Translational Level Research Articles

Erlotinib Treatment in Colorectal Cancer Suppresses Autophagy Based on KRAS Mutation.

The KRAS gene is mutated in approximately 45% of colorectal cancer patients. There are currently very few targeted treatments or therapies equipped to directly inhibit KRAS due to its unusual structural intricacies. Erlotinib, an EGFR inhibitor, has previously been demonstrated to reduce cell viability by inducing autophagy in lung cancer cell lines with varying EGFR mutations. In contrast to lung cancer cells, evidence is provided herein for the first time that erlotinib treatment in colorectal cancer (CRC) cell lines reduces autophagy and still results in decreased cell viability. However, the effects of erlotinib in CRC cell lines containing a wildtype KRAS gene were different than in cells carrying a mutant KRAS gene. We show that there is significantly more downregulation of autophagy in KRAS mutant CRC cells compared to KRAS wildtype cells, both at transcriptional and translational levels, suggesting that the KRAS mutation is advantageous for cancer growth, even in the presence of erlotinib. Cell viability results determined that KRAS wildtype CRC cells had significantly more cell death compared to KRAS mutant cells. Using patient mRNA datasets, we showed that there was a significant correlation between the presence of the KRAS mutation and the expression of autophagy proteins. Additionally, through molecular dynamics simulations, we develop a blueprint for KRAS and autophagy protein interaction and the impact of the KRAS mutation on autophagy protein regulation. Overall, this is the first report of erlotinib treatment in CRC cells that assesses autophagy, and we demonstrate that autophagy activity is downregulated in these cells. This effect is not only greater in cells carrying a KRAS mutation compared to wildtype cells, but the KRAS mutant cells also have increased cell viability compared to wildtype cells. We hypothesize that the difference in cell viability and autophagy expression between KRAS mutant and KRAS wildtype cells after treatment with erlotinib can be of therapeutic value to treat CRC patients carrying KRAS mutations.

FKBP38 deletion exacerbates ConA-induced hepatitis by promoting the immune response through the MCP-1/p38 pathway

Autoimmune hepatitis (AIH) is a chronic liver disease characterized by immune dysregulation and hepatocyte damage. FKBP38, a member of the immunophilin family, has been implicated in immune regulation and the modulation of intracellular signaling pathways; however, its role in AIH pathogenesis remains poorly understood. In this study, we aimed to investigate the effects of hepatic FKBP38 deletion on AIH using a hepatic FKBP38 knockout (LKO) mouse model created via cre-loxP technology. We compared the survival rates, incidence, and severity of AIH in LKO mice with those in control mice. Our findings revealed that hepatic FKBP38 deletion resulted in an unfavorable prognosis in LKO mice with AIH. Specifically, LKO mice exhibited heightened liver inflammation and extensive hepatocyte damage compared to control mice, with a significant decrease in anti-apoptotic proteins and a marked increase in pro-apoptotic proteins. Additionally, transcriptional and translational levels of pro-inflammatory cytokines and chemokines were significantly increased in LKO mice compared to control mice. Immunoblot analysis showed that MCP-1 expression was significantly elevated in LKO mice. Furthermore, the phosphorylation of p38 was increased in LKO mice with AIH, indicating that FKBP38 deletion promotes liver injury in AIH by upregulating p38 phosphorylation and increasing MCP-1 expression. Immune cell profiling demonstrated elevated populations of T, NK, and B cells, suggesting a dysregulated immune response in LKO mice with AIH. Overall, our findings suggest that FKBP38 disruption exacerbates AIH severity by augmenting the immune response by activating the MCP-1/p38 signaling pathway.

