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Background: Argonaute 2 (AGO2), the only protein with catalytic activity in the human Argonaute family, is considered as a key component of RNA interference (RNAi) pathway. Here we performed a yeast two-hybrid screen using the human Argonaute 2 PIWI domain as bait to screen for new AGO2-interacting proteins and explored the specific mechanism through a series of molecular biology and biochemistry experiments., Methods: The yeast two-hybrid system was used to screen for AGO2-interacting proteins. Co-immunoprecipitation and immunofluorescence assays were used to further determine interactions and co-localization. Truncated plasmids were constructed to clarify the interaction domain. EGFP fluorescence assay was performed to determine the effect of PSMC3 on RNAi. Regulation of AGO2 protein expression and ubiquitination by PSMC3 and USP14 was examined by western blotting. RT-qPCR assays were applied to assess the level of AGO2 mRNA. Rescue assays were also performed., Results: We identified PSMC3 (proteasome 26S subunit, ATPase, 3) as a novel AGO2 binding partner. Biochemical and bioinformatic analysis demonstrates that this interaction is performed in an RNA-independent manner and the N-terminal coiled-coil motif of PSMC3 is required. Depletion of PSMC3 impairs the activity of the targeted cleavage mediated by small RNAs. Further studies showed that depletion of PSMC3 decreased AGO2 protein amount, whereas PSMC3 overexpression increased the expression of AGO2 at a post-translational level. Cycloheximide treatment indicated that PSMC3 depletion resulted in a decrease in cytoplasmic AGO2 amount due to an increase in AGO2 protein turnover. The absence of PSMC3 promoted ubiquitination of AGO2, resulting in its degradation by the 26S proteasome. Mechanistically, PSMC3 assists in the interaction of AGO2 with the deubiquitylase USP14(ubiquitin specific peptidase 14) and facilitates USP14-mediated deubiquitination of AGO2. As a result, AGO2 is stabilized, which then promotes RNAi., Conclusion: Our findings demonstrate that PSMC3 plays an essential role in regulating the stability of AGO2 and thus in maintaining effective RNAi., (© 2022. The Author(s).)

Over the past decade, non-coding RNA-based therapeutics have proven as a great potential for the development of targeted therapies for cancer and other diseases. The discovery of the critical function of microRNAs (miRNAs) has generated great excitement in developing miRNA-based therapies. The dysregulation of miRNAs contributes to the pathogenesis of various human diseases and cancers by modulating genes that are involved in critical cellular processes, including cell proliferation, differentiation, apoptosis, angiogenesis, metastasis, drug resistance, and tumorigenesis. miRNA (miRNA mimic, anti-miRNA/antagomir) and small interfering RNA (siRNA) can inhibit the expression of any cancer-related genes/mRNAs with high specificity through RNA interference (RNAi), thus representing a remarkable therapeutic tool for targeted therapies and precision medicine. siRNA and miRNA-based therapies have entered clinical trials and recently three novel siRNA-based therapeutics were approved by the Food and Drug Administration (FDA), indicating the beginning of a new era of targeted therapeutics. The successful clinical applications of miRNA and siRNA therapeutics rely on safe and effective nanodelivery strategies for targeting tumor cells or tumor microenvironment. For this purpose, promising nanodelivery/nanoparticle-based approaches have been developed using a variety of molecules for systemic administration and improved tumor targeted delivery with reduced side effects. In this review, we present an overview of RNAi-based therapeutics, the major pharmaceutical challenges, and the perspectives for the development of promising delivery systems for clinical translation. We also highlight the passive and active tumor targeting nanodelivery strategies and primarily focus on the current applications of nanoparticle-based delivery formulations for tumor targeted RNAi molecules and their recent advances in clinical trials in human cancers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors., (Published by Elsevier B.V.)

Multiple antiviral immunities were developed to defend against viral infection in hosts. RNA interference (RNAi)-based antiviral innate immunity is evolutionarily conserved in eukaryotes and plays a vital role against all types of viruses. During the arms race between the host and virus, many viruses evolve viral suppressors of RNA silencing (VSRs) to inhibit antiviral innate immunity. Here, we reviewed the mechanism at different stages in RNAi-based antiviral innate immunity in plants and the counteractions of various VSRs, mainly upon infection of RNA viruses in model plant Arabidopsis. Some critical challenges in the field were also proposed, and we think that further elucidating conserved antiviral innate immunity may convey a broad spectrum of antiviral strategies to prevent viral diseases in the future.

P0 proteins encoded by poleroviruses Brassica yellows virus (BrYV) and Potato leafroll virus (PLRV) are viral suppressors of RNA silencing (VSR) involved in abolishing host RNA silencing to assist viral infection. However, other roles that P0 proteins play in virus infection remain unclear. Here, we found that C-terminal truncation of P0 resulted in compromised systemic infection of BrYV and PLRV. C-terminal truncation affected systemic but not local VSR activities of P0 proteins, but neither transient nor ectopic stably expressed VSR proteins could rescue the systemic infection of BrYV and PLRV mutants. Moreover, BrYV mutant failed to establish systemic infection in DCL2/4 RNAi or RDR6 RNAi plants, indicating that systemic infection might be independent of the VSR activity of P0. Partially rescued infection of BrYV mutant by the co-infected PLRV implied the functional conservation of P0 proteins within genus. However, although C-terminal truncation mutant of BrYV P0 showed weaker interaction with its movement protein (MP) when compared to wild-type P0, wild-type and mutant PLRV P0 showed similar interaction with its MP. In sum, our findings revealed the role of P0 in virus systemic infection and the requirement of P0 carboxyl terminal region for the infection.

Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. Here, from an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), we identify a factor, Yun, required for proliferation of normal and transformed ISCs. Yun is mainly expressed in progenitors; our genetic and biochemical evidence suggest that it acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. We demonstrate that the Yun/PHB complex is regulated by and acts downstream of EGFR/MAPK signaling. Importantly, the Yun/PHB complex interacts with and positively affects the levels of the transcription factor E2F1 to regulate ISC proliferation. In addition, we find that the role of the PHB complex in cell proliferation is evolutionarily conserved. Thus, our study uncovers a Yun/PHB-E2F1 regulatory axis in stem cell proliferation., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Glioblastoma (GBM) is a malignant and aggressive brain tumor with a median survival of ∼15 months. Resistance to treatment arises from the extensive cellular and molecular heterogeneity in the three major components: glioma tumor cells, glioma stem cells, and tumor-associated microglia and macrophages. Within this triad, there is a complex network of intrinsic and secreted factors that promote classic hallmarks of cancer, including angiogenesis, resistance to cell death, proliferation, and immune evasion. A regulatory node connecting these diverse pathways is at the posttranscriptional level as mRNAs encoding many of the key drivers contain adenine- and uridine rich elements (ARE) in the 3' untranslated region. Human antigen R (HuR) binds to ARE-bearing mRNAs and is a major positive regulator at this level. This review focuses on basic concepts of ARE-mediated RNA regulation and how targeting HuR with small molecule inhibitors represents a plausible strategy for a multi-pronged therapeutic attack on GBM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Background: The Asian tiger mosquito Aedes albopictus is a competent vector of several viral arboviruses including yellow fever, dengue fever, and chikungunya. Several vital mosquito behaviors (e.g., feeding, host-seeking, mating, and oviposition) are primarily dependent on the olfactory system for semiochemicals detection and discrimination. However, the limited number of studies hampers our understanding of the relationships between the Ae. albopictus olfactory system and the complex chemical world., Methods: We performed RT-qPCR assay on antennae of Ae. albopictus mosquitoes of different sexes, ages and physiological states, and found odorant receptor 11 (AalbOr11) enriched in non-blood-fed female mosquitoes. Then, we examined the odorant preference with a panel of physiologically and behaviorally relevant odorants in Xenopus oocytes., Results: The results indicated that AalbOr11 could be activated by ten aromatics, seven terpenes, six heterocyclics, and three alcohols. Furthermore, using post-RNA interference (RNAi) hand-in-cage assay, we found that reducing the transcript level of AalbOr11 affected the repellency activity mediated by (+)-fenchone at a lower concentration (0.01% v/v)., Conclusions: Using in vitro functional characterization, we found that AalbOr11 was a broadly tuned receptor. Moreover, we found that AalbOr11 shared a conserved odorant reception profile with homologous Anopheles gambiae Or11. In addition, RNAi and bioassay suggested that AablOr11 might be one of the receptors mediating (+)-fenchone repellency activity. Our study attempted to link odor-induced behaviors to odorant reception and may lay the foundation for identifying active semiochemicals for monitoring or controlling mosquito populations., (© 2022. The Author(s).)

