A Mutation in the Schizosaccharomyces pombe rae1 Gene Causes Defects in Poly(A)+ RNA Export and in the Cytoskeleton

Abstract

A collection of fission yeast Schizosaccharomyces pombe conditional mutants was screened for defective nucleocytoplasmic transport of poly(A)+ RNA by fluorescence in situ hybridization. We identified a temperature-sensitive mutant that accumulated poly(A)+ RNA in the nucleus and have named it rae1-1, for ribonucleic acid export. All rae1-1 cells exhibit the defect in poly(A)+ RNA export within 30 min following a shift to the non-permissive temperature. In addition, in the rae1-1 mutant, actin and tubulin become disorganized, and cells undergo an irreversible cycle arrest. Results from experiments in which rae1-1 cells were arrested in various phases of the cell division cycle and then shifted to nonpermissive temperature suggest that cells are particularly vulnerable to loss of rae1 function during G2/M. However, the inability to export RNA from the nucleus to the cytoplasm was not limited to a particular phase of the cell division cycle. The rae1 gene was isolated by complementation and encodes a predicted protein of 352 amino acids with four beta-transducin/WD40 repeats.

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

“…Gle2p possesses striking amino acid sequence similarity to the protein encoded by RAE1, a gene required for normal poly(A)+ RNA export in S. pombe (Brown et al, 1995) It has been suggested that the rael-1 mutant RNA export phenotype in S. pombe cells is an indirect consequence of a role for Raelp in the cell cycle or cytoskeletal machinery (Brown et al, 1995). Our characterization studies in S. cerevisiae demonstrate both a genetic and physical basis for Gle2p function at the NPC.…”

“…Alignment of the sequences gave an overall match of 82% (with 52% identical and 32% similar). The four putative ,B-transducin/WD repeats in Raelp (Brown et al, 1995) are largely conserved in Gle2p (underlined, Figure 2B). Mutation of the glycine residue at position 219 in Raelp to glutamic acid results in a temperaturesensitive RNA export mutant (Brown et al, 1995).…”

“…The four putative ,B-transducin/WD repeats in Raelp (Brown et al, 1995) are largely conserved in Gle2p (underlined, Figure 2B). Mutation of the glycine residue at position 219 in Raelp to glutamic acid results in a temperaturesensitive RNA export mutant (Brown et al, 1995). By site-directed mutagenesis, the glycine residue at position 222 in Gle2p was changed to an aspartic acid, and the mutated gene was expressed in a gle2A background (see below).…”

“…In S. pombe, RAE1 is an essential gene (Brown et al, 1995). To test whether GLE2 encoded an essential gene product, the chromosomal copy of GLE2 was replaced by homologous recombination with HIS3.…”

“…The localization of Raelp in S. pombe cells is reported as cytoplasmic (Brown et al, 1995). The subcellular localization of epitope-tagged-Gle2p was determined by indirect immunofluorescence microscopy.…”