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MicroRNA-139-5p (miR-139-5p) is one of the most differentially expressed miRNAs in the brain between healthy people and depressed patients. However, its function in depression is unclear. Therefore, we investigated the function of miR-139-5p in depression. Here, miR-139-5p expression was found to be upregulated in the model group. MiR-139-5p inhibition could increase sucrose preference and decrease mice immobility time after chronic corticosterone (CORT) injection. Furthermore, compared with the antago-NC group, 3 weeks of antagomiR-139-5p treatment significantly decreased miR-139-5p level in model group hippocampus, increased sucrose preference index, reduced neuron damages, and enhanced the levels of nuclear receptor subfamily 3 group C member 1 (NR3C1), brain-derived neurotrophic factor (BDNF), phosphorylated/total tyrosine kinase receptor B (p-TrkB/TrkB), phosphorylated/total cAMP-response element-binding protein (p-CREB/CREB) and phosphorylated/total extracellular regulated protein kinases (p-ERK/ERK). Moreover, as a potential target for miR-139-5p, NR3C1 level was reduced by miR-139-5p mimic. Altogether, by activating the BDNF-TrkB signaling pathway, miR-139-5p inhibition plays an antidepressant-like role and might serve as an effective depression target (Fig. graphical abstract).

Background/Aim: Meningiomas represent the main intracranial primary central nervous system (CNS) tumour in adults worldwide. Oncogenes’ over-activation combined with suppressor genes’ silencing affect negatively the biological behavior of these neoplasms. This study aimed to explore the impact of p53 suppressor gene expression in meningiomas’ clinic-pathological features based on a combination of sophisticated techniques. Materials and Methods: Fifty (n=50) meningiomas were included in the study, comprising a broad spectrum of histopathological sub-types. An immunohistochemistry assay was applied on tissue microarray cores followed by digital image analysis. Results: p53 protein over-expression (high staining intensity levels) was observed in 27/50 (54%) cases, whereas the rest (23/50-/46%) demonstrated moderate to low levels of the protein. p53 over-expression was statistically significantly correlated to the mitotic index of the examined cases (p-value=0.001). Interestingly, the atypical/anaplastic group of histotypes demonstrated the strongest p53 expression rates compared to the others (p-value=0.001). Conclusion: p53 over-expression is observed in a broad spectrum of meningiomas. High expression levels lead to an aggressive biological behavior of the malignancy (combined with increased mitotic rates), especially in atypical and anaplastic sub-types that also have a high recurrence rate.

Wilms' tumor is the most-frequent malignant-kidney tumor in children under 3-4 years of age and is caused by genetic alterations of oncogenes (OG) and tumor-suppressor genes (TG). Wilms' tumor has been linked to many OG--TG. However, only WT1 has a proven role in the development of this embryonic-tumor.The study investigates the level of mRNA expression of 16 OGs and 20 TGs involved in key-signaling pathways, including chromatin modification; RAS; APC; Cell Cycle/Apoptosis; Transcriptional Regulation; PI3K; NOTCH--HH; PI3KRAS of 24-fresh Wilms'-tumor cases by capture-and-reporter probe Code-Sets chemistry, as CNVs in these pathway genes have been reported.Upon extensively investigating, MEN1, MLL2, MLL3, PBRM1, PRDM1, SMARCB1, SETD2, WT1, PTPN11, KRAS, HRAS, NF1, APC, RB1, FUBP1, BCOR, U2AF1, PIK3CA, PTEN, EBXW7, SMO, ALK, CBL, EP300-and-GATA1 were found to be significantly up-regulated in 58.34, 62.5, 79.17, 91.67, 58, 66.66,54, 58.34, 66.67, 75, 62.5, 62.5, 58, 79.17, 79.17, 75, 70.84, 50, 50, 75, 66.66, 62.50, 61.66, 58.34-and-62.50% of cases respectively, whereas BRAF, NF2, CDH1, BCL2, FGFR3, ERBB2, MET, RET, EGFR-and-GATA2 were significantly down regulated in 58, 87.50, 79.16, 54.16, 79.17, 91.66, 66.66, 58.33, 91.66-and-62.50% of cases, respectively. Interestingly, the WT1 gene was five-fold down regulated in 41.66% of cases only.Hence, extensive profiling of OGs and TGs association of major-signaling pathways in Wilms' tumor cases may aid in disease diagnosis. PBRM1 (up-regulated in 91.67% of cases), ERBB2 and EGFR (down-regulated in 91.66 and 91.66% of cases, respectively) could be marker genes. However, validation of all relevant results in a larger number of samples is required.

The status of DNA methylation in primary tumor tissue and adjacent tumor-free tissue is associated with the occurrence of aggressive colorectal cancer (CRC) and can aid personalized cancer treatments at early stages. Tumor tissue and matched adjacent nontumorous tissue were extracted from 208 patients with CRC, and the correlation between the methylation levels of PTGER4 and ZNF43 at certain CpG loci and the prognostic factors of CRC was determined using the MassARRAY System testing platform. The Wilcoxon signed-rank test, a Chi-square test, and McNemar’s test were used for group comparisons, and Kaplan–Meier curves and a log-rank test were used for prediction. The hypermethylation of PTGER4 at the CpG_4, CpG_5, CpG_15, and CpG_17 tumor tissue sites was strongly correlated with shorter recurrence-free survival (RFS), progression-free survival (PFS), and overall survival (OS) [hazard ratio (HR) = 3.26, 95% confidence interval (CI) = 1.38–7.73 for RFS, HR = 2.35 and 95% CI = 1.17–4.71 for PFS, HR = 4.32 and 95% CI = 1.8–10.5 for OS]. By contrast, RFS and PFS were significantly longer in the case of increased methylation of ZNF43 at the CpG_5 site of normal tissue [HR = 2.33, 95% CI = 1.07–5.08 for RFS, HR = 2.42 and 95% CI = 1.19–4.91 for PFS]. Aberrant methylation at specific CpG sites indicates tissue with aggressive behavior. Therefore, the differential methylation of PTGER4 and ZNF43 at specific loci can be employed for the prognosis of patients with CRC.