The Influence of miR-3149 on the Malignancy Progression of Gastric Cancer by Negatively Regulating CEACAM5

Background: There are some reports indicating that carcinoembryonic antigen associated cell adhesion molecule 5 (CEACAM5) can act as a diagnostic indicator or molecular marker for GC, but its upstream regulatory pathway still needs to be explored. microRNAs (miRNAs) regulate gene expression at the translation level and fulfil a crucial role in GC physiologyin, whether miR-3149 in family members can affect GC progression by regulating CEACAM5 is still unclear. Methods: miR-3149 and CEACAM5 expressions in GC tissues and cells (N87, AGS, and HGC-27) were observed by RT-qPCR or Western blot. Intervened the miR-3149 and CEACAM5 expression in GC cells, CCK8, Wound healing, Transwell and flow cytometry assays evaluated cell activity and function separately, while detecting related proteins. Predicted the target of miR-3149 and the expression of CEACAM5 in tumor tissue through bioinformatics analysis and evaluated the correlation between both using Spearman analysis. Verified the targeting relationship between the two through the Luciferase reporter assay. Conducted another rescue experiment on CEACAM5 overexpression to further verify this relationship. Results: In GC tissues and cells, miR-3149 and CEACAM5 expression levels were respectively down-regulated and up-regulated. Transfection of miR-3149 mimics or inhibitors respectively reduced or increased GC cell apoptosis and inhibited or encouraged cell proliferation, migration and invasion, which were shown to be enhanced or inhibited respectively by transfection of OE-CEACAM5/Si-CEACAM5, while their apoptosis rate decreased or increased. miR-3149 could target regulating CEACAM5, and showed an inverse correlation with the expression of CEACAM5. The impacts of miR-3149 mimics on the malignancy progression of GC could be partially reversed by OE-CEACAM5. Conclusion: The results indicate that miR-3149 can downregulate the expression of CEACAM5 to inhibit the malignancy progression, as a GC suppressor,which identify a new miRNA mediated tumor suppressor mechanism in GC.

Glutamine enhances pneumococcal growth under methionine semi-starvation by elevating intracellular pH.

Bacteria frequently encounter nutrient limitation in nature. The ability of living in this nutrient shortage environment is vital for bacteria to preserve their population and important for some pathogenic bacteria to cause infectious diseases. Usually, we study how bacteria survive after nutrient depletion, a total starvation condition when bacteria almost cease growth and try to survive. However, nutrient limitation may not always lead to total starvation. Bacterial adaptation to nutrient shortage was studied by determining bacterial growth curves, intracellular pH, intracellular amino acid contents, gene transcription, protein expression, enzyme activity, and translation and replication activities. No exogenous supply of methionine results in growth attenuation of Streptococcus pneumoniae, a human pathogen. In this paper, we refer to this inhibited growth state between ceased growth under total starvation and full-speed growth with full nutrients as semi-starvation. Similar to total starvation, methionine semi-starvation also leads to intracellular acidification. Surprisingly, it is intracellular acidification but not insufficient methionine synthesis that causes growth attenuation under methionine semi-starvation. With excessive glutamine supply in the medium, intracellular methionine level was not changed, while bacterial intracellular pH was elevated to ~ 7.6 (the optimal intracellular pH for pneumococcal growth) by glutamine deamination, and bacterial growth under semi-starvation was restored fully. Our data suggest that intracellular acidification decreases translation level and glutamine supply increases intracellular pH to restore translation level, thus restoring bacterial growth. This growth with intracellular pH adjustment by glutamine is a novel strategy we found for bacterial adaptation to nutrient shortage, which may provide new drug targets to inhibit growth of pathogenic bacteria under semi-starvation.

Widespread changes to the translational landscape in a maize microRNA biogenesis mutant.

MicroRNAs are short, non-coding RNAs that repress gene expression in both plants and animals and have diverse functions related to growth, development, and stress responses. The ribonuclease, DICER-LIKE1 (DCL1) is required for two steps in plant miRNA biogenesis: cleavage of the primary miRNAs (pri-miRNAs) to release a hairpin structure, called the precursor miRNA (pre-miRNA) and cleavage of the pre-miRNA to generate the miRNA/miRNA* duplex. The mature miRNA guides the RNA-induced silencing complex to target RNAs with complementary sequences, resulting in translational repression and/or RNA cleavage of target mRNAs. However, the relative contribution of translational repression versus mRNA degradation by miRNAs remains unknown at the genome-level in crops, especially in maize. The maize fuzzy tassel (fzt) mutant contains a hypomorphic mutation in DCL1 resulting in broad developmental defects. While most miRNAs are reduced in fzt, the levels of miRNA-targeted mRNAs are not dramatically increased, suggesting that translational regulation by miRNAs may be common. To gain insight into the repression mechanism of plant miRNAs, we combined ribosome profiling and RNA-sequencing to globally survey miRNA activities in maize. Our data indicate that translational repression contributes significantly to regulation of most miRNA targets and that approximately one-third of miRNA targets are regulated primarily at the translational level. Surprisingly, ribosomes appear altered in fzt mutants suggesting that DCL1 may also have a role in ribosome biogenesis. Thus, DICER-LIKE1 shapes the translational landscape in plants through both miRNA-dependent and miRNA-independent mechanisms.

