Splicing promotes rapid and efficient mRNA export in mammalian cells

Abstract

The numerous steps in protein gene expression are extensively coupled to one another through complex networks of physical and functional interactions. Indeed, >25 coupled reactions, often reciprocal, have been documented among such steps as transcription, capping, splicing, and polyadenylation. Coupling is usually not essential for gene expression, but instead enhances the rate and/or efficiency of reactions and, physiologically, may serve to increase the fidelity of gene expression. Despite numerous examples of coupling in gene expression, whether splicing enhances mRNA export still remains controversial. Although splicing was originally reported to promote export in both mammalian cells and Xenopus oocytes, it was subsequently concluded that this was not the case. These newer conclusions were surprising in light of the observations that the mRNA export machinery colocalizes with splicing factors in the nucleus and that splicing promotes recruitment of the export machinery to mRNA. We therefore reexamined the relationship between splicing and mRNA export in mammalian cells by using FISH, in combination with either transfection or nuclear microinjection of plasmid DNA. Together, these analyses indicate that both the kinetics and efficiency of mRNA export are enhanced 6- to 10-fold (depending on the construct) for spliced mRNAs relative to their cDNA counterparts. We conclude that splicing promotes mRNA export in mammalian cells and that the functional coupling between splicing and mRNA export is a conserved and general feature of gene expression in higher eukaryotes.

Fig. 1.

Splicing results in a 10-fold increase in the C/N ratio of β-globin mRNA. (A) Schematic of β-globin constructs. The CMV promoter and BGH poly(A) site are indicated. The WT and cDNA constructs are isogenic except for the presence or absence of introns. The positions of the FISH and RPA probes are shown. (B) HeLa cells were transiently transfected with 900 ng of β-globin WT or cDNA constructs. FISH was performed to visualize β-globin mRNA, and DAPI staining was used to identify the cell nucleus. (Magnification: ×630.) (C) The C/N ratio of β-globin mRNA was determined for a minimum of 70 cells per construct. The graph shows the average C/N ratio for spliced mRNAs and cDNA transcripts, and error bars indicate standard error.

Fig. 2.

Splicing enhances the C/N ratio for Smad and TPI-RL mRNAs. (A) Schematic of Smad constructs. The positions of the FISH and RPA probes are indicated. (B) HeLa cells were transiently transfected with 900 ng of Smad WT or Smad cDNA constructs. FISH was performed to visualize Smad mRNA, and DAPI staining was used to identify the cell nucleus. (C) The C/N ratio of Smad mRNA was determined for a minimum of 90 cells per construct. The graph shows the average C/N ratio for spliced mRNAs and cDNA transcripts, and error bars indicate standard error. (D, E, and F) The same as A, B, and C, respectively, except TPI-RL was used. (Magnification: B and E, ×630.)

Fig. 3.

RPA analysis of β-globin spliced mRNAs versus cDNA transcripts. (A) HeLa cells were transiently transfected with 900 ng of β-globin WT or β-globin cDNA constructs and 900 ng of a transfection control plasmid, xpSER, which encodes the human tRNA^SER gene with a tag at the 5′ end. Total RNA was extracted 24 h after transfection and RPA was performed. Reactions in lanes 1 and 3 contained 250 ng of cellular RNA, and reactions in lanes 2 and 4 contained 1 μg of cellular RNA. β-globin probe (24 fmol) and 2 fmol of xpSER probe were used. Lane 5 was carried out in the absence of target RNA. Lane 6 shows the probe in the absence of target RNA and RNase. (B) Graph of the fold difference in mRNA levels for each gene. mRNA levels were normalized to the levels of xpSER, and the ratio of mRNA generated from intron-containing genes to mRNA from intronless genes was calculated. Data represent the average of three experiments, and error bars indicate standard errors.

Fig. 4.

Evidence that β-globin-spliced mRNAs are exported more rapidly and efficiently than cDNA transcripts. (A) β-Globin WT and cDNA constructs (50 ng/μl) shown in Fig. 1A were microinjected into HeLa cell nuclei along with FITC-conjugated dextran as an injection marker. α-Amanitin (50 μg/ml) was added 15 min after injection to inhibit transcription. Cells were incubated for the indicated times before fixation. Representative pictures are shown for each time point. Large pictures show FISH signal; inset pictures show the injection marker. (Magnification: ×630.) (B) The ln-total FISH florescence was determined for a minimum of 20 cells per construct per time point. The graph shows the average ln-total fluorescence for spliced mRNAs and cDNA transcripts at each time point. (C) The same as B, except C/N ratios were calculated.