The Brassica-specific gene MS5 mediates early meiotic progression, and its allelic variants contribute to a valuable genic male sterility three-line hybrid production system in rapeseed (Brassica napus L.). However, the underlying mechanisms of its triallelic inheritance are poorly understood. Herein, we show that the restorer allele MS5a and the maintainer allele MS5c are both necessary for male fertility in B. napus. The functional divergence of MS5a and MS5c is strongly related to sequence variations in their coding regions and less strongly to their promoter regions. The male-sterile allele MS5b encodes a chimeric protein containing only the complete MS5 coiled-coil (CC) domain, having lost the MS5 superfamily domain. Both MS5a and MS5c can form homodimers in the nucleus via the CC domain. MS5b can interact competitively with MS5a or MS5c to form non-functional heterodimers. Owing to the close transcript levels of MS5b and MS5c in MS5b MS5c , these heterodimers induced a dominant-negative effect of MS5b on MS5c , resulting in a male-sterile phenotype. The extremely high transcript abundance of MS5a maintains sufficient MS5a homodimers in MS5a MS5b , causing the recovery of male sterility. These findings provide substantial genetic and molecular evidence to improve our understanding of the mechanisms underlying the multiallelic inheritance of MS5, and enable the construction of a solid foundation for improved use of the MS5-controlled GMS system in Brassica species.

Betasatellites (DNA β) are circular ssDNA molecules that are associated with monopartite geminiviruses and exert a positive effect on the viral infection. Betasatellites encode one protein, named βC1, on the complementary strand; βC1 functions as a pathogenicity factor and RNA silencing suppressor. In this report, we describe the identification of another betasatellite-encoded protein, βVl, which also contributes to symptom development. The βVl open reading frame can be found on the viral strand of approximately 40% of reported betasatellite sequences, and is conserved in position and sequence. The presence of the βVl transcript was observed in plants infected with Tomato yellow leaf curl China virus (TYTCCNV) along with its associated betasatellite Tomato yellow leaf curl China betasatellite (TYTCCNB). Mutant viruses unable to produce βVl showed reduced virulence and decreased viral load. Ectopic expression of the TYTCCNB-PV1 gene in Nicotiana benthamiana plants from a PVX-based vector resulted in leaf mosaic and chlorosis. We further demonstrated that the βVl protein could elicit hypersensitive response (HR)-type cell death in N. benthamiana leaves. Our results uncover a novel betasatellite-encoded protein that contributes to the virus infection, and this discover gives us a more complete view of the plant-geminivirus interaction landscape.

This study aimed to elucidate how SPINK5 affects the malignant phenotypes of NSCLC and the molecular mechanism. NSCLC and adjacent normal tissues were collected to detect the differential level of SPINK5. The influence of SPINK5 on pathological indicators of NSCLC was analyzed. Cellular functions of NSCLC cells overexpressing SPINK5 were assessed by CCK-8, EdU, and transwell assay. By confirming the downstream target of SPINK5, its molecular mechanism on regulating NSCLC was finally explored through rescue experiments. SPINK5 was lowly expressed in NSCLC tissues, and it predicted tumor staging and lymphatic metastasis. In vitro overexpression of SPINK5 declined proliferative and migratory rates in NSCLC cells. PSIP1 was verified as the target gene binding SPINK5, and they displayed a negative correlation in NSCLC tissues. Overexpression of PSIP1 was able to reverse the inhibited proliferative and migratory potentials in NSCLC cells overexpressing SPINK5. SPINK5 level has a close relation to tumor staging and lymphatic metastasis in NSCLC. It serves as a tumor-suppressor gene that inhibits proliferation and migration of NSCLC through negatively regulating PSIP1.

In plants and some animal lineages, RNA silencing is an efficient and adaptable defense mechanism against viruses. To counter it, viruses encode suppressor proteins that interfere with RNA silencing. Phloem-restricted viruses are spreading at an alarming rate and cause substantial reduction of crop yield, but how they interact with their hosts at the molecular level is still insufficiently understood. Here, we investigate the antiviral response against phloem-restricted turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana. Using a combination of genetics, deep sequencing, and mechanical vasculature enrichment, we show that the main axis of silencing active against TuYV involves 22-nt vsiRNA production by DCL2, and their preferential loading into AGO1. Moreover, we identify vascular secondary siRNA produced from plant transcripts and initiated by DCL2-processed AGO1-loaded vsiRNA. Unexpectedly, and despite the viral encoded VSR P0 previously shown to mediate degradation of AGO proteins, vascular AGO1 undergoes specific post-translational stabilization during TuYV infection. Collectively, our work uncovers the complexity of antiviral RNA silencing against phloem-restricted TuYV and prompts a re-assessment of the role of its suppressor of silencing P0 during genuine infection.

