The hair coat is available for the yak to live in the harsh environment of the plateau. Besides, improving the hair production of yak is necessary for its textile industry development. Hair grows from hair follicles (HFs). The HFs undergo periodic growth after birth and are regulated by the complex gene regulatory network. However, the molecular mechanism of HFs regeneration in the Tianzhu white yak remains unclear. RNA editing is a post-transcriptional mechanism that regulates gene expression and produces new transcripts. Hence, we investigated the influence of the A-to-I RNA editing events on the HFs cycle of the Tianzhu white yak.FAS, APCDD1, WWOX, MPZL3, RUNX1, KANK2, DCN, DSC2, LEPR, HEPHL1, and PTK2B were suggested as the potential RESs involving HFs development.We finally identified 54,707 adenosine-to-inosine (A-to-I) RNA editing sites (RESs) from RNA sequencing data of the HFs cycle in the Tianzhu white yak. Annotation results showed RESs caused missense amino acid changes in 7 known genes. And 202 A-to-I editing sites altered 23 target genes of 140 microRNAs. A total of 1,722 differential RESs were identified during the HFs cycle of Tianzhu white yak. GO and KEGG enrichment analysis revealed several signaling pathways and GO terms involved skin development, hair growth, and HFs cycle. Such as genes with differential RNA editing levels were significantly enriched in the peroxisome, metabolic pathways, Notch signaling pathway, and PPAR signaling pathway. Besides, the editing sites in HFs development-related genes This study investigated the global A-to-I RNA editing events during the HFs cycle of yak skin tissue and expanded the knowledge of A-to-I RNA editing on the HFs cycle. Furthermore, this study revealed that RNA editing-influenced genes may regulate the HFs cycle by participating in the HFs development-related pathways. The findings might provide new insight into the regulation of RNA editing in hair growth.The online version contains supplementary material available at 10.1186/s12864-022-08951-5. Hair grows from hair follicles (HFs), and the HFs of animals could be divided into primer HFs and secondary HFs. After birth, the HFs distinctively undergo cyclical transformations, progressing through stages of anagen, catagen, and telogen . BesidesBos grunniens) is an indigenous yak breed mainly distributed in the Gansu province of China, which is well known for its pure white coat. The well-proportioned coarse and fine hairs on the surface allow them to survive in the harsh environment of high altitude, characterized by strong wind, ultraviolet radiation, and low temperature [Tianzhu white yak  was considered complementary to the antisense strand (-) because the transcriptome data containing antisense transcripts in this study, such as A-to-G + from the sense strand correspond to T-to-C- type from the antisense strand. And the statistics result showed the percentage of editing types was roughly equal between complementary editing types . We know RNA editing events in the protein-coding regions may alter the amino acid sequences and influence the protein functions, then, we further analyzed the editing events in the CDS region. Among these editing sites, 69.64% of editing events caused missense changes in protein sequence Fig. .Genome distribution analysis showed that 0.88% of editing sites occurred in 3′UTR Fig. e. The edConservation analysis showed 101 editing sites were conserved between the human and yak, and only 2 editing sites were conserved between the mouse and yak  of the editing level to analyze sample similarities Fig. a. AccordTo ensure the reliability of identified A-to-I editing events, we randomly selected two types of RESs (A-to-G + and T-to-C-) for validation. The sequence result showed that most nucleotide variations existed in the cDNA sequence but not in the corresponding position of the DNA sequence  were missense variants, which cause amino acid sequences to change in 22 protein-coding genes including 7 known genes and 15 novel genes. The transcriptomes work of humans and mice has also identified thousands of RESs located in the CDS region . Althougperature . A previem cells . In thisem cells . In addiem cells . Hence, APCDD1, WWOX, MPZL3, RUNX1, KANK2, DCN, DSC2, LEPR, and HEPHL1. Previous studies revealed that the mutation of APCDD1 was associated with hair loss in hereditary hypotrichosis simplex patient, and suggested APCDD1 as an inhibitor of WNT pathway may cause HFs miniaturization and hair loss [APCDD1 was also considered a potential factor that caused the various wool characteristics [Wwox-/- mice. And the HFs stem cell numbers were also reduced in Wwox-/- mice. Cao et al. revealed the rough coat mice have a missense mutation in the Mpzl3 gene [Mpzl3 involves a skin disorder. The Mpzl3-/- mouse appeared with sebaceous hypertrophy and hair loss soon after birth [Mpzl3 is involved in epidermal differentiation by interacting with mitochondrial protein [Mpzl3 controls HFs cycling may be related to mitochondria protein activity [Runx1 could