Sodium Houttuyfonate Ameliorates DSS-induced Colitis Aggravated by Candida albicans through Dectin-1/NF-κB/miR-32-5p/NFKBIZ Axis Based on Intestinal microRNA Profiling.

Our previous research indicated that Sodium houttuyfonate (SH) can effectively ameliorate dextran sulfate sodium (DSS)-induced colitis exacerbated by Candida albicans. However, the underlying protective mechanism of SH remains unclear. Therefore, in this study, a mice colitis model was infected with C. albicans, and the total colonic miRNAs were assessed. Furthermore, the differentially expressed miRNAs were enriched, clustered, and analyzed. Moreover, based on the dual luciferase analysis of NFKBIZ modulation by miR-32-5p, the in vitro and in vivo therapeutic effects of SH on inflammatory response, fungal burden, oxidative stress, and apoptosis were assessed at transcriptional and translational levels in the presence of agonist and antagonist. A total of 1157 miRNAs were identified, 84 of which were differentially expressed. Furthermore, qRT-PCR validated that SH treatment improved 17 differentially expressed miRNAs with > fourfold upregulation or > sixfold downregulation. Similar to most differentially altered miRNA, C. albicans significantly increased Dectin-1, NF-κB, TNF-α, IL-1β, IL-17A, and decreased miR-32-5p which negatively targeted NFKBIZ. In addition, SH treatment reduced inflammatory response and fungal burden in a colitis model with C. albicans infection. Further analyses indicated that in C. albicans infected Caco2 cells, SH inhibited fungal growth, oxidative stress, and apoptosis by increasing Dectin-1, NF-κB, NFKBIZ, TNF-α, IL-1β, IL-17A, and decreasing miR-32-5p. Therefore, SH can ameliorate the severity of colitis aggravated by C. albicans via the Dectin-1/NF-κB/miR-32-5p/NFKBIZ axis.

Lysine Methylation and Histone Modifications during Cold Stress of Insects: Freeze-Tolerant Eurosta solidaginis and Freeze-Avoiding Epiblema scudderiana.

Overwintering survival by insects, whether of the freeze-tolerant or freeze-avoiding types, is typically associated with a strong suppression of metabolic rate (e.g., entry into diapause) that involves the differential expression of many genes with regulation at the transcriptional, translational or post-translational levels. Epigenetic modifications have been suggested to play a vital role in regulating cold responses of insects. However, knowledge of the roles of epigenetic mechanisms in modulating gene expression for winter survival of the larvae of two goldenrod gall formers, the freeze-tolerant dipteran Eurosta solidaginis and the freeze-avoiding lepidopteran Epiblema scudderiana, remain unknown. The current study evaluates the role of cold-induced lysine methylation and histone modifications, with enzymes of lysine methylation (SETD8, SETD7, SUV39H1, SMYD2 and ASH2L), as well as relative levels of histone H3 acetylation (H3K9ac, H3K18ac, H3K27ac, H3K56ac) and methylation (H3K4me1, H3K9me3, H3K36me2) examined in two insects. Significant (p < 0.05) reductions were observed in most of the targets of histone methylation/acetylation for decreasing temperatures of Ep. scudderiana larvae, whereas selected histone methylation/acetylation targets were conversely elevated (p < 0.05) in E. solidaginis, particularly under conditions of 5 °C for 4 h. Histone H3 expression was found to be variable without statistical differences in larval goldenrod gall moths and gall flies. These results provide basic information on the patterns of epigenetic regulation involved in insect cold hardiness.

