Alternative polyadenylation factor CPSF6 regulates temperature compensation of the mammalian circadian clock
Fig 1
Knockdown of CPSF6 impairs circadian temperature compensation.
(A) Circadian free-running period of U-2 OS cells lengthens upon shRNA-mediated CPSF6 knockdown (red) in comparison with control cells (blue), as accessed by a Bmal1-luciferase reporter construct. Raw time series data have been normalized by its baseline trend (magnitude). (B) Temperature compensation is significantly (p<0.001; n = 32; see S2A Fig for details) reduced upon CPSF6 knockdown. (C) Relative CPSF6 mRNA expression (n = 3−4 per condition) in control (blue) as well as CPSF6-depleted (red) cells at 3 different temperatures. Expression values are normalized to the average expression of control cells at 37°C. (D) Schematic drawing of experimental setup for determination of alternative PAS usage, transcript expression, as well as transcript half-lives. (E) Dynamics of CLOCK (left), NR1D2 (middle), and PER3 (right) mRNA expression after application of transcription inhibitor triptolide in wild-type (blue) and CPSF6 knockdown cells (red). Straight lines depict linear fits to the logarithmic data as used for the determination of half-lives. (F) Bar plots, comparing the total mRNA half-life (left) as determined from the fits in panel (E), total mRNA expression (middle), and distal PAS usage (right) in CLOCK, NR1D2, and PER3 in wild-type versus CPSF6 knockdown cells. Each dot denotes results from an individual experiment where values of 3 technical replicates have been averaged. Bars denote the corresponding averages of these individual experiments. Please note that results in panel (E) show dynamics of the 3 technical replicates within 1 individual experiment, thus corresponding to 1 dot in panel F (left). P values for a statistical comparison (t test) between the total mRNA half-life, total mRNA expression, and distal PAS usage between control and CPSF6-depleted cells are pa =0.187, pb = 0.002, pc = 0.580, pd = 0.225, pe = 0.012, pf<0.001, pg = 0.014, ph = 0.023, pi =0.003. Raw data underlying panels A, B, C, E, and F can be found in S1 and S5 Data. CPSF6, cleavage and polyadenylation specificity factor subunit 6; PAS, polyadenylation site; shRNA, short hairpin RNA.