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Myeloid malignancies with DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5ʹ splice site (5ʹSS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5ʹSS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.

The discovery and useful application of natural products can help improve human life. Chemicals that inhibit plant growth are broadly utilized as herbicides to control weeds. As various types of herbicides are required, the identification of compounds with novel modes of action is desirable. In the present study, we discovered a novel N-alkoxypyrrole compound, kumamonamide from Streptomyces werraensis MK493-CF1 and established a total synthesis procedure. Resulted in the bioactivity assays, we found that kumamonamic acid, a synthetic intermediate of kumamonamide, is a potential plant growth inhibitor. Further, we developed various derivatives of kumamonamic acid, including a kumamonamic acid nonyloxy derivative (KAND), which displayed high herbicidal activity without adverse effects on HeLa cell growth. We also detected that kumamonamic acid derivatives disturb plant microtubules; and additionally, that KAND affected actin filaments and induced cell death. These multifaceted effects differ from those of known microtubule inhibitors, suggesting a novel mode of action of kumamonamic acid, which represents an important lead for the development of new herbicides.

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 C2H2 Zn finger (ZnF). These NBTSs act synergistically to localize RBM10 to S1-1 NBs. The C2H2 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 C2H2 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.Note that the previous title of this manuscript was Targeting of RBM10 to S1-1 Nuclear Bodies: Targeting Sequences and its Biological Significance.

Chromosome segregation is strictly regulated for the proper distribution of genetic material to daughter cells. During this process, mitotic chromosomes are pulled to both poles by bundles of microtubules attached to kinetochores that are assembled on the chromosomes. Centromeres are specific regions where kinetochores assemble. Although these regions were previously considered to be silent, some experimental studies have demonstrated that transcription occurs in these regions to generate non-coding RNAs (ncRNAs). These centromeric ncRNAs (cenRNAs) are involved in centromere functions. Here, we describe the currently available information on the functions of cenRNAs in several species.

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 NESRnc1 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.

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.

Noncoding (nc) RNA called satellite I is transcribed from the human centromere region. Depletion of this ncRNA results in abnormal nuclear morphology because of defects in chromosome segregation. Some protein factors interact with this ncRNA and function as a component of a nc ribonucleoprotein (RNP) complex in mitotic regulation. Here, we found that DHX38, a pre-mRNA splicing-related DEAH box RNA helicase, interacts with satellite I ncRNA. Depletion of DHX38 resulted in defective chromosome segregation similar to knockdown of satellite I ncRNA. Interaction between DHX38 and ncRNA was interphase-specific, but DHX38 depletion affected the function of Aurora B, which associated with satellite I ncRNA at mitotic phase. Based on these findings, we suggest that DHX38 has a role in mitotic regulation as a component of the satellite I ncRNP complex at interphase.

Satellite I RNA, a noncoding (nc)RNA transcribed from repetitive regions in human centromeres, binds to Aurora kinase B and forms a ncRNP complex required for chromosome segregation. To examine its function in this process, we purified satellite I ncRNP complex from nuclear extracts prepared from asynchronized or mitotic (M) phase-arrested HeLa cells and then carried out LC/MS to identify proteins bound to satellite I RNA. RBMX (RNA-binding motif protein, X-linked), which was isolated from M phase-arrested cells, was selected for further characterization. We found that RBMX associates with satellite I RNA only during M phase. Knockdown of RBMX induced premature separation of sister chromatid cohesion and abnormal nuclear division. Likewise, knockdown of satellite I RNA also caused premature separation of sister chromatids during M phase. The amounts of RBMX and Sororin, a cohesion regulator, were reduced in satellite I RNA-depleted cells. These results suggest that satellite I RNA plays a role in stabilizing RBMX and Sororin in the ncRNP complex to maintain proper sister chromatid cohesion.

