Search Results (18)

Background/objectives: Euchromatic histone lysine methyltransferase 2 (EHMT2, also known as G9a) is a mammalian histone methyltransferase that catalyzes the dimethylation of histone 3 lysine 9 (H3K9). On human chromosome 15, the parental-specific expression of Prader–Willi Syndrome (PWS)-related genes, such as SNRPN and SNORD116, are regulated through the genetic imprinting of the PWS imprinting center (PWS-IC). On the paternal allele, PWS genes are expressed whereas the epigenetic maternal silencing of PWS genes is controlled by the EHMT2-mediated methylation of H3K9 in PWS-IC. Here, we measured the effects of RNA interference of EHMT2 on the maternal expression of genes deficient in PWS in mouse model and patient iPSC-derived cells. Methods: We used small interfering RNA (siRNA) oligonucleotides and lentiviral short harpin RNA (shRNA) to reduce Ehtm2/EHMT2 expression in mouse Snord116 deletion primary neurons, PWS patient-derived induced pluripotent stem cell (iPSC) line and PWS iPSC-derived neurons. We then measured the expression of transcript or protein (if relevant) of PWS genes normally silenced on the maternal allele. Results: With an approximate reduction of 90% in EHMT2 mRNA and more than 80% of the EHMT2 protein, we demonstrated close to a 2-fold increase in the expression of maternal transcripts for SNRPN and SNORD116 in PWS iPSCs treated with siEHMT2 compared to PWS iPSC siControl. A similar increase in SNORD116 and SNRPN RNA expression was observed in PWS iPSC-derived neurons treated with shEHMT2. Conclusions: RNAi reduction in EHMT2 activates maternally silenced PWS genes. Further studies are needed to determine whether the increase is therapeutically relevant. This study confirms the role of EHMT2 in the epigenetic regulation of PWS genes. Full article

Transgenerational nanoplastic toxicity could be detected in Caenorhabditis elegans after exposure at the parental generation (P0-G); however, the underlying mechanisms remain largely unclear. We aimed to examine the role of germline nuclear hormone receptors (NHRs) in controlling the transgenerational toxicity of polystyrene nanoparticles (PS-NPs) based on gene expression screening and functional analysis. Among germline NHR genes, daf-12, nhr-14, and nhr-47 expressions were increased and nhr-12 expression was decreased by PS-NPs (1 and 10 μg/L). Transgenerational alterations in expressions of these four NHR genes were also induced by PS-NPs (1 and 10 μg/L). RNAi of daf-12, nhr-14, and nhr-47 caused resistance, whereas RNAi of nhr-12 conferred susceptibility to transgenerational PS-NP toxicity. After PS-NP exposure, expressions of ins-3, daf-28, and ins-39 encoding insulin ligands, efn-3 encoding Ephrin ligand, and lin-44 encoding Wnt ligand, as well as expressions of their receptor genes (daf-2, vab-1, and/or mig-1), were dysregulated by the RNAi of daf-12, nhr-14, nhr-47, and nhr-12. Therefore, alteration in certain germline NHRs could mediate the induction of transgenerational nanoplastic toxicity by affecting secreted ligands and their receptors in the offspring of exposed organisms. Full article