The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants., Pathogenic fungi rapidly develop resistance to the antifungal agent 5-fluorocytosine (5-FC). Here, Chang et al. explore the mechanisms by which Cryptococcus develops 5-FC resistance at a high frequency, including mutations in several genes and altered levels of key metabolites.

Gross Chromosomal Rearrangements (GCRs) play an important role in human diseases, including cancer. Although most of the nonessential Genome Instability Suppressing (GIS) genes in Saccharomyces cerevisiae are known, the essential genes in which mutations can cause increased GCR rates are not well understood. Here 2 S. cerevisiae GCR assays were used to screen a targeted collection of temperature-sensitive mutants to identify mutations that caused increased GCR rates. This identified 94 essential GIS (eGIS) genes in which mutations cause increased GCR rates and 38 candidate eGIS genes that encode eGIS1 protein-interacting or family member proteins. Analysis of TCGA data using the human genes predicted to encode the proteins and protein complexes implicated by the S. cerevisiae eGIS genes revealed a significant enrichment of mutations affecting predicted human eGIS genes in 10 of the 16 cancers analyzed.

Sensing misfolded proteins in the endoplasmic reticulum (ER), cells initiate the ER stress response and, when overwhelmed, undergo apoptosis. However, little is known about how cells prevent excessive ER stress response and cell death to restore homeostasis. Here, we report the identification and characterization of cellular suppressors of ER stress-induced apoptosis. Using a genome-wide CRISPR library, we screen for genes whose inactivation further increases ER stress-induced up-regulation of C/EBP homologous protein 10 (CHOP)—the transcription factor central to ER stress-associated apoptosis. Among the top validated hits are two interacting components of the polycomb repressive complex (L3MBTL2 [L(3)Mbt-Like 2] and MGA [MAX gene associated]), and microRNA-124-3 (miR-124-3). CRISPR knockout of these genes increases CHOP expression and sensitizes cells to apoptosis induced by multiple ER stressors, while overexpression confers the opposite effects. L3MBTL2 associates with the CHOP promoter in unstressed cells to repress CHOP induction but dissociates from the promoter in the presence of ER stress, whereas miR-124-3 directly targets the IRE1 branch of the ER stress pathway. Our study reveals distinct mechanisms that suppress ER stress-induced apoptosis and may lead to a better understanding of diseases whose pathogenesis is linked to overactive ER stress response.

Background RASSF1A, a tumor suppressor gene, is frequently inactivated in lung cancer leading to a YAP-dependent epithelial-mesenchymal transition (EMT). Such effects are partly due to the inactivation of the anti-migratory RhoB GTPase via the inhibitory phosphorylation of GEF-H1, the GDP/GTP exchange factor for RhoB. However, the kinase responsible for RhoB/GEF-H1 inactivation in RASSF1A-depleted cells remained unknown. Methods NDR1/2 inactivation by siRNA or shRNA effects on epithelial-mesenchymal transition, invasion, xenograft formation and growth in SCID−/− Beige mice, apoptosis, proliferation, cytokinesis, YAP/TAZ activation were investigated upon RASSF1A loss in human bronchial epithelial cells (HBEC). Results We demonstrate here that depletion of the YAP-kinases NDR1/2 reverts migration and metastatic properties upon RASSF1A loss in HBEC. We show that NDR2 interacts directly with GEF-H1 (which contains the NDR phosphorylation consensus motif HXRXXS/T), leading to GEF-H1 phosphorylation. We further report that the RASSF1A/NDR2/GEF-H1/RhoB/YAP axis is involved in proper cytokinesis in human bronchial cells, since chromosome proper segregation are NDR-dependent upon RASSF1A or GEF-H1 loss in HBEC. Conclusion To summarize, our data support a model in which, upon RASSF1A silencing, NDR2 gets activated, phosphorylates and inactivates GEF-H1, leading to RhoB inactivation. This cascade induced by RASSF1A loss in bronchial cells is responsible for metastasis properties, YAP activation and cytokinesis defects. Electronic supplementary material The online version of this article (10.1186/s13046-019-1145-8) contains supplementary material, which is available to authorized users.

The genetics underlying autism spectrum disorder (ASD) are complex. Approximately 3–5% of ASD cases arise from maternally inherited duplications of 15q11.2-q13.1, termed Duplication 15q syndrome (Dup15q). 15q11.2-q13.1 includes the gene UBE3A which is believed to underlie ASD observed in Dup15q syndrome. UBE3A is an E3 ubiquitin ligase that targets proteins for degradation and trafficking, so finding UBE3A substrates and interacting partners is critical to understanding Dup15q ASD. In this study, we take an unbiased genetics approach to identify genes that genetically interact with Dube3a, the Drosophila melanogaster homolog of UBE3A. We conducted an enhancer/suppressor screen using a rough eye phenotype produced by Dube3a overexpression with GMR-GAL4. Using the DrosDel deficiency kit, we identified 3 out of 346 deficiency lines that enhanced rough eyes when crossed to two separate Dube3a overexpression lines, and subsequently identified IA2, GABA-B-R3, and lola as single genes responsible for rough eye enhancement. Using the FlyLight GAL4 lines to express uas-Dube3a + uas-GFP in the endogenous lola pattern, we observed an increase in the GFP signal compared to uas-GFP alone, suggesting a transcriptional co-activation effect of Dube3a on the lola promoter region. These findings extend the role of Dube3a/UBE3A as a transcriptional co-activator, and reveal new Dube3a interacting genes.