promote the proliferation/activation of HFs stem cells and regulate HFs cells from telogen to anagen transition [Runx1 was associated with the differentiation and dedifferentiation of HFs bulge stem cells and maintained a balance between HFs stem cells and early progenitor populations during normal HFs catagen [KANK2 [DCN has been suggested as a candidate factor that is related to age-regulated hair loss [DSC2) has been demonstrated plays a vital role in regulating epithelial morphogenesis [Dsc2 reduction could lead to defective hair morphogenesis in nude mice [DSC2 is also the candidate factor related to woolly or straight hair [LEP and its receptor (LEPR) were observed expressed in hair follicles of bovine skin [Lepr expressed in dermal condensates during HFs morphogenesis, and Lepr could as a marker for dermal papilla cell isolation [HEPHL1 is a member of the multicopper oxidase family, it has been reported that two mutations in HEPHL1 caused abnormal hair phenotype, and the Hephl1 knockout mice appeared with curly whiskers [PTK2B was a hub gene among differential RESs edited genes. The normal growth of hair is closely related to the development of skin. Koppel et al. revealed that function loss of Ptk2b caused wound repair delays in mice [In the previous study, the RNA editing ratio was dynamically changed to respond to the different biological situations or stages. In this study, we analyzed the RESs with dynamically changed editing levels during the HFs cycle. Enrichment analysis results of differential RESs showed that three study groups were all enriched in the peroxisome, metabolic pathways, notch signaling pathway, and PPAR signaling pathway. And these signaling pathways were reported to be essential for HFs development. For example, Notch signaling determines the HFs stem cell fate . And Perair loss . In the eristics . The subzl3 gene . The norer birth . In addi protein . Nicu etactivity . Runx1 iansition , 51. Lee catagen . A previn [KANK2 . HFs steair loss . The devogenesis . The expude mice . In goatght hair . Leptin ght hair . LEP andine skin . It has solation . HEPHL1 whiskers . The pro in mice . All eviIn conclusion, this study detected the amount of A-to-I editing events of Tianzhu white yak during the HFs cycle using RNA-Seq data, and systematically analyzed the protein-coding region, interaction relation between miRNA and mRNA, and the differential RESs during the HFs cycle. GO and KEGG enrichment results of genes with dynamically changed editing level during the HFs cycle suggested several signaling pathways related to skin development, hair growth, and HFs cycling. The findings of this study might provide new insight into the study of RNA editing regulation and mechanism in hair growth and HFs cycle.Bos grunniens reference genome (LU_Bosgru_v3.0) and used BWA with default parameters. Subsequently, the sequence alignment/map format (sam) files were sorted and converted to BAM files by Samtools (1.9).To investigate the RNA editing events during the HFs cycle of Tianzhu white yak, we retrieved 15 publicly available paired-end RNA-Seq data. The data were generated from the skin tissue of Tianzhu white yak from three stages of the HFs cycle containing five-time points , and each time point has three replicates. . The detailed information of samples was shown in  using the default parameters. The SPRINT software could identify RNA editing events without the need to filter out single nucleotide polymorphism (SNPs) according to the difference in distribution characteristics between RESs and SNPs [The editing sites were detected by the SPRINT software (and SNPs . To ensuand SNPs . While thttp://kobas.cbi.pku.edu.cn/anno_iden.php).To investigate the impact of RNA editing on miRNA-mRNA binding regions, we used MiRanda (3.3a) and RNAhybrid (2.1.2) to predict miRNA-mRNA 3′UTR interactions. Based on the unedited reference targets regions (3′UTR) of miRNAs as control. Edited sequence caused new interaction of miRNA was defined as the gain of miRNA binding targets supported by two software, on the contrary, the loss of miRNA binding targets in edited sequence caused referential interaction of miRNA binding targets loss. Then, the altered target genes of KEGG and GO were enriched by KOBAS 3.0 (http://srv00.recas.ba.infn.it/atlas/) databases [To analyze the conservation of editing sites across the yak, humans, and mice. We aligned the 25- base sequence upstream and downstream of A-to-I editing sites against 50 bp flanking regions of the reported human sites by using the BLASTn tools. Human and mouse RNA editing site data were downloaded from REDIportal (atabases . We deemhttps://cn.string-db.org/) and Cytoscape_3.7.2 software to construct the interaction network of the differential RESs edited genes-corresponding proteins.The dynamically changed editing levels during the HFs cycle may with an important function. A-I editing level was quantified as the ratio of the number of G (C) reads to the total number of A (T) and G (C) reads covering the site. The method of