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RBM10 is an RNA-binding protein that regulates alternative splicing (AS). It localizes to the extra-nucleolar nucleoplasm and S1-1 nuclear bodies (NBs) in the nucleus. We investigated the biological significance of this localization in relation to its molecular function. Our analyses, employing deletion mutants, revealed that RBM10 possesses two S1-1 NB-targeting sequences (NBTSs), one in the KEKE motif region and another in the C 2 H 2 Zn finger (ZnF). These NBTSs act synergistically to localize RBM10 to S1-1 NBs. The C 2 H 2 ZnF not only acts as an NBTS, but is also essential for AS regulation by RBM10. Moreover, RBM10 does not participate in S1-1 NB formation, and without alterations of RBM10 protein levels, its NB-localization changes, increasing as cellular transcriptional activity declines, and vice versa. These results indicate that RBM10 is a transient component of S1-1 NBs and is sequestered in NBs via its NBTSs when cellular transcription decreases. We propose that the C 2 H 2 ZnF exerts its NB-targeting activity when RBM10 is unbound by pre-mRNAs, and that NB-localization of RBM10 is a mechanism to control its AS activity in the nucleus.

RNA-binding proteins (RBPs) play important roles in the posttranscriptional regulation of gene expression, including mRNA stability, transport and translation. Fission yeast rnc1 + encodes a K Homology (KH)-type RBP, which binds and stabilizes the Pmp1 MAPK phosphatase mRNA thereby suppressing the Cl - hypersensitivity of calcineurin deletion and MAPK signaling mutants. Here, we analyzed the spatial regulation of Rnc1 and discovered a putative nuclear export signal (NES) Rnc1 , which dictates the cytoplasmic localization of Rnc1 in a Crm1-independent manner. Notably, mutations in the NES Rnc1 altered nucleocytoplasmic distribution of Rnc1 and abolished its function to suppress calcineurin deletion, although the Rnc1 NES mutant maintains the ability to bind Pmp1 mRNA. Intriguingly, the Rnc1 NES mutant destabilized Pmp1 mRNA, suggesting the functional importance of the Rnc1 cytoplasmic localization. Mutation in Rae1, but not Mex67 deletion or overproduction, induced Rnc1 accumulation in the nucleus, suggesting that Rnc1 is exported from the nucleus to the cytoplasm via the mRNA export pathway involving Rae1. Importantly, mutations in the Rnc1 KH-domains abolished the mRNA-binding ability and induced nuclear localization, suggesting that Rnc1 may be exported from the nucleus together with its target mRNAs. Collectively, the functional Rae1-dependent mRNA export system may influence the cytoplasmic localization and function of Rnc1., (© 2017 John Wiley & Sons Ltd.)

RNA localization is an important event that is essential for the polarization and differentiation of a cell. Although several methods are currently used to detect localized RNAs, a simplified detection system has not yet been developed for Schizosaccharomyces pombe. In the present study, we describe a new vector system for the visualization of localized RNAs in S. pombe using a U1A-tag-GFP system. A pREP1-U1A-tag vector plasmid to express U1A-tagged RNA and a pREP2-U1AGFP plasmid to produce a U1A-GFP fusion protein were constructed for this system. Since the U1A-GFP protein binds U1A-tagged RNA, fluorescence is observed at the location of U1A-tagged RNA in cells expressing both of these. The nucleolar localization of U3 snoRNA was successfully detected using this system, and a novel RNA localized at the DNA region of the nucleus was found by screening localized RNAs. This system will accelerate the study of localized RNAs in S. pombe.

To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A)(+) RNA transport] 1 to 11, which accumulate poly(A)(+) RNA in the nucleus at a nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr5-1 mutant shows dots- or a ring-like accumulation of poly(A)(+) RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5(+) gene and found that it encodes a component of the nuclear pore complex (NPC), nucleoporin 85 (Nup85). The ptr5-1 mutant shows no defects in protein transport, suggesting the specific involvement of Ptr5p/Nup85p in nuclear mRNA export in S. pombe. We identified Seh1p, a nucleoporin interacting with Nup85p, an mRNA-binding protein Mlo3p, and Sac3p, a component of the TREX-2 complex involved in coupling of nuclear mRNA export with transcription, as multi-copy suppressors for the ptr5-1 mutation. In addition, we found that the ptr5-1 mutation is synthetically lethal with a mutation of the mRNA export factor Rae1p, and that the double mutant exaggerates defective nuclear mRNA export, suggesting that Ptr5p/Nup85p is involved in nuclear mRNA export through Rae1p. Interestingly, the ptr5-1 mutation also showed synthetic effects with several prp pre-mRNA splicing mutations, suggesting a functional linkage between the NPCs and the splicing apparatus in the yeast nucleus., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Transport of mRNA from the nucleus to the cytoplasm is an essential process for gene expression in eukaryotic cells. In this unit, methods for monitoring nuclear mRNA export are described. Visualization of cellular mRNAs by fluorescence in situ hybridization with oligo(dT) probes is effectively applied to monitoring mRNA export from the nucleus in yeast and mammalian cells. In addition to the protocols for fluorescence in situ hybridization, this unit includes an alternate method that the authors have been developing for visual analysis of nuclear mRNA export in living mammalian cells by microinjection of fluorescently labeled pre-mRNA into the nuclei., (Copyright 2008 by John Wiley & Sons, Inc.)

