Isolation of a novel rmn1 gene genetically linked to spnab2 with respect to mRNA export in fission yeast

In fission yeast, Schizosaccharomyces pombe, the spnab2 gene encodes an ortholog of the budding yeast nuclear abundant poly(A)⁺ RNA-binding protein 2 (Nab2) that is an essential protein required for both mRNA biogenesis and nuclear export of mRNA to the cytoplasm. We have previously isolated three mutants (SLnab1–3) that showed synthetic lethality under the repressed condition of spnab2 expression. In this study, we isolated a novel rmn1 gene as a multicopy suppressor that complemented the defects in growth and mRNA export of SLnab1 mutant cells. The rmn1 gene contained three introns and encoded a 589 amino-acid protein with the RNA recognition motif (RRM) in the central region. The Δrmn1 null mutant was viable but showed a s light mRNA export defect. However, its over-expression caused a deleterious effect on growth accompanied by intense accumulation of poly(A)⁺ RNA in the nucleus. The combination of Δrmn1 with Δspnab2 or Δspmex67 also inhibited growth. In addition, Rmn1p was associated with Rae1p in vivo. These results suggest that rmn1 is a novel gene that is functionally linked to spnab2.     

Isolation of synthetic lethal mutations in the rsm1-null mutant of fission yeast

To identify mutations in genes that are genetically linked to rsm1, we performed a synthetic lethal genetic screen in the fission yeast, Schizosaccharomyces pombe. Four mutations that showed synthetic lethality in combination with the rsm1null allele were isolated from approximately 320,000 colonies and defined in three complementation groups. One mutant (SLrsm1) exhibited a significant accumulation of poly(A)⁺ RNA in the nucleus under synthetic lethal conditions, while the rest had no mRNA export defects. In addition, some genes (spmex67, rae1, or mlo3) required for mRNA export complemented the growth defects of the identified mutants. These results suggest that the isolated mutants contain mutations in genes that are involved in mRNA export and/or pre-mRNA retention.     

Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.     

Dss1 associating with the proteasome functions in selective nuclear mRNA export in yeast

Dss1p is an evolutionarily conserved small protein that interacts with BRCA2, a tumor suppressor protein, in humans. The Schizosaccharomyces pombe strain lacking the dss1⁺ gene (Δdss1) shows a temperature-sensitive growth defect and accumulation of bulk poly(A)⁺ RNA in the nucleus at a nonpermissive temperature. In situ hybridization using probes for several specific mRNAs, however, revealed that the analyzed mRNAs were exported normally to the cytoplasm in Δdss1, suggesting that Dss1p is required for export of some subsets of mRNAs. We identified the pad1⁺ gene, which encodes a component of the 26S proteasome, as a suppressor for the ts⁻ phenotype of Δdss1. Unexpectedly, overexpression of Pad1p could suppress neither the defect in nuclear mRNA export nor a defect in proteasome function. In addition, loss of proteasome functions does not cause defective nuclear mRNA export. Dss1p seems to be a multifunctional protein involved in nuclear export of specific sets of mRNAs and the ubiquitin-proteasome pathway in fission yeast.     

Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe

New prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe were isolated from a bank of 700 mutants that were either temperature sensitive (ts^-) or cold sensitive (cs^-) for growth. The bank was screened by Northern blot analysis with probes complementary to S. pombe U6 small nuclear RNA (sn RNA), the gene for which has a splicesomal (mRNA-type) intron. We identified 12 prp mutants that accumulated the U6 snRNA precursor at the nonpermissive temperature. All such mutants were also found to have defects in an early step of TFIID pre-mRNA splicing at the nonpermissive temperature. Complementation analyses showed that seven of the mutants belong to six new complementation groups designated as prp8 and prp10-prp14, whereas the five other mutants were classified into the known complementation groups prp1, prp2 and prp3. Interestingly, some of the isolated prp mutants produced elongated cells at the nonpermissive temperature, which is a phenotype typical of cell division cycle (cdc) mutants. Based on these findings, we propose that some of the wild-type products from these prp ⁺ genes play important roles in the cellular processes of pre-mRNA splicing and cell cycle progression.     

