Terminal regions of UAP56 and URH49 are required for their distinct complex formation functioning to an essential role in mRNA processing and export.

UAP56 and URH49 are closely related RNA helicases that function in selective mRNA processing and export pathways to fine-tune gene expression through distinct complex formations. The complex formation of UAP56 and URH49 is believed to play a crucial role in regulating target mRNAs. However, the mechanisms underlying this complex formation have not been fully elucidated. Here we identified the regions essential for the complex formation of both helicases. The terminal regions of UAP56 and the C-terminal region of URH49 were indispensable for their respective complex formation. Further analysis revealed that a specific amino acid at the C-terminus of UAP56 is critical for its complex formation. Alanine substitution of this amino acid impairs its complex formation and subsequently affected its mRNA processing and export activity. Our study provides a deeper understanding of the basis for the complex formation between UAP56 and URH49.

Structural differences between the closely related RNA helicases, UAP56 and URH49, fashion distinct functional apo-complexes.

mRNA export is an essential pathway for the regulation of gene expression. In humans, closely related RNA helicases, UAP56 and URH49, shape selective mRNA export pathways through the formation of distinct complexes, known as apo-TREX and apo-AREX complexes, and their subsequent remodeling into similar ATP-bound complexes. Therefore, defining the unidentified components of the apo-AREX complex and elucidating the molecular mechanisms underlying the formation of distinct apo-complexes is key to understanding their functional divergence. In this study, we identify additional apo-AREX components physically and functionally associated with URH49. Furthermore, by comparing the structures of UAP56 and URH49 and performing an integrated analysis of their chimeric mutants, we exhibit unique structural features that would contribute to the formation of their respective complexes. This study provides insights into the specific structural and functional diversification of these two helicases that diverged from the common ancestral gene Sub2.

Inhibition of mitochondrial complex III or dihydroorotate dehydrogenase (DHODH) triggers formation of poly(A)+ RNA foci adjacent to nuclear speckles following activation of ATM (ataxia telangiectasia mutated).

Intracellular and intercellular signalling networks play an essential role in optimizing cellular homoeostasis and are thought to be partly reflected in nuclear mRNA dynamics. However, the regulation of nuclear mRNA dynamics by intracellular and intercellular signals remains largely unexplored, and research tools are lacking. Through an original screening based on the mRNA metabolic mechanism, we discovered that eight well-known inhibitors cause significant nuclear poly(A)+ RNA accumulation. Among these inhibitors, we discovered a new mRNA metabolic response in which the addition of antimycin A, an inhibitor of mitochondrial respiratory-chain complex III (complex III), resulted in a marked accumulation of poly(A)+ RNA near the nuclear speckles. Furthermore, dihydroorotate dehydrogenase (DHODH) inhibitors, a rate-limiting enzyme in the intracellular de novo pyrimidine synthesis reaction that specifically exchanges electrons with complex III, also caused a remarkable accumulation of nuclear poly(A)+ RNA adjacent to the nuclear speckles, which was abolished by extracellular uridine supply, indicating that the depletion of intracellular pyrimidine affects poly(A)+ RNA metabolism. Further analysis revealed that ataxia telangiectasia mutated (ATM), a serine and threonine kinase and a master regulator of DNA double-strand break (DSB) and nucleolar stress, is required for this poly(A)+ RNA nuclear accumulation phenomenon. This study reports new insights into novel aspects of nuclear poly(A)+ RNA metabolism, especially the relationship between mitochondrial respiratory-chain functions, pyrimidine metabolism, and nuclear RNA metabolism.

Epigenetic regulation of ecotype-specific expression of the heat-activated transposon ONSEN.

Transposable elements are present in a wide variety of organisms; however, our understanding of the diversity of mechanisms involved in their activation is incomplete. In this study, we analyzed the transcriptional activation of the ONSEN retrotransposon, which is activated by high-temperature stress in Arabidopsis thaliana. We found that its transcription is significantly higher in the Japanese ecotype Kyoto. Considering that transposons are epigenetically regulated, DNA methylation levels were analyzed, revealing that CHH methylation was reduced in Kyoto compared to the standard ecotype, Col-0. A mutation was also detected in the Kyoto CMT2 gene, encoding a CHH methyltransferase, suggesting that it may be responsible for increased expression of ONSEN. CHH methylation is controlled by histone modifications through a self-reinforcing loop between DNA methyltransferase and histone methyltransferase. Analysis of these modifications revealed that the level of H3K9me2, a repressive histone marker for gene expression, was lower in Kyoto than in Col-0. The level of another repressive histone marker, H3K27me1, was decreased in Kyoto; however, it was not impacted in a Col-0 cmt2 mutant. Therefore, in addition to the CMT2 mutation, other factors may reduce repressive histone modifications in Kyoto.

