Pre-mRNAs undergo splicing to remove introns, and the spliced mRNA is exported to the cytoplasm for translation. Here we investigated the mechanism for recruitment of the conserved mRNA export machinery (TREX complex) to mRNA. We show that the human TREX complex is recruited to a region near the 5' end of mRNA, with the TREX component Aly bound closest to the 5' cap. Both TREX recruitment and mRNA export require the cap, and these roles for the cap are splicing dependent. CBP80, which is bound to the cap, associates efficiently with TREX, and Aly mediates this interaction. Together, these data indicate that the CBP80-Aly interaction results in recruitment of TREX to the 5' end of mRNA, where it functions in mRNA export. As a consequence, the mRNA would be exported in a 5' to 3' direction through the nuclear pore, as observed in early electron micrographs of giant Balbiani ring mRNPs.

In eukaryotic cells, most mRNAs are exported from the nucleus by the transcription export (TREX) complex, which is loaded onto mRNAs after their splicing and capping. We have studied in mammalian cells the nuclear export of mRNAs that code for secretory proteins, which are targeted to the endoplasmic reticulum membrane by hydrophobic signal sequences. The mRNAs were injected into the nucleus or synthesized from injected or transfected DNA, and their export was followed by fluorescent in situ hybridization. We made the surprising observation that the signal sequence coding region (SSCR) can serve as a nuclear export signal of an mRNA that lacks an intron or functional cap. Even the export of an intron-containing natural mRNA was enhanced by its SSCR. Like conventional export, the SSCR-dependent pathway required the factor TAP, but depletion of the TREX components had only moderate effects. The SSCR export signal appears to be characterized in vertebrates by a low content of adenines, as demonstrated by genome-wide sequence analysis and by the inhibitory effect of silent adenine mutations in SSCRs. The discovery of an SSCR-mediated pathway explains the previously noted amino acid bias in signal sequences and suggests a link between nuclear export and membrane targeting of mRNAs.

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. D uring gene expression, pre-mRNA undergoes several processing steps in the nucleus, including capping, splicing, and polyadenylation, and the mature mRNA is then exported to the cytoplasm for translation. All of the steps in gene expression are carried out by distinct multicomponent machines, but it is now clear that there is extensive physical and functional coupling among them (1).Initial studies on the role of splicing in gene expression revealed that splicing of the simian virus 40 (SV40) intron was required for expression of late viral genes (2, 3). Similarly, splicing was required for efficient ␤-globin expression from SV40 expression vectors (4, 5). Since then, numerous studies have reported a splicing-dependent enhancement of gene expression for multiple genes (6-15). Furthermore, this effect has been observed in Arabidopsis, Drosophila, maize cells, transgenic mice, and a variety of cell lines, indicating that it is a conserved and important feature of gene expression (7,9,11,13,15). The specific mechanisms by which splicing promotes gene expression have also been extensively investigated, revealing that splicing affects several steps, including transcription, total mRNA levels, and 3Ј end processing (1,(16)(17)(18)(19)(20). In addition, splicing enhances both translation and localization of mRNA (13,14,21).Although splicing has also been reported to promote mRNA export, this functional connection has been controversial. In early studies, R...

The TREX complex, which functions in mRNA export, is recruited to mRNA during splicing. Both the splicing machinery and the TREX complex are concentrated in 20–50 discrete foci known as nuclear speckle domains. Using a model system where CMV-DNA constructs were microinjected into HeLa cell nuclei, we have followed the fates of the transcripts. Here we show that transcripts lacking functional splice sites, which are inefficiently exported, do not associate with nuclear speckle domains but are instead distributed throughout the nucleoplasm. In contrast, pre-mRNAs containing functional splice sites accumulate in nuclear speckles, and our data suggest that splicing occurs in these domains. When the TREX components UAP56 or Aly are knocked down, spliced mRNA, as well as total polyA+ RNA, accumulates in nuclear speckle domains. Together, our data raise the possibility that pre-mRNA undergoes splicing in nuclear speckle domains, before release by TREX components for efficient export to the cytoplasm.