Screening for long-lived genes identifies Oga1, a guanine-quadruplex associated protein that affects the chronological lifespan of the fission yeast Schizosaccharomyces pombe

Schizosaccharomyces pombe and Saccharomyces cerevisiae are excellent model organisms to study lifespan. We conducted screening to identify novel genes that, when overexpressed, extended the chronological lifespan of fission yeast. We identified seven genes, among which we focused on SPBC16A3.08c. The gene product showed similarity to Ylr150w of S. cerevisiae, which has affinity for guanine-quadruplex nucleic acids (G4). The SPBC16A3.08c product associated with G4 in vitro and complemented the phenotype of an S. cerevisiae Ylr150w deletion mutant. From these results, we proposed that SPBC16A3.08c encoded for a functional homolog of Ylr150w, which we designated ortholog of G4-associated protein (oga1 ⁺). oga1 ⁺ overexpression extended the chronological lifespan and also decreased mating efficiency and caused both high and low temperature-sensitive growth. Deleting oga1 ⁺ resulted in caffeine-sensitive and canavanine-resistant phenotypes. Based on these results, we discuss the function of Oga1 on the chronological lifespan of fission yeast.     

NTF2-like domain of Tap plays a critical role in cargo mRNA recognition and export

Metazoan Tap-p15 (also called Nxf1-Nxt1) and yeast Mex67-Mtr2 heterodimers are the general mRNA export receptors. The RNA binding activity of Tap-p15, which is essential for mRNA nuclear export, has been attributed to the amino-terminal RNA binding module of Tap consists of RNA recognition motif (RRM) and leucine-rich repeat. In this study, we identified a novel RNA interaction surface in the NTF2-like (NTF2L) domain of Tap, which is analogous to the rRNA binding platform of Mex67-Mtr2. Tap-p15 uses the three domains to tightly bind the retroviral constitutive transport element. The RNA binding through the NTF2L domain is functionally relevant as introduction of mutations in this region reduced CTE-containing mRNA export activity. In contrast, only when the RRM and NTF2L domains were mutated simultaneously, bulk poly (A)⁺ RNA export and in vivo poly (A)⁺ RNA binding activities of Tap-p15 were significantly attenuated. Moreover, an engineered human cell line harboring the NTF2L domain mutation in the NXF1 gene showed a synthetic growth phenotype and severe mRNA export defect under Aly/REF and Thoc5 depleted condition. These data suggest that Tap-p15 recognizes bulk mRNAs through combinatorial use of the distinct RNA binding domains.     

Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe

 New prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe were isolated from a bank of 700 mutants that were either temperature sensitive (ts^-) or cold sensitive (cs^-) for growth. The bank was screened by Northern blot analysis with probes complementary to S. pombe U6 small nuclear RNA (sn RNA), the gene for which has a splicesomal (mRNA-type) intron. We identified 12 prp mutants that accumulated the U6 snRNA precursor at the nonpermissive temperature. All such mutants were also found to have defects in an early step of TFIID pre-mRNA splicing at the nonpermissive temperature. Complementation analyses showed that seven of the mutants belong to six new complementation groups designated as prp8 and prp10-prp14, whereas the five other mutants were classified into the known complementation groups prp1, prp2 and prp3. Interestingly, some of the isolated prp mutants produced elongated cells at the nonpermissive temperature, which is a phenotype typical of cell division cycle (cdc) mutants. Based on these findings, we propose that some of the wild-type products from these prp ⁺ genes play important roles in the cellular processes of pre-mRNA splicing and cell cycle progression. Received: 15 April 1996/Accepted: 9 July 1996     

Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis

By examining cytological phenotypes of 587 temperature-sensitive mutants of the fission yeast Schizosaccharomyces pombe, we obtained 18 mutants which cause cell division in the absence of nuclear division. By genetic analyses, these novel nuclear division arrest mutants can be classified into nine complementation groups (designated cut1 – cut9). The cytological phenotype of cut mutants is similar but not identical to that of DNA topoisomerase II mutants (top2). The cut1⁺ gene was cloned by transformation and shown to complement cut2 as well as cut1, indicating a functional relationship between the two genes. The cut genes are required for nuclear division, but their mutant phenotypes differ from most of the previously identified mutants which block nuclear division and also the subsequent cytokinesis. Fluorescence microscopy indicates that the mitotic chromosomes formed in cut mutant cells are abnormal and fail to separate properly. We suggest that cut mutations, like top2, block mitotic chromosome formation and concomitantly nuclear division, but that cytokinesis proceeds independently of the defects in nuclear division, demonstrating uncoordinated mitotic pathways. A novel mutant nuc1 is also described which shows a cytological phenotype similar to the double mutant of DNA topoisomerases I and II but contains normal levels of both DNA topoisomerase activities.     

Mitotic checkpoint gene expression is tuned by codon usage bias

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half‐lives and long protein half‐lives supports stable SAC protein levels. For the SAC genes mad2 ⁺ and mad3 ⁺, their short mRNA half‐lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1 ⁺ mRNA has a short half‐life despite a higher frequency of optimal codons, and despite the lack of known RNA‐destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co‐translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine‐tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.     

Fission Yeast Scp3 Potentially Maintains Microtubule Orientation through Bundling

Microtubules play important roles in organelle transport, the maintenance of cell polarity and chromosome segregation and generally form bundles during these processes. The fission yeast gene scp3 ⁺ was identified as a multicopy suppressor of the cps3-81 mutant, which is hypersensitive to isopropyl N-3-chlorophenylcarbamate (CIPC), a poison that induces abnormal multipolar spindle formation in higher eukaryotes. In this study, we investigated the function of Scp3 along with the effect of CIPC in the fission yeast Schizosaccharomyces pombe. Microscopic observation revealed that treatment with CIPC, cps3-81 mutation and scp3 ⁺ gene deletion disturbed the orientation of microtubules in interphase cells. Overexpression of scp3 ⁺ suppressed the abnormal orientation of microtubules by promoting bundling. Functional analysis suggested that Scp3 functions independently from Ase1, a protein largely required for the bundling of the mitotic spindle. A strain lacking the ase1 ⁺ gene was more sensitive to CIPC, with the drug affecting the integrity of the mitotic spindle, indicating that CIPC has a mitotic target that has a role redundant with Ase1. These results suggested that multiple systems are independently involved to ensure microtubule orientation by bundling in fission yeast.     

Division-site selection, cell separation, and formation of anucleate minicells inSchizosaccharomyces pombe mutants resistant to cell-wall lytic enzymes

Summary In most eukaryotic organisms that have cell walls, cell separation or cytokinesis is a degradative enzymatic process. In the fission yeastSchizosaccharomyces pombe, it is a post-M-phase event that includes the degradation of part of the cell wall and the primary septum. We describe the isolation of mutants partially defective in cytokinesis by enrichment of clones resistant to cell-wall lytic enzymes. The mutations confer mycelial morphology (chains of non-separated cells) and define four genes.Sep2-SA2 was subjected to detailed genetic and cytological analysis. Its cells frequently form complex septa composed of multiple layers, which appear as twin septa separated by anucleate minicells if the cell length is extended. This suggests that a polar signal-like mechanism may also operate inS. pombe during division-site selection andsep2 ⁺ takes part in it.Sep2 ⁺ seems to be involved in several cell cycle functions because its mutation can transiently block cell-cycle progression after nuclear division and provoke a transition from haploidy to diploidy in the double mutantsep2-SA2 cexl-SA2. Cexl-SA2 is another novel mutation which causes cell-length extension.Abbreviations DAPI 4,6-diamidino-2-phenylindole - YEA yeast extract agar - YEL yeast extract liquid - SMA synthetic minimal agar - MEA malt extract agar     

Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake

Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)⁺ mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)⁺ mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.