The Nuclear Export Signal of Splicing Factor Uap56p Interacts with Nuclear Pore-associated Protein Rae1p for mRNA Export in Schizosaccharomyces pombe

Abstract

Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNAbinding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.Mammalian UAP56 (Saccharomyces cerevisiae Sub2p) and its functional homologs belong to the conserved DECD box class of ATP-dependent RNA helicases that play important functional roles in multiple aspects of DNA and RNA metabolism (1). They are essential in a number of organisms, including yeast, nematode, and fruit fly (2). UAP56/Sub2p is directly involved in pre-messenger RNA splicing and nuclear export of messenger RNAs in S. cerevisiae and in metazoan cells (3). Recently, in Schizosaccharomyces pombe, we found that uap56 is an essential gene for growth and that Uap56p is critical for mRNA export (4). Mammalian UAP56 functions in both ATP-independent and ATP-dependent steps of the spliceosome assembly process (5). Uap56p homologs typically contain a characteristic ATP-binding site, a catalytic DECD box, and an RNA interaction motif. Mutations within the ATP-binding pocket or the catalytic DECD box residues abolished the splicing functions of UAP56 (5).

The Cellular DExD/H-Box RNA-Helicases UAP56 and URH49 Exhibit a CRM1-Independent Nucleocytoplasmic Shuttling Activity

“…Nucleocytoplasmic shuttling appears to be conserved from yeast to human UAP56, as it has been reported that Uap56p of Schizosaccharomyces pombe ( S.pombe) is also able to exit the nucleus [36]. The NES of Uap56p was mapped to its amino acids 250 to 350, a region that coincided with a domain required for binding of the nuclear pore-associated protein Rae1p.…”

“…The NES of Uap56p was mapped to its amino acids 250 to 350, a region that coincided with a domain required for binding of the nuclear pore-associated protein Rae1p. This interaction was critical for both, nucleocytoplasmic shuttling as well as UAP56p-mediated mRNA export [36]. Importantly, two single mutations, Q297R and F320A, within the UAP56p NES-region, partially abolished export, while a combination of both mutations led to a total loss of UAP56p shuttling.…”

“…This suggests that the Rae1-interaction motif of human UAP56 was different from that detected in S.pombe. Since Thakurta and colleagues had demonstrated that two amino acid substitutions within the NES of Uap56p abolished Rae1-binding and shuttling [36], site directed mutagenesis was performed to construct human UAP56, carrying the amino acid substitutions Q289R and F312A (construct named UAP56-QF). After confirmation that UAP56-QF was expressed like wildtype UAP56 in immunofluorescence (data not shown) and Western blot analyses (compare Fig.…”

Schizosaccharomyces pombe Dss1p Is a DNA Damage Checkpoint Protein That Recruits Rad24p, Cdc25p, and Rae1p to DNA Double-strand Breaks

“…Rad24p and Rae1p Are Enriched at DSB Sites in S. pombe Cells-We previously showed that Rae1p was not recruited to active genes (5,24). Also, it is not known whether Rad24 or any of its homologs recruit to genes.…”

“…pombe Rae1p is essential for the nuclear export of mRNA and functions in cell cycle progression from G 2 to M (23,24). However, the role of Rae1p in the G 2 /M transition was shown to be independent of its role in mRNA export (25).…”