In eukaryotic cells, the process of the nuclear export of messenger RNA (mRNA) physically and functionally couple nuclear gene transcription and mRNA splicing with the translation apparatus in the cytoplasm. Fully mature transcripts are exported as ribonucleoprotein (RNP) complexes through specialized pores embedded within the nuclear envelope. It is thought that one or more proteins within the RNP act as carriers to target the mRNA to the pores and mediate its exit from the nucleus by interacting with various pore-associated proteins. In the cytoplasm, the RNP complex is disassembled to release the mRNA for translation; the proteins involved in export are returned to the nucleus for initiating another round of mRNA export. We use the fission yeast Schizosaccharomyces pombe (S. pombe) and employ genetic and biochemical approaches to understand the functions of two key conserved mRNA export factors, Rae1p (Gle2 in metazoan or S. cerevisiae) and the NXF/NXT (Mex67/p15 in S. pombe or Tap/p15 in metazoan) heterodimer. In S. pombe, the role of Rae1p in mRNA export is essential. The functions of NXF/NXT homolog in various organisms appear to be essential in mRNA export, but surprisingly in S. pombe, the role of their homolog Mex67p/p15 is redundant. Both Rae1p and Mex67p/p15 are thought to function at the terminal nuclear steps of mRNA export presumably for linking RNP complexes to the pore and for mediating RNP export through the nuclear pore complexes (NPC). In the Mex67p/Tap mediated pathway, an early mRNA export factor Sub2p (metazoan UAP56) is recruited to activated genes and the transcripts and Sub2p in turn recruits RNA annealing protein Yra1p (Mlo3 in S. pombe and Aly in metazoan) to the nascent messages. Mex67/Tap is believed to displace Sub2p (UAP56) from the RNP complex to bind Yra1p (Aly). Interactions between Mex67p/Tap with ?FG (Phelylalanine-Glycine repeat) containing nucleoporins are believed to underlie pore targeting as well as the passage of the RNP cargo through the NPC. No similar mechanistic details are known about how Rae1p functions. We have identified esl49 as a gene whose functions are crucial for mediating mRNA export via Rae1p. The encoded protein is conserved across evolution. We have found that Els49p interacts directly with Mlo3p (Yra1p) and with Rae1p while Mlo3p does not interact with Rae1p directly. A ternary complex containing Mlo3p-Esl49p-Rae1p readily forms in vitro demonstrating Esl49p could link Mlo3p to Rae1p. Immuno-precipitation experiments suggest that Esl49p is associated with Mlo3p and with Rae1p in complexes in vivo. These studies have uncovered the mRNA export components functioning in the Rae1p-mediated essential mRNA export pathway of S. pombe. To understand how NXF proteins could be targeted to the nuclear pore, we have identified additional pore targeting domains in analogous regions in spMex67p and hTap by genetic protein export assays in S. pombe and in the HeLa cells. These domains are not part of the two FG interacting pockets known in Mex67p/hTap. We have further found direct in vitro binding of ?FG containing regions of Nup98 and Nup159 with these pore-targeting domains. Mutations that abolished nuclear export properties of these domains in vivo concomitantly led to loss of interaction with the ?FG proteins. These results lead us to propose the existence of new and conserved pore targeting domains in human and S. pombe NXF homologs.
| Thakurta, Anjan G; Gopal, Ganesh; Yoon, Jin Ho et al. (2004) Conserved nuclear export sequences in Schizosaccharomyces pombe Mex67 and human TAP function in mRNA export by direct nuclear pore interactions. J Biol Chem 279:17434-42 |
