Major circRNAs in HeLa cells are not associated with polysomes.
(A) Polysome gradient profiles of HeLa cytoplasmic extracts after treatment with cycloheximide or puromycin. The RNA distribution (A260nm) and the positions of ribosomal size markers are indicated (40S/60S ribosomal subunits, 80S initiation complex/monosomes and polysome region). (B) Analysis of circRNA fractionation on polysome gradients, comparing profiles after cycloheximide (CHX) or puromycin (Puro) treatment. After RNA extraction, fractions were pooled (#1, #2–3, #4–5, #6–7, #8–9 and #10–11), followed by RT-PCR analysis of ten abundant circRNAs across the gradient. HIPK3 linear mRNA was used as a positive control for puromycin-induced mRNA release from polysomes (top two panels); the ten circRNAs (from GSE1, 219 nt, to HIPK3, 1099 nt) are ordered according to their sizes. Mispriming products corresponding to the linear mRNA of CAMSAP1 in polysome fractions (CAMSAP1 circ, #6–11) are marked by asterisks.