Background Information: The RNA-binding protein S1-1, also called RBM10 (RNA-binding motif 10), is a paralogue of putative tumour suppressor RBM5 and has been correlated with cancer proliferation and apoptosis. In the present study, we have investigated the cell biology of S1-1., Results: In the extranucleolar nucleoplasm, S1-1 occurred in hundreds of punctate and irregular domains. Some 10-40 of these domains were larger than 0.5 mum and prominent for S1-1 immunostaining. These domains (S1-1 nuclear bodies) were commonly present in tissue cells and in cultured cells. When cellular transcription was globally reduced by heat shock, serum starvation, culture at high cell densities or inhibition with RNA polymerase II inhibitors, small S1-1 domains (S1-1 granules), with weak immunostaining signals, reduced in number, whereas S1-1 nuclear bodies became prominent and increased in size. These altered S1-1 domains were returned to initial states when the cells were placed under normal conditions. Similar to paraspeckles, S1-1 nuclear bodies occurred closely adjacent to nuclear speckles or IGCs (interchromatin granule clusters), as determined by immunoelectron microscopy. However, the S1-1 nuclear bodies did not correspond to paraspeckles or IGAZs (interchromatin-granule-associated zones), but coincided with TIDRs (transcription-inactivation-dependent RNA domains), which we had characterized previously at the RNA level. The enlarged S1-1 nuclear bodies/TIDRs accumulated the S1-1 protein and microinjected primary and spliced mRNAs, presumably for later elevation of gene expression. In addition, electron microscopy revealed that S1-1 was also present on perichromatin fibrils, suggesting the structure of S1-1 granules seen at higher resolution., Conclusions: S1-1 constitutes hundreds of nuclear domains, which dynamically change their structures in a reversible manner. Upon globally reducing RNA polymerase II transcription, S1-1 nuclear bodies enlarge and decrease in number. They are novel domains different from paraspeckles or IGAZs, despite their similar occurrence adjacent to nuclear speckles. We discuss S1-1 granules in terms of their association with gene expression. In addition, this is the first report of a TIDR-localized protein.

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 (Deltadss1) 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 Deltadss1, 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 Deltadss1. 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.

Nuclear retention of pre-mRNAs is tightly regulated by several security mechanisms that prevent pre-mRNA export into the cytoplasm. Recently, spliceostatin A, a methylated derivative of a potent antitumor microbial metabolite FR901464, was found to cause pre-mRNA accumulation and translation in mammalian cells. Here we report that spliceostatin A also inhibits splicing and nuclear retention of pre-mRNA in a fission yeast strain that lacks the multidrug resistance protein Pmd1. As observed in mammalian cells, spliceostatin A is bound to components of the SF3b complex in the spliceosome. Furthermore, overexpression of nup211, a homolog of Saccharomyces cerevisiae MLP1, suppresses translation of pre-mRNAs accumulated by spliceostatin A. These results suggest that the SF3b complex has a conserved role in pre-mRNA retention, which is independent of the Mlp1 function.

To identify novel factors involved in nuclear mRNA export in Schizosaccharomyces pombe, we isolated and characterized the ptr8(+) gene, mutation of which causes nuclear accumulation of poly (A)(+) RNA. The ptr8(+) gene encodes an S. pombe homologue of human XPB, a component of TFIIH involved in nucleotide excision repair (NER) and transcription. A temperature-sensitive mutant of ptr8(+) (ptr8-1) was highly sensitive to UV irradiation, as are human XPB cells. Northern blot analysis demonstrated that the amount of total poly (A)(+) mRNAs does not decrease significantly at the nonpermissive temperature in ptr8-1 cells, whereas a pulse-labeling assay using (35)S-methionine showed that protein synthesis decreases rapidly after incubation of cells at the nonpermissive temperature, suggesting that ptr8-1 cells have a defect in nuclear mRNA export. In Saccharomyces cerevisiae, a mutation in the SSL2 gene encoding a homologue of Ptr8p also causes a block of mRNA export at the nonpermissive temperature. In addition, expression of human XPB in ptr8-1 cells rescued the ts phenotype and the mRNA export defects, suggesting that human XPB may also play a role in mRNA export. Furthermore, we revealed a functional interaction between Ptr8p and Tho2p, a component of the TREX complex involved in mRNA export. These results suggest that XPB/Ptr8p plays roles not only in NER and transcription, but also plays a conserved role in mRNA export.