MSA1 mRNA encodes Msa1p, a protein associated with the SCB-binding factor (SBF) and MCB-binding factor (MBF) complex. Msa1p promotes the transcription of G1 phase-specific genes, and is subjected to cell cycle-dependent regulation for its abundance and subcellular localization. MSA1 mRNA and Msa1p levels oscillate in the cell cycle with peaks at the late M/early G1 phase and early G1 phase, respectively. Phosphorylation by CDK1 negatively regulates the nuclear localization of Msa1p. In the present study, we identified MSA1 mRNA as a bud tip-localized mRNA in screening using a Tag-GFP system. A fragmentation analysis revealed a sequence of ~145 bases for the bud tip localization. Endogenous MSA1 mRNA localized at the bud tip in a manner that depended on SHE2. Msa1p levels were also affected by SHE2 in cells constitutively expressing MSA1 mRNA. These results suggest the existence of a regulatory mechanism for Msa1p through the localized control of MSA1 mRNA.

In fission yeast, the formation of centromeric heterochromatin is induced through the RNA interference (RNAi)-mediated pathway. Some pre-mRNA splicing mutants (prp) exhibit defective formation of centromeric heterochromatin, suggesting that splicing factors play roles in the formation of heterochromatin, or alternatively that the defect is caused by impaired splicing of pre-mRNAs encoding RNAi factors. Herein, we demonstrate that the splicing factor spPrp16p is enriched at the centromere, and associates with Cid12p (a factor in the RNAi pathway) and the intron-containing dg ncRNA. Interestingly, removal of the dg intron, mutations of its splice sites, or replacement of the dg intron with an euchromatic intron significantly decreased H3K9 dimethylation. We also revealed that splicing of dg ncRNA is repressed in cells and its repression depends on the distance from the transcription start site to the intron. Inefficient splicing was also observed in other intron-containing centromeric ncRNAs, dh and antisense dg, and splicing of antisense dg ncRNA was repressed in the presence of the RNAi factors. Our results suggest that the introns retained in centromeric ncRNAs work as facilitators, co-operating with splicing factors assembled on the intron and serving as a platform for the recruitment of RNAi factors, in the formation of centromeric heterochromatin., Author summary Formation of centromeric heterochromatin is required for correct segregation of sister chromatids during mitosis. In fission yeast, formation of heterochromatin at centromeres is performed through the RNA interference (RNAi) system, which involves processing of noncoding RNAs transcribed from the centromeres. We found that the intron in the centromeric dg ncRNAs facilitates formation of centromeric heterochromatin in fission yeast. We showed that the splicing factor spPrp16p associates with the RNAi factor and intron-containing dg ncRNA. Removal of or mutations in the dg intron significantly decreased H3K9 dimethylation, suggesting that the intron and associated splicing factors serve as a platform for recruitment of RNAi factors. Inefficient splicing is a hallmark of intron-containing centromeric ncRNAs. Such repression of splicing seems to be important for facilitation of heterochromatin formation. Introns in euchromatic regions are removed by splicing to generate functional RNAs, whereas centromeric introns are retained in ncRNAs by splicing repression and play roles in gene silencing. Our findings shed light on the novel roles of introns in epigenetic regulation of gene expression and heterochromatin formation.

RNA-binding proteins (RBPs) play important roles in the posttranscriptional regulation of gene expression, including mRNA stability, transport and translation. Fission yeast rnc1

In response to environmental stress, cytoplasmic mRNAs aggregate to form stress granules (SGs). SGs have mainly been studied indirectly using protein markers, but the real-time behavior of endogenous mRNAs in SGs remains uncertain. Here, we visualized endogenous cytoplasmic poly(A)+ mRNAs in living mammalian cells using a linear antisense 2′-O-methyl RNA probe. In arsenite-stressed cells, endogenous mRNAs aggregated in granules that colocalized with SGs marked by TIA-1–GFP. Moreover, analysis of mRNA dynamics using fluorescence recovery after photobleaching showed that approximately one-third of the endogenous mRNAs in SGs was immobile, another one-third was diffusive, and the remaining one-third was in equilibrium between binding to and dissociating from SGs, with a time constant of approximately 300 seconds. These dynamic characteristics of mRNAs were independent of the duration of stress and microtubule integrity. Similar characteristics were also observed from fos mRNA labeled with an antisense 2′-O-methyl RNA probe. Our results revealed the behavior of endogenous mRNAs, and indicated that SGs act as dynamic harbors of untranslated poly(A)+ mRNAs.