Application of Free Translation Method in Lexical and Syntactic Level of Translation of Investigating Classroom Discourse

Based on practical experience translating Steve Walsh s Investigating Classroom Discourse, this paper explores the application of free translation in academic contexts. Despite high scholarly regard for classroom discourse analysis, this study delves into the role of free translation in this field, highlighting its theoretical and practical feasibility. This approach aims to make such analysis more accessible to Chinese educators, enhancing domestic teaching practices and deepening insights into bilingual education.

Molecular mechanisms of neutron radiation dose effects on M1 generation peas

The dose effect of radiation has long been a topic of concern, but the molecular mechanism behind it is still unclear. In this study, dried pea seeds were irradiated with 252Cf fission neutron source. Through analyzing the transcriptome and proteome of M1 generation pea (Pisum sativum L.) leaves, we studied the molecular rule and mechanism of neutron dose effect. Our results showed three important rules of global gene expression in the studied dose range. The rule closely related to the neutron absorbed dose at the transcription and translation levels is: the greater the difference in neutron absorbed dose between two radiation treatment groups, the greater the difference in differential expression between the two groups and the control group. We also obtained important sensitive metabolic pathways of neutron radiation, as well as related key genes. Furthermore, the overall molecular regulation mechanism of dose effect was revealed based on the main functional items obtained. Our research results can be applied to appropriate radiation dose estimation and agricultural production practice.

O-299 Exposure of antral follicles to progestins during stimulation does not cause molecular perturbations in gonadotropin-responsiveness and steroidogenic function of granulosa cells in progestin-primed IVF cycles

Abstract Study question Does progestin exposure cause any molecular perturbations in the steroidogenic features and gonadotropin responsiveness of the granulosa cells in progestin-primed ovarian stimulation (PPOS) cycles? Summary answer No. PPOS cycles are identical to antagonist cycles for gonadotropin receptor expression, response to gonadotropins, steroidogenic function at molecular level in normal responding IVF patients. What is known already PPOS is commonly used in IVF cycles as an alternative to antagonist cycles. Although accumulating evidence from clinical studies did not indicate any particular differences between these two types of stimulation protocol for IVF cycle characteristics, it is still unknown if exposure of antral follicle cohorts to synthetic progesterone derivatives during ovarian stimulation causes any subtle molecular aberrations in PPOS cycles in terms of steroidogenesis and gonadotropin responsiveness. To address this issue, detailed molecular analyses were conducted in the luteinized mural granulosa cells (GCs) obtained from the normal responding patients undergoing PPOS vs. antagonist IVF cycles. Study design, size, duration A translational research study in IVF patients. Participants/materials, setting, methods 55 normal responding IVF patients underwent ovarian stimulation either with PPOS using medroxy progesterone acetate (5mg twice daily) or, with GnRH antagonist cetrorelix acetate were included in the study. Recombinant forms of FSH and hCG were used for ovarian stimulation and ovulation trigger, respectively. Luteinized mural granulosa cells obtained during oocyte retrieval procedure were used for the experiments. Cell culture, quantitative real-time PCR, immunoblotting, confocal time-lapse live cell imaging and hormone assays were used. Main results and the role of chance Demographic and IVF characteristics of the patients undergoing ovarian stimulation with PPOS vs. GnRH antagonist were similar including ovarian-response, mature oocyte yield and fertilization rates. Molecular analyses revealed that the expression of the enzymes involved in sex-steroid synthesis (StAR, aromatase, 3B-HSD, 17B-HSD) and uptake/storage/utilization of cholesterol (LDL receptor, Hormone-sensitive lipase, hydroxy-methyl glutaryl Co-enzyme-A reductase and Sterol O-acyltransferase1) in the GCs of the PPOS cycles were comparable to the antagonist IVF cycles. Both cycles were also similar for the expression of FSH and LH receptors at transcriptional and translational level. Basal and hCG-induced increases in 3B-HSD expression and P4 production, and basal and FSH-induced increases in aromatase expression and E2 output of the GCs of the PPOS patients did not show any meaningful differences from the antagonist cycles. Similarly, basal and hCG-induced up-regulation in the LDL receptor expression and cholesterol uptake rate did not differ between the groups. Confocal imaging also revealed similar patterns of expression for the steroidogenic enzymes and their co-localization with mitochondria. Lastly, the expression of the other genes regulating cumulus expansion, ovulation and luteal function (Relaxin, ADAMTS-1 and EGF-like growth factor amphiregulin (AREG)) in the GCs of the PPOS cycles were comparable to the antagonist cycles. Limitations, reasons for caution Caution should be exercised when interpreting our data, which was produced in normal responders. It is unclear if the molecular parameters assessed here vary according to infertility etiologies, ovarian response type, mode of trigger and any other underlying ovarian pathology or systemic diseases that are concomitantly present. Wider implications of the findings This study provides reassuring molecular evidence that exposure of antral cohorts to progestins follicular growth phase does not appear to have any detrimental effect on their steroidogenic, ovulatory and luteal functions, suggesting that PPOS IVF cycles are not different from traditional antagonist cycles. Trial registration number not applicable