Recently, the transgenerational toxicity of nanoplastics has received increasing attention. Caenorhabditis elegans is a useful model to assess the transgenerational toxicity of different pollutants. In nematodes, the possibility of early-life exposure to sulfonate-modified polystyrene nanoparticle (PS-S NP) causing transgenerational toxicity and its underlying mechanisms were investigated. After exposure at the L1-larval stage, transgenerational inhibition in both locomotion behavior (body bend and head thrash) and reproductive capacity (number of offspring and fertilized egg number in uterus) was induced by 1–100 μg/L PS-S NP. Meanwhile, after exposure to 1–100 μg/L PS-S NP, the expression of germline lag-2 encoding Notch ligand was increased not only at the parental generation (P0-G) but also in the offspring, and the transgenerational toxicity was inhibited by the germline RNA interference (RNAi) of lag-2. During the transgenerational toxicity formation, the parental LAG-2 activated the corresponding Notch receptor GLP-1 in the offspring, and transgenerational toxicity was also suppressed by glp-1 RNAi. GLP-1 functioned in the germline and the neurons to mediate the PS-S NP toxicity. In PS-S NP-exposed nematodes, germline GLP-1 activated the insulin peptides of INS-39, INS-3, and DAF-28, and neuronal GLP-1 inhibited the DAF-7, DBL-1, and GLB-10. Therefore, the exposure risk in inducing transgenerational toxicity through PS-S NP was suggested, and this transgenerational toxicity was mediated by the activation of germline Notch signal in organisms. Full article

DNA double-strand breaks (DSBs) caused by platinum drugs are dangerous lesions that kill cancer cells in chemotherapy. Repair of DSB by homologous recombination (HR) and nonhomologous end joining (NHEJ) is frequently associated with platinum resistance in ovarian cancer. While the role of the HR pathway and HR-targeting strategy in platinum resistance is well studied, dissecting and targeting NHEJ machinery to overcome platinum resistance in ovarian cancer remain largely unexplored. Here, through an NHEJ pathway-focused gene RNAi screen, we found that the knockdown of XRCC4 significantly sensitized cisplatin treatment in the platinum-resistant ovarian cancer cell lines. Moreover, upregulation of XRCC4 is observed in a panel of platinum-resistant cell lines relative to the parental cell lines, as well as in ovarian cancer patients with poor progression-free survival. Mechanistically, the increased sensitivity to cisplatin upon XRCC4 knockdown was caused by accumulated DNA damage. In cisplatin-resistant ovarian cancer, the JNK-cJUN complex, activated by cisplatin, translocated into the nucleus and promoted the transcription of XRCC4 to confer cisplatin resistance. Knockdown of XRCC4 or treatment of the JNK inhibitor led to the attenuation of cisplatin-resistant tumor growth in the xenograft mouse models. These data suggest targeting XRCC4 is a potential strategy for ovarian cisplatin resistance in ovarian cancer. Full article

In nematode Caenorhabditis elegans, exposure to polystyrene nanoparticles (PS-NPs) at predicted environmental concentrations can cause induction of transgenerational toxicity. However, the underlying mechanisms for toxicity formation of PS-NP in the offspring remain largely unknown. In this study, based on high-throughput sequencing, Ephrin ligand EFN-3 was identified as a target of KSR-1/2 (two kinase suppressors of Ras) in the germline during the control of transgenerational PS-NP toxicity. At parental generation (P0-G), exposure to 0.1–10 μg/L PS-NP caused the increase in expression of germline efn-3, and this increase in germline efn-3 expression could be further detected in the offspring, such as F1-G and F2-G. Germline RNAi of efn-3 caused a resistance to transgenerational PS-NP toxicity, suggesting that the activation of germline EFN-3 at P0-G mediated transgenerational PS-NP toxicity. In the offspring, Ephrin receptor VAB-1 was further activated by the increased EFN-3 caused by PS-NP exposure at P0-G, and RNAi of vab-1 also resulted in resistance to transgenerational PS-NP toxicity. VAB-1 acted in both the neurons and the germline to control toxicity of PS-NP in the offspring. In the neurons, VAB-1 regulated PS-NP toxicity by suppressing expressions of DBL-1, JNK-1, MPK-1, and GLB-10. In the germline, VAB-1 regulated PS-NP toxicity by increasing NDK-1 and LIN-23 expressions and decreasing EGL-1 expression. Therefore, germline Ephrin ligand EFN-3 and its receptor VAB-1 acted together to mediate the formation of transgenerational PS-NP toxicity. Our data highlight the important role of activation in germline Ephrin signals in mediating transgenerational toxicity of nanoplastics at predicted environmental concentrations in organisms. Full article