In eukaryotic cells, pre-mRNAs are transcribed in the nucleus, processed by 5' capping, 3'-polyadenylation, and splicing, and exported to the cytoplasm for translation. To examine the nuclear mRNA transport mechanism, intron-deficient mRNAs of truncated beta-globin and EGFP were synthesized, fluorescently labeled in vitro, and injected into the nucleus of living Xenopus A6 cells. The trajectories of single mRNA molecules in the nucleus were visualized using video-rate confocal microscopy. Approximately half the mRNAs moved by Brownian motion in the nucleoplasm, except the nucleoli, with an apparent diffusion coefficient of 0.2microm(2)/s, about 1/150 of that in water. The slow diffusion could not be explained by simple diffusion obeying the Stokes-Einstein equation, suggesting interactions of the mRNAs with nuclear components. The remaining mRNAs were stationary with an average residence time of about 30s, comparable to the time required for mRNA diffusion from the site of synthesis to nuclear pores.

Export of mRNA from the nucleus to the cytoplasm is one of the essential steps for eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, heat shock stress at 42 degrees C causes block of mRNA export from the nucleus. We now report that saline and ethanol stresses also inhibit nuclear mRNA export, resulting in accumulation of bulk poly (A)+ RNA, as well as a specific mRNA, in the nucleus. Under stressed conditions, an mRNA export receptor Mex67p relocates to the nucleolus from the nuclear periphery and this relocation is closely correlated with inhibition of mRNA export by the stresses. Pretreatment of cells with a mild saline stress induced thermotolerance in mRNA export in a similar manner seen with mild heat pretreatment and protected mRNA export machinery from the subsequent severe heat shock. In contrast, mild ethanol stress could not induce the thermotolerance in mRNA export, suggesting that the stress response induced by ethanol differs from that induced by saline and heat shock stresses. In addition, we found that the Spc1p MAPK pathway is involved in induction of thermotolerance in mRNA export. Of the downstream targets for Spc1p, the Atf1p transcription factor was essential for induction of thermotolerance in mRNA export. We also found that Hsp16p, the expression of which is controlled by Atf1p, is involved in acquisition of thermotolerance in mRNA export in S. pombe.

To elucidate the mechanism of mRNA export from the nucleus, we isolated five novel temperature-sensitive mutants (ptr7 to ptr11) that accumulate poly(A)(+) RNA in the nuclei at the nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr11 mutation was found in the top2(+) gene encoding DNA topoisomerase II. In addition to the nuclear accumulation of poly(A)(+) RNA, ptr11 exhibited the cut (cell untimely torn) phenotype at the nonpermissive temperature, like the previously isolated mutant, ptr4. In these two mutants, cytokinesis occurred without prior nuclear division, resulting in cleavage of the undivided nuclei by the septum. To investigate the relationship between mRNA export defects and the cut phenotype observed in ptr4 and ptr11, we analyzed 11 other mutants displaying the cut phenotype and found that all these tested mutants accumulate poly(A)(+) mRNA in the aberrantly cleaved nuclei. Interestingly, nuclear accumulation of poly(A)(+) mRNA was observed only in the anucleolate nuclei produced by aberrant cytokinesis. In addition, nuc1, the S. pombe mutant exhibiting a collapsed nucleolus, trapped poly(A)(+) mRNA in the nucleolar region at the nonpermissive temperature. In ptr11 and nuc1, mRNA transcribed from the intron-containing TBP gene showed nuclear accumulation, but not transcripts from the intron-less TBP cDNA, suggesting that the export pathway differs between the spliced and unspliced TBP mRNAs. These findings support the notion that a subset of mRNAs in yeast is exported from the nucleus through transient association with the nucleolus.

Fission yeast ptr1-1 is one of the mRNA transport mutants that accumulate poly(A)+ RNA in the nuclei at the nonpermissive temperature. We found that the ptr1+ gene encodes a homolog of Saccharomyces cerevisiae Tom1p, a hect type ubiquitin ligase. In ptr1-1, a conserved amino acid in the hect domain of Ptr1p is mutated. The ptr1+ gene is essential for growth and its mutation did not affect nuclear protein export. A ptr1-1 rae1-167 double mutant showed a synthetic effect on a growth defect, indicating that Ptr1p functionally interacts with an essential mRNA export factor Rae1p. We also isolated a multi-copy suppressor for ptr1-1 and found that it is the mpd2+ gene isolated as a multi-copy suppressor of cdc7-PD1.

Fission yeast ptr4-1 is one of the mRNA transport mutants that accumulate poly(A)(+) RNA in the nuclei at the nonpermissive temperature. We cloned the ptr4(+) gene and found that it is identical with the cut1(+) gene essential for chromosome segregation during mitosis. ptr4/cut1 has no defects in nucleocytoplasmic transport of a protein, indicative of a specific blockage of mRNA export by this mutation. A mutant of Cut2p cooperating with Cut1p in sister chromatid separation also showed defective mRNA export at the nonpermissive temperature. Our results suggest a novel linkage between the cell division cycle and nuclear mRNA export in eukaryotic cells.