Daytime warm temperature elicits thermomorphogenesis in Arabidopsis by stabilizing the central thermoregulator PHYTOCHROME INTERACTING transcription FACTOR 4 (PIF4), whose degradation is otherwise promoted by the photoreceptor and thermosensor phytochrome B. PIF4 stabilization in the light requires a transcriptional activator, HEMERA (HMR), and is abrogated when HMR’s transactivation activity is impaired in hmr-22. Here, we report the identification of a hmr-22 suppressor mutant, rcb-101, which surprisingly carries an A275V mutation in REGULATOR OF CHLOROPLAST BIOGENESIS (RCB). rcb-101/hmr-22 restores thermoresponsive PIF4 accumulation and reverts the defects of hmr-22 in chloroplast biogenesis and photomorphogenesis. Strikingly, similar to hmr, the null rcb-10 mutant impedes PIF4 accumulation and thereby loses the warm-temperature response. rcb-101 rescues hmr-22 in an allele-specific manner. Consistently, RCB interacts directly with HMR. Together, these results unveil RCB as a novel temperature signaling component that functions collaboratively with HMR to initiate thermomorphogenesis by selectively stabilizing PIF4 in the daytime., The Arabidopsis PIF4 transcription factor is stabilized during the daytime in response to warm temperature and regulates thermomorphogenesis. Here the authors show that the response to warm temperature depends on the concerted action of the HMR and RCB proteins that act collaboratively to stabilize PIF4.

Collagen type IV alpha 1 and alpha 2 (COL4A1 and COL4A2) are major components of almost all basement membranes. COL4A1 and COL4A2 mutations cause a multisystem disorder that can affect any organ but typically involves the cerebral vasculature, eyes, kidneys and skeletal muscles. In recent years, patient advocacy and family support groups have united under the name of Gould syndrome. The manifestations of Gould syndrome are highly variable, and animal studies suggest that allelic heterogeneity and genetic context contribute to the clinical variability. We previously characterized a mouse model of Gould syndrome caused by a Col4a1 mutation in which the severities of ocular anterior segment dysgenesis (ASD), myopathy and intracerebral hemorrhage (ICH) were dependent on genetic background. Here, we performed a genetic modifier screen to provide insight into the mechanisms contributing to Gould syndrome pathogenesis and identified a single locus [modifier of Gould syndrome 1 (MoGS1)] on Chromosome 1 that suppressed ASD. A separate screen showed that the same locus ameliorated myopathy. Interestingly, MoGS1 had no effect on ICH, suggesting that this phenotype could be mechanistically distinct. We refined the MoGS1 locus to a 4.3 Mb interval containing 18 protein-coding genes, including Fn1, which encodes the extracellular matrix component fibronectin 1. Molecular analysis showed that the MoGS1 locus increased Fn1 expression, raising the possibility that suppression is achieved through a compensatory extracellular mechanism. Furthermore, we found evidence of increased integrin-linked kinase levels and focal adhesion kinase phosphorylation in Col4a1 mutant mice that is partially restored by the MoGS1 locus, implicating the involvement of integrin signaling. Taken together, our results suggest that tissue-specific mechanistic heterogeneity contributes to the variable expressivity of Gould syndrome and that perturbations in integrin signaling may play a role in ocular and muscular manifestations., Summary: We performed a genetic screen in Col4a1 mutant mice to elucidate the mechanisms contributing to Gould syndrome. Our results implicate FN1 as a genetic modifier of some, but not all, aspects of Gould syndrome.

A missense mutant, unc-17(e245), which affects the Caenorhabditis elegans vesicular acetylcholine transporter UNC-17, has a severe uncoordinated phenotype, allowing efficient selection of dominant suppressors that revert this phenotype to wild-type. Such selections permitted isolation of numerous suppressors after EMS (ethyl methanesulfonate) mutagenesis, leading to demonstration of delays in mutation fixation after initial EMS treatment, as has been shown in T4 bacteriophage but not previously in eukaryotes. Three strong dominant extragenic suppressor loci have been defined, all of which act specifically on allele e245, which causes a G347R mutation in UNC-17. Two of the suppressors (sup-1 and sup-8/snb-1) have previously been shown to encode synaptic proteins able to interact directly with UNC-17. We found that the remaining suppressor, sup-2, corresponds to a mutation in erd-2.1, which encodes an endoplasmic reticulum retention protein; sup-2 causes a V186E missense mutation in transmembrane helix 7 of ERD-2.1. The same missense change introduced into the redundant paralogous gene erd-2.2 also suppressed unc-17(e245). Suppression presumably occurred by compensatory charge interactions between transmembrane helices of UNC-17 and ERD-2.1 or ERD-2.2, as previously proposed in work on suppression by SUP-1(G84E) or SUP-8(I97D)/synaptobrevin. erd-2.1(V186E) homozygotes were fully viable, but erd-2.1(V186E); erd-2.2(RNAi) exhibited synthetic lethality [like erd-2.1(RNAi); erd-2.2(RNAi)], indicating that the missense change in ERD-2.1 impairs its normal function in the secretory pathway but may allow it to adopt a novel moonlighting function as an unc-17 suppressor.

