Polysome Profiling: Ribosomal Subunit Analysis

Polysome profiling was performed following the protocols described in (19 ,20 (link)). To obtain the cytoplasmic lysates, cells were treated with cycloheximide (10 μg ml⁻¹) for 3–4 min and then lysed in 300 μl of cold hypotonic lysis buffer (19 ). To remove nuclei, mitochondria and cellular debris, the lysates were centrifuged at 4°C for 5 min at 20 000 g. To separate ribosomal subunits, ribosomes and polysomes from other cytoplasmic molecules, the supernatant was loaded on a 10–40% (w/v) sucrose gradient and centrifuged for 1 h 30 min at 260 000 g at 4°C in a SW41 rotor using a Beckman Optima LE-80 Ultracentrifuge. Twelve 1 ml fractions were collected and the absorbance at 254 nm was monitored with the UA-6 UV/VIS detector (Teledyne Isco). RNA was purified fraction by fraction using the phenol/chloroform extraction method described in (21 (link)). The retro-transcription reaction was performed using the same volume of RNA for all polysomal fractions. The co-sedimentation profile of mRNAs was obtained by calculating the percentage (or fraction) of mRNAs in each fraction by qPCR as described in (22 (link)).