Sucrose synthase (SuSy) and fructokinase (FRK) work together to control carbohydrate flux in sink tissues. SuSy cleaves sucrose into fructose and UDP-glucose; whereas FRK phosphorylates fructose. Previous results have shown that suppression of the SUS1,3&4 genes by SUS-RNAi alters auxin transport in the shoot apical meristems of tomato plants and affects cotyledons and leaf structure; whereas antisense suppression of FRK2 affects vascular development. To explore the joint developmental roles of SuSy and FRK, we crossed SUS-RNAi plants with FRK2-antisense plants to create double-mutant plants. The double-mutant plants exhibited novel phenotypes that were absent from the parent lines. About a third of the plants showed arrested shoot apical meristem around the transition to flowering and developed ectopic meristems. Use of the auxin reporter DR5::VENUS revealed a significantly reduced auxin response in the shoot apical meristems of the double-mutant, indicating that auxin levels were low. Altered inflorescence phyllotaxis and significant disorientation of vascular tissues were also observed. In addition, the fruits and the seeds of the double-mutant plants were very small and the seeds had very low germination rates. These results show that SUS1,3&4 and FRK2 enzymes are jointly essential for proper meristematic and vascular development, and for fruit and seed development. Full article

Recent studies on nuclear-encoded mitochondrial genes (N-mt genes) in Drosophila melanogaster have shown a unique pattern of expression for newly duplicated N-mt genes, with many duplicates having a testis-biased expression and playing an essential role in spermatogenesis. In this study, we investigated a newly duplicated N-mt gene—i.e., Cytochrome c oxidase 4-like (COX4L)—in order to understand its function and, consequently, the reason behind its retention in the D. melanogaster genome. The COX4L gene is a duplicate of the Cytochrome c oxidase 4 (COX4) gene of OXPHOS complex IV. While the parental COX4 gene has been found in all eukaryotes, including single-cell eukaryotes such as yeast, we show that COX4L is only present in the Brachycera suborder of Diptera; thus, both genes are present in all Drosophila species, but have significantly different patterns of expression: COX4 is highly expressed in all tissues, while COX4L has a testis-specific expression. To understand the function of this new gene, we first knocked down its expression in the D. melanogaster germline using two different RNAi lines driven by the bam-Gal4 driver; second, we created a knockout strain for this gene using CRISPR-Cas9 technology. Our results showed that knockdown and knockout lines of COX4L produce partial sterility and complete sterility in males, respectively, where a lack of sperm individualization was observed in both cases. Male infertility was prevented by driving COX4L-HA in the germline, but not when driving COX4-HA. In addition, ectopic expression of COX4L in the soma caused embryonic lethality, while overexpression in the germline led to a reduction in male fertility. COX4L-KO mitochondria show reduced membrane potential, providing a plausible explanation for the male sterility observed in these flies. This prominent loss-of-function phenotype, along with its testis-biased expression and its presence in the Drosophila sperm proteome, suggests that COX4L is a paralogous, specialized gene that is assembled in OXPHOS complex IV of male germline cells and/or sperm mitochondria. Full article

The doublesex (dsx) gene, which encodes a transcription factor, regulates sexual differentiation in insects. Sex-specific splicing of dsx occurs to yield male- and female-specific isoforms, which promote male and female development, respectively. Thus, functional disruption of dsx leads to an intersexual phenotype in both sexes. We previously identified a dsx ortholog in the sawfly, Athalia rosae. Similar to dsx in other insects, dsx in the sawfly yields different isoforms in males and females as a result of alternative splicing. The sawfly exploits a haplodiploid mode of reproduction, in which fertilized eggs develop into diploid females, whereas unfertilized eggs parthenogenetically develop into haploid males. In the present study, we knocked down the A. rosae ortholog of dsx (Ardsx) during several developmental stages with repeated double-stranded RNA (dsRNA) injections. Knockdown of Ardsx via parental RNA interference (RNAi), which enables knockdown of genes in offspring embryos, led to a lack of internal and external genitalia in haploid male progeny. Additional injection of dsRNA targeting Ardsx in these animals caused almost complete male-to-female sex reversal, but the resulting eggs were infertile. Notably, the same knockdown approach using diploid males obtained by sib-crossing caused complete male-to-female sex reversal; they were morphologically and behaviorally females. The same RNAi treatment did not affect female differentiation. These results indicate that dsx in the sawfly is essential for male development and its depletion caused complete male-to-female sex reversal. This is the first demonstration of functional depletion of dsx not causing intersexuality but inducing total sex reversal in males instead. Full article

