Compartmentalization of the eukaryotic cell necessitates the movement of macromolecules through the nuclear envelope. In particular, mRNAs must be transported from the site of transcription in the nucleus to the cytoplasm for translation to occur. Export of mRNAs requires processing, packaging by RNA-binding proteins, recognition by export factors, and translocation through the nuclear pore complex (NPC) into the cytoplasm.
mRNAs transcribed by RNA Pol II undergo rapid processing in the nucleus. Pre-mRNA processing steps include addition of a 5' monomethyl cap, splicing of introns, and 3' cleavage and polyadenylation. 5' capping occurs on nascent transcripts soon after transcription initiation (Salditt-Georgieff et al. 1980; Jove and Manley 1984; Rasmussen and Lis 1993). Furthermore, capping enzyme is associated with Pol II and can be found associated with promoters of transcribed genes (Cho et al. 1997; McCracken et al. 1997; Komarnitsky et al. 2000; Schroeder et al. 2000). Additionally, the transcription machinery interacts with splicing and 3' cleavage and polyadenylation factors, suggesting that these events occur either cotranscriptionally or soon after transcription termination (Mortillaro et al. 1996; Yuryev et al. 1996; Dantonel et al. 1997). Coordination of mRNA processing events and transcription is thought to be primarily through Pol II and its carboxy-terminal domain (CTD) in a manner that can be dependent on the state of CTD phosphorylation (for review, see Hirose and Manley 2000).
In the nucleus, hnRNPs and other RNA-binding proteins package mRNAs into ribonucleoprotein particles (RNPs), and a subset of these proteins remain in the nucleus whereas others accompany the RNP into the cytoplasm, dissociate, and move back into the nucleus for further rounds of export. In the visually tractable Chironomus tentans, it is possible to study the very large (35-40 kb) Balbiani ring (BR) RNA throughout its various maturation stages, including transcription, nuclear export, and translation. Previous immunoelectron microscopy studies showed that the hnRNPs hrp36, hrp45, and hrp23 associate with the BR particle during its transcription (Alzhanova-Ericsson et al. 1996; Visa et al. 1996a; Sun et al. 1998). hrp45 and hrp23 dissociate from the BR particle at the nuclear pore whereas hrp36 can accompany the mRNP into the cytoplasm and remain associated during translation. Furthermore, CBP20, which together with CBP80 forms the mRNA cap-binding complex, was also shown to associate with the nascent BR particle (Visa et al. 1996b).
Recent studies have suggested that mechanisms exist to mark mRNAs as fully processed and export competent. In metazoans, splicing can enhance mRNA export (Luo and Reed 1999). The splicing machinery deposits a complex of proteins that mark exon-exon boundaries, and two members of this complex, Aly/REF and Y14, have been suggested to act as markers of spliced and export-competent RNPs (Kataoka et al. 2000; Le Hir et al. 2000; Zhou et al. 2000). Although it remains to be seen whether these proteins directly mediate the export process, both Aly and Y14 shuttle between the nucleus and cytoplasm, and the yeast homolog of Aly, Yra1, is essential for mRNA export (Sträßer and Hurt 2000; Zhou et al. 2000).
Recognition of the RNP for export may be mediated by a number of factors. A strong candidate for a metazoan mRNA export receptor is TAP, which binds and exports the constitutive transport element (CTE)-containing RNA produced by simian type D retroviruses (Gruter et al. 1998; Kang and Cullen 1999). Its yeast homolog, Mex67, along with the small protein Mtr2, is essential for mRNA export (Kadowaki et al. 1994; Santos-Rosa et al. 1998). Both TAP and Mex67 bind mRNA and contact components of the NPC (Katahira et al. 1999; Bachi et al. 2000; Sträßer et al. 2000).
Translocation of RNPs through the NPC undoubtedly requires the action of multiple nucleoporins as various mutations in nucleoporin genes result in blocked mRNA export (for review, see Fabre and Hurt 1997). The actual translocation process may be aided by the action of the ATP-dependent RNA helicase Dbp5/Rat8 and associated protein Gle1, which localize to the cytoplasmic side of the NPC and specifically contact Nup159 (Snay-Hodge et al. 1998; Hodge et al. 1999; Strahm et al. 1999). It has long been postulated and only recently shown that a helicase is able to remodel an RNA-protein complex, and this activity may serve as the mechanism for unwinding and translocating RNPs as well as releasing nonshuttling proteins from the mRNA at the NPC (Jankowsky et al. 2001). In summary, multiple factors are implicated in the translocation step of mRNA export.
Npl3 (also termed Mtr13/Mts1/Nab1/Nop3), a shuttling hnRNP that contains two RNA-recognition motifs (RRMs) and an RS-like domain, is a major mRNA-binding protein in S. cerevisiae (Wilson et al. 1994). Mutations in Npl3 cause nuclear accumulation of mRNA, indicating a role for Npl3 in the mRNA export process (Singleton et al. 1995; Lee et al. 1996). It has been postulated that along with other proteins, Npl3 packages pre-mRNA into an export-competent RNP and escorts it through the NPC (Lee et al. 1996; Shen et al. 1998). On arrival in the cytoplasm, Npl3 dissociates from mRNA and is transported back into the nucleus by the importin Mtr10 to allow further rounds of mRNA export (Pemberton et al. 1997; Senger et al. 1998). These characteristics of Npl3 make it an attractive paradigm for the function of hnRNPs with respect to nuclear export of mRNA.
In an effort to study Npl3 and mRNA export, we found that combined mutations in Npl3 and TATA-binding protein (TBP) block mRNA export. Moreover, we show that Npl3 is found in a complex with RNA Pol II and is associated with genes in a transcription dependent manner. Furthermore, the mRNA export factor Yra1 also associates with transcribed chromatin but is recruited at a later step than Npl3. These results suggest that cotranscriptional recruitment of mRNA export factors may be critical for proper mRNA export.