Figure 3.

Effect of mRNA shortening on RNA export. (A) A ³²P-labeled RNA mixture containing shortened DHFR mRNAs (300 nt, 200 nt, 121 nt, 80 nt, and 50 nt), full-length DHFR mRNA, U1 ΔSm, and U6 Δss RNAs was microinjected with either PHAX WT (lanes 5,6), PHAX ΔNES (lanes 7,8), BSA–NES (lanes 9,10), or BSA-mut (lanes 11,12), or without proteins (buffer; lanes 3,4). All the RNAs except U6 were m7G-capped. RNA was extracted immediately (0 h; lanes 1,2) or 1 h (1 h; lanes 3–12) after injection, and analyzed as in Figure 1. (B,C) Quantitation of RNA export from three independent experiments carried out as in A. (D–F) The same as in A–C except that β-globin mRNA derivatives (360 nt, 300 nt, 200 nt, 130 nt, 80 nt, and 50 nt) were used. (G) Effect of the cap structure on export of the shortened β-globin mRNAs. The same RNA mixture as in D was prepared by primed-transcription with either dinucleotide ApppG (A-cap; lanes 1–4) or m⁷GpppG (m⁷G-cap; lanes 5–8) and RNA export was analyzed as in D. Note that U6 Δss RNA was always uncapped. (H) Effect of an mRNA competitor on RNA export. The same RNA mixture as in D was microinjected either with 0.5 pmole of unlabeled uncapped DHFR mRNA (DHFR comp., 500 times excess over labeled RNAs) or without it (buffer). RNA was extracted and analyzed as in D.