DEAH box RNA helicase DHX38 associates with satellite I noncoding RNA involved in chromosome segregation.

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.

The intron in centromeric noncoding RNA facilitates RNAi-mediated formation of heterochromatin.

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.

RBMX is a component of the centromere noncoding RNP complex involved in cohesion regulation.

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.

Involvement of satellite I noncoding RNA in regulation of chromosome segregation.

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.

U7 small nuclear ribonucleoprotein represses histone gene transcription in cell cycle-arrested cells.

Histone gene expression is tightly coordinated with DNA replication, as it is activated at the onset of S phase and suppressed at the end of S phase. Replication-dependent histone gene expression is precisely controlled at both transcriptional and posttranscriptional levels. U7 small nuclear ribonucleoprotein (U7 snRNP) is involved in the 3'-end processing of nonpolyadenylated histone mRNAs, which is required for S phase-specific gene expression. The present study reports a unique function of U7 snRNP in the repression of histone gene transcription under cell cycle-arrested conditions. Elimination of U7 snRNA with an antisense oligonucleotide in HeLa cells as well as in nontransformed human lung fibroblasts resulted in elevated levels of replication-dependent H1, H2A, H2B, H3, and H4 histone mRNAs but not of replication-independent H3F3B histone mRNA. An analogous effect was observed upon depletion of Lsm10, a component of the U7 snRNP-specific Sm ring, with siRNA. Pulse-chase experiments revealed that U7 snRNP acts to repress transcription without remarkably altering mRNA stability. Mass spectrometric analysis of the captured U7 snRNP from HeLa cell extracts identified heterogeneous nuclear (hn)RNP UL1 as a U7 snRNP interaction partner. Further knockdown and overexpression experiments revealed that hnRNP UL1 is responsible for U7 snRNP-dependent transcriptional repression of replication-dependent histone genes. Chromatin immunoprecipitation confirmed that hnRNP UL1 is recruited to the histone gene locus only when U7 snRNP is present. These findings support a unique mechanism of snRNP-mediated transcriptional control that restricts histone synthesis to S phase, thereby preventing the potentially toxic effects of histone synthesis at other times in the cell cycle.

MENepsilon/beta noncoding RNAs are essential for structural integrity of nuclear paraspeckles.

Recent transcriptome analyses have shown that thousands of noncoding RNAs (ncRNAs) are transcribed from mammalian genomes. Although the number of functionally annotated ncRNAs is still limited, they are known to be frequently retained in the nucleus, where they coordinate regulatory networks of gene expression. Some subnuclear organelles or nuclear bodies include RNA species whose identity and structural roles are largely unknown. We identified 2 abundant overlapping ncRNAs, MENepsilon and MENbeta (MENepsilon/beta), which are transcribed from the corresponding site in the multiple endocrine neoplasia (MEN) I locus and which localize to nuclear paraspeckles. This finding raises the intriguing possibility that MENepsilon/beta are involved in paraspeckle organization, because paraspeckles are, reportedly, RNase-sensitive structures. Successful removal of MENepsilon/beta by a refined knockdown method resulted in paraspeckle disintegration. Furthermore, the reassembly of paraspeckles disassembled by transcriptional arrest appeared to be unsuccessful in the absence of MENepsilon/beta. RNA interference and immunoprecipitation further revealed that the paraspeckle proteins p54/nrb and PSF selectively associate with and stabilize the longer MENbeta, thereby contributing to the organization of the paraspeckle structure. The paraspeckle protein PSP1 is not directly involved in either MENepsilon/beta stabilization or paraspeckle organization. We postulate a model for nuclear paraspeckle body organization where specific ncRNAs and RNA-binding proteins cooperate to maintain and, presumably, establish the structure.

DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells.

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 5'SS, 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.

Efficient oligonucleotide-mediated degradation of nuclear noncoding RNAs in mammalian cultured cells.

Recent large-scale transcriptome analyses have revealed that large numbers of noncoding RNAs (ncRNAs) are transcribed from mammalian genomes. They include small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and longer ncRNAs, many of which are localized to the nucleus, but which have remained functionally elusive. Since ncRNAs are only known to exist in mammalian species, established experimental systems, including the Xenopus oocyte system and yeast genetics, are not available for functional analysis. RNA interference (RNAi), commonly used for analysis of protein-coding genes, is effective in eliminating cytoplasmic mRNAs, but not nuclear RNAs. To circumvent this problem, we have refined the system for knockdown of nuclear ncRNAs with chemically modified chimeric antisense oligonucleotides (ASO) that were efficiently introduced into the nucleus by nucleofection. Under optimized conditions, our system appeared to degrade at least 20 different nuclear ncRNA species in multiple mammalian cell lines with high efficiency and specificity. We also confirmed that our method had greatly improved knockdown efficiency compared with that of the previously reported method in which ASOs are introduced with transfection reagents. Furthermore, we have confirmed the expected phenotypic alterations following knockdown of HBII295 snoRNA and U7 snRNA, which resulted in a loss of site-specific methylation of the artificial RNA and the appearance of abnormal polyadenylated histone mRNA species with a concomitant delay of the cell cycle S phase, respectively. In summary, we believe that our system is a powerful tool to explore the biological functions of the large number of nuclear ncRNAs with unknown function.