Gene silencing is the epigenetic regulation of any gene in order to prevent gene expression at the transcription or translation levels. Among various gene silencing techniques, RNA silencing (RNAi) is notable gene regulation technique that involves sequence-specific targeting and RNA degradation. However, the effectiveness of transgene-induced RNAi in F₁ generation of chrysanthemum has not been studied yet. In the current study, we used RNAi-constructed CmTFL1 (white-flowered) and CmSVP overexpressed (yellow flowered) transgenic plants of previously conducted two studies for our experiment. Cross hybridization was performed between these intergeneric transgenic and non-transgenic plants of the winter-growing chrysanthemum selection “37” (light pink flowered). The transgene CmSVP was confirmed in F₁ hybrids by RT-PCR analysis, whereas hybrids of CmTFL1 parental plants were non-transgenic. Besides this, quantitative real-time PCR (qPCR) was used to explain the molecular mechanism of flower development using reference genes. Intergeneric and interspecific hybrids produced different colored flowers unlike their respective parents. These results suggest that generic traits of CmSVP overexpressed plants can be transferred into F₁ generations when crossed with mutant plants. This study will aid in understanding the breeding phenomenon among intergeneric hybrids of chrysanthemum plants at an in vivo level, and such transgenics will also be more suitable for sustainable flower yield under a low-light production system. Full article

Chitin synthase 1 (CHS1) is an essential gene regulating chitin during different developmental stages of arthropods. In the current study, we explored for the first time the role of CHS1 gene regulation in the citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae), by silencing its expression using (RNA interference) RNAi-based strategies. The results reveal that P. citri tested in different developmental stages, including larvae, protonymphs, deutonymphs, and adults fed on sweet orange leaves dipped in various concentrations (200, 400, 600, and 800 ng/μL) of dsRNA-PcCHS1, resulted in a continuous reduction in their gene expression, and the extent of transcript knockdown was positively correlated with the concentration of dsRNA. Concentration–mortality response assays revealed a mortality of more than 50% among all the studied developmental stages, except for adulthood. Furthermore, the target gene dsRNA-PcCHS1 treatment of larvae, protonymphs, deutonymphs, and females at a treatment rate of 800 ng/mL of dsRNA significantly decreased the egg-laying rates by 48.50%, 43.79%, 54%, and 39%, respectively, and the hatching rates were also considerably reduced by 64.70%, 70%, 64%, and 52.90%, respectively. Moreover, using the leaf dip method, we found that the RNA interference effectively reduced the PcCHS1 transcript levels by 42.50% and 42.06% in the eggs and adults, respectively. The results of this study demonstrate that the RNAi of PcCHS1 can dramatically reduce the survival and fecundity of P. citri, but the dsRNA concentrations and developmental stages can significantly influence the RNAi effects. These findings indicate the potential utility of the PcCHS1 gene in causing developmental irregularities, which could aid in the development of effective and novel RNAi-based strategies for controlling P. citri. Full article

