A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif.

Abstract

The RNA1 gene from Saccharomyces cerevisiae is defined by the temperature-sensitive rna1-1 mutation that interferes with the maturation and/or nucleocytoplasmic transport of RNA. We describe the purification of a 44-kDa protein from the evolutionary distant fission yeast Schizosaccharomyces pombe and the cloning and sequence analysis of the corresponding gene. Although this protein shares only 42% sequence identity with the RNA1 gene product, it represents a functional homologue because the expression of the S. pombe gene in S. cerevisiae complements the rna1-1 defect. Disruption in S. pombe of the gene encoding the 44-kDa protein, for which we propose the name S. pombe rna1p, reveals that it is essential for growth. Our analysis of purified S. pombe rna1p represents the first biochemical characterization of an RNA1 gene product and reveals that it is a monomeric protein of globular shape. Cell fractionation and immunofluorescence microscopy indicate that rna1p is a cytoplasmic protein possibly enriched in the nuclear periphery. We identify a sequence motif of 29 residues, which is rich in leucine and repeated eight times both in S. pombe and in S. cerevisiae rna1p. Similar leucine-rich repeats present in a series of other proteins, e.g., the mammalian ribonuclease/angiogenin inhibitor, adenylyl cyclase from S. cerevisiae, the toll protein from Drosophila melanogaster, and the sds22 protein phosphatase regulatory subunit from S. pombe, are thought to be involved in protein-protein interactions. Thus rna1p may act as a scaffold protein possibly interacting in the nuclear periphery with a protein ligand that could be associated with exported RNA.

The Acidic C-terminal Domain of rna1p Is Required for the Binding of Ran·GTP and for RanGAP Activity

“…GST-cDNAs encoding full-length S. pombe rna1p and S. cerevisiae Rna1p were isolated by PCR using a S. pombe RNA1 BspMI-XhoI cDNA fragment (29) or a genomic S. cerevisiae RNA1 NcoI-BamHI fragment (31) as template. In the case of S. pombe RNA1 PCR employed oligonucleotide SP1 (5Ј-CTTTCAACAACGAATTCCATGTCGCGT-3Ј, an EcoRI restriction site is underlined) as a sense primer.…”

“…The following antisense primers were used to generate wild-type and mutant cDNAs (a PstI restriction site is underlined): oligonucleotide SP2 (5Ј-CGGAAA-CAGCGCTGCAGGGTTCGAGAGGG-3Ј) resulted in a cDNA encoding the full-length protein, whereas oligonucleotides SP3 (5Ј-GATAGCTCT-GCAGCAAGTTCTTAGTCTTC-3Ј), SP4 (5Ј-GAAGTCTCCGCTGCAGG-GCTTTAAGACTCAGC-3Ј), SP5 (5Ј-GGTAAGTTCCTGCAGATCTTAA-AGCTCATC-3Ј), SP6 (5Ј-CACCACGACCTGCAGTTCAAAAGACTTCA-CG-3Ј), and SP7 (5Ј-GAAGTCTCCGCTGCAGGGCTTTAAGACTCAGC-TTCTTCTTCTTGATCCTGCTGCTCCTCGTCGGTAAG-3Ј) yielded the truncated cDNAs ⌬SP375-386, ⌬SP361-386, ⌬SP341-386, ⌬SP330 -386, and ⌬SP361-386/QQDQ, respectively. The numbers indicate amino acid positions in the rna1 protein sequence (29) that are deleted in the mutant proteins encoded by the different cDNAs. In ⌬SP361-386/QQDQ, the cDNA encodes a three amino acid replacement (the glutamic acid residues at positions 351, 352, and 354 are substituted by glutamine) in the background of the ⌬SP361-386 deletion.…”

“…The PCR employed a BspMI-XhoI fragment of the S. pombe rna1p cDNA (29) as template and the oligonucleotides SP8 (5Ј-CGGAATTCTTGATGAAATACGTGAAGTCTTTA-GC-3Ј; the EcoRI site introduced is underlined) and SP9 (5Ј-CGAGAGGGAAAGTAGGAGCTCTAAATATGAGC-3Ј; the SacI site introduced is underlined) as sense and antisense primers, respectively. The resulting plasmid pGEX-SPCT was transformed into E. coli BL21(DE3)pLysS cells.…”

“…In Saccharomyces cerevisiae and Schizosaccharomyces pombe, these genes encode the Ran homologues Gsp1p and spi1p (25,26), the RCC1 homologues Prp20p and pim1p (26,27), and the RanGAP homologues Rna1p and rna1p, respectively (28,29). Often, mutations in these genes cause a number of different defects including an * The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…In the yeast proteins, the acidic sequence is followed by a C-terminal stretch of 12 (S. pombe) or 15 (S. cerevisiae) residues capable of forming an amphipathic ␣-helix, whereas the C-terminal sequences in the mammalian proteins are much longer (189 and 191 residues in RanGAP and Fug1, respectively) and not related to Rna1p/rna1p (20,28,29,33). Limited proteolysis revealed that in S. pombe rna1p the LRRs form a relatively stable domain, whereas the acidic region appears more flexible and susceptible to limited digestion (34).…”