Figure 4
Depletion of the splicing factor SRSF3 stabilizes intronless mRNAs in the nucleus. (a) Immunoblot: Western blotting analysis of HeLa cells transfected with the pTRE2-BDLF1 construct without or with an EB2 expression plasmid as indicated. Cells were previously transfected with either a control siRNA (lanes 1 and 2) or a siRNA specific for SRSF3 (lanes 3 and 4). The western blots were probed with either an anti-Flag antibody to detect Flag-tagged EB2, an anti-SRSF3 or an anti-Tubulin antibody as a loading control. (b) and (c) Nuclear and cytoplasmic BDLF1 mRNA accumulation: Quantification by RT-qPCR of nuclear (b) and cytoplasmic (c) BDLF1 mRNA from HeLa cells transfected as described in (a). Error bars represent the s.d. from three independent experiments (n = 3). * Indicates a P-value of <0.05 and ** indicates a P-value of <0.01. (d) Nuclear BDLF1 mRNA stability: Relative nuclear BDLF1 mRNA level after transcription inhibition (as a percentage of the amount at time point 0) was determined by RT-qPCR from HeLa cells transfected as described in (a). Numbers above the panel refer to hours after doxycycline-mediated transcriptional shutoff of the BDLF1 intronless gene. Half-lives (t1/2) and s.d were calculated from three independent experiments (n = 3). (e) RNA immunoprecipitation: HeLa cells were transfected with the pCMV-BDLF1 or the pCMV-RLuc reporter constructs together with a HA-SRSF3 expression plasmid as indicated. SRSF3 containing complexes were immunoprecipitated using an anti-HA antibody. RNA was then purified and the amount of specific BDLF1 and RLuc transcripts co-immunoprecipitated was evaluated by RT-qPCR. The amount of SRSF3 expressed and immunoprecipitated in the samples was evaluated by western blot analysis. Error bars represent the s.d. from two independent experiments (n = 2). * Indicates a P-value of <0.05 and ** indicates a P-value of <0.01.