The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b's nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b's C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b's nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b's efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm., Competing Interests: The authors have declared that no competing interests exist.

Eukaryotic genomes harbor invading transposable elements that are silenced by PIWI-interacting RNAs (piRNAs) to maintain genome integrity in animal germ cells. However, whether piRNAs also regulate endogenous gene expression programs remains unclear. Here, we show that C. elegans piRNAs trigger the transcriptional silencing of hundreds of spermatogenic genes during spermatogenesis, promoting sperm differentiation and function. This silencing signal requires piRNA-dependent small RNA biogenesis and loading into downstream nuclear effectors, which correlates with the dynamic reorganization of two distinct perinuclear biomolecular condensates present in germ cells. In addition, the silencing capacity of piRNAs is temporally counteracted by the Argonaute CSR-1, which targets and licenses spermatogenic gene transcription. The spatial and temporal overlap between these opposing small RNA pathways contributes to setting up the timing of the spermatogenic differentiation program. Thus, our work identifies a prominent role for piRNAs as direct regulators of endogenous transcriptional programs during germline development and gamete differentiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Odorant-binding proteins (OBPs) play important roles in many physiological processes of mosquitoes. Previous high-throughput sequencing studies have revealed that some OBPs of Culex quinquefasciatus might be involved in the development of resistance to insecticides., Methods: Based on the results of sequencing analyses, the OBP28 gene was selected for evaluation in this study. Three laboratory strains of Cx. quinquefasciatus [susceptible strain (SS), deltamethrin-resistant strain 1 (HN) and deltamethrin-resistant strain 2 (RR)] were first examined by using the Centers for Disease Control and Prevention bottle bioassay, after which the expression level of the OBP28 gene in the susceptible and deltamethrin-resistant strains was determined by real-time quantitative polymerase chain reaction. The OBP28 gene in deltamethrin-resistant strain RR was silenced using RNA interference technology. The expression level of OBP28 and the resistance level were tested in the silenced strain and control strain after microinjection of double-stranded RNA for a 48-h interference period. Four field-collected strains (henceforth 'field strains') of Cx. quinquefasciatus were also examined for their resistance to deltamethrin and levels of OBP28 expression. Finally, a correlation analysis between deltamethrin resistance and gene expression was carried out for all seven strains, i.e. the four field strains and the three laboratory strains., Results: In the bioassay, the mortality of SS, HN and RR was 100%, 21.33% and 1.67%, respectively. The relative expression levels of OBP28 in strains HN and RR were 6.30- and 6.86-fold higher, respectively, than that of strain SS. After silencing of the OBP28 gene, the mortality of strain RR was 72.20% and that of the control strain 26.32%. The mortality of strain RR increased significantly after interference compared to that of the control strain. There was a negative correlation between OBP28 gene expression and mortality in adult mosquitoes after exposure to deltamethrin., Conclusions: To our knowledge, this study shows for the first time a correlation between the expression of a gene coding for OBP and insecticide resistance in mosquitoes. The potential resistance mechanism that was elucidated provides a new target gene for the surveillance of resistance in mosquitoes., (© 2021. The Author(s).)

There is an unmet clinical need to identify patients with early-stage non-small cell lung cancer (NSCLC) who are likely to develop recurrence and to predict their therapeutic responses. Our previous study developed a qRT-PCR-based seven-gene microfluidic assay to predict the recurrence risk and the clinical benefits of chemotherapy. This study showed it was feasible to apply this seven-gene panel in RNA sequencing profiles of The Cancer Genome Atlas (TCGA) NSCLC patients ( n = 923) in randomly partitioned feasibility-training and validation sets ( p < 0.05, Kaplan-Meier analysis). Using Boolean implication networks, DNA copy number variation-mediated transcriptional regulatory network of the seven-gene signature was identified in multiple NSCLC cohorts ( n = 371). The multi-omics network genes, including PD-L1 , were significantly correlated with immune infiltration and drug response to 10 commonly used drugs for treating NSCLC. ZNF71 protein expression was positively correlated with epithelial markers and was negatively correlated with mesenchymal markers in NSCLC cell lines in Western blots. PI3K was identified as a relevant pathway of proliferation networks involving ZNF71 and its isoforms formulated with CRISPR-Cas9 and RNA interference (RNAi) profiles. Based on the gene expression of the multi-omics network, repositioning drugs were identified for NSCLC treatment.

The pluripotent transcription factor NANOG is essential for maintaining embryonic stem cells and driving tumorigenesis. We previously showed that PKC activity is involved in the regulation of NANOG expression. To explore the possible involvement of microRNAs in regulating the expression of key pluripotency factors, we performed a genome-wide analysis of microRNA expression in the embryonal carcinoma cell line NT2/D1 in the presence of the PKC activator, PMA. We found that MIR630 was significantly upregulated in PMA-treated cells. Experimentally, we showed that transfection of MIR630 mimic into embryonal carcinoma cell lines directly targeted the 3'UTR of OCT4, SOX2, and NANOG and markedly suppressed their expression. RNAhybrid and RNA22 algorithms were used to predict miRNA target sites in the NANOG 3'UTR, four possible target sites of MIR630 were identified. To examine the functional interaction between MIR630 and NANOG mRNA, the predicted MIR630 target sites in the NANOG 3'UTR were deleted and the activity of the reporters were compared. After targeted mutation of the predicted MIR630 target sites, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. It is worth noting that mutation of a single putative binding site in the 3'UTR of NANOG did not completely abolish MIR630-mediated suppression, suggesting that MIR630 in the NANOG 3'UTR may have multiple binding sites and act together to maximally repress NANOG expression. Interestingly, MIR630 mimics significantly downregulated NANOG gene transcription. Exogenous expression of OCT4, SOX2, and NANOG lacking the 3'UTR almost completely rescued the reduced transcriptional activity of MIR630. MIR630 mediated the expression of differentiation markers in NT2/D1 cells, suggesting that MIR630 leads to the differentiation of NT2/D1 cell. Our findings show that MIR630 represses NANOG through transcriptional and post-transcriptional regulation, suggesting a direct link between core pluripotency factors and MIR630.