Background:Brain amyloid-β (Aβ) peptide is released into the interstitial fluid (ISF) in a neuronal activity-dependent manner, and Aβ deposition in Alzheimer's disease (AD) is linked to baseline neuronal activity. Although the intrinsic mechanism for Aβ generation remains to be elucidated, interleukin-like epithelial-mesenchymal transition inducer (ILEI) is a candidate for an endogenous Aβ suppressor., Objective:This study aimed to access the mechanism underlying ILEI secretion and its effect on Aβ production in the brain., Methods:ILEI and Aβ levels in the cerebral cortex were monitored using a newly developed ILEI-specific ELISA and in vivo microdialysis in mutant human Aβ precursor protein-knockin mice. ILEI levels in autopsied brains and cerebrospinal fluid (CSF) were measured using ELISA., Results:Extracellular release of ILEI and Aβ was dependent on neuronal activation and specifically on tetanus toxin-sensitive exocytosis of synaptic vesicles. However, simultaneous monitoring of extracellular ILEI and Aβ revealed that a spontaneous fluctuation of ILEI levels appeared to inversely mirror that of Aβ levels. Selective activation and inhibition of synaptic receptors differentially altered these levels. The evoked activation of AMPA-type receptors resulted in opposing changes to ILEI and Aβ levels. Brain ILEI levels were selectively decreased in AD. CSF ILEI concentration correlated with that of Aβ and were reduced in AD and mild cognitive impairment., Conclusion:ILEI and Aβ are released from distinct subpopulations of synaptic terminals in an activity-dependent manner, and ILEI negatively regulates Aβ production in specific synapse types. CSF ILEI might represent a surrogate marker for the accumulation of brain Aβ.

A population of more than six million people worldwide at high risk of Alzheimer’s disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of β-amyloid-(Aβ)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aβ deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical β and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aβ-preventing (Aβ1–19) and Aβ-degradation products (Aβ1–20 and Aβ1–34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis-cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability. IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

The gaseous hormone ethylene regulates a diverse range of plant development and stress responses. Ethylene biosynthesis is tightly regulated by the transcriptional and posttranscriptional regulation of ethylene biosynthetic enzymes. ACC synthase (ACS) is the rate-limiting enzyme that controls the speed of ethylene biosynthesis in plant tissues, thus serving as a primary target for biotic and abiotic stresses to modulate ethylene production. Despite the critical role of ACS in ethylene biosynthesis, only a few regulatory components regulating ACS stability or ACS transcript levels have been identified and characterized. Here we show a genetic approach for identifying novel regulatory components in ethylene biosynthesis by screening EMS-mutagenized Arabidopsis seeds.

Structural maintenance of chromosomes (SMC) complexes, cohesin, condensin and Smc5/6, are essential for viability and participate in multiple processes, including sister chromatid cohesion, chromosome condensation, and DNA repair. Here we show that SUMO chains target all three SMC complexes and are antagonized by the SUMO protease Ulp2 to prevent their turnover. We uncover that the essential role of the cohesin-associated subunit Pds5 is to counteract SUMO chains jointly with Ulp2. Importantly, fusion of Ulp2 to kleisin Scc1 supports viability ofPDS5null cells and protects cohesin from proteasomal degradation mediated by the SUMO-targeted ubiquitin ligase Slx5/Slx8. The lethality ofPDS5deleted cells can also be bypassed by simultaneous loss of the PCNA unloader, Elg1, and the cohesin releaser, Wpl1, but only when Ulp2 is functional. Condensin and Smc5/6 complex are similarly guarded by Ulp2 against unscheduled SUMO-chain assembly, which we propose to time the availability of SMC complexes on chromatin.

Essential genes tend to be highly conserved across eukaryotes, but, in some cases, their critical roles can be bypassed through genetic rewiring. From a systematic analysis of 728 different essential yeast genes, we discovered that 124 (17%) were dispensable essential genes. Through whole‐genome sequencing and detailed genetic analysis, we investigated the genetic interactions and genome alterations underlying bypass suppression. Dispensable essential genes often had paralogs, were enriched for genes encoding membrane‐associated proteins, and were depleted for members of protein complexes. Functionally related genes frequently drove the bypass suppression interactions. These gene properties were predictive of essential gene dispensability and of specific suppressors among hundreds of genes on aneuploid chromosomes. Our findings identify yeast's core essential gene set and reveal that the properties of dispensable essential genes are conserved from yeast to human cells, correlating with human genes that display cell line‐specific essentiality in the Cancer Dependency Map (DepMap) project., A systematic analysis of 728 different essential yeast genes identifies 124 (17%) dispensable essential genes. Whole‐genome sequencing is used to identify the genome alterations underlying the bypass suppression.

Mediator 12 (MED12) and MED13 are components of the Mediator multi-protein complex, that facilitates the initial steps of gene transcription. Here, in an Arabidopsis mutant screen, we identify MED12 and MED13 as positive gene regulators, both of which contribute broadly to morc1 de-repressed gene expression. Both MED12 and MED13 are preferentially required for the expression of genes depleted in active chromatin marks, a chromatin signature shared with morc1 re-activated loci. We further discover that MED12 tends to interact with genes that are responsive to environmental stimuli, including light and radiation. We demonstrate that light-induced transient gene expression depends on MED12, and is accompanied by a concomitant increase in MED12 enrichment during induction. In contrast, the steady-state expression level of these genes show little dependence on MED12, suggesting that MED12 is primarily required to aid the expression of genes in transition from less-active to more active states., Mediator is a multiprotein complex required to activate gene transcription by RNAPII. Here, the authors report that MED12 and MED13 are conditional positive regulators that facilitate the expression of genes depleted in active chromatin marks and the induction of gene expression in response to environmental stimuli in Arabidopsis.