editing-level computing was described by Cai. An unpaired student’s t-test method was used for the differential RNA-editing sites analysis  between different periods of the HFs cycle. Then, the KEGG and GO results of differential RESs annotated genes were enriched by KOBAS 3.0. GO terms and KEGG pathways were defined as significant with Fisher’s exact test P-value < 0.05. Then, we used the online STRING database  for the validation experiment were collected from the Tianzhu Tibetan Autonomous County, Gansu Province of China at Jun (Telogen). A total of 19 skin samples and 19 venous blood samples were collected from the anesthetized yaks. The blood and skin samples were collected from the same individual. The operation of sample collection was allowed by the herdsman. We used the skin biopsy punches (8-mm-diameter) to collect skin samples of yak after injecting a local anesthetic (2% lidocaine) into the subcutaneous of the shoulder. We had made all efforts to minimize the suffering of the animals studied here. The animal study is approved by The Animal Administration and Ethics Committee of Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS (Permit No. SYXK-2014-0002). The study is in accordance with all the guidelines and regulations. The study is in accordance with the ARRIVE Guidelines. Total RNA of skins tissue was extracted using TRIzol reagent , and then the total RNA was used as templates for cDNA generation by TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix . The· blood genomic DNA was extracted using the BloodZol kit . PCR reactions were performed using GoTaq (Promega), gDNA was amplified by the mixed template gDNA, and cDNA was amplified by the mixed template cDNA. The primer and RESs information of amplification was shown in (Additional file Below is the link to the electronic supplementary material.Additional file 1: Identified A-to-I editing sites of hair follicles cycle in Tianzhu white yakAdditional file 2: The information of protein-coding genes with missense RNA editing sites Additional file 3: The influence of RNA editing on the relationship between miRNA and mRNAAdditional file 4: The information of conserved RNA editing sitesAdditional file 5: The validation result of RNA editing sitesAdditional file 6: The information of 15 skin samplesAdditional file 7: The information of the primer and validated RNA editing sites


Saccharomyces pastorianus G03H8 with a high RNA yield was developed via ARTP mutagenesis and fed-batch fermentation was applied to optimize production capacity. Genome sequencing analysis was used to reveal the underlying mechanism of higher RNA production genetic differences in the preferred mutant; (3) Results: Compared with the highest RNA content of the mutant strain, G03H8 increased by 40% compared with the parental strain G03 after response surface model optimization. Meanwhile, in fed-batch fermentation, G03H8′s dry cell weight (DCW) reached 60.58 g/L in 5 L fermenter by molasses flowing and RNA production reached up to 3.58 g/L. Genome sequencing showed that the ribosome biogenesis, yeast meiosis, RNA transport, and longevity regulating pathway were closely related to the metabolism of high RNA production; (4) Conclusion: S. pastorianus G03H8 was developed for RNA production and had the potential to greatly reduce the cost of RNA production and shorten the fermentation cycle. This work lays the foundation for efficient RNA content using S. pastorianus.(1) Background: The degradation products of ribonucleic acid (RNA)are widely used in the food and pharmaceutical industry for their flavoring and nutritional enhancement functions. Yeast is the main source for commercial RNA production, and an efficient strain is the key to reducing production costs; (2) Methods: A mutant  Ribonucleic acid (RNA) is an important biological macromolecule .Its degrCandida tropicalis is widely used in industries to produce RNA for its higher RNA content [Saccharomyces cerevisiae that produces a large amount of RNA [ content ,6 but wi content . Researct of RNA ,9. Brewet of RNA  for its t of RNA , easy tot of RNA , and higt of RNA . Additiot of RNA . Howevert of RNA . Comparet of RNA . For exat of RNA .S. cerevisiae increased to 253% by continuously feeding cysteine during fed-batch fermentation [S. cerevisiae and C. tropicalis were achieved with the fed-batch fermentation strategy [The response surface methodology (RSM) has proven to be an effective method in medium optimization . Fed-batentation . Higher strategy ,21.S. pastorianus strain to develop a mutant yeast strain with a higher RNA yield. RSM and fed-batch fermentation were further applied to improve RNA production. Furthermore, genomic sequencing analysis helps us understand the mechanism for higher RNA accumulation in a cell.In this study, ARTP mutagenesis was conducted on the S. pastorianus G03 obtained relatively higher RNA content in the cell 2SO4, (NH4)2C2O4, tryptone) added in YPD medium were used to observe the growth of the strain under different medium conditions. In the tolerance