Role of purine-rich exonic splicing enhancers in nuclear retention of pre-mRNAs

Abstract

Intron-containing pre-mRNAs are normally retained in the nucleus until they are spliced to produce mature mRNAs that are exported to the cytoplasm. Although the detailed mechanism is not well understood, the formation of splicing-related complexes on pre-mRNAs is thought to be responsible for the nuclear retention. Therefore, pre-mRNAs containing suboptimal splice sites should tend to leak out to the cytoplasm. Such pre-mRNAs often contain purine-rich exonic splicing enhancers (ESEs) that stimulate splicing of the adjacent intron. Here, we show that ESEs per se possess an activity to retain RNAs in the nucleus through a saturable nuclear retention factor. Cross-competition experiments revealed that intron-containing pre-mRNAs (without ESEs) used the same saturable nuclear retention factor as ESEs. Interestingly, although intronless mRNAs containing ESEs were also poorly exported, spliced mRNAs produced from ESE-containing pre-mRNAs were efficiently exported to the cytoplasm. Thus, the splicing reaction can reset the nuclear retention state caused by ESEs, allowing nuclear export of mature mRNAs. Our results reveal a novel aspect of ESE activity that should contribute to gene expression and RNA quality control.

Fig. 1.

Effect of ESEs on U1 RNA export. (A) Schematic representation of U1-ESE derivatives used for the export analysis. One or multiple copies of purine-rich ESE sequences from various genes were inserted into U1ΔSm RNA. (B) A mixture of in vitro-transcribed ³²P-labeled RNAs containing U1-ESE, U1ΔSm, and U6Δss RNAs was injected into the nucleus of Xenopus oocytes. U6Δss RNA was uncapped, and all of the other RNAs were m7G-capped. RNA was extracted from nuclear (N) and cytoplasmic (C) fractions immediately (0 h; lanes 1, 2, 5, 6, 9, 10, 13, and 14) or 1 h (1 h; lanes 3, 4, 7, 8, 11, 12, 15, and 16) after injection and analyzed by 8% denaturing PAGE. (C) Quantitation of RNA export from three independent experiments as in B. Averages and standard deviations are shown. (D) The same as in B except that one to three copies of ASLV ESE were used. (E) Quantitation of D.

Fig. 2.

Cross-competition experiments with U1-ESE RNAs. The same as in Fig. 1B except that the RNA mixture was injected either alone (A, lanes 1–4) or with 100 fmol per oocyte of unlabeled, uncapped U1-(GAA)₁₂ (GAA; A, lanes 5 and 6; B–D, lanes 1 and 2) or U1-(CAA)₁₂ (CAA; A, lanes 7 and 8; B–D, lanes 3 and 4) competitor RNA. RNA was analyzed immediately (0 h; A, lanes 1 and 2) or 1 h (1.5 h; lanes 3–8) after injection. N, nuclear fractions; C, cytoplasmic fractions.

Fig. 3.

Effect of U1-(GAA)₁₂ competitor on the nuclear retention of pre-ftz. (A) An antisense oligo against U2 snRNA was injected into the cytoplasm of Xenopus oocytes to destroy endogenous U2 RNA (ΔU2). Water was injected as a control (−). After 20 h of incubation, RNA was recovered, and the integrity of endogenous U1 and U2 snRNAs was examined by Northern blot analysis. (B) A mixture of ³²P-labeled ftz pre-mRNA without ESE (pre-ftz), DHFR mRNA, U1-(GAA)₁₂ RNA, U1ΔSm RNA, U6Δss RNA, and tRNA^phe was injected into the nucleus of control (lanes 1–4) or ΔU2 (lanes 5–10) oocytes, either alone (lanes 1–6) or with GAA (lanes 7 and 8) or CAA (lanes 9 and 10) competitor as in Fig. 2. RNA was analyzed immediately (0 h; lanes 1 and 2) or 2 h (2 h; lanes 3–10) after injection. N, nuclear fractions; C, cytoplasmic fractions.

Fig. 4.

Effect of splicing on the nuclear retention by ESEs. (A) Schematic representation of β-globin derivatives. GAA or CAA repeat sequence was fused to either β-globin pre-mRNA (pre-beta; Upper) or intronless β-globin mRNA (intronless beta; Lower). The sizes of the exons and an intron are indicated. (B) ³²P-labeled β-globin pre-mRNA (pre-beta) or intronless β-globin mRNA (intronless beta), fused to either GAA (lanes 1–12) or CAA (lanes 13–24) repeat sequence, was injected into the nucleus of Xenopus oocytes, together with ³²P-labeled DHFR mRNA and U6Δss RNA. RNA was analyzed immediately (0 h; lanes 1, 2, 7, 8, 13, 14, 19, and 20), 1 h (1 h; lanes 3, 4, 9, 10, 15, 16, 21, and 22), or 3 h (3 h; lanes 5, 6, 11, 12, 17, 18, 23, and 24) after injection. (C) Export kinetics of intronless beta or spliced beta RNA derivatives from B. (D) The same as in Fig. 1 except that an intronless β-globin mRNA instead of U1 was fused to various ESEs and the incubation was 0 or 1.5 h. (E) Quantitation of RNA export from three independent experiments as in D. N, nuclear fractions; C, cytoplasmic fractions.

Fig. 5.

Model of action of ESEs. How ESEs may contribute to gene expression and RNA quality control is illustrated. See Discussion for details.