MPP6 stimulates both RRP6 and DIS3 to degrade a specified subset of MTR4-sensitive substrates in the human nucleus.

Recent in vitro reconstitution analyses have proven that the physical interaction between the exosome core and MTR4 helicase, which promotes the exosome activity, is maintained by either MPP6 or RRP6. However, knowledge regarding the function of MPP6 with respect to in vivo exosome activity remains scarce. Here, we demonstrate a facilitative function of MPP6 that composes a specific part of MTR4-dependent substrate decay by the human exosome. Using RNA polymerase II-transcribed poly(A)+ substrate accumulation as an indicator of a perturbed exosome, we found functional redundancy between RRP6 and MPP6 in the decay of these poly(A)+ transcripts. MTR4 binding to the exosome core via MPP6 was essential for MPP6 to exert its redundancy with RRP6. However, at least for the decay of our identified exosome substrates, MTR4 recruitment by MPP6 was not functionally equivalent to recruitment by RRP6. Genome-wide classification of substrates based on their sensitivity to each exosome component revealed that MPP6 deals with a specific range of substrates and highlights the importance of MTR4 for their decay. Considering recent findings of competitive binding to the exosome between auxiliary complexes, our results suggest that the MPP6-incorporated MTR4-exosome complex is one of the multiple alternative complexes rather than the prevailing one.

CHERP Regulates the Alternative Splicing of pre-mRNAs in the Nucleus.

Calcium homeostasis endoplasmic reticulum protein (CHERP) is colocalized with the inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum or perinuclear region, and has been involved in intracellular calcium signaling. Structurally, CHERP carries the nuclear localization signal and arginine/serine-dipeptide repeats, like domain, and interacts with the spliceosome. However, the exact function of CHERP in the nucleus remains unknown. Here, we showed that poly(A)+ RNAs accumulated in the nucleus of CHERP-depleted U2OS cells. Our global analysis revealed that CHERP regulated alternative mRNA splicing events by interaction with U2 small nuclear ribonucleoproteins (U2 snRNPs) and U2 snRNP-related proteins. Among the five alternative splicing patterns analyzed, intron retention was the most frequently observed event. This was in accordance with the accumulation of poly(A)+ RNAs in the nucleus. Furthermore, intron retention and cassette exon choices were influenced by the strength of the 5' or 3' splice site, the branch point site, GC content, and intron length. In addition, CHERP depletion induced anomalies in the cell cycle progression into the M phase, and abnormal cell division. These results suggested that CHERP is involved in the regulation of alternative splicing.

Analyses of putative anti-cancer potential of three STAT3 signaling inhibitory compounds derived from Salvia officinalis.

The extract of Salvia officinalis (Common Sage) exhibited inhibitory activity of STAT3 signal after screening of several plants extracts using the STAT3-responsive reporter system. Cirsiliol, luteolin, and carnosol were identified from the methanol extract of Silvia officinalis as inhibitors of STAT3 signaling and the effects of these three compounds on STAT3 protein or growth inhibition on cancer cells was compared. Luteolin at the dose of 90 µM clearly suppressed the phosphorylation of STAT3 induced by IL-6, while carnosol was prone to decrease total STAT3 proteins at high doses (>90 µM). Cirsiliol had almost no effect. Since the three compounds exhibited similar concentration-dependent suppression patterns in the reporter assay except for cirsiliol became plateau beyond 30 µM, these compounds appeared to function as STAT3 inhibitory factors in different ways. The direct anti-proliferative activity of three compounds was examined with or without the anti-cancer drug gefitinib using HepG2 and A549 cells. The anti-proliferative effect of the three compounds was additively enhanced by gefitinib. At the doses of 3.6 µM, statistically significant suppression of proliferation was observed in HepG2 cells only by cirsiliol among the three compounds in the absence of gefitinib but all three compounds were prone to suppress the proliferation of HepG2 cells and A549 cells dose-dependently although cirsiliol showed a modest dose-dependency and this suppression of proliferation was enhanced by the addition of gefitinib. Cirsiliol, a dimethyoxylated flavone, activated the natural killer activity of KHYG-1 cells against erythroleukemia K562 cells like a hexamethoxylated flavone, nobiletin, suggesting that it may also have an indirect anti-cancer potential through activation of NK cells. These results shed light on the putative anti-cancer potential of Salvia officinalis.

Regulating Divergent Transcriptomes through mRNA Splicing and Its Modulation Using Various Small Compounds.

