Connections between the processing and nuclear export of mRNA: evidence for an export license?

A lthough the mechanisms regulating the nuclear export of eukaryotic mRNAs remain poorly understood, it has been known for some time that premRNAs, i.e., mRNAs that retain functional splice sites, are actively retained in the nucleus by what are termed splicing commitment factors (1, 2). The subsequent nuclear export of mature mRNAs therefore could be perceived as being largely caused by the removal, concomitantly with the completion of splicing, of these nuclear retention factors. However, in a recent issue of PNAS, Luo and Reed (3) provide evidence that nuclear mRNA export also involves the selective recruitment to fully spliced transcripts of factors that target these mRNAs for nuclear export (Fig. 1). Although the identification of cellular factors involved in mediating nuclear RNA export has been the subject of intense research interest, this issue is at best partly resolved (reviewed by refs. 4 and 5). However, it has been known for some time, based on in vivo competition assays, that different classes of RNAs, i.e., tRNAs, U small nuclear RNAs (snRNA), ribosomal RNAs (rRNAs), and mRNAs, exit the nucleus via distinct pathways that depend on different cofactors (6). Although rRNA nuclear export, which occurs as part of assembled ribosomal subunits, has not been extensively characterized, both tRNA export and U snRNA export are believed to depend on distinct members of the importin b family of nucleocytoplasmic factors, i.e., exportin t in the case of tRNA and Crm1 in the case of U snRNAs (5). Although Crm1 is capable of mediating nuclear mRNA export, and is indeed recruited for this purpose by retroviral RNA export factors such as the HIV-1 Rev protein, neither Crm1 nor exportin t appears to play any role in cellular mRNA export (5, 7). Although it initially was thought that mRNA export therefore might depend on yet a third, unidentified member of the importin b protein family, it now appears likely that an entirely unrelated export mechanism might be involved. The first proteins that were proposed to play a role in mRNA export were the heterogenous nuclear RNA-binding proteins (hnRNPs). The hnRNPs are a highly abundant family of proteins that associate with pre-mRNAs during transcription and remain associated with nuclear mRNAs after splicing is completed (4). Although some hnRNPs, such as hnRNP C, are removed from mRNA molecules at the nuclear pore during export, others, including particularly hnRNP A1 and hnRNP K, leave the nucleus with mRNAs and then dissociate from the mRNA in the cytoplasm, whereupon they are reimported into the nucleus. This nucleocytoplasmic shuttling is not passive, as both hnRNP A1 and hnRNP K contain nuclear export signals that can mediate protein nuclear export in the absence of RNA binding (8, 9). This observation therefore led to the suggestion that these shuttling hnRNPs might in fact be directly required for nuclear mRNA export (4), a hypothesis that gained support from data in yeast showing that the mutational inactivation of Npl3p, a likely functional homolog of vertebrate hnRNP A1, resulted in a defect in mRNA export (10). However, binding to hnRNP A1 andyor hnRNP K is clearly not sufficient for mRNA export as these proteins are also bound to premRNAs that are not capable of leaving the nucleus. The finding that a fusion of hnRNP C, a nonshuttling hnRNP, to hnRNP A1 blocked hnRNP A1 nuclear export raised the possibility that mRNA export was regulated by the interplay of hnRNPs that promoted nuclear retention (e.g., hnRNP C) and hnRNPs that could mediate nuclear export (e.g., hnRNP A1) (4). Although this scenario remains an attractive one, recent data have strongly implicated yet another protein in the mRNA export pathway, i.e., the Tap nuclear RNA export factor. Tap first was identified as the target for a retroviral constitutive transport element (CTE), an RNA element that permits the nuclear export of pre-mRNAs when present in cis (11). The Tap protein contains not only a CTE-specific RNA binding domain but also an essential carboxylterminal sequence that functions both as a nuclear export signal and as a binding domain for nucleoporins, a group of proteins that constitute the nuclear pores, the portal used for all nuclear import and export (5, 12, 13). Two lines of evidence implicate Tap as a critical constituent in global nuclear mRNA export. First, titration of Tap by nuclear microinjection of high levels of CTE RNA inhibits nuclear mRNA export but does not affect the export of other RNAs, such as tRNAs or U snRNAs (14, 15). Second, the yeast homolog of Tap, termed Mex67, is clearly essential for poly(A)1 RNA export (13). Remarkably, human Tap can rescue mRNA export in Mex67-deficient yeast cells if expressed together with an essential human cofactor termed p15, thus strongly suggesting that a critical role for Tap in nuclear mRNA export has been conserved across a considerable evolutionary distance (13). It therefore has been suggested that recruitment of a Tapyp15 complex might be the critical, final step that induces cellular mRNA export from the nucleus (11). However, how Tapyp15 is recruited to mature mRNAs, and how access to Tapyp15 is regulated to prevent an inappropriate interaction with pre-mRNAs, remains unknown. This important issue, i.e., how do mRNAs receive an export license from the nucleus, is addressed by the Luo and Reed manuscript (3). These authors report two novel findings. First, injection of intron containing pre-mRNAs into the Xenopus oocyte nucleus results in the efficient splicing and subsequent nuclear export of these mRNAs. In contrast, however, nuclear injection of the fully spliced forms of these same mRNAs, what the authors term Di-mRNAs, results in very inefficient nuclear mRNA export and primarily leads to the nuclear degradation of the DimRNA (Fig. 1). Second, these authors present compelling evidence that mRNA splicing can result not only in the removal of nuclear retention factors but also in the selective recruitment of nuclear RNA ex-