Discovery, characterization and functional improvement of kumamonamide as a novel plant growth inhibitor that disturbs plant microtubules.

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.

Centromeric Non-Coding RNAs: Conservation and Diversity in Function.

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.

Tissue-specific splicing regulator Fox-1 induces exon skipping by interfering E complex formation on the downstream intron of human F1gamma gene.

Fox-1 is a regulator of tissue-specific splicing, via binding to the element (U)GCAUG in mRNA precursors, in muscles and neuronal cells. Fox-1 can regulate splicing positively or negatively, most likely depending on where it binds relative to the regulated exon. In cases where the (U)GCAUG element lies in an intron upstream of the alternative exon, Fox-1 protein functions as a splicing repressor to induce exon skipping. Here we report the mechanism of exon skipping regulated by Fox-1, using the hF1gamma gene as a model system. We found that Fox-1 induces exon 9 skipping by repressing splicing of the downstream intron 9 via binding to the GCAUG repressor elements located in the upstream intron 8. In vitro splicing analyses showed that Fox-1 prevents formation of the pre-spliceosomal early (E) complex on intron 9. In addition, we located a region of the Fox-1 protein that is required for inducing exon skipping. Taken together, our data show a novel mechanism of how RNA-binding proteins regulate alternative splicing.

Introns play an essential role in splicing-dependent formation of the exon junction complex.

Pre-mRNA splicing specifically deposits the exon junction complex (EJC) onto spliced mRNA, which is important for downstream events. Here, we show that EJC components are primarily recruited to the spliceosome by association with the intron via the intron-binding protein, IBP160. This initial association of EJC components occurs in the absence of the final EJC-binding site on the exon. RNA interference (RNAi) knockdown of IBP160 arrested EJC association with cytoplasmic RNAs following nonsense-mediated decay. We propose that the intron has a crucial role in the early steps of EJC formation and is indispensable for the subsequent formation of a functional EJC.

Identification and characterization of human non-coding RNAs with tissue-specific expression.

We have examined the expression profile of selected non-coding RNAs (ncRNAs) in 11 human tissues. Among 5489 full-length cDNA clones annotated as non-protein-coding transcripts in the H-Invitational database, we chose 150 clones for further analysis based on their gene structure and EST information. Expression profiling using quantitative RT-PCR and Northern blot hybridization revealed that the majority of the selected ncRNAs exhibited tissue specificity: 67% are predominantly expressed in a restricted subset of tissues. The absolute quantification of representative ncRNAs revealed that the majority of ncRNAs are expressed as low abundance transcripts. A comparative genomic analysis revealed that only 27% of the selected ncRNAs have mouse counterparts. Since the expression patterns of the human ncRNAs having no mouse counterparts remain to be similar to those of the mouse ncRNAs, the expression patterns of the selected ncRNAs may be conserved between human and mouse.

A spliceosomal intron binding protein, IBP160, links position-dependent assembly of intron-encoded box C/D snoRNP to pre-mRNA splicing.

Pre-mRNA splicing in vertebrates is molecularly linked to other processes. We previously reported that splicing is required for efficient assembly of intron-encoded box C/D small nucleolar ribonucleoprotein (snoRNP). In the spliceosomal C1 complex, snoRNP proteins efficiently assemble onto snoRNA sequences if they are located about 50 nt upstream of the intron branchpoint. Here, we identify the splicing factor responsible for coupling snoRNP assembly to intron excision. Intron binding protein (IBP) 160, a helicase-like protein previously detected in the spliceosomal C1 complex, binds the pre-mRNA in a sequence-independent manner, contacting nucleotides 33-40 upstream of the intron branch site, regardless of whether a snoRNA is present. Depletion of IBP160 abrogates snoRNP assembly in vitro. IBP160 binding directly to a snoRNA located too close to the intron branch site interferes with snoRNP assembly. Thus, IBP160 is the key factor linking snoRNP biogenesis and perhaps other postsplicing events to pre-mRNA splicing.

A mutation in the gene involved in sister chromatid separation causes a defect in nuclear mRNA export in fission yeast.

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.

The nucleolus is involved in mRNA export from the nucleus in fission yeast.

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.