RNA localization is a common mechanism for recruiting proteins to specific regions of a cell, which causes cell polarization and sometimes asymmetric division. We found that EGD1 mRNA accumulates dose-dependently as a cytoplasmic granule in Saccharomyces cerevisiae. EGD1 encodes a β-subunit of the nascent polypeptide-associated complex (NAC). NAC is a heterodimer consisting of α- and β-subunits, associated with ribosomes and thought to be involved in the folding of nascent polypeptide chains. Analysis of deletion constructs showed that the localization of EGD1 mRNA requires both an upstream region and an ORF of EGD1, suggesting that the translation of Egd1p is important for localization. We also showed that Egd1p and P-body components are co-localized with EGD1 mRNA. This granule, named the EGD1 granule, has features similar to cellular inclusions containing aggregated proteins. Disruption of microtubules by treatment with a drug, benomyl, resulted in loss of the EGD1 granule. When the expression level of EGD2 encoding the αNAC increased, the percentage of cells showing the EGD1 granule decreased, suggesting that the granular distribution of EGD1 depends on the quantitative balance between α- and β-subunits of NAC. Taken together, we propose a novel microtubule-dependent mechanism for controlling NAC through RNA localization.

prp13-1 is one of the mutants isolated in a screen for defective pre-mRNA splicing at a nonpermissive temperature in fission yeast Schizosaccharomyces pombe. We cloned the prp13(+) gene and found that it encodes U4 small nuclear RNA (snRNA) involved in the assembly of the spliceosome. The prp13-1 mutant produced elongated cells, a phenotype similar to cell division cycle mutants, and displays a high incidence of lagging chromosomes on anaphase spindles. The mutant is hypersensitive to the microtubule-destabilizing drug thiabendazole, supporting that prp13-1 has a defect in chromosomal segregation. We found that the prp13-1 mutation resulted in expression of the ura4(+) gene inserted in the pericentromeric heterochromatin region and reduced recruitment of the heterochromatin protein Swi6p to that region, indicating defects in the formation of pericentromeric heterochromatin, which is essential for the segregation of chromosomes, in prp13-1. The formation of centromeric heterochromatin is induced by the RNA interference (RNAi) system in S. pombe. In prp13-1, the processing of centromeric noncoding RNAs to siRNAs, which direct the heterochromatin formation, was impaired and unprocessed noncoding RNAs were accumulated. These results suggest that U4 snRNA is required for the RNAi-directed heterochromatic gene silencing at the centromeres. In relation to the linkage between the spliceosomal U4 snRNA and the RNAi-directed formation of heterochromatin, we identified a mRNA-type intron in the centromeric noncoding RNAs. We propose a model in which the assembly of the spliceosome or a sub-spliceosome complex on the intron-containing centromeric noncoding RNAs facilitates the RNAi-directed formation of heterochromatin at centromeres, through interaction with the RNA-directed RNA polymerase complex.

Recruitment of RAD18 to stalled replication forks facilitates monoubiquitination of PCNA during S-phase, promoting translesion synthesis at sites of UV irradiation-induced DNA damage. In this study, we show that RAD18 is also recruited to ionizing radiation (IR)-induced sites of DNA double-strand breaks (DSBs) forming foci which are co-localized with 53BP1, NBS1, phosphorylated ATM, BRCA1 and gamma-H2AX. RAD18 associates with 53BP1 and is recruited to DSB sites in a 53BP1-dependent manner specifically during G1-phase, RAD18 monoubiquitinates KBD domain of 53BP1 at lysine 1268 in vitro. A monoubiquitination-resistant 53BP1 mutant harboring a substitution at lysine 1268 is not retained efficiently at the chromatin in the vicinity of DSBs. In Rad18-null cells, retention of 53BP1 foci, efficiency of DSB repair and post-irradiation viability are impaired compared with wild-type cells. Taken together, these results suggest that RAD18 promotes 53BP1-directed DSB repair by enhancing retention of 53BP1, possibly through an interaction between RAD18 and 53BP1 and the modification of 53BP1.