Anthocyanin pigments furnish a powerful visual output of the stress and metabolic status of Arabidopsis thaliana plants. Essential for pigment accumulation is TRANSPARENT TESTA19 (TT19), a glutathione S-transferase proposed to bind and stabilize anthocyanins, participating in their vacuolar sequestration, a function conserved across the flowering plants. Here, we report the identification of genetic suppressors that result in anthocyanin accumulation in the absence of TT19. We show that mutations in RDR6, SGS3, or DCL4 suppress the anthocyanin defect of tt19 by pushing carbon towards flavonoid biosynthesis. This effect is not unique to tt19 and extends to at least one other anthocyanin pathway gene mutant. This synergy between mutations in components of the RDR6-SGS3-DCL4 siRNA system and the flavonoid pathway reveals genetic/epigenetic mechanisms regulating metabolic fluxes., TRANSPARENT TESTA19 (TT19) encodes a glutathione S-transferase which functions in anthocyanin stabilization and vacuolar transport. Here, by tt19 suppressor screening, the authors show that RDR6/SGS3/DCL4 siRNA pathway constituents synergistically interact with components of the flavonoid pathway to control carbon metabolism.

Zhang and Ye preview work from the Li group highlighting direct recognition of transmembrane helices by a membrane ubiquitin ligase adaptor in regulated degradation of an unwanted lysine transporter., Elimination of membrane proteins often requires recognition of their transmembrane domains (TMDs) in the lipid bilayer. In this issue, Arines et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.202001116) show that in Saccharomyces cerevisiae, the vacuole-associated Rsp5 ubiquitin ligase uses a TMD in substrate adaptor Ssh4 to recognize membrane helices in Ypq1, which targets this lysine transporter for lysosomal degradation during lysine starvation.

Nodule primordia induced by rhizobial glycan mutants often remain uninfected. To identify processes involved in infection and organogenesis we used forward genetics to identify plant genes involved in perception and responses to bacterial glycans. To dissect the mechanisms underlying the negative plant responses to the Mesorhizobium loti R7AexoU and ML001cep mutants, a screen for genetic suppressors of the nodulation phenotypes was performed on a chemically mutagenized Lotus population. Two mutant lines formed infected nitrogen-fixing pink nodules, while five mutant lines developed uninfected large white nodules, presumably altered in processes controlling organogenesis. Genetic mapping identified a mutation in the cytokinin receptor Lhk1 resulting in an alanine to valine substitution adjacent to a coiled-coil motif in the juxta-membrane region of LHK1. This results in a spontaneous nodulation phenotype and increased ethylene production. The allele was renamed snf5, and segregation studies of snf5 together with complementation studies suggest that snf5 is a gain-of-function allele. This forward genetic approach to investigate the role of glycans in the pathway synchronizing infection and organogenesis shows that a combination of plant and bacterial genetics opens new possibilities to study glycan responses in plants as well as identification of mutant alleles affecting nodule organogenesis.

Tra1 is an essential component of the SAGA/SLIK and NuA4 complexes in S. cerevisiae, recruiting these co-activator complexes to specific promoters. As a PIKK family member, Tra1 is characterized by a C-terminal phosphoinositide 3-kinase (PI3K) domain. Unlike other PIKK family members (e.g., Tor1, Tor2, Mec1, Tel1), Tra1 has no demonstrable kinase activity. We identified three conserved arginine residues in Tra1 that reside proximal or within the cleft between the N- and C-terminal subdomains of the PI3K domain. To establish a function for Tra1's PI3K domain and specifically the cleft region, we characterized a tra1 allele where these three arginine residues are mutated to glutamine. The half-life of the Tra1[Formula: see text] protein is reduced but its steady state level is maintained at near wild-type levels by a transcriptional feedback mechanism. The tra1[Formula: see text] allele results in slow growth under stress and alters the expression of genes also regulated by other components of the SAGA complex. Tra1[Formula: see text] is less efficiently transported to the nucleus than the wild-type protein. Likely related to this, Tra1[Formula: see text] associates poorly with SAGA/SLIK and NuA4. The ratio of Spt7SLIK to Spt7SAGA increases in the tra1[Formula: see text] strain and truncated forms of Spt20 become apparent upon isolation of SAGA/SLIK. Intragenic suppressor mutations of tra1[Formula: see text] map to the cleft region further emphasizing its importance. We propose that the PI3K domain of Tra1 is directly or indirectly important for incorporating Tra1 into SAGA and NuA4 and thus the biosynthesis and/or stability of the intact complexes.

Heterologous tRNA:aminoacyl tRNA synthetase pairs are often employed for noncanonical amino acid incorporation in the quest for an expanded genetic code. In this work, we investigated one possible mechanism by which directed evolution can improve orthogonal behavior for a suite of Methanocaldococcus jannaschii (Mj) tRNATyr-derived amber suppressor tRNAs. Northern blotting demonstrated that reduced expression of heterologous tRNA variants correlated with improved orthogonality. We suspected that reduced expression likely minimized nonorthogonal interactions with host cell machinery. Despite the known abundance of post-transcriptional modifications in tRNAs across all domains of life, few studies have investigated how host enzymes may affect behavior of heterologous tRNAs. Therefore, we measured tRNA orthogonality using a fluorescent reporter assay in several modification-deficient strains, demonstrating that heterologous tRNAs with high expression are strongly affected by some native E. coli RNA-modifying ...

