Figure 3.
UIF and REF Are Required for Bulk Poly(A)+ RNA Export
(A) Schematic of the mRNA export reporter vector. The splice donor (SD) and acceptor (SA) sites are indicated.
(B and C) Tethering of REF and UIF to the reporter RNA triggers luciferase expression (B) and increases cytoplasmic levels of the reporter RNA, assayed by reverse transcriptase-polymerase chain reaction (qRT-PCR) (C). Error bars represent the standard error of the mean for three or more experiments.
(D) Overexpression of GFP-REF and GFP-UIF in COS-7 cells leads to nuclear accumulation of poly(A)+ RNA. Panels are shown at the same exposure. Poly(A)+ RNA was detected via fluorescence in situ hybridization (FISH) with a Cy3-oligo-dT probe.
(E) Western analysis of stable cell lines following tetracycline-induced expression of miRNAs for mRNA export factors. Proteins were detected with antibodies to the endogenous proteins indicated on the right of each panel.
(F) Localization of poly(A)+ RNA following induction of miRNAs targeting export factors for 48, 72, or 96 hr. UAP56/DDX39 knockdown leads to cell death by 72 hr. All equivalent panels are shown at the same exposure.
(G) Growth of stable cell lines following induction of miRNAs targeting the indicated genes. Error bars represent the standard deviation based on three independent experiments.
(H) Quantitative RT-PCR analysis of the RNA distribution in stable 293 cells expressing miRNAs to the indicated genes. The ratio of cytoplasmic to total RNA normalized to U1 small nuclear RNA (snRNA) levels is displayed at 48 hr and 96 hr with values for control samples set at 100%. Error bars represent standard error of the mean for three or more experiments.