Functional roles of folic acid in alleviating dexamethasone‐induced fatty liver syndrome in laying hens

AbstractFatty liver syndrome (FLS) poses a threat to the poultry industry due to its high occurrence and mortality rate. Folic acid (FA) is a coenzyme crucial for one‐carbon metabolism. However, the mechanism by which FA mitigates FLS in laying hens remains elusive. In this study, 60 21‐week‐old Hy‐Line Brown layers were divided into three groups: the Control (Con) group, the dexamethasone (DXM) group, and the DXM + FA group. Results showed that liver index was significantly increased in the DXM group. H&amp;E and oil red O staining showed the accumulation of lipid droplets in the liver was intensified, confirming the successful establishment of an early fatty liver model without inflammation. FA significantly reversed hepatic lipid deposition, and 57 differentially expressed genes affected by FA were identified in the transcriptome analysis. Their transcriptional and translational levels indicate that in the early FLS, insulin‐like growth factor 2/phosphatidylinositol‐3‐kinase/protein kinase B pathway related to lipid metabolism was activated; folate cycling was inhibited, while endoplasmic reticulum (ER) stress and apoptosis‐related protein abundance were elevated. Dietary FA enhanced the folate circulation, reduced lipogenesis and ER stress, and apoptosis‐related protein expression, thereby mitigating the lipid metabolism disturbance in FLS. Metabolomics identified 151 differential metabolites involved in early FLS occurrence, 34 of which were reversed by FA. Metabolites were also enriched in pathways related to lipid metabolism and hepatic damage. Collectively, these findings can be concluded that FA can alleviate early FLS by affecting lipogenesis, ER stress and apoptosis, which may be mediated by enhanced folate metabolism.

Opportunity for genome engineering to enhance phosphate homeostasis in crops.

Plants maintain cellular homeostasis of phosphate (Pi) through an integrated response pathway regulated by different families of transcription factors including MYB, WRKY, BHLH, and ZFP. The systemic response to Pi limitation showed the critical role played by inositol pyrophosphate (PP-InsPs) as signaling molecule and SPX (SYG1/PHO81/XPR1) domain proteins as sensor of cellular Pi status. Binding of SPX to PP-InsPs regulates the transcriptional activity of the MYB-CC proteins, phosphate starvation response factors (PHR/PHL) as the central regulator of Pi-deficiency response in plants. Vacuolar phosphate transporter, VPT may sense the cellular Pi status by its SPX domain, and vacuolar sequestration is activated under Pi replete condition and the stored Pi is an important resource to be mobilized under Pi deficiency. Proteomic approaches led to new discoveries of proteins associated with Pi-deficient response pathways and post-translational events that may influence plants in achieving Pi homeostasis. This review provides current understanding on the molecular mechanisms at the transcriptional and translational levels for achieving Pi homeostasis in plants. The potential strategies for employing the CRISPR technology to modify the gene sequences of key regulatory and response proteins for attaining plant Pi homeostasis are discussed.

Translational control in the spinal cord regulates gene expression and pain hypersensitivity in the chronic phase of neuropathic pain.