experiment, there were 10, 15, 20, 30, 40, 50, 60 and 70% (w/w) glucose, 4, 6, 8, 10, 12, 14, 15 and 16% (w/w) potassium chloride, 2, 4, 6, 8, 10, 12, 14 and 16% (v/v) ethanol contained in YPD medium or with a different pH .YPD solid medium containing 1% potassium chloride (KCl) were used to select mutants with high RNA yield . Additio7 CFU/mL by cell counter . The output power was 100 W, and the flow rate of the carrier gas (helium) was 10.0 L/min. The mutagenic exposure time was set at 0 to 80 s. The mutants with high RNA yield obtained in each round of mutagenesis were used as the parents for the next round mutation. The fatality rate was calculated with Equation (1) (A was the colony number of the control and mA was the colony number after ARTP treatment).Target strains were screened after several rounds’ selections, as shown in A260). Dry cell weight (DCW) of 1 mL culture was measured after drying the cells at 100 ± 5 °C to meet constant weight. Total RNA content was calculated by the following equation:By measuring the RNA content vs. the biomass of the mutants, we were able to discriminate the candidates and confirm the target mutants. The genetic stability of the mutants was measured by analyzing the changes in RNA content after eight generations’ culture. RNA of yeast cell were measured with Sasano’s methods . After c600 was measured, and the growth conditions of the cells were measured in different mediums containing different levels of tolerant parameters including glucose, KCl, ethanol or different pH.The fermentation ability of the strains were studied with the Duchenne tubule method . Gas proThe contents of 5’-nucleotides (5′-GMP and 5′-IMP) were determined by high performance liquid chromatography (HPLC) with modifications . RNA wasTM HSS T3 5 μm 4.6 × 250 mm Column.Column: Waters XSELECT2PO4, adjusted to pH 5.8 with phosphoric acid; B—Methanol.Mobile phase: A—20 mmol/L KHGradient: 0–10 min 100% A, 10–15 min B increases to 5%, 15–15.1 min was the same, 15.1–20 min 100% A.2 = 0.9992; 5′-GMP, y = 121,634x − 22,559, R2 = 0.9997).The nucleotides were quantified using external calibration curves. A mixed nucleotide calibration standard solution ranging from 0 to 25 μg/mL (GMP and IMP) was prepared  were aligned to the reference genome, S. pastorianus.CBS.1483 genome sequence. The KEGG database  was used to analyze the enrichment of differentially expressed genes. The DAVID bioinformatics resources 6.8 database  was used for GO functional analysis of mutant genes.The yeast was collected after centrifugation at 8000 rpm for 5 min, washed twice with sterile water, treated with liquid nitrogen and stored at −80 °C for genome sequencing. The samples were sent to the BGI Genomics Co., Ltd. . The ligation products were amplified via PCR and sequenced using Illumina HiSeq 2500. The clean data  was applied to optimize the fermentation medium. Plackett–Burman (PB) designs were applied to quickly screen multiple factors and identify the impact of each factor . Each vap-values of these factors were considered as the significant factors which were used to enhance RNA yield by Design Expert 11.0 in central composite design (CCD). The three independent factors were observed at five different levels , as shown in The three smallest v/v) was transferred into a 5 L fermenter with 3 L optimal medium. The ventilation was 1.8 L/min at the beginning and then manually increased when the dissolved oxygen (DO) dropped below 5%. The rotating speed was increased from 180 r/min to 250 r/min, 300 r/min at 20 h and 24 h according to the dissolved oxygen (DO). The initial pH of the medium was adjusted to the optimal pH. For fed-batch fermentation, throughout the fermentation period, the pH was automatically controlled at 5.0 when RNA content was the highest in batch fermentation by using 20% v/v H3PO4 or 20 g/L of NaOH if needed. Ventilation during incubation was constantly adjusted and peanut oil was used as an antifoam agent. Molasses began to be added in the logarithmic phase (8–12 h). The concentration (c = 570 g/L) and flow rate (V = 40 mL/h) of molasses was based on the biggest and fastest consumption in batch fermentation. The flow rate was reduced to 20 mL/h when the dissolved oxygen increased. The samples were taken every 4 h to measure the reduction in sugar content, DCW and RNA production. Fermentation stopped until DO greatly increased and growth kinetic parameters’ calculations were referred to the research [In batch fermentation—conditions changes are shown in research .v:v = 1:1) and the reaction was carried out for 15 min at 70 °C in a water bath. The reaction was stopped by adding 600 μL 200 g/L NaOH. Then, 540 nm (A) was measured when it cooled. Then, 500 μL of water was added to 700 µL solution and the system was tested under the same conditions, which was blank (A0). The standard curve was drawn using 10 g/L glucose after dilution; the glucose content was the abscissa and the absorbance value was the ordinate . The formula for calculating the reduction in sugar content is as