In eukaryote cells, lipid droplets (LDs) are key intracellular organelles that dynamically regulate cellular energy homeostasis. LDs originate from the ER and continuously contact the ER during their growth. How the ER affects LD growth is largely unknown. Here, we show that RNAi knockdown of acs-1, encoding an acyl-CoA synthetase required for the biosynthesis of monomethyl branched-chain fatty acids C15iso and C17iso, remarkably prevented LD growth in Caenorhabditis elegans. Dietary C17iso, or complex lipids with C17iso including phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol, could fully restore the LD growth in the acs-1RNAi worms. Mechanistically, C17iso may incorporate into phospholipids to ensure the membrane integrity of the ER so as to maintain the function of ER-resident enzymes such as SCD/stearoyl-CoA desaturase and DGAT2/diacylglycerol acyltransferase for appropriate lipid synthesis and LD growth. Collectively, our work uncovers a unique fatty acid, C17iso, as the side chain of phospholipids for determining the ER homeostasis for LD growth in an intact organism, C. elegans., (© 2021 Zhang et al.)

Dicer-like proteins (DCLs) play a vital role in RNA interference (RNAi), by cleaving RNA filament into small RNAs. Although DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in mycoparasitic interactions is yet to be investigated. In this study, we deleted dcl genes in the mycoparasitic fungus Clonostachys rosea and characterize the functions of DCL-dependent RNAi in mycoparasitism. Deletion of dcl2 resulted in a mutant with reduced secondary metabolite production, antagonism toward the plant-pathogenic fungus Botrytis cinerea , and reduced ability to control Fusarium foot rot disease on wheat, caused by Fusarium graminearum. Transcriptome sequencing of the in vitro interaction between the C. rosea Δ dcl2 strain and B. cinerea or F. graminearum identified the downregulation of genes coding for transcription factors, membrane transporters, hydrolytic enzymes, and secondary metabolites biosynthesis enzymes putatively involved in antagonistic interactions, in comparison with the C. rosea wild-type interaction. A total of 61 putative novel microRNA-like RNAs (milRNAs) were identified in C. rosea , and 11 were downregulated in the Δ dcl2 mutant. In addition to putative endogenous gene targets, these milRNAs were predicted to target B . cinerea and F. graminearum virulence factor genes, which showed an increased expression during interaction with the Δ dcl2 mutant incapable of producing the targeting milRNAs. In summary, this study constitutes the first step in elucidating the role of RNAi in mycoparasitic interactions, with important implications for biological control of plant diseases, and poses the base for future studies focusing on the role of cross-species RNAi regulating mycoparasitic interactions. IMPORTANCE Small RNAs mediated RNA interference (RNAi) known to regulate several biological processes. Dicer-like endoribonucleases (DCLs) play a vital role in the RNAi pathway by generating sRNAs. In this study, we investigated a role of DCL-mediated RNAi in interference interactions between mycoparasitic fungus Clonostachys rosea and the two fungal pathogens Botrytis cinerea and Fusarium graminearum (here called mycohosts). We found that the dcl mutants were not able to produce 11 sRNAs predicted to finetune the regulatory network of genes known to be involved in production of hydrolytic enzymes, antifungal compounds, and membrane transporters needed for antagonistic action of C. rosea . We also found C . rosea sRNAs putatively targeting known virulence factors in the mycohosts, indicating RNAi-mediated cross-species communication. Our study expanded the understanding of underlying mechanisms of cross-species communication during interference interactions and poses a base for future works studying the role of DCL-based cross-species RNAi in fungal interactions.

Alternative RNA splicing is an important regulatory process used by genes to increase their diversity. This process is mainly executed by specific classes of RNA binding proteins that act in a dosage-dependent manner to include or exclude selected exons in the final transcripts. While these processes are tightly regulated in cells and tissues, little is known on how the dosage of these factors is achieved and maintained. Several recent studies have suggested that alternative RNA splicing may be in part modulated by microRNAs (miRNAs), which are short, non-coding RNAs (~22 nt in length) that inhibit translation of specific mRNA transcripts. As evidenced in tissues and in diseases, such as cancer and neurological disorders, the dysregulation of miRNA pathways disrupts downstream alternative RNA splicing events by altering the dosage of splicing factors involved in RNA splicing. This attractive model suggests that miRNAs can not only influence the dosage of gene expression at the post-transcriptional level but also indirectly interfere in pre-mRNA splicing at the co-transcriptional level. The purpose of this review is to compile and analyze recent studies on miRNAs modulating alternative RNA splicing factors, and how these events contribute to transcript rearrangements in tissue development and disease.

Depression is an effect of complex interactions between genetic, epigenetic and environmental factors. It is well established that stress responses are associated with multiple modest and often dynamic molecular changes in the homeostatic balance, rather than with a single genetic factor that has a strong phenotypic penetration. As depression is a multifaceted phenotype, it is important to study biochemical pathways that can regulate the overall allostasis of the brain. One such biological system that has the potential to fine-tune a multitude of diverse molecular processes is RNA interference (RNAi). RNAi is an epigenetic process showing a very low level of evolutionary diversity, and relies on the posttranscriptional regulation of gene expression using, in the case of mammals, primarily short (17-23 nucleotides) noncoding RNA transcripts called microRNAs (miRNA). In this review, our objective was to examine, summarize and discuss recent advances in the field of biomedical and clinical research on the role of miRNA-mediated regulation of gene expression in the development of depression. We focused on studies investigating post-mortem brain tissue of individuals with depression, as well as research aiming to elucidate the biomarker potential of miRNAs in depression and antidepressant response.

The transformer (tra) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina. Sex-specific tra RNA splicing is controlled by Sex lethal (Sxl) in Drosophila melanogaster but is auto-regulated in L. cuprina. Sxl also represses X chromosome dosage compensation in female D. melanogaster. We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L. cuprina. In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L. cuprina. However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos., Competing Interests: The authors have declared that no competing interests exist