Sensitization of spinal nociceptive circuits plays a crucial role in neuropathic pain. This sensitization depends on new gene expression that is primarily regulated via transcriptional and translational control mechanisms. The relative roles of these mechanisms in regulating gene expression in the clinically relevant chronic phase of neuropathic pain are not well understood. Here, we show that changes in gene expression in the spinal cord during the chronic phase of neuropathic pain are substantially regulated at the translational level. Downregulating spinal translation at the chronic phase alleviated pain hypersensitivity. Cell-type-specific profiling revealed that spinal inhibitory neurons exhibited greater changes in translation after peripheral nerve injury compared to excitatory neurons. Notably, increasing translation selectively in all inhibitory neurons or parvalbumin-positive (PV+) interneurons, but not excitatory neurons, promoted mechanical pain hypersensitivity. Furthermore, increasing translation in PV+ neurons decreased their intrinsic excitability and spiking activity, whereas reducing translation in spinal PV+ neurons prevented the nerve injury-induced decrease in excitability. Thus, translational control mechanisms in the spinal cord, particularly in inhibitory neurons, play a role in mediating neuropathic pain hypersensitivity.

High Polymorphism Levels of De Novo ORFs in a Yoruba Human Population.

During evolution, new open reading frames (ORFs) with the potential to give rise to novel proteins continuously emerge. A recent compilation of noncanonical ORFs with translation signatures in humans has identified thousands of cases with a putative de novo origin. However, it is not known which is their distribution in the population. Are they universally translated? Here, we use ribosome profiling data from 65 lymphoblastoid cell lines from individuals of Yoruba origin to investigate this question. We identify 2,587 de novo ORFs translated in at least one of the cell lines. In line with their de novo origin, the encoded proteins tend to be smaller than 100 amino acids and encode positively charged proteins. We observe that the de novo ORFs are more polymorphic in the population than the set of canonical proteins, with a substantial fraction of them being translated in only some of the cell lines. Remarkably, this difference remains significant after controlling for differences in the translation levels. These results suggest that variations in the level translation of de novo ORFs could be a relevant source of intraspecies phenotypic diversity in humans.

Oxidative stress-mediated deleterious effects of hypoxia in the brackish water flea Diaphanosoma celebensis

In this study, we investigated the acute toxicity, in vivo effects, oxidative stress, and gene expression changes caused by hypoxia on the brackish water flea Diaphanosoma celebensis. The no-observed-effect concentration (NOEC) of 48 h of hypoxia exposure was found to be 2 mg/L O2. Chronic exposure to NOEC caused a significant decline in lifespan but had no effect on total fecundity. The induction of reactive oxygen species increased in a time-dependent manner over 48 h, whereas the content of antioxidant enzymes (superoxide dismutase and catalase) decreased. The transcription and translation levels were modulated by hypoxia exposure. In particular, a significant increase in hemoglobin level was followed by up-regulation of hypoxia-inducible factor 1α gene expression and activation of the mitogen-activated protein kinase pathway. In conclusion, our findings provide a better understanding of the molecular mechanism of the adverse effects of hypoxia in brackish water zooplankton.

Sequencing-based analysis of microbiomes.

Microbiomes occupy a range of niches and, in addition to having diverse compositions, they have varied functional roles that have an impact on agriculture, environmental sciences, and human health and disease. The study of microbiomes has been facilitated by recent technological and analytical advances, such as cheaper and higher-throughput DNA and RNA sequencing, improved long-read sequencing and innovative computational analysis methods. These advances are providing a deeper understanding of microbiomes at the genomic, transcriptional and translational level, generating insights into their function and composition at resolutions beyond the species level.