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.

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.

In eukaryotic cells, export of mRNA from the nucleus to the cytoplasm is one of the essential steps in gene expression. To examine mechanisms involved in the nucleocytoplasmic transport of mRNA, we microinjected fluorescently labeled fushi tarazu (ftz) pre-mRNA into the nuclei of HeLa cells. The injected intron-containing ftz pre-mRNA was distributed to the SC35 speckles and exported to the cytoplasm after splicing by an energy-requiring active process. In contrast, the injected intron-less ftz mRNA was diffusely distributed in the nucleus and then presumably degraded. Interestingly, export of the ftz pre-mRNA was inhibited by treatment with transcriptional inhibitors (actinomycin D, alpha-amanitin or DRB). Cells treated with transcriptional inhibitor showed foci enriched with the injected mRNA, which localize side by side with SC35 speckles. Those nuclear foci, referred to as TIDRs (transcriptional-inactivation dependent RNA domain), do not overlap with paraspeckles. In addition, in situ hybridization analysis revealed that the export of endogenous poly(A)+ mRNA is also affected by transcriptional inactivation. These results suggest that nuclear mRNA export is coupled to ongoing gene transcription in mammalian cells.

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.

We provide evidence that Prp4p kinase activity is required for pre-mRNA splicing in vivo and show that loss of activity impairs G1-S and G2-M progression in the cell cycle. Prp4p interacts genetically with the non-SR (serine/arginine) splicing factors Prp1p and Prp5p. Bacterially produced Prp1p is phosphorylated by Prp4p in vitro. Prp4p and Prp1p also interact in the yeast two-hybrid system. In vivo labelling studies using a strain with a mutant allele of the prp4 gene in the genetic background indicate a change in phosphorylation of the Prp1p protein. These results are consistent with the notion that Prp4p kinase is involved in the control of the formation of active spliceosomes, targeting non-SR splicing factors.

The prp2 gene of fission yeast has previously been shown to encode the large subunit of the splicing factor spU2AF. SpU2AF(59) is an evolutionarily conserved protein that has an arginine/serine-rich region and three RNA recognition motifs (RRMs). We have sequenced three temperature-sensitive alleles of prp2 and determined that the mutations result in single amino acid changes within one of the RRMs or between RRMs. All mutant alleles of prp2 have pre-mRNA splicing defects at the non-permissive temperature. Although the mutant strains are growth-arrested at 37 degrees C, they do not elongate like typical fission yeast cell cycle mutants. The DNA of the prp2(-) strains stains more intensely than a wild-type strain, suggesting that the chromatin may be condensed. Ultrastructural studies show differences in the mutant nuclei including a prominent distinction between the chromatin- and non-chromatin-enriched regions compared to the more homogenous wild-type nucleus. Two-hybrid assays indicate that some of the wild-type protein interactions are altered in the mutant strains. These results suggest that normal functioning of spU2AF(59) may be essential not only for pre-mRNA splicing but also for the maintenance of proper nuclear structure and normal cell cycle progression.

We report here that a defect of the interleukin common gamma subunit (gamma c) in X-linked severe combined immunodeficiency (XSCID) previously known as a missense mutation resulted instead in exon skipping in a Japanese XSCID patient. The phenotype of the patient was consistent with that of typical XSCID, and his Epstein-Barr virus-transformed B cells accordingly entirely lacked surface expression of gamma c . On analysis by the reverse transcription-polymerase chain reaction (RT-PCR), a single but small gamma c mRNA species was detected. Exon 6, which encodes the transmembrane domain of gamma c, was skipped in the mRNA. A G to A mutation was found at the last nucleotide of exon 6 of the gamma c gene (868G--A). The predicted consequence of the exon skipping is a frameshift resulting in a premature stop codon, and the mutated gamma c presumably loses association with the cell membrane. In XSCID, this mutation (868G--A) is known as a missense mutation that results in R285Q [corrected]. Previously reported patients with the same mutation apparently had no aberrant or alternative splicing but did have the R285Q [corrected] exchange. Similar mutations at the last nucleotide of an outskipped exon have been reported. However, such mutations do not always cause exon skipping. Analyses of RNA structural changes induced by the mutations supported the variability of consequences of the mutations. Taken together, our findings suggest that the 868G--A mutation of the gamma c gene may affect gamma c transcripts differently, i.e., generating missense or exon skipping, in XSCID patients with the same mutation. Patient-specific variation in splicing thus appears to occur.

Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A)(+) RNAs comprising mRNAs and poly (A)(+) non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culture extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A)(+) RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.

Human centromeres consist of repetitive sequences from which satellite I noncoding RNAs are transcribed. We found that knockdown of satellite I RNA causes abnormal chromosome segregation and generation of nuclei with a grape-shape phenotype. Co-immunoprecipitation experiments showed that satellite I RNA associates with Aurora B, a component of the chromosome passenger complex (CPC) regulating proper attachment of microtubules to kinetochores, in mitotic HeLa cells. Satellite I RNA was also shown to associate with INCENP, another component of the CPC. In addition, depletion of satellite I RNA resulted in up-regulation of kinase activity of Aurora B and delocalization of the CPC from the centromere region. These results suggest that satellite I RNA is involved in chromosome segregation through controlling activity and centromeric localization of Aurora B kinase.

The prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature. One of the prp mutants, prp1-4, also has a defect in poly(A)+ RNA transport. The prp1 + gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions. The amino acid sequence of Prplp shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly. No functional complementation was observed between S. pombe prp1 + and S. cerevisiae PRP6. We examined synthetic lethality of prp1-4 with the other known prp mutations in S. pombe. The results suggest that Prp1p interacts either physically or functionally with Prp4p, Prp6p and Prp13p. Interestingly, the prp1 + gene was found to be identical with the zer1 + gene that functions in cell cycle control. These results suggest that Prp1p/Zer1p is either directly or indirectly involved in cell cycle progression and/or poly(A)+ RNA nuclear export, in addition to pre-mRNA splicing.

Nucleocytoplasmic transport of mRNA is essential for eukaryotic gene expression. However, how mRNA is exported from the nucleus is mostly unknown. To elucidate the mechanisms of mRNA transport, we took a genetic approach to identify genes, the products of which play a role in that process. From about 1000 temperature -sensitive (ts- or cs-) mutants, we identified five ts- mutants that are defective in poly(A)+ RNA transport by using a situ hybridization with an oligo(dT)50 as a probe. These mutants accumulate poly(A)+ RNA in the nuclei when shifted to a nonpermissive temperature. All five mutations are tightly linked to the ts- growth defects, are recessive, and fall into four different groups designated as ptr 1-4 (poly(A)+ RNA transport). Interestingly, each group of mutants has a differential localization pattern of poly(A)+ RNA in the nuclei at the nonpermissive temperature, suggesting that they have defects at different steps of the mRNA transport pathway. Localization of a nucleoplasmin-green fluorescent protein fusion suggests that ptr2 and ptr3 have defects also in nuclear protein import. Among the isolated mutants, only ptr2 showed a defect in pre-mRNA splicing. We cloned the ptr2+ and ptr3+ genes and found that they encode Schizosaccharomyces pombe homologues of the mammalian RCC1, a guanine nucleotide exchange factor for RAN/TC4, and the ubiquitin-activating enzyme E1 involved in ubiquitin conjugation, respectively. The ptr3+ gene is essential for cell viability, and Ptr3p tagged with green fluorescent protein was localized in both the nucleus and the cytoplasm. This is the first report suggesting that the ubiquitin system plays a role in mRNA export.

