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

Anjan G Thakurta,Saravana P Selvanathan,Andrew D Patterson,Ganesh Gopal,Ravi Dhar

doi:10.1074/jbc.m609727200

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 RNA-binding 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.

Highlights

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]
Because Mex67p is not essential in wild type S. pombe cells for mRNA export, these results led us to propose that Uap56p was not removed from mRNPs, rather it played a critical role in nuclear pore complex (NPC) targeting and the export of mRNAs
MRNA Export in S. pombe in vivo experiments, we proposed that Uap56p and Dss1p-mediated links are critical for targeting mature mRNPs to the NPC [4]

Summary

EXPERIMENTAL PROCEDURES

Strains and Culture—Basic cell culture techniques used in this study were as described previously [14, 15]. Plasmid Constructions—The coding sequence for uap was amplified from the genomic DNA and inserted into pREP42X vector [18, 19]. To get a genomic construct, the gene for uap along with its promoter and 3Ј-untranslated region were inserted into an S. pombe vector pRL1 that carried the auxotropic marker LEU2 gene of S. cerevisiae. This plasmid was further used to make mutations within the uap coding sequences. S. pombe NES Assay—The construction of the S. pombe vector, pAG177, for in vivo nuclear export assay has been described previously [24]. The details of the export assay are described in the figure legends

RESULTS

DISCUSSION

By combining functions of

Ravi Dhar