Circular RNAs (circRNAs) are essential regulators associated with many cardiac conditions, including myocardial infarction (MI). This study aimed to explore circRNA expression during MI development in an animal model and in hypoxia/reoxygenation (H/R)-treated cardiomyocytes. Microarray and real-time quantitative PCR showed that the circRNA PVT1 (circPVT1) was expressed at high levels in MI tissues and H/R-triggered cardiomyocytes. Loss-of-function assays were utilized for examining the influence of circPVT1 on cardiac function and cardiomyocyte properties. Cardiac function was measured by echocardiography at 7 d after MI. Reduced circPVT1 expression significantly decreased MI-triggered myocardial infarct size by 60% and prevented MI-triggered reductions in fractional shortening (%FS) and ejection fraction (EF%). Results of LDH, CCK-8, EdU staining, colony formation assays, and flow cytometry showed that circPVT1 silencing restored cell viability and proliferation while decreased apoptosis. Mechanistic experiments indicated that microRNAs (miR)-125b and miR-200a associated with circPVT1. We demonstrated that circPVT1 functioned as a competitive endogenous RNA (ceRNA) to sponge both miR-125b and miR-200a. Gain-of-function assays showed that miR-125b and miR-200a upregulation partially eliminated the effects of circPVT1 on cardiomyocyte properties. In addition, we found that the previously reported p53/TRAF6, SIRT7, Keap1/Nrf2, and PDCD4 pathways were regulated by the circPVT1/miR-125b/miR-200a axis. In conclusion, our study suggests that circPVT1 protects the myocardium from MI and H/R injury by preventing miR-125b- and miR-200a-mediated apoptotic signaling., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The proper regulation of RNA processing is critical for muscle development and the fine-tuning of contractile ability among muscle fiber-types. RNA binding proteins (RBPs) regulate the diverse steps in RNA processing, including alternative splicing, which generates fiber-type specific isoforms of structural proteins that confer contractile sarcomeres with distinct biomechanical properties. Alternative splicing is disrupted in muscle diseases such as myotonic dystrophy and dilated cardiomyopathy and is altered after intense exercise as well as with aging. It is therefore important to understand splicing and RBP function, but currently, only a small fraction of the hundreds of annotated RBPs expressed in muscle have been characterized. Here, we demonstrate the utility of Drosophila as a genetic model system to investigate basic developmental mechanisms of RBP function in myogenesis. We find that RBPs exhibit dynamic temporal and fiber-type specific expression patterns in mRNA-Seq data and display muscle-specific phenotypes. We performed knockdown with 105 RNAi hairpins targeting 35 RBPs and report associated lethality, flight, myofiber and sarcomere defects, including flight muscle phenotypes for Doa , Rm62 , mub , mbl , sbr , and clu . Knockdown phenotypes of spliceosome components, as highlighted by phenotypes for A-complex components SF1 and Hrb87F (hnRNPA1), revealed level- and temporal-dependent myofibril defects. We further show that splicing mediated by SF1 and Hrb87F is necessary for Z-disc stability and proper myofibril development, and strong knockdown of either gene results in impaired localization of kettin to the Z-disc. Our results expand the number of RBPs with a described phenotype in muscle and underscore the diversity in myofibril and transcriptomic phenotypes associated with splicing defects. Drosophila is thus a powerful model to gain disease-relevant insight into cellular and molecular phenotypes observed when expression levels of splicing factors, spliceosome components and splicing dynamics are altered.

Eukaryote-eukaryote endosymbiosis was responsible for the spread of chloroplast (plastid) organelles. Stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms that act to stabilize emergent endosymbioses-between two fundamentally selfish biological organisms-are unclear. Theory suggests that enforcement mechanisms, which punish misbehavior, may act to stabilize such interactions by resolving conflict. However, how such mechanisms can emerge in a facultative endosymbiosis has yet to be explored. Here, we propose that endosymbiont-host RNA-RNA interactions, arising from digestion of the endosymbiont population, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model facultative endosymbiosis between Paramecium bursaria and Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) system. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived messenger RNA (mRNA) released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We predict multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate through delivery of synthetic endosymbiont sRNA that exposure to these regions can knock down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knockdown in response to endosymbiont-derived RNA processing by host RNAi factors, which we term "RNAi collisions," represents a mechanism that can promote stability in a facultative eukaryote-eukaryote endosymbiosis. Specifically, by imposing a cost for breakdown of the endosymbiosis, endosymbiont-host RNA-RNA interactions may drive maintenance of the symbiosis across fluctuating ecological conditions., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Epithelial ovarian cancer (EOC) outpaces all the other forms of the female reproductive system malignancies. MicroRNAs have emerged as promising predictive biomarkers to therapeutic treatments as their expression might characterize the tumor stage or grade. In EOC, miR-200c is considered a master regulator of oncogenes or tumor suppressors. To investigate novel miR-200c-3p target genes involved in EOC tumorigenesis, we evaluated the association between this miRNA and the mRNA expression of several potential target genes by RNA-seq data of both 46 EOC cell lines from Cancer Cell line Encyclopedia (CCLE) and 456 EOC patient bio-specimens from The Cancer Genome Atlas (TCGA). Both analyses showed a significant anticorrelation between miR-200c-3p and the protein phosphatase 3 catalytic subunit γ of calcineurin (PPP3CC) levels involved in the apoptosis pathway. Quantitative mRNA expression analysis in patient biopsies confirmed the inverse correlation between miR-200c-3p and PPP3CC levels. In vitro regulation of PPP3CC expression through miR-200c-3p and RNA interference technology led to a concomitant modulation of BCL2- and p-AKT-related pathways, suggesting the tumor suppressive role of PPP3CC in EOC. Our results suggest that inhibition of high expression of miR-200c-3p in EOC might lead to overexpression of the tumor suppressor PPP3CC and subsequent induction of apoptosis in EOC patients.

SARS-CoV-2 has mutually illuminated our collective knowledge and knowledge gaps, particularly in antiviral defense and therapeutic strategies. A recent study in Science (Poirier et al., 2021) uncovers an ancient antiviral mechanism that mammals utilize to suppress viruses, including SARS-CoV-2 and Zika virus, that could have broad implications for therapeutic strategies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

RNA interference (RNAi) is an across-kingdom gene regulatory and defense mechanism. However, little is known about how organisms sense initial cues to mobilize RNAi. Here, we show that wounding to Nicotiana benthamiana cells during virus intrusion activates RNAi-related gene expression through calcium signaling. A rapid wound-induced elevation in calcium fluxes triggers calmodulin-dependent activation of calmodulin-binding transcription activator-3 (CAMTA3), which activates RNA-dependent RNA polymerase-6 and Bifunctional nuclease-2 (BN2) transcription. BN2 stabilizes mRNAs encoding key components of RNAi machinery, notably AGONAUTE1/2 and DICER-LIKE1, by degrading their cognate microRNAs. Consequently, multiple RNAi genes are primed for combating virus invasion. Calmodulin-, CAMTA3-, or BN2-knockdown/knockout plants show increased susceptibility to geminivirus, cucumovirus, and potyvirus. Notably, Geminivirus V2 protein can disrupt the calmodulin-CAMTA3 interaction to counteract RNAi defense. These findings link Ca 2+ signaling to RNAi and reveal versatility of host antiviral defense and viral counter-defense., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Regeneration requires the coordination of stem cells, their progeny and distant differentiated tissues. Here, we present a comprehensive atlas of whole-body regeneration in Schmidtea mediterranea and identify wound-induced cell states. An analysis of 299,998 single-cell transcriptomes captured from regeneration-competent and regeneration-incompetent fragments identified transient regeneration-activated cell states (TRACS) in the muscle, epidermis and intestine. TRACS were independent of stem cell division with distinct spatiotemporal distributions, and RNAi depletion of TRACS-enriched genes produced regeneration defects. Muscle expression of notum, follistatin, evi/wls, glypican-1 and junctophilin-1 was required for tissue polarity. Epidermal expression of agat-1/2/3, cyp3142a1, zfhx3 and atp1a1 was important for stem cell proliferation. Finally, expression of spectrinβ and atp12a in intestinal basal cells, and lrrk2, cathepsinB, myosin1e, polybromo-1 and talin-1 in intestinal enterocytes regulated stem cell proliferation and tissue remodelling, respectively. Our results identify cell types and molecules that are important for regeneration, indicating that regenerative ability can emerge from coordinated transcriptional plasticity across all three germ layers., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Spodoptera exigua is a worldwide pest afflicting edible vegetables and has developed varying levels of resistance to insecticides. Methoxyfenozide (MET), an ecdysteroid agonist, is effective against lepidopteran pests such as S. exigua . However, the mechanism of MET to S. exigua remains unclear. In this study, we analyzed the expression patterns of genes related to the ecdysone signaling pathway in transcriptome data treated with sublethal doses of MET and analyzed how expression levels of key genes affect the toxicity of MET on S. exigua . Our results demonstrated that 2639 genes were up-regulated and 2512 genes were down-regulated in S. exigua treated with LC 30 of MET. Of these, 15 genes were involved in the ecdysone signaling pathway. qPCR results demonstrated that ecdysone receptor A ( EcRA ) expression levels significantly increased in S. exigua when treated with different doses of MET, and that the RNAi-mediated silencing of EcRA significantly increased mortality to 55.43% at 72 h when L3 S. exigua larvae were exposed to MET at the LC 30 dose. Additionally, knocking down EcRA suppressed the most genes expressed in the ecdysone signaling pathway. The combination of MET and ds EcRA affected the expression of E74 and enhanced the expression of TREA . These results demonstrate that the adverse effects of sublethal MET disturb the ecdysone signaling pathway in S. exigua , and EcRA is closely related to MET toxic effect. This study increases our collective understanding of the mechanisms of MET in insect pests.