Metabolic dysregulation leading to sugar-phosphate accumulation is toxic in organisms ranging from bacteria to humans. By comparing two models of sugar-phosphate toxicity in Saccharomyces cerevisiae, we demonstrate that toxicity occurs, at least in part, through multiple, isomer-specific mechanisms, rather than a single general mechanism., Toxicity resulting from accumulation of sugar-phosphate molecules is an evolutionarily conserved phenomenon, observed in multiple bacterial and eukaryotic systems, including a number of human diseases. However, the molecular mechanisms involved in sugar-phosphate toxicity remain unclear. Using the model eukaryote Saccharomyces cerevisiae, we developed two systems to accumulate human disease-associated sugar-phosphate species. One system utilizes constitutive expression of galactose permease and galactose kinase to accumulate galactose-1-phosphate, while the other system utilizes constitutive expression of a mammalian ketohexokinase gene to accumulate fructose-1-phosphate. These systems advantageously dissociate sugar-phosphate toxicity from metabolic demand for downstream enzymatic products. Using them, we characterized the pathophysiological effects of sugar-phosphate accumulation, in addition to identifying a number of genetic suppressors that repair sugar-phosphate toxicity. By comparing the effects of different sugar-phosphates, and examining the specificity of genetic suppressors, we observed a number of striking similarities and significant differences. These results suggest that sugar-phosphates exert toxic effects, at least in part, through isomer-specific mechanisms rather than through a single general mechanism common to accumulation of any sugar-phosphate.

The functional impact of most genomic alterations found in cancer, alone or in combination, remains largely unknown. Here we integrate tumor barcoding, CRISPR/Cas9-mediated genome editing and ultra-deep barcode sequencing to interrogate pairwise combinations of tumor suppressor alterations in autochthonous mouse models of human lung adenocarcinoma. We map the tumor suppressive effects of 31 common lung adenocarcinoma genotypes and identify a landscape of context dependence and differential effect strengths.

Human RNPS1 protein was first identified as a pre-mRNA splicing activator in vitro and RNPS1 regulates alternative splicing in cellulo. RNPS1 was also known as a peripheral factor of the exon junction complex (EJC). Here we show that cellular knockdown of RNPS1 induced a reduction of the wild-type aurora kinase B (AURKB) protein due to the induced aberrant pre-mRNA splicing events, indicating that the fidelity of AURKB pre-mRNA splicing was reduced. The major aberrant AURKB mRNA was derived from the upstream pseudo 5' and 3' splice sites in intron 5, which resulted in the production of the non-functional truncated AURKB protein. AURKB, is an essential mitotic factor, whose absence is known to cause multiple nuclei, and this multinucleation phenotype was recapitulated in RNPS1-knockdown cells. Importantly this RNPS1-knockdown phenotype was rescued by ectopic expression of AURKB, implying it is a major functional target of RNPS1. We found RNPS1 protein, not as a component of the EJC, binds directly to a specific element in the AURKB exon upstream of the authentic 5' splice site, and this binding is required for normal splicing. RNPS1-knockdown induces a parallel aberrant splicing pattern in a fully distinct pre-mRNA, MDM2, suggesting that RNPS1 is a global guardian of splicing fidelity. We conclude that RNPS1 is a key factor for the quality control of mRNAs that is essential for the phenotypes including cell division.

The transcription of many genes is regulated through alternative splicing, with over 60% of genes in Arabidopsis ( Arabidopsis thaliana ) producing more than one mRNA ([Marquez et al., 2012][1]). The most common forms of alternative splicing are intron retention and the use of alternative

Tumor-suppressor genes can be inactivated by several mechanisms and, in a majority of cases, both alleles need to be affected. One of the mechanisms of inactivation is due to deletions ranging from dozen to hundreds of nucleotides; such deletions are often missed by variant callers. HomDelDetect is a method to detect such homozygous deletions in cancer models, such as cancer cell lines and potentially patient tumor-derived xenografts. This method can be applied to partial exome, whole-exome sequencing, whole-genome sequencing, and RNA-seq data. We applied our method across a panel of CCLE cancer cell lines and observed good concordance with SNP array-based analysis and also detected deletions that have been missed by variant callers and by SNP arrays, demonstrating the ability of HomDelDetect to improve the annotations of tumor-suppressor genes in cancer models.

We have developed a new functional complementation approach to clone modifier genes which overexpression is able to suppress the biochemical defects caused by mtDNA mutations (suppressor genes). This strategy consists in transferring human genes into respiratory chain-deficient fibroblasts, followed by a metabolic selection in a highly selective medium. We used a normalized expression cDNA library in an episomal vector (pREP4) to transfect the fibroblasts, and a medium with glutamine and devoid of any carbohydrate source to select metabolically. Growing the patient's fibroblasts in this selective medium, the deficient cells rapidly disappear unless they are rescued by the cDNA of a suppressor gene. The use of an episomal vector allows us to carry out several rounds of transfection/selection (cyclical phenotypic rescue) to enrich the rescue with true clones of suppressor genes. Using fibroblasts from a patient with epileptic encephalopathy with the m.3946G>A (p.E214K) mutation in the MT-ND1 gene, several candidate genes were identified and one of them was characterized functionally. Thus, overexpression of MRPS18C gene (that encode for bS18m protein) suppressed the molecular defects produced by this mtDNA mutation, recovering the complex I activity and reducing the ROS produced by this complex to normal levels. We suggest that modulation of bS18m expression may be an effective therapeutic strategy for the patients with this mutation.