Human transcriptomes are more divergent than genes and contribute to the sophistication of life. This divergence is derived from various isoforms arising from alternative splicing. In addition, alternative splicing regulated by spliceosomal factors and RNA structures, such as the RNA G-quadruplex, is important not only for isoform diversity but also for regulating gene expression. Therefore, abnormal splicing leads to serious diseases such as cancer and neurodegenerative disorders. In the first part of this review, we describe the regulation of divergent transcriptomes using alternative mRNA splicing. In the second part, we present the relationship between the disruption of splicing and diseases. Recently, various compounds with splicing inhibitor activity were established. These splicing inhibitors are recognized as a biological tool to investigate the molecular mechanism of splicing and as a potential therapeutic agent for cancer treatment. Food-derived compounds with similar functions were found and are expected to exhibit anticancer effects. In the final part, we describe the compounds that modulate the messenger RNA (mRNA) splicing process and their availability for basic research and future clinical potential.

URH49 exports mRNA by remodeling complex formation and mediating the NXF1-dependent pathway.

The TREX complex integrates information from nuclear mRNA processing events to ensure the timely export of mRNA to the cytoplasm. In humans, UAP56 and its paralog URH49 form distinct complexes, the TREX complex and the AREX complex, respectively, which cooperatively regulate the expression of a specific set of mRNA species on a genome wide scale. The difference in the complex formation between UAP56 and URH49 are thought to play a critical role in the regulation of target mRNAs. To date, the underlying mechanism remains poorly understood. Here we characterize the formation of the TREX complex and the AREX complex. In the ATP depleted condition, UAP56 formed an Apo-TREX complex containing the THO subcomplex but not ALYREF and CIP29. URH49 formed an Apo-AREX complex containing CIP29 but not ALYREF and the THO subcomplex. However, with the addition of ATP, both the Apo-TREX complex and the Apo-AREX complex were remodeled to highly similar ATP-TREX complex containing the THO subcomplex, ALYREF and CIP29. The knockdown of URH49 caused a reduction in its target mRNAs and a cytokinesis failure. Similarly, cytokinesis abnormality was observed in CIP29 knockdown cells, suggesting that CIP29 belongs to the URH49 regulated mRNA export pathway. Lastly, we confirmed that the export of mRNA in URH49-dependent pathway is achieved by NXF1, which is also observed in UAP56-dependent pathway. Our studies propose an mRNA export model that the mRNA selectivity depends on the Apo-form TREX/AREX complex, which is remodeled to the highly similar ATP-form complex upon ATP loading, and integrated to NXF1.

Stimulation of insulin secretion by acetylenic fatty acids in insulinoma MIN6 cells through FFAR1.

We examined whether the acetylenic fatty acids 6-octadecynoic acid (6-ODA) and 9-octadecynoic acid (9-ODA) perform as ligands for free fatty acid receptors of medium- and long-chain fatty acids FFAR1 and FFAR4, previously called GPR40 and GPR120, respectively. Phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 was increased through FFAR1 but not through FFAR4 expressed in HEK 293 cells, suggesting that 6-ODA and 9-ODA function as an FFAR1 ligand, but not as an FFAR4 ligand. Activation of ERK in FFAR1-expressing HEK293 cells by 6-ODA and 9-ODA peaked at 10 min after stimulation followed by a slow decrease, similar to ERK activation by rosiglitazone, which peaked at 10 min after stimulation and lasted longer. Glucose-dependent production of insulin from MIN6 insulinoma cells was induced by 6-ODA and 9-ODA in an FFAR1-dependent manner. In this process, 6-ODA and 9-ODA stimulated the production of insulin not in the first phase that occurred within 10 min after stimulation but in the second phase. F-actin-remodeling that reflects insulin granule recruiting to the plasma membrane in the second phase of insulin secretion by 6-ODA and 9-ODA suggested that they have an FFAR1-dependent function in insulin secretion from MIN6 cells.

Food-Derived Compounds Apigenin and Luteolin Modulate mRNA Splicing of Introns with Weak Splice Sites.

Cancer cells often exhibit extreme sensitivity to splicing inhibitors. We identified food-derived flavonoids, apigenin and luteolin, as compounds that modulate mRNA splicing at the genome-wide level, followed by proliferation inhibition. They bind to mRNA splicing-related proteins to induce a widespread change of splicing patterns in treated cells. Their inhibitory activity on splicing is relatively moderate, and introns with weak splice sites tend to be sensitive to them. Such introns remain unspliced, and the resulting intron-containing mRNAs are retained in the nucleus, resulting in the nuclear accumulation of poly(A)+ RNAs in these flavonoid-treated cells. Tumorigenic cells are more susceptible to these flavonoids than nontumorigenic cells, both for the nuclear poly(A)+ RNA-accumulating phenotype and cell viability. This study illustrates the possible mechanism of these flavonoids to suppress tumor progression in vivo that were demonstrated by previous studies and provides the potential of daily intake of moderate splicing inhibitors to prevent cancer development.