ASPECTS OF TRANSLATION QUALITY PROBLEM

Translation quality evaluation is generally referred to as one the mechanisms for translation industry development and the most important tool in the field of translator training, which determines the relevance of the stated topic. The purpose of this study is to review the problems and approaches to translation quality evaluation highlighting the differences in their treatment. The paper focuses on the study of the cognitive-terminological and system-activity aspects of translation evaluation, with special emphasis on the borderline between the process of quality evaluation and its result. An analysis of the term translation quality as used in meta-translation discourse demonstrated significant differences in the conceptualization of this notion. In translation theory, the term is generally used as an evaluative indicator of translation results, although in a broader sense it also refers to one of the main categories of translation studies that requires systematic research and description. In meta-translation discourse, translation quality may refer to a fairly high level of translation per se, but in combination with additional descriptive characteristics the term can also indicate different levels of quality, down to the lowest. To assess the results of translation using classical evaluative terms, namely equivalence and adequacy, it is generally necessary to clarify their conceptual content, which corresponds to a particular scientific paradigm. The existence of different and/or opposing points of view on the theory and practice of translation quality evaluation is accounted for by the complex and multidimensional nature of the phenomenon under consideration. The novelty of the conducted research lies in the distinction between translation evaluation as a result and translation evaluation as a process considered as a type of intellectual activity. Finally, we developed a model which reflects the structure of evaluation activities in the field of translation. The main components of this model are: the evaluation sphere, the evaluation actor, the object under evaluation (i.e. translation results), the purpose of evaluation activity, the evaluation method, the criteria and the parameters of evaluation. Following the system-activity approach one can take into account various facets of the problem of translation quality evaluation as well as the complex of motives and individual independent steps that determine the end result.

WTAP and m6A-modified circRNAs modulation during stress response in acute myeloid leukemia progenitor cells

N6-methyladenosine (m6A) is one of the most prevalent and conserved RNA modifications. It controls several biological processes, including the biogenesis and function of circular RNAs (circRNAs), which are a class of covalently closed-single stranded RNAs. Several studies have revealed that proteotoxic stress response induction could be a relevant anticancer therapy in Acute Myeloid Leukemia (AML). Furthermore, a strong molecular interaction between the m6A mRNA modification factors and the suppression of the proteotoxic stress response has emerged. Since the proteasome inhibition leading to the imbalance in protein homeostasis is strictly linked to the stress response induction, we investigated the role of Bortezomib (Btz) on m6A regulation and in particular its impact on the modulation of m6A-modified circRNAs expression. Here, we show that treating AML cells with Btz downregulated the expression of the m6A regulator WTAP at translational level, mainly because of increased oxidative stress. Indeed, Btz treatment promoted oxidative stress, with ROS generation and HMOX-1 activation and administration of the reducing agent N-acetylcysteine restored WTAP expression. Additionally, we identified m6A-modified circRNAs modulated by Btz treatment, including circHIPK3, which is implicated in protein folding and oxidative stress regulation. These results highlight the intricate molecular networks involved in oxidative and ER stress induction in AML cells following proteotoxic stress response, laying the groundwork for future therapeutic strategies targeting these pathways.

Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria

Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery.

Editing of eIF(iso)4E.c confers resistance against Turnip mosaic virus in Brassica rapa

Turnip mosaic virus (TuMV) constitutes one of the primary diseases affecting Brassica rapa, severely impacting its production and resulting in crop failures in various regions worldwide. Recent research has demonstrated the significance of plant translation initiation factors, specifically the eIF4E and eIF4G family genes, as essential recessive disease resistance genes. In our study, we conducted evolutionary and gene expression studies, leading us to identify eIF(iso)4E.c as a potential TuMV-resistant gene. Leveraging CRISPR/Cas9 technology, we obtained mutant B. rapa plants with edited eIF(iso)4E.c gene. We confirmed eIF(iso)4E.c confers resistance against TuMV through phenotypic observations and virus content evaluations. Furthermore, we employed ribosome profiling assays on eif(iso)4e.c mutant seedlings to unravel the translation landscape in response to TuMV. Interestingly, we observed a moderate correlation between the fold changes in gene expression at the transcriptional and translational levels (R2 = 0.729). Comparative analysis of ribosome profiling and RNA-seq data revealed that plant–pathogen interaction, and MAPK signaling pathway–plant pathways were involved in eIF(iso)4E.c-mediated TuMV resistance. Further analysis revealed that sequence features, coding sequence length, and normalized minimal free energy, influenced the translation efficiency of genes. Our study highlights that the loss of eIF(iso)4E.c can result in a highly intricate translation mechanism, acting synergistically with transcription to confer resistance against TuMV.