for 6 min, gently washed with 1 M sorbitol, transferred to 1.5 ml tubes, and sedimented for 1 min at 1500 rcf and 4°C. Pellets were resuspended in 200 μl cold 0.4 M sorbitol and lysed on ice for 30 min by the addition of 700 μl lysis buffer (25 mM HEPES/KOH [pH 8], 50 mM KCl, 10 mM MgSO4, 0.25% Triton X-100, 1 mM PMSF, 3 mM DTT, 1 × complete EDTA-free protease inhibitors), supplemented with 100 μg/ml RNase A and 300 mM NaCl. Cell extracts were obtained by spinning the lysed spheroplasts at 12,000 rcf and 4°C for 5 min.
Cleared lysates (450 μl) were loaded on sucrose gradients prepared in Biocomp gradient station and sedimented in SW41 rotor (Beckman Optima L-100 XP Preparative Ultracentrifuge) at 18,000 rpm for 4 hr. Gradients were fractionated using Gilson FC203B fractionator, collecting 15 drops/fraction.