Foot-and-mouth disease virus (FMDV) is one of the most important animal pathogens in the world. FMDV naturally infects swine, cattle, and other cloven-hoofed animals. FMD is not adequately controlled by vaccination. An alternative strategy is to develop swine that are genetically resistant to infection. Here, we generated FMDV-specific shRNA transgenic cells targeting either nonstructural protein 2B or polymerase 3D of FMDV. The shRNA-positive transgenic cells displayed significantly lower viral production than that of the control cells after infection with FMDV (P < 0.05). Twenty-three transgenic cloned swine (TGCS) and nine non-transgenic cloned swine (Non-TGCS) were produced by somatic cell nuclear transfer (SCNT). In the FMDV challenge study, one TGCS was completely protected, no clinical signs, no viremia and no viral RNA in the tissues, no non-structural antibody response, another one TGCS swine recovered after showing clinical signs for two days, whereas all of the normal control swine (NS) and Non-TGCS developed typical clinical signs, viremia and viral RNA was determined in the tissues, the non-structural antibody was determined, and one Non-TGCS swine died. The viral RNA load in the blood and tissues of the TGCS was reduced in both challenge doses. These results indicated that the TGCS displayed resistance to the FMDV infection. Immune cells, including CD3 + , CD4 + , CD8 + , CD21 + , and CD172 + cells, and the production of IFN-γ were analyzed, there were no significant differences observed between the TGCS and NS or Non-TGCS, suggesting that the FMDV resistance may be mainly derived from the RNAi-based antiviral pathway. Our work provides a foundation for a breeding approach to preventing infectious disease in swine., (© 2021. The Author(s).)

The approval of the first small interfering RNA (siRNA) drug Patisiran by FDA in 2018 marks a new era of RNA interference (RNAi) therapeutics. MicroRNAs (miRNA), an important post-transcriptional gene regulator, are also the subject of both basic research and clinical trials. Both siRNA and miRNA mimics are ~21 nucleotides RNA duplexes inducing mRNA silencing. Given the well performance of siRNA, researchers ask whether miRNA mimics are unnecessary or developed siRNA technology can pave the way for the emergence of miRNA mimic drugs. Through comprehensive comparison of siRNA and miRNA, we focus on (1) the common features and lessons learnt from the success of siRNAs; (2) the unique characteristics of miRNA that potentially offer additional therapeutic advantages and opportunities; (3) key areas of ongoing research that will contribute to clinical application of miRNA mimics. In conclusion, miRNA mimics have unique properties and advantages which cannot be fully matched by siRNA in clinical applications. MiRNAs are endogenous molecules and the gene silencing effects of miRNA mimics can be regulated or buffered to ameliorate or eliminate off-target effects. An in-depth understanding of the differences between siRNA and miRNA mimics will facilitate the development of miRNA mimic drugs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Pseudomonas aeruginosa ( Pae ) is an opportunistic pathogen showing a high intrinsic resistance to a wide variety of antibiotics. It causes nosocomial infections that are particularly detrimental to immunocompromised individuals and to patients suffering from cystic fibrosis. We provide a snapshot on regulatory RNAs of Pae that impact on metabolism, pathogenicity and antibiotic susceptibility. Different experimental approaches such as in silico predictions, co-purification with the RNA chaperone Hfq as well as high-throughput RNA sequencing identified several hundreds of regulatory RNA candidates in Pae . Notwithstanding, using in vitro and in vivo assays, the function of only a few has been revealed. Here, we focus on well-characterized small base-pairing RNAs, regulating specific target genes as well as on larger protein-binding RNAs that sequester and thereby modulate the activity of translational repressors. As the latter impact large gene networks governing metabolism, acute or chronic infections, these protein-binding RNAs in conjunction with their cognate proteins are regarded as global post-transcriptional regulators.

Drosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct-processive or distributive-modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2's cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated., (© 2021. The Author(s).)

The treatment of complex diseases often relies on combinatorial therapy, a strategy where drugs are used to target multiple genes simultaneously. Promising candidate genes for combinatorial perturbation often constitute epistatic genes, i.e., genes which contribute to a phenotype in a non-linear fashion. Experimental identification of the full landscape of genetic interactions by perturbing all gene combinations is prohibitive due to the exponential growth of testable hypotheses. Here we present a model for the inference of pairwise epistatic, including synthetic lethal, gene interactions from siRNA-based perturbation screens. The model exploits the combinatorial nature of siRNA-based screens resulting from the high numbers of sequence-dependent off-target effects, where each siRNA apart from its intended target knocks down hundreds of additional genes. We show that conditional and marginal epistasis can be estimated as interaction coefficients of regression models on perturbation data. We compare two methods, namely glinternet and xyz, for selecting non-zero effects in high dimensions as components of the model, and make recommendations for the appropriate use of each. For data simulated from real RNAi screening libraries, we show that glinternet successfully identifies epistatic gene pairs with high accuracy across a wide range of relevant parameters for the signal-to-noise ratio of observed phenotypes, the effect size of epistasis and the number of observations per double knockdown. xyz is also able to identify interactions from lower dimensional data sets (fewer genes), but is less accurate for many dimensions. Higher accuracy of glinternet, however, comes at the cost of longer running time compared to xyz. The general model is widely applicable and allows mining the wealth of publicly available RNAi screening data for the estimation of epistatic interactions between genes. As a proof of concept, we apply the model to search for interactions, and potential targets for treatment, among previously published sets of siRNA perturbation screens on various pathogens. The identified interactions include both known epistatic interactions as well as novel findings., Competing Interests: JJ is an employee of Novartis Institutes for BioMedical Research, which provided support in the form of the salary, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Geminiviruses are plant viruses with limited coding capacity. Geminivirus-encoded proteins are traditionally identified by applying a 10-kDa arbitrary threshold; however, it is increasingly clear that small proteins play relevant roles in biological systems, which calls for the reconsideration of this criterion. Here, we show that geminiviral genomes contain additional ORFs. Using tomato yellow leaf curl virus, we demonstrate that some of these small ORFs are expressed during the infection, and that the encoded proteins display specific subcellular localizations. We prove that the largest of these additional ORFs, which we name V3, is required for full viral infection, and that the V3 protein localizes in the Golgi apparatus and functions as an RNA silencing suppressor. These results imply that the repertoire of geminiviral proteins can be expanded, and that getting a comprehensive overview of the molecular plant-geminivirus interactions will require the detailed study of small ORFs so far neglected., (© 2021. The Author(s).)

