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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.

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.

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.

Exons are ligated in an ordered manner without the skipping of exons in the constitutive splicing of pre-mRNAs with multiple introns. To identify factors ensuring ordered exon joining in constitutive pre-mRNA splicing, we previously screened for exon skipping mutants in Schizosaccharomyces pombe using a reporter plasmid, and characterized three exon skipping mutants named ods1 (ordered splicing 1), ods2, and ods3, the responsible genes of which encode Prp2/U2AF59, U2AF23, and SF1, respectively. They form an SF1-U2AF59-U2AF23 complex involved in recognition of the branch and 3' splice sites in pre-mRNA. In the present study, we identified a fourth ods mutant, ods4, which was isolated in an exon-skipping screen. The ods4+ gene encodes Cwf16p, which interacts with the NineTeen Complex (NTC), a complex thought to be involved in the first catalytic step of the splicing reaction. We isolated two multi-copy suppressors for the ods4-1 mutation, Srp2p, an SR protein essential for pre-mRNA splicing, and Tif213p, a translation initiation factor, in S. pombe. The overexpression of Srp2p suppressed the exon-skipping phenotype of all ods mutants, whereas Tif213p suppressed only ods4-1, which has a mutation in the translational start codon of the cwf16 gene. We also showed that the decrease in the transcriptional elongation rate induced by drug treatment suppressed exon skipping in ods4-1. We propose that Cwf16p/NTC participates in the early recognition of the branch and 3' splice sites and cooperates with the SF1-U2AF59-U2AF23 complex to maintain ordered exon joining.

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.

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.

DNA is frequently damaged by genotoxic stresses such as ionizing radiation, reactive oxygen species, and nitrogen species. DNA damage is a key contributor to cancer initiation and progression, and thus the precise and timely repair of these harmful lesions is required. Recent studies revealed transcription as a source of genome instability, and transcription-coupled DNA damage has been a focus in cancer research. Impaired mRNA export is closely related to DNA damage through R-loop formation. The molecular machineries of transcription-coupled DNA damage have been extensively analyzed in Saccharomyces cerevisiae. However, the molecular basis of these phenomena in higher eukaryotes remains elusive. In this review, we focus on the relationship between deregulated mRNA export through the transcription-export-2 (TREX-2) complex and cancer development. Particularly, the expression of germinal center-associated nuclear protein (GANP), a molecular scaffold in the TREX-2 complex, is highly associated with tumorigenesis in mice and humans. Although the deregulated expression of other components in the TREX-2 complex might affect cancer development, we have directly demonstrated the significance of GANP in tumorigenesis using genetically modified mice. Additionally, we describe recent evidence for medical applications demonstrating that the downregulation of the other components may be a good candidate for a chemotherapeutic target in terms of reducing the side effects.

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 5′ splice site (5′SS) 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.

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.

We provide evidence that Prp4p kinase activity is required for pre‐mRNA splicing in vivo and show that loss of activity impairs G 1 –S and G 2 –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.