Lung cancer (LC) and malignant mesothelioma (MM) are malignancies linked to environmental/occupational exposure, which are increasing in incidence. Despite advances in chemotherapy, radiation therapy and surgical management of LC and MM, the median survival remains less than 12 months. Early detection represents one of the most promising approaches to reducing the growing cancer burden by increasing chemotherapy treatment efficiency. At present, early diagnosis is rather difficult and depends on invasive sampling of pleural fluid or tissue. Currently the most widely used screening method for the surveillance of exposed subjects is computed tomography (CT), which is costly, exposes patients to repeated high doses of radiation, and typically detects the malignancy at its advanced stage. Recently, a virtually non-invasive 'liquid biopsy' has emerged as source to characterize tumour heterogeneity. The genetic/epigenetic changes during tumour evolution can be detected in fluids and used as cancer biomarkers. Therefore, increasingly interest has been paid to circulating (cell-free) nucleic acids (cfDNA/cfmiRNAs) epigenetically modulated during cell transformation. Hypermethylation of tumour suppressor genes is frequently observed in cancers, and such epigenetic changes are potential markers for detecting and monitoring tumours. The same predictive biomarkers can be used as therapy targets.

While most missense suppressors have very narrow specificities and only suppress the allele against which they were isolated, the sumA missense suppressor from Salmonella enterica serovar Typhimurium is a promiscuous or broad-acting missense suppressor that suppresses numerous missense mutants. The sumA missense suppressor was identified as a glyV tRNA Gly3(GAU/C) missense suppressor that can recognize GAU or GAC aspartic acid codons and insert a glycine amino acid instead of aspartic acid. In addition to rescuing missense mutants caused by glycine to aspartic acid changes as expected, sumA could also rescue a number of other missense mutants as well by changing a neighboring (contacting) aspartic acid to glycine, which compensated for the other amino acid change. Thus the ability of sumA to rescue numerous missense mutants was due in part to the large number of glycine codons in genes that can be mutated to an aspartic acid codon and in part to the general tolerability and/or preference for glycine amino acids in proteins. Because the glyV tRNA Gly3(GAU/C) missense suppressor has also been extensively characterized in Escherichia coli as the mutA mutator, we demonstrated that all gain-of-function mutants isolated in a glyV tRNA Gly3(GAU/C) missense suppressor are transferable to a wild-type background and thus the increased mutation rates, which occur in glyV tRNA Gly3(GAU/C) missense suppressors, are not due to the suppression of these mutants.

Protein folding is a complex cellular process often assisted by chaperones, but it can also be facilitated by interactions with lipids. Disulfide bond formation is a common mechanism to stabilize a protein. This can help maintain functionality amid changes in the biochemical milieu, including those relating to energy-transducing membranes. Plastidic Type I Signal Peptidase 1 (Plsp1) is an integral thylakoid membrane signal peptidase that requires an intramolecular disulfide bond for in vitro activity. We have investigated the interplay between disulfide bond formation, lipids, and pH in the folding and activity of Plsp1. By combining biochemical approaches with a genetic complementation assay using Arabidopsis thaliana plants, we provide evidence that interactions with lipids in the thylakoid membrane have reconstitutive chaperoning activity toward Plsp1. Further, the disulfide bridge appears to prevent an inhibitory conformational change resulting from proton motive force-mimicking pH conditions. Broader implications related to the folding of proteins in energy-transducing membranes are discussed.

The eye imaginal disc-specific knockdown of dFIG4, a Drosophila homolog of FIG4 that is one of the Charcot-Marie-Tooth disease (CMT)-causing genes, induces an aberrant adult compound eye morphology, the so-called rough eye phenotype. We previously performed modifier screening on the dFIG4 knockdown-induced rough eye phenotype and identified several genes, including CR18854, encoding a long non-coding RNA (lncRNA) as genetic interactants with dFIG4. In the present study, in more extensive genetic screening, we found that the deletion of a gene locus encoding both Odorant rector 46a (Or46a) and lncRNA CR43467 effectively suppressed the rough eye phenotype induced by the knockdown of dFIG4. Both genes were located on the same locus, but oriented in opposite directions. In order to identify which of these genes is responsible for the suppression of the rough eye phenotype, we established a CR43467-specific knockdown line using the CRISPR-dCas9 system. By using this system, we demonstrated that the CR43467 gene, but not the Or46a gene, genetically interacted with the dFIG4 gene. The knockdown of CR43467 rescued the reductions in the length of synaptic branches and number of boutons at neuromuscular junctions induced by the knockdown of dFIG4. The vacuole enlargement phenotype induced by the fat body-specific dFIG4 knockdown was also effectively suppressed by the knockdown of CR43467. The knockdown of CR43467 also suppressed the rough eye phenotype induced by other peripheral neuropathy-related genes, such as dCOA7, dHADHB, and dPDHB. We herein identified another gene encoding lncRNA, CR43467 as a genetic interactant with the CMT-causing gene.