Figure 4.

RNAi of Prp19 complex components or U2AF2 blocks export of naturally intronless IFNβ1 mRNAs. (A) Schematic of CMV-IFNβ1 construct used for microinjection. The length of the 5′-UTR, coding region and 3′-UTR are indicated. Vector sequences are shown as gray lines. BGH pA: bovine growth hormone polyA site. (B, D, J) Western blots showing levels of XAB2 (B), AQR (D) or U2AF2 (J) after transfecting HeLa cells with targeting or non-targeting (Cntl) siRNAs as indicated. Tubulin was used as a loading control. (F and H) RT–PCR showing levels of ISY1 or CRNKL1 mRNAs after transfecting HeLa cells with targeting or non-targeting (Cntl) siRNAs as indicated. Endogenous DNAJB1 was amplified by PCR as a loading control. Reverse transcription was carried out in reactions that contained (+) or lacked (−) reverse transcriptase (RTase) followed by PCR. (C, E, G, I, K) CMV-IFNβ1 naturally intronless construct was microinjected into nuclei of the indicated knockdown or control cells. FISH was carried out to determine the nucleocytoplasmic distribution of the RNAs. Dextran 70 kDa was used an injection marker. Scale bar: 10 µm.