Correction: Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect.

[This corrects the article DOI: 10.1371/journal.pone.0197165.].

Inhibition of mRNA processing activity from ginger-, clove- and cinnamon-extract, and by two ginger constituents, 6-gingerol and 6-shogaol.

Inhibition of mRNA processing, including splicing in the nucleus, is a potential anti-cancer candidate. To obtain mRNA processing inhibitors, we have screened for active constituents from spices. Ginger, clove, and cinnamon showed an inhibitory effect on mRNA processing in the nucleus. Two components in ginger, 6-gingerol and 6-shogaol, exhibited the inhibition of mRNA processing.

Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect.

DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNß-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism.

Characterization of a heat-activated retrotransposon in natural accessions of Arabidopsis thaliana.

Natural accessions are used for studying intraspecies genetic variation in the model plant Arabidopsis thaliana in order to address fundamental questions of evolution. Transposable elements are responsible for a wide range of mutations and play significant roles in shaping a genome over evolutionary time. In the present study, we aimed to characterize ONSEN, a heat-activated long terminal repeat (LTR) retrotransposon, in natural A. thaliana accessions. Southern blot analysis demonstrated that ONSEN was present in all the studied accessions, but the copy number was diverse. Olympia-1 contained a single ONSEN copy, located in the centromere of Chromosome 3. A premature stop codon in Olympia-1 ONSEN presumably abolishes integrase activity, which in turn presumably renders the retrotransposon non-functional. Hybridization of Col-0 with Olympia-1 showed that several ONSEN copies in Col-0 were activated by heat stress and maintained their transpositional activity in the progeny.

The isoflavone fraction from soybean presents mRNA maturation inhibition activity.

Recent findings indicate that mRNA splicing inhibitors can be potential anticancer candidates. We have previously established a screening system which monitors mRNA processing in order to identify mRNA processing inhibitors. Among a number of dietary resources, isoflavone fractions showed an inhibitory effect of mRNA processing. These findings demonstrate that a variety of dietary sources have an impact on mRNA biogenesis.

Inducible Transposition of a Heat-Activated Retrotransposon in Tissue Culture.

A transposition of a heat-activated retrotransposon named ONSEN required compromise of a small RNA-mediated epigenetic regulation that includes RNA-directed DNA methylation (RdDM) machinery after heat treatment. In the current study, we analyzed the transcriptional and transpositional activation of ONSEN to better understand the underlying molecular mechanism involved in the maintenance and/or induction of transposon activation in plant tissue culture. We found the transposition of heat-primed ONSEN during tissue culture independently of RdDM mutation. The heat activation of ONSEN transcripts was not significantly up-regulated in tissue culture compared with that in heat-stressed seedlings, indicating that the transposition of ONSEN was regulated independently of the transcript level. RdDM-related genes were up-regulated by heat stress in both tissue culture and seedlings. The level of DNA methylation of ONSEN did not show any change in tissue culture, and the amount of ONSEN-derived small RNAs was not affected by heat stress. The results indicated that the transposition of ONSEN was regulated by an alternative mechanism in addition to the RdDM-mediated epigenetic regulation in tissue culture. We applied the tissue culture-induced transposition of ONSEN to Japanese radish, an important breeding species of the family Brassicaceae. Several new insertions were detected in a regenerated plant derived from heat-stressed tissues and its self-fertilized progeny, revealing the possibility of molecular breeding without genetic modification.

Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line.

Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation.

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation.

Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.

A Stress-Activated Transposon in Arabidopsis Induces Transgenerational Abscisic Acid Insensitivity.

Transposable elements (TEs), or transposons, play an important role in adaptation. TE insertion can affect host gene function and provides a mechanism for rapid increases in genetic diversity, particularly because many TEs respond to environmental stress. In the current study, we show that the transposition of a heat-activated retrotransposon, ONSEN, generated a mutation in an abscisic acid (ABA) responsive gene, resulting in an ABA-insensitive phenotype in Arabidopsis, suggesting stress tolerance. Our results provide direct evidence that a transposon activated by environmental stress could alter the genome in a potentially positive manner. Furthermore, the ABA-insensitive phenotype was inherited when the transcription was disrupted by an ONSEN insertion, whereas ABA sensitivity was recovered when the effects of ONSEN were masked by IBM2. These results suggest that epigenetic mechanisms in host plants typically buffered the effect of a new insertion, but could selectively "turn on" TEs when stressed.