Zinc-finger domains are found in many nucleic acid-binding proteins in both prokaryotes and eukaryotes. Proteins carrying zinc-finger domains have important roles in various nuclear transactions, including transcription, mRNA processing and mRNA export; however, for many individual zinc-finger proteins in eukaryotes, the exact function of the protein is not fully understood. Here, we report that Red5 is involved in efficient suppression of specific mRNAs during vegetative growth of Schizosaccharomyces pombe. Red5, which contains five C3H1-type zinc-finger domains, localizes to the nucleus where it forms discrete dots. A red5 point mutation, red5-2, results in the upregulation of specific meiotic mRNAs in vegetative mutant red5-2 cells; northern blot data indicated that these meiotic mRNAs in red5-2 cells have elongated poly(A) tails. RNA-fluorescence in situ hybridization results demonstrate that poly(A)(+) RNA species accumulate in the nucleolar regions of red5-deficient cells. Moreover, Red5 genetically interacts with several mRNA export factors. Unexpectedly, three components of the nuclear pore complex also suppress a specific set of meiotic mRNAs. These results indicate that Red5 function is important to meiotic mRNA degradation; they also suggest possible connections among selective mRNA decay, mRNA export and the nuclear pore complex in vegetative fission yeast.

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.

U1, U2, U4, U5, and U6 small nuclear RNAs are essential for precursor mRNA splicing. We previously found one and four mRNA-type, or spliceosomal introns, in the U2 and U6 genes, respectively, of yeast Rhodotorula hasegawae (Erythrobasidium hasegawianum). We proposed that U2 and U6 RNAs form a catalytic core for precursor mRNA splicing and that the introns in those small nuclear RNA genes may have been acquired through reverse splicing of an intron from a precursor mRNA into a catalytic site in small nuclear RNAs. In the present study, we analyzed U1, U4, and U5 genes in R. hasegawae. One spliceosomal intron was found in the U1 region forming base-pairs with a 5' splice site of a precursor mRNA. The U5 gene has two spliceosomal introns in the region that interacts with 5' and 3' splice sites. In contrast, the gene for U4 RNA, which is released from the spliceosome prior to the first step of the splicing reaction, has no intron. These results lend a further support to the proposed relation between presence and position of an intron in an small nuclear RNA gene and the function of the encoded small nuclear RNA.

The fission-yeast gene cdc28+ was originally identified in a screen for temperature-sensitive mutants that exhibit a cell-division cycle arrest and was found to be required for mitosis. We undertook a study of this gene to understand more fully the general requirements for entry into mitosis. Cells carrying the conditional lethal cdc28-P8 mutation divide once and arrest in G2 after being shifted to the restrictive temperature. We cloned the cdc28+ gene by complementation of the temperature-sensitive growth arrest in cdc28-P8. DNA sequence analysis indicated that cdc28+ encodes a member of the DEAH-box family of putative RNA-dependent ATPases or helicases. The Cdc28 protein is most similar to the Prp2, Prp16, and Prp22 proteins from budding yeast, which are required for the splicing of mRNA precursors. Consistent with this similarity, the cdc28-P8 mutant accumulates unspliced precursors at the restrictive temperature. Independently, we isolated a temperature-sensitive pre-mRNA splicing mutant prp8-1 that exhibits a cell-cycle phenotype identical to that of cdc28-P8. We have shown that cdc28 and prp8 are allelic. These results suggest a connection between pre-mRNA splicing and progression through the cell cycle.

In eukaryotic cells, there are two major compartments separated by the nuclear membrane: the nucleus, where transcription and replication of DNA occur, and the cytoplasm, where translation of mRNAs to proteins occurs. Interestingly, the interphase nucleus is further divided into a dozen subnuclear compartments, such as nucleoli, speckles, Cajal bodies, paraspeckles, promyelocytic leukemia (PML) bodies, and gems (for gemini of Cajal bodies) (Fig. 1) [1].