Stable epigenetic changes appear uncommon, suggesting that changes typically dissipate or are repaired. Changes that stably alter gene expression across generations presumably require particular conditions that are currently unknown. Here we report that a minimal combination of cis-regulatory sequences can support permanent RNA silencing of a single-copy transgene and its derivatives in C. elegans simply upon mating. Mating disrupts competing RNA-based mechanisms to initiate silencing that can last for >300 generations. This stable silencing requires components of the small RNA pathway and can silence homologous sequences in trans. While animals do not recover from mating-induced silencing, they often recover from and become resistant to trans silencing. Recovery is also observed in most cases when double-stranded RNA is used to silence the same coding sequence in different regulatory contexts that drive germline expression. Therefore, we propose that regulatory features can evolve to oppose permanent and potentially maladaptive responses to transient change., (© 2021. The Author(s).)

The neurohormone octopamine regulates many crucial physiological processes in insects and exerts its activity via typical G-protein coupled receptors. The roles of octopamine receptors in regulating behavior and physiology in Coleoptera (beetles) need better understanding. We used the red flour beetle, Tribolium castaneum , as a model species to study the contribution of the octopamine receptor to behavior and physiology. We cloned the cDNA of a β-adrenergic-like octopamine receptor ( TcOctβ2R ). This was heterologously expressed in human embryonic kidney (HEK) 293 cells and was demonstrated to be functional using an in vitro cyclic AMP assay. In an RNAi assay, injection of dsRNA demonstrated that TcOctβ2R modulates beetle locomotion, mating duration, and fertility. These data present some roles of the octopaminergic signaling system in T. castaneum . Our findings will also help to elucidate the potential functions of individual octopamine receptors in other insects.

Physiologically based pharmacokinetic (PBPK) modeling is a powerful tool with many demonstrated applications in various phases of drug development and regulatory review. RNA interference (RNAi)-based therapeutics are a class of drugs that have unique pharmacokinetic properties and mechanisms of action. With an increasing number of RNAi therapeutics in the pipeline and reaching the market, there is a considerable amount of active research in this area requiring a multidisciplinary approach. The application of PBPK models for RNAi therapeutics is in its infancy and its utility to facilitate the development of this new class of drugs is yet to be fully evaluated. From this perspective, we briefly discuss some of the current computational modeling approaches used in support of efficient development and approval of RNAi therapeutics. Considerations for PBPK model development are highlighted both in a relative context between small molecules and large molecules such as monoclonal antibodies and as it applies to RNAi therapeutics. In addition, the prospects for drawing upon other recognized avenues of PBPK modeling and some of the foreseeable challenges in PBPK model development for these chemical modalities are briefly discussed. Finally, an exploration of the potential application of PBPK model development for RNAi therapeutics is provided. We hope these preliminary thoughts will help initiate a dialogue between scientists in the relevant sectors to examine the value of PBPK modeling for RNAi therapeutics. Such evaluations could help standardize the practice in the future and support appropriate guidance development for strengthening the RNAi therapeutics development program., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The genome could be considered as raw data expressed in proteins and various types of noncoding RNAs (ncRNAs). However, a large portion of the genome is dedicated to ncRNAs, which in turn represent a considerable amount of the transcriptome. ncRNAs are modulated on levels of type and amount whenever any physiological process occurs or as a response to external modulators. ncRNAs, typically forming complexes with other partners, are key molecules that influence diverse cellular processes. Based on the knowledge of mammalian biology, ncRNAs are known to regulate and control diverse trafficking pathways and cellular activities. Long noncoding RNAs (lncRNAs) notably have diverse and more regulatory roles than microRNAs. Expanding these studies on fish has derived the same conclusion with relevance to other species, including invertebrates, explored the potentials to harness such types of RNA to further understand the biology of such organisms, and opened gates for applying recent technologies, such as RNA interference and delivering micromolecules as microRNAs to living cells and possibly to target organs. These technologies should improve aquaculture productivity and fish health, as well as help understand fish biology.

Context: Healthy hyperplasic (many but smaller fat cells) white adipose tissue (WAT) expansion is mediated by recruitment, proliferation and/or differentiation of new fat cells. This process (adipogenesis) is controlled by transcriptional programs that have been mostly identified in rodents., Objective: A systemic investigation of adipogenic human transcription factors (TFs) that are relevant for metabolic conditions has not been revealed previously., Methods: TFs regulated in WAT by obesity, adipose morphology, cancer cachexia, and insulin resistance were selected from microarrays. Their role in differentiation of human adipose tissue-derived stem cells (hASC) was investigated by RNA interference (RNAi) screen. Lipid accumulation, cell number, and lipolysis were measured for all screened factors (148 TFs). RNA (RNAseq), protein (Western blot) expression, insulin, and catecholamine responsiveness were examined in hASC following siRNA treatment of selected target TFs., Results: Analysis of TFs regulated by metabolic conditions in human WAT revealed that many of them belong to adipogenesis-regulating pathways. The RNAi screen identified 39 genes that affected fat cell differentiation in vitro, where 11 genes were novel. Of the latter JARID2 stood out as being necessary for formation of healthy fat cell metabolic phenotype by regulating expression of multiple fat cell phenotype-specific genes., Conclusion: This comprehensive RNAi screening in hASC suggests that a large proportion of WAT TFs that are impacted by metabolic conditions might be important for hyperplastic adipose tissue expansion. The screen also identified JARID2 as a novel TF essential for the development of functional adipocytes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

In eukaryotes, histones and their variants are essential for chromatin structure and function; both play important roles in the regulation of gene transcription, as well as the development of tumors. We aimed to explore the genomics data of hepatocellular carcinoma (HCC), combined with literature analysis, in terms of the histone variant H2A.Z. Cell phenotype assay confirmed the effect of H2A.Z on the proliferation, metastasis, apoptosis, and cell cycle of HCC cells. H2A.Z was shown to function via the tumor dysregulation signaling pathway, with BCL6 as its interacting protein. In addition, the acetylation level of H2A.Z was higher in HCC and was related to tumor formation. We found the acetylation of H2A.Z to be related to and regulated by lincZNF337-AS1. LincZNF337-AS1 was found to bind to H2A.Z and KAT5 at different sites, promoting the acetylation of H2A.Z through KAT5. We concluded that, in HCC, H2A.Z is an oncogene, whose acetylation promotes the transcription of downstream genes, and is regulated by lincZNF331-AS1.

