Figure 4. The Ars2 bound RNA 7SK promotes proliferation yet opposes proper replication-dependent histone mRNA 3′ end processing.

(A) HeLa cells were crosslinked and immunoprecipitation was performed using control or Ars2 antibodies. RNA was isolated from the precipitate and Northern blotting was performed with a probe specific for 7SK RNA. tRNA^Tyr was probed to demonstrate the specificity of the Ars2-7SK interaction. Relative quantification of bands is displayed below each blot with input set to 1. 100 ng of total RNA was used for input.

(B) HeLa cells were transfected with siRNAs targeting Ars2 (Ars2-2), 7SK, or a control siRNA. Two days later cells were collected, counted and re-seeded in triplicate into 6 well plates at equal densities. Cell counts were performed over the course of the next four days (day 0–4). Data points represent the average number of cells per well from two independent experiments +/− standard deviation. Inset - HeLa cells were transfected with siRNA targeting 7SK RNA or control siRNA and three days later RNA was isolated. Northern blotting was performed to confirm knockdown of 7SK. tRNA^Met was probed as a loading control.

(C) HeLa cells were transfected with siRNA targeting 7SK or control siRNA and RNA was isolated three days later. cDNA synthesis was performed using either oligo(dT) or random hexamer primers and TaqMan®-based qPCR was used to determine changes in the levels of polyadenylated (poly(A)) and total HIST3H2A mRNA. Bars represent relative quantification using the ΔΔC_t method normalized to control siRNA transfection +/− 95% confidence interval of three replicates. Human ACTB was used as an endogenous control.

(D) Hela cells were transfected with siRNA targeting Ars2 (Ars2-2), 7SK or a control siRNA and RNA was isolated three days later. cDNA synthesis was performed using oligo(dT) primers and TaqMan®-based qPCR was used to determine changes in the levels of polyadenylated HIST2H2BE (H2BE) and HIST1H3H (H3H) mRNA. Bars represent relative quantification using the ΔΔC_t method normalized to control siRNA transfection +/− 95% confidence interval of three replicates. Human ACTB was used as an endogenous control.

(E) Northern blotting was used to determine the effect of 7SK depletion on total levels of histone H2B transcript.