Hexokinase (HK) is a key enzyme in chitin biosynthesis in insects and plays an important role in development and energy regulation. It also performs a crucial role in the synthesis of Glucose-6-phosphate and its putative functions are studied via injection of dsRNA corresponding to the hexokinase gene from Cnaphalocrocis medinalis (CmHK). This study was designed to analyze the characteristics and expression patterns of HK-related genes in various tissues of C. medinalis at different developmental stages. The CmHK ORF is a 1359 bp in length, encoding a protein of 452 amino acids, with homology and cluster analysis showing that CmHK shares an 85.11% sequence similarity with hexokinase from Ostrinia furnacalis.CmHK was highly expressed in the ovary and in the fifth instar larvae. Injection of dsCmHK significantly suppressed mRNA expression (73.6%) 120 h post-dsRNA injection as compared to a control group. The results demonstrated an increased incidence of larval and pupal mortality of 80% and 78%, respectively, with significant variation in the sex ratio between males (68.33%) and females (35%), overt larval deformities, and a reduction in average weight gain observed 120 h post-dsRNA injection. In addition, dsCmHK-injected C. medinalis showed a significant reduction in ovulation per female and larval hatching rate, along with increased larval and pupal mortality and variation in male and female emergence over three generations (G1, G2, and G3). Taken together, the outcomes of the study provide a foundation to study gene function and a new dimension to control C. medinalis by transgenic RNAi technology. Full article

Arabidopsis thaliana MYB43 (AtMYB43) is suggested to be involved in cell wall lignification. PtrMYB152, the Populus orthologue of AtMYB43, is a transcriptional activator of lignin biosynthesis and vessel wall deposition. In this research, MYB43 genes from Brassica napus (rapeseed) and its parental species B. rapa and B. oleracea were molecularly characterized, which were dominantly expressed in stem and other vascular organs and showed responsiveness to Sclerotinia sclerotiorum infection. The BnMYB43 family was silenced by RNAi, and the transgenic rapeseed lines showed retardation in growth and development with smaller organs, reduced lodging resistance, fewer silique number and lower yield potential. The thickness of the xylem layer decreased by 28%; the numbers of sclerenchymatous cells, vessels, interfascicular fibers, sieve tubes and pith cells in the whole cross section of the stem decreased by 28%, 59%, 48%, 34% and 21% in these lines, respectively. The contents of cellulose and lignin decreased by 17.49% and 16.21% respectively, while the pectin content increased by 71.92% in stems of RNAi lines. When inoculated with S. sclerotiorum, the lesion length was drastically decreased by 52.10% in the stems of transgenic plants compared with WT, implying great increase in disease resistance. Correspondingly, changes in the gene expression patterns of lignin biosynthesis, cellulose biosynthesis, pectin biosynthesis, cell cycle, SA- and JA-signals, and defensive pathways were in accordance with above phenotypic modifications. These results show that BnMYB43, being a growth-defense trade-off participant, positively regulates vascular lignification, plant morphology and yield potential, but negatively affects resistance to S. sclerotiorum. Moreover, this lignification activator influences cell biogenesis of both lignified and non-lignified tissues of the whole vascular organ. Full article

Changes in plant architecture, such as leaf size, leaf shape, leaf angle, plant height, and floral organs, have been major factors in improving the yield of cereal crops. Moreover, changes in grain size and weight can also increase yield. Therefore, screens for additional factors affecting plant architecture and grain morphology may enable additional improvements in yield. Among the basic Helix-Loop-Helix (bHLH) transcription factors in rice (Oryza sativa), we found an enhancer-trap T-DNA insertion mutant of OsbHLH079 (termed osbhlh079-D). The osbhlh079-D mutant showed a wide leaf angle phenotype and produced long grains, similar to the phenotypes of mutants with increased brassinosteroid (BR) levels or enhanced BR signaling. Reverse transcription-quantitative PCR analysis showed that BR signaling-associated genes are largely upregulated in osbhlh079-D, but BR biosynthesis-associated genes are not upregulated, compared with its parental japonica cultivar ‘Dongjin’. Consistent with this, osbhlh079-D was hypersensitive to BR treatment. Scanning electron microscopy revealed that the expansion of cell size in the adaxial side of the lamina joint was responsible for the increase in leaf angle in osbhlh079-D. The expression of cell-elongation-associated genes encoding expansins and xyloglucan endotransglycosylases/hydrolases increased in the lamina joints of leaves in osbhlh079-D. The regulatory function of OsbHLH079 was further confirmed by analyzing 35S::OsbHLH079 overexpression and 35S::RNAi-OsbHLH079 gene silencing lines. The 35S::OsbHLH079 plants showed similar phenotypes to osbhlh079-D, and the 35S::RNAi-OsbHLH079 plants displayed opposite phenotypes to osbhlh079-D. Taking these observations together, we propose that OsbHLH079 functions as a positive regulator of BR signaling in rice. Full article