We have previously identified the RNA recognition motif (RRM)-type RNA-binding protein Nrd1 as an important regulator of the posttranscriptional expression of myosin in fission yeast. Pmk1 MAPK-dependent phosphorylation negatively regulates the RNA-binding activity of Nrd1. Here, we report the role of Nrd1 in stress-induced RNA granules. Nrd1 can localize to poly(A)-binding protein (Pabp)-positive RNA granules in response to various stress stimuli, including heat shock, arsenite treatment, and oxidative stress. Interestingly, compared with the unphosphorylatable Nrd1, Nrd1(DD) (phosphorylation-mimic version of Nrd1) translocates more quickly from the cytoplasm to the stress granules in response to various stimuli; this suggests that the phosphorylation of Nrd1 by MAPK enhances its localization to stress-induced cytoplasmic granules. Nrd1 binds to Cpc2 (fission yeast RACK) in a phosphorylation-dependent manner and deletion of Cpc2 affects the formation of Nrd1-positive granules upon arsenite treatment. Moreover, the depletion of Nrd1 leads to a delay in Pabp-positive RNA granule formation, and overexpression of Nrd1 results in an increased size and number of Pabp-positive granules. Interestingly, Nrd1 deletion induced resistance to sustained stresses and enhanced sensitivity to transient stresses. In conclusion, our results indicate that Nrd1 plays a role in stress-induced granule formation, which affects stress resistance in fission yeast.

Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

The mitotic checkpoint is essential for maintaining genomic stability in differentiating B cells undergoing genetic alterations of the Ig gene. In this study, using real-time RT-PCR and in situ RNA hybridization, we demonstrated that MAD2 mRNA export is selectively regulated by Pcid2/Thp1. Pcid2 small interfering RNA induced a cell-cycle abnormality with increased apoptosis and polyploidy, as previously observed in MAD2-knockdown cells. Pcid2 small interfering RNA reduced MAD2 expression, but not the expression of other cell-cycle checkpoint proteins, such as MAD1 and BUBR1, or the cell-cycle–associated proteins, cyclin A, cyclin B1, and cyclin-dependent kinase 1. In mouse B lineage cells, Pcid2 transcripts appeared in a stage-dependent manner at high levels in bone marrow pre-B and immature B cells, and in spleen transitional 1 and follicular B cells, but at lower levels in pro-B, transitional 2, and marginal zone B cells, suggesting a stage-dependent requirement for MAD2 regulation. Cd19-cre–derived targeting of the Pcid2 gene induced a mature B cell deficiency in mice. These findings indicate that Pcid2 is essential for B cell survival through the regulation of MAD2 expression during B cell differentiation.

U6 small nuclear RNA is one of the spliceosomal RNAs essential for pre-mRNA splicing. Discovery of mRNA-type introns in the highly conserved region of the U6 snRNA genes led to the hypothesis that U6 snRNA functions as a catalytic element during pre-mRNA splicing. The highly conserved region of U6 snRNA has a structural similarity with the catalytic domain of the negative strand of the satellite RNA of tobacco ring spot virus [(-)sTRSV], suggesting that the highly conserved region of U6 snRNA forms the catalytic center. We examined whether synthetic RNAs consisting of the sequence of the highly conserved region of U6 snRNA or various chimeric RNAs between the U6 region and the catalytic RNA of (-)sTRSV could cleave a substrate RNA that can partially base-pair with them and have a GU sequence. Chimeric RNAs with 70 to 83% sequence identity with the conserved region of S. pombe U6 snRNA cleaved the substrate RNA at the 5' side of the GU sequence, which is shared by the 5' end of an intron in a pre-mRNA. We found that the highly conserved region of U6 snRNA and the catalytic domain of (-)sTRSV are strikingly similar in structure to the catalytic core region of the group I self-splicing intron in cyanobacteria. These results suggest that U6 snRNA, (-)sTRSV and the group I self-splicing intron originated from a common ancestral RNA, and support the hypothesis that U6 snRNA catalyzes pre-mRNA splicing reaction.

The homologue of the rat pancreatic elastase I gene was found in the human genome, but its transcription was completely suppressed in the adult human pancreas as we reported previously. In this study, we characterized the complete structure of the eight putative exons of the silent gene for human elastase I. A genotype analysis of the exon 1 DNA sequence revealed that at least two allelic elastase I genes are present in human genomes. A primate-specific repetitive DNA element (MER1) was identified in the 3'-flanking region of the human elastase I gene. The primary structure of human preproelastase I, deduced from the sequences of the eight exons, showed an 89% identity with that of porcine or rat pancreatic preproelastase I. The amino acid residues of the serine protease catalytic triad and the eight cysteine residues conserved in the elastase family were present at positions equivalent to those observed in porcine and rat elastase I, suggesting that the gene product may function as an elastolytic enzyme if this gene is expressed in any tissue.