In late 2019, the betacoronavirus SARS-CoV-2 was identified as the viral agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. Coronaviruses Spike proteins are responsible for their ability to interact with host membrane receptors and different proteins have been identified as SARS-CoV-2 interactors, among which Angiotensin-converting enzyme 2 (ACE2), and Basigin2/EMMPRIN/CD147 (CD147). CD147 plays an important role in human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, Kaposi's sarcoma-associated herpesvirus, and severe acute respiratory syndrome coronavirus infections. In particular, SARS-CoV recognizes the CD147 receptor expressed on the surface of host cells by its nucleocapsid protein binding to cyclophilin A (CyPA), a ligand for CD147. However, the involvement of CD147 in SARS-CoV-2 infection is still debated. Interference with both the function (blocking antibody) and the expression (knock down) of CD147 showed that this receptor partakes in SARS-CoV-2 infection and provided additional clues on the underlying mechanism: CD147 binding to CyPA does not play a role; CD147 regulates ACE2 levels and both receptors are affected by virus infection. Altogether, these findings suggest that CD147 is involved in SARS-CoV-2 tropism and represents a possible therapeutic target to challenge COVID-19.

Transgenes that are stably expressed in plant genomes over many generations could be assumed to behave epigenetically the same as endogenous genes. Here, we report that whereas the histone H3K9me2 demethylase IBM1, but not the histone H3K4me3 demethylase JMJ14, counteracts DNA methylation of Arabidopsis endogenous genes, JMJ14, but not IBM1, counteracts DNA methylation of expressed transgenes. Additionally, JMJ14-mediated specific attenuation of transgene DNA methylation enhances the production of aberrant RNAs that readily induce systemic post-transcriptional transgene silencing (PTGS). Thus, the JMJ14 chromatin modifying complex maintains expressed transgenes in a probationary state of susceptibility to PTGS, suggesting that the host plant genome does not immediately accept expressed transgenes as being epigenetically the same as endogenous genes.

Spinal muscular atrophy (SMA) is a debilitating neurological disorder marked by degeneration of spinal motor neurons and muscle atrophy. SMA results from mutations in survival motor neuron 1 (SMN1), leading to deficiency of survival motor neuron (SMN) protein. Current therapies increase SMN protein and improve patient survival but have variable improvements in motor function, making it necessary to identify complementary strategies to further improve disease outcomes. Here, we perform a genome-wide RNAi screen using a luciferase-based activity reporter and identify genes involved in regulating SMN gene expression, RNA processing, and protein stability. We show that reduced expression of Transcription Export complex components increases SMN levels through the regulation of nuclear/cytoplasmic RNA transport. We also show that the E3 ligase, Neurl2, works cooperatively with Mib1 to ubiquitinate and promote SMN degradation. Together, our screen uncovers pathways through which SMN expression is regulated, potentially revealing additional strategies to treat SMA., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Ticks are the primary vector of arboviruses in temperate climates worldwide. They are both the vector of these pathogens to humans and an integral component of the viral sylvatic cycle. Understanding the tick-pathogen interaction provides information about the natural maintenance of these pathogens and informs the development of countermeasures against human infection. In this review, we discuss currently available information on tick-viral interactions within the broader scope of general tick immunology. While the tick immune response to several pathogens has been studied extensively, minimal work centres on responses to viral infection. This is largely due to the high pathogenicity of tick-borne viruses; this necessitates high-containment laboratories or low-pathogenicity substitute viruses. This has biased most research towards tick-borne flaviviruses. More work is required to fully understand the role of tick-virus interaction in sylvatic cycling and transmission of diverse tick-borne viruses., (© 2020 John Wiley & Sons Ltd.)

Double-stranded RNAs (dsRNAs) represent a promising class of biosafe insecticidal compounds. We examined the ability to induce RNA interference (RNAi) in the pollen beetle Brassicogethes aeneus via anther feeding, and compared short-term (3 d) to chronic (17 d) feeding of various concentrations of dsRNA targeting αCOP (dsαCOP). In short-term dsαCOP feeding, only the highest concentration resulted in significant reductions in B. aeneus survival; whereas in chronic dsαCOP feeding, all three concentrations resulted in significant mortality. Chronic dsαCOP feeding also resulted in significantly greater mortality compared to short-term feeding of equivalent dsαCOP concentrations. Our results have implications for the economics and development of dsRNA spray approaches for managing crop pests, in that multiple lower-concentration dsRNA spray treatments across crop growth stages may result in greater pest management efficacy, compared to single treatments using higher dsRNA concentrations. Furthermore, our results highlight the need for research into the development of RNAi cultivars for oilseed rape protection, given the enhanced RNAi efficacy resulting from chronic, compared to short-term, dsRNA feeding in B. aeneus.

The viruses utilize multiple cellular proteins to facilitate their proliferation. The Heat Shock Protein (HSP), the highly conserved protein in eukaryotes and prokaryotes, plays a critical role in facilitating viral proliferation. However, less is known about the role of the HSPs in the life cycles of the Baculoviruses. We constructed recombinant Bombyx mori nucleopolyhedrovirus and discovered the Heat Shock Protein 75 (TRAP1) in the B. mori ovary (BmN) cells by the co-immunoprecipitation experiment using the GP64 (glycoprotein 64) as the bait protein. Tissue expression profile analysis of B. mori indicated that the TRAP1 gene has higher expression levels in the ovary, midgut, and hemolymph. Down-regulation of TRAP1 via RNA interference (RNAi) and geldanamycin (GA, a TRAP1 inhibitor) treatment can reduce the expression level of the major capsid protein VP39 (viral protein 39) of BmNPV. In contrast, the up-regulation of TRAP1 via overexpression can increase the expression level of the VP39. These results indicated that the TRAP1 of B. mori could facilitate the proliferation of the BmNPV. This study provided new insights into the function of TRAP1, and the basic mechanisms of the baculoviruses life cycle for disease prevention., Competing Interests: Declaration of competing interest Authors declare that they have no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

Prescription opioids are powerful pain-controlling medications that have both benefits and potentially serious risks. Morphine is one of the preferred analgesics that are widely used to treat chronic pain. However, chronic morphine exposure has been found to cause both functional and structural changes in several brain regions, including the medial prefrontal cortex (mPFC), ventral tegmental area (VTA), and hippocampus (HPC), which lead to addictive behavior. Caveolin-1 (Cav-1), a scaffolding protein of membrane lipid rafts (MLRs), has been shown to organize GPCRs and multiple synaptic signaling proteins within the MLRs to regulate synaptic signaling and neuroplasticity. Previously, we showed that in vitro morphine treatment significantly elevates Cav-1 expression and causes neuroplasticity changes. In this study, we confirmed that chronic morphine exposure can significantly increase Cav-1 expression (P < 0.05) and microtubule-associated protein (MAP-2)-positive neuronal dendritic growth in the hippocampus. Moreover, the rewarding effect and dendritic growth in the HPC induced by chronic morphine exposure were significantly inhibited by hippocampal Cav-1 knockdown. Together, these data suggest that Cav-1 in the hippocampus plays an essential role in the neuroplasticity changes that underlie morphine addiction behaviors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aims: Polo-like kinase 2 (PLK2) belongs to a family of serine/threonine kinases, and it is involved in tumorigenesis. The present study aimed to explore the potential clinical significance of PLK2 in the development of gliomas., Main Methods: Immunohistochemistry (IHC) was performed to detect the expression of PLK2 in glioma tissues. Cell proliferation and apoptosis were determined by Cell Counting Kit 8 (CCK8) and flow cytometry analysis, respectively., Key Findings: PLK2 expression gradually increased with the degree of glioma malignancy. High PLK2 expression was associated with a poor prognosis in glioma. Short hairpin RNAs targeting PLK2 (shPLK2) inhibited the viability and induced apoptosis of glioma cells, both in vitro and in vivo. Ring finger protein 180 (RNF180), an E3 ubiquitin ligase, interacted with PLK2 and induced the ubiquitination of PLK2. Overexpression of PLK2 in glioma cells significantly inhibited RNF180 upregulation-induced cell apoptosis. The expression level of RNF180 gradually decreased with the degree of glioma malignancy., Significance: Knocking down of PLK2 may suppress the glioma development through cancer cell proliferation inhibition and cell apoptosis promotion. Furthermore, RNF180 may mediate the ubiquitination of PLK2. The present findings may help improve the clinical management of glioma in the future., (Copyright © 2021. Published by Elsevier Inc.)