Background: The aim of this study is to elucidate the mechanisms of acquired resistance to pemetrexed in echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearranged non-small cell lung cancer. Methods: We analyzed the sensitivity to pemetrexed and the expression patterns of various proteins after pemetrexed treatment in the cell lines, A549, NCI-H460, NCI-H2228 harboring EML4-ALK variant 3, and NCI-H3122 harboring EML4-ALK variant 1. Pemetrexed-resistant cell lines were also generated through long-term exposure to pemetrexed. Results: The EML4-ALK variant 1 rearranged NCI-H3122 was found to be more sensitive than the other cell lines. Cell cycle analysis after pemetrexed treatment showed that the fraction of cells in the S phase increased in A549, NCI-H460, and NCI-H2228, whereas the fraction in the apoptotic sub-G₁ phase increased in NCI-H3122. The pemetrexed-resistant NCI-H3122 cell line showed increased expression of EGFR and HER2 compared to the parent cell line, whereas A549 and NCI-H460 did not show this change. The pan-HER inhibitor afatinib inhibited this alternative signaling pathway, resulting in a superior cytotoxic effect in pemetrexed-resistant NCI-H3122 cell lines compared to that in the parental cells line. Conclusion: The activation of EGFR-HER2 contributes to the acquisition of resistance to pemetrexed in EML4-ALK rearranged non-small cell lung cancer. However, the inhibition of this alternative survival signaling pathway with RNAi against EGFR-HER2 and with afatinib overcomes this resistance. Full article

In the ciliate Stylonychia, somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly, over 95% of micronuclear sequences adopt a heterochromatin structure characterized by the histone variant H3.4 and H3K27me3. RNAi-related genes and histone variants dominate the list of developmentally expressed genes. Simultaneously, 27nt-ncRNAs that match sequences retained in new macronuclei are synthesized and bound by PIWI1. Recently, we proposed a mechanistic model for ‘RNA-induced DNA replication interference’ (RIRI): during polytene chromosome formation PIWI1/27nt-RNA-complexes target macronucleus-destined sequences (MDS) by base-pairing and temporarily cause locally stalled replication. At polytene chromosomal segments with ongoing replication, H3.4K27me3-nucleosomes become selectively deposited, thus dictating the prospective heterochromatin structure of these areas. Consequently, these micronucleus-specific sequences become degraded, whereas 27nt-RNA-covered sites remain protected. However, the biogenesis of the 27nt-RNAs remains unclear. It was proposed earlier that in stichotrichous ciliates 27nt-RNA precursors could derive from telomere-primed bidirectional transcription of nanochromosomes and subsequent Dicer-like (DCL) activity. As a minimalistic explanation, we propose here that the 27nt-RNA precursor could rather be mRNA or pre-mRNA and that the transition of coding RNA from parental macronuclei to non-coding RNAs, which act in premature developing macronuclei, could involve RNA-dependent RNA polymerase (RDRP) activity creating dsRNA intermediates prior to a DCL-dependent pathway. Interestingly, by such mechanism the partition of a parental somatic genome and possibly also the specific nanochromosome copy numbers could be vertically transmitted to the differentiating nuclei of the offspring. Full article