Figure 4.

CircPABPN1 suppresses PABPN1 translation. (A) 48 h after transfecting HeLa cells with either control siRNA or HuR siRNA (left) or with pcDNA3 or pCircPABPN1 (right), the levels of PABPN1, HuR, and the loading control HSP90 were assessed by Western blot analysis. Following quantification of the bands on Western blots, the relative signal intensities were represented as the means ± SEM from three independent experiments. *, P <0.05 (Student's t-test)., (B, C) HeLa cells prepared as in (A) were size-separated through sucrose gradients into 12 fractions (arrow, direction of sedimentation). Unbound RNA was in fractions 1 and 2; 40S, 60S, and 80S were in fractions 3–5; and low- and high-molecular-weight polysomes (LMWP and HMWP) were in fractions 6–8 and 9–12, respectively (B right, C right) and Methods. After isolating RNA from each fraction, the relative distribution (%) of PABPN1 and GAPDH mRNAs on the sucrose gradients was quantified by RT-qPCR analysis (B left, C left). (D) Proposed model whereby CircPABPN1 sequesters HuR away from PABPN1 mRNA, in turn suppressing PABPN1 mRNA translation.