U6 small nuclear RNA is one of the spliceosomal RNAs involved in pre-mRNA splicing. In the fission yeast Schizosaccharomyces pombe, the U6 RNA gene was found to have an intron similar to a nuclear pre-mRNA intron, and it was proposed that the U6 intron might be inserted erroneously during pre-mRNA splicing. Using the polymerase chain reaction, we analyzed the U6 RNA genes of 52 organisms. In addition to the five species of Schizosaccharomyces, we found that the yeast species Rhodotorula hasegawae and Rhodosporidium dacryoidum also have mRNA-type introns in their U6 genes; however, in all the other organisms tested, we found no intron within the region of the U6 gene examined. Four introns and one intron are present in the R. hasegawae and R. dacryoidum U6 genes, respectively; and these introns are located at sites differing from the location of the Schizosaccharomyces U6 intron. Most of the U6 introns locate within the conserved domain, which is strikingly similar in structure to the catalytic center of the negative strand of the satellite RNA of tobacco ring spot virus. The introns of the S. pombe and R. dacryoidum U6 genes are located immediately adjacent to the nucleotides that were shown to be essential for the second step of the splicing reaction. These results support the notion that U6 RNA has a catalytic role in pre-mRNA splicing and that U6 introns originated from insertion of an excised intron during pre-mRNA splicing.

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. Curr. Protoc. Cell Biol. 41:22.13.1-22.13.20. © 2008 by John Wiley & Sons, Inc. Keywords: mRNA; transport; in situ hybridization; microinjection; yeast; mammalian cells

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 μm 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.

Speckles are subnuclear domains where pre-mRNA splicing factors accumulate in the interchromatin space. To investigate the dynamics of mRNAs at speckles, fluorescently labeled Drosophila Fushitarazu (ftz) pre-mRNAs were microinjected into the nuclei of Cos7 cells and the dissociation kinetics of pre-mRNAs from speckles was analyzed using photobleaching techniques. The microinjected ftz pre-mRNAs accumulated in speckles in an intron-dependent manner and were spliced and exported to the cytoplasm with a half-time of about 10 min. Dissociation of the accumulated pre-mRNAs in speckles exhibited rapid diffusion and slow-dissociation of about 100 s. The slow-dissociation required metabolic energy of ATP. Two types of splice-defective mutated mRNAs dissociated from the speckle with a time constant similar to that of wild-type mRNA, indicating that slow-dissociation was not coupled to the splicing reaction. Furthermore, some pre-mRNAs shuttled between speckles and nucleoplasm, suggesting that pre-mRNAs repeatedly associated with and dissociated from speckles until introns were removed. Next, endogenous poly(A)+ RNA was visualized by injecting Cy3-labeled 2'O-methyl oligo(U)22 probes. Some poly(A)+ RNA distributed diffusely within the nucleus, but some of them accumulated in speckles and dissociated at time constant of about 100 s.

The removal of intervening sequences from transcripts is catalyzed by the spliceosome, a multicomponent complex that assembles on the newly synthesized pre-mRNA. Pre-mRNA translation in the cytoplasm leads to the generation of aberrant proteins that are potentially harmful. Therefore, tight control to prevent undesired pre-mRNA export from the nucleus and its subsequent translation is an essential requirement for reliable gene expression. Here, we show that the natural product FR901464 (1) and its methylated derivative, spliceostatin A (2), inhibit in vitro splicing and promote pre-mRNA accumulation by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. Importantly, treatment of cells with these compounds resulted in leakage of pre-mRNA to the cytoplasm, where it was translated. Knockdown of SF3b by small interfering RNA induced phenotypes similar to those seen with spliceostatin A treatment. Thus, the inhibition of pre-mRNA splicing during early steps involving SF3b allows unspliced mRNA leakage and translation.

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.