FIG 3.

Mutation of the LC8 binding site is lethal. (A) Schematic of the CITFA2 locus (left; not to scale) in BF cell line smC2-PTP in which one allele has been replaced with a hygromycin resistance cassette (HYG), while the remaining allele has been fused to the PTP tag sequence by integration of pPURO-PTP-CITFA2, which harbors a puromycin resistance cassette (PURO). Gray rectangles indicate gene flanks with essential RNA processing signals. Doxycycline-induced expression of PTP dsRNA specifically targets PTP-CITFA2 mRNA. An smC2-PTP culture growth curve in the presence (+dox) and absence (−dox) of doxycycline is shown (middle). Immunoblot monitoring of the CITFA2 knockdown using both anti-CITFA2 and anti-PTP antibodies, with TFIIB serving as a loading control, was performed (right). Note that the absence of an ∼55-kDa band in anti-CITFA2 antibody probing confirms exclusive expression of PTP-tagged CITFA2 in smC2-PTP cells. (B) At top is a schematic depiction (not to scale) of the construct that harbored the CITFA2-HA transgene and was targeted to the silent RRNA intergenic region to conditionally express RNAi-resistant CITFA2-HA mRNA and to rescue the PTP-CITFA2 knockdown. Culture growth curves of representative smC2-PTP cell lines whose PTP-targeted CITFA2 knockdown was rescued with wild-type (WT), 3Amut, or NDel CITFA2-HA expression are shown below. BLE, phleomycin resistance cassette. (C) RNA analysis of the rescue cell lines after either no induction (ni) or induction with 2 days of doxycycline treatment. (D) Immunoblotting of PTP-CITFA2 and CITFA2-HA proteins during a time course of doxycycline-induced PTP-CITFA2 knockdown (KD) and CITFA2-HA expression, with TFIIB serving as a loading control. Arrows indicate coreductions of 3Amut and NDel CITFA2-HA proteins with PTP-CITFA2 in the corresponding cell lines.