Insect pest control by RNA interference (RNAi)-mediated gene expression knockdown can be undermined by many factors, including small sequence differences between double-stranded RNA (dsRNA) and the target gene. It can also be compromised by effects that are independent of the dsRNA sequence on non-target organisms (known as sequence-non-specific effects). This study investigated the species-specificity of RNAi in plant sap-feeding hemipteran pests. We first demonstrated sequence-non-specific suppression of aphid feeding by dsRNA at dietary concentrations ≥0.5 µg µL -1 . Then we quantified the expression of NUC (nuclease) genes in insects administered homologous dsRNA (with perfect sequence identity to the target species) or heterologous dsRNA (generated against a related gene of non-identical sequence in a different insect species). For the aphids Acyrthosiphon pisum and Myzus persicae , significantly reduced NUC expression was obtained with the homologous but not heterologous dsRNA at 0.2 µg µL -1 , despite high ds NUC sequence identity. Follow-up experiments demonstrated significantly reduced expression of NUC genes in the whitefly Bemisia tabaci and mealybug Planococcus maritimus administered homologous dsNUCs , but not heterologous aphid ds NUC s. Our demonstration of inefficient expression knockdown by heterologous dsRNA in these insects suggests that maximal dsRNA sequence identity is required for RNAi targeting of related pest species, and that heterologous dsRNAs at appropriate concentrations may not be a major risk to non-target sap-feeding hemipterans.

Rationale: Estrogen-dependent cancers (e.g., breast, endometrial, and ovarian cancers) are among the leading causes of morbidity and mortality in women worldwide. Recently, exosomes released by tumor-infiltrating CD8 + T cells have been under the spotlight in the field of cancer immunotherapy. Our study aims at elucidating the underlying mechanisms of the crosstalk between estrogen signaling and CD8 + T cells, and possible intervention values in uterine corpus endometrial cancer (UCEC). Methods: Micro RNA-seq was conducted to screen differentially expressed micro RNA in UCEC. Bioinformatic analysis was processed to predict the target of miR-765. RNA silencing or overexpressing and pharmacologic inhibitors were used to assess the functions of ERβ/miR-765/PLP2/Notch axis in UCEC cell proliferation and invasion in vivo and in vitro . In vivo imaging was performed to evaluate the metastasis of tumor in mice. Combined fluorescent in situ hybridization for miR-765 and immunofluorescent labeling for CD8 was carried out to prove the co-localization between miR-765 and CD8 + T cells. Exosomes derived from CD45RO - CD8 + T cells were isolated to detect the regulatory effects on UCEC. Results: miR-765 is characterized as the most downregulated miRNA in UCEC, and there is a negative correlation between miR-765 and Proteolipid protein 2 (PLP2) in UCEC lesion. Estrogen significantly down-regulates miR-765 level, and facilitates the development of UCEC by estrogen receptor (ER) β. Mechanistically, this process is mediated through the miRNAs (e.g., miR-3584-5p, miR-7-5p, miR-150-5p, and miR-124-3p) cluster-controlled regulation of the PLP2, which further regulates Ki-67 and multiple epithelial-mesenchymal transition (EMT)-related molecules (e.g, E-cadherin and Vimentin) in a Notch signaling pathway-dependent manner. Interestingly, the selective ER degrader Fulvestrant alleviates estrogen-mediated miR-765/PLP2 expression regulation and UCEC development in ERβ-dependent and -independent manners. Additionally, CD45RO - CD8 + T cell-derived exosomes release more miR-765 than that from CD45RO + CD8 + T cells. In therapeutic studies, these exosomes limit estrogen-driven disease development via regulation of the miR-765/PLP2 axis. Conclusions: This observation reveals novel molecular mechanisms underlying estrogen signaling and CD8 + T cell-released exosomes in UCEC development, and provides a potential therapeutic strategy for UCEC patients with aberrant ERβ/miR-765/PLP2/Notch signaling axis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Heat shock 70kDa proteins (HSP70s) and their cochaperones DNAJs are ubiquitous molecular chaperones, which function as the "HSP70/DNAJ machinery" in a myriad of biological processes. At present, a number of HSP70s have been classified in many species, but studies on DNAJs, especially in insects, are lacking. Here, we first systematically identified and characterized the HSP70 and DNAJ family members in the brown planthopper (BPH), Nilaparvata lugens , a destructive rice pest in Asia. A total of nine HSP70 and 31 DNAJ genes were identified in the BPH genome. Sequence and phylogenetic analyses revealed the high diversity of the NlDNAJ family. Additionally, spatio-temporal expression analysis showed that most NlHSP70 and NlDNAJ genes were highly expressed in the adult stage and gonads. Furthermore, RNA interference (RNAi) revealed that seven NlHSP70s and 10 NlDNAJs play indispensable roles in the nymphal development, oogenesis, and female fertility of N. lugens under physiological growth conditions; in addition, one HSP70 ( NlHSP68 ) was found to be important in the thermal tolerance of eggs. Together, our results in this study shed more light on the biological roles of HSP70/DNAJ in regulating life cycle, coping with environmental stresses, and mediating the interactions within, or between, the two gene families in insects.

Chloroplasts cannot develop normally without the coordinated action of various proteins and signaling connections between the nucleus and the chloroplast genome. Many questions regarding these processes remain unanswered. Here, we report a novel P-type pentatricopeptide repeat (PPR) factor, named Albino Cotyledon Mutant1 (ACM1), which is encoded by a nuclear gene and involved in chloroplast development. Knock-down of ACM1 transgenic plants displayed albino cotyledons but normal true leaves, while knock-out of the ACM1 gene in seedlings was lethal. Fluorescent protein analysis showed that ACM1 was specifically localized within chloroplasts. PEP-dependent plastid transcript levels and splicing efficiency of several group II introns were seriously affected in cotyledons in the RNAi line. Furthermore, denaturing gel electrophoresis and Western blot experiments showed that the accumulation of chloroplast ribosomes was probably damaged. Collectively, our results indicate ACM1 is indispensable in early chloroplast development in Arabidopsis cotyledons.