Fig 2.
Effects of TCERG1 on Bcl-x splicing depend on the SB1 regulatory element. (A) Structure of the Bcl-x gene and X2 and X2.13 (ΔSB1) minigenes. (B and C) Analysis of the effects of SB1 deletion on Bcl-x splice site selection in response to TCERG1 overexpression (OE) (B) and knockdown (C). HEK293T cells were cotransfected with Bcl-X2 or Bcl-X2.13 together with empty vector (-) or TCERG1 expression plasmid. For the RNAi experiments, HEK293T cells were cotransfected with the Bcl-x minigene together with siTCERG1 or control (siEGFP). RT-PCR was performed to analyze the alternatively spliced forms of Bcl-x. The graphs show the densitometric analysis results as the ratio of Bcl-xL to Bcl-xS isoforms from three independent experiments (means ± standard deviations [SD]). *, P < 0.05; **, P < 0.01. A fraction of the cell lysates was analyzed by immunoblotting with the indicated antibodies to detect the TCERG1 and CDK9 proteins. (D) RNA coimmunoprecipitation of the Bcl-x pre-mRNA with antibodies against TCERG1 is dependent on the SB1 region. The X2 or X2.13 transcripts were incubated in HeLa nuclear extracts under splicing conditions and then immunoprecipitated using the indicated antibodies. After five washes, the RNA was precipitated and quantified. The data are presented as the percentage of the bound input (means ± SD). **, P < 0.01. (E) TCERG1 associates with transcripts derived from the endogenous Bcl-x gene. In vivo immunoprecipitation assays were carried out with specific antibodies against TCERG1. Transcripts derived from the endogenous Bcl-x gene were detected by qPCR (see Materials and Methods). The data are presented as percentages of the bound input (means ± SD). **, P < 0.01. (F) Diagrammatic representation of the deleted sequences (in bold) in the Bcl-x minigene tested in the experiments. The numbers below the lines indicate the deleted regions. (G) HEK293T cells were cotransfected with 0.5 μg HIV-2 reporter minigene (X2, wild type; X2.13, carrying a complete deletion of the SB1 element; Δ9, Δ11, Δ13, Δ16, Δ17, Δ23, carrying 10-nucleotide deletions in the SB1 element) together with 0.5 μg empty vector (-) or 0.5 μg TCERG1 expression vector (+). RT-PCR was performed to analyze the alternatively spliced forms of Bcl-x. The bar graph shows the densitometric analysis results as the ratio of Bcl-xL to Bcl-xS isoforms from three independent experiments. (H) HEK293T cells were cotransfected with 0.5 μg of the indicated Bcl-x minigenes together with siRNAs against enhanced green fluorescent protein (siEGFP) or TCERG1 (siTCERG1). RT-PCR was performed to determine alternatively spliced forms of Bcl-x. The bar graph shows the densitometric analysis results as the ratio of Bcl-xL to Bcl-xS isoforms from three independent experiments. (I) RNA coimmunoprecipitation was carried out using the Δ12 and Δ23 transcripts as described in the legend for panel D. Data are presented as percentages of the bound input (means ± SD). *, P < 0.05; **, P < 0.01.