follows :The reduction in sugar content was measured with 3,5-dinitrosalicylic acid (DNS) method . The sucp value < 0.05 were considered a statistically significant difference. The experiments were performed in triplicates. The data presented in the tables and figures are the average values of the triplicate experiments with the standard deviations.Statistical analysis was carried out with the standard deviation calculation using Origin Pro 2017. Differences with a S. pastorianus G03H8 was selected as the optimum strain for further study.The optimum exposure time with ARTP was studied  and 80 sCompared to the parental strain G03, the mutant G03H8 was found to start fermentation earlier  and haveFungal resequencing can analyze the types of genomic variation such as single nucleotide polymorphisms (SNPs), insertions, and deletions between closely related strains . This caS. cerevisiae, such as ribosome biogenesis, yeast meiosis, RNA transport, MAPK signaling pathway, tryptophan metabolism, carbon metabolism, and longevity regulating pathway. The results showed that RNA biosynthesis is associated with cell growth. Ribosome is composed of four RNAs (>5400 nucleotides), which are all rRNA and 79 different proteins [Some genes with a significant number of differences were considered in accordance with the KEGG pathway . For eacproteins . Ribosomproteins . The MAPproteins . Carbon proteins ; the lon4)2SO4 were selected as the nitrogen source in RSM optimization. After comprehensive consideration, molasses was chosen as the carbon source, and the yeast was cultured at 28 °C, with an initial pH 6.The optimum growth temperature for yeast growth and RNA accumulation was 28 °C a, where 2+, Fe2+, Zn2+ are very useful for RNA production [4)2SO4, KH2PO4, yeast extract, MgSO4, ZnSO4 and FeSO4. A 12-run PB design was used  with R2 of 0.9491, indicating that 94.91% of the variability in the response could be explained by the model. Additionally, the confidence level of other variables was lower than 95%, which was considered insignificant. Based on the statistical analysis, the factors which had the greatest impact on the yield of RNA were identified as molasses, (NH4)2SO4 and KH2PO4. These variables were ranked as follows: molasses > (NH4)2SO4 > KH2PO4.The regression coefficients for the linear regression model of RNA yield  showed t2PO4 was positive, while the coefficient of (NH4)2SO4 was negative. This meant that increasing the concentration of molasses and KH2PO4 and decreasing the concentrations of (NH4)2SO4 would have positive effects on RNA yield. Therefore, these factors were considered in the experimental design of the steepest ascent  for further optimization.Based on regression results of PB design, the coefficient of molasses and KHt ascent  and the p-value of model was 0.019, was significant, as shown in A highly significant predicted model obtained by CCD was very useful in determining the optimal concentration of the medium components which had a significant effect on RNA yield. A set of 20 experiments  in whichw/w), (NH4)2SO4 0.62% (w/w), KH2PO4 0.72% (w/w), MgSO4·7H2O 2% (w/w), FeSO4·7H2O 0.06 g/L, ZnSO4·7H2O 0.06 g/L and yeast extract 1%. The predicted maximum RNA yield using this model was 158.40 mg/g DCW, which has 23% improvement compared with the original culture conditions. Validation experiments were carried out under the model-predicted medium composition. The RNA content obtained from was 158.03 (mg/g DCW), which was in excellent agreement with the predicted value.All the predicted values of the RSM model were close to the experimental values, as shown in the plot of predicted values versus experimental values a. Three-Scale-up fermentation is necessary for the further application of yeast strains in the industry. Fed-batch fermentation is a common strategy used in the fermentation industry. Here, in this study, fed-batch fermentation was carried out in a 5 L fermenter containing 3 L optimum culture medium. In batch fermentation a, it couIn order to obtain higher biomass during continuous fermentation, molasses was continuously fed to the fermentation at 8 h. With the rapid cell growth and consumption of substrate, the dilution rate increased gradually. On the one hand, the addition of fresh medium could maintain the concentration of the substrate and provide nutrient substance for G03H8. On the other hand, it could reduce the problem of catabolite toxicity and maintain the regular growth of the cell. The pH was automatically maintained at 5, and the temperature was set at 28 °C. The dry weight of the G03H8 reached 60.58 g/L and the RNA yield reached 3.58 g/L after 44 h of fermentation b.Fed-batch fermentation showed obvious advantages over single batch fermentation. The cell biomass increased from 6.17 to 60 g/L, the utilization rate of the substrate increased from 0.26 to 1.58 g/L/h, cell yield coefficient increased from 0.19 to 0.43 g/g and the production efficiency increased from 33.33 to 75.44% in fed-batch fermentation as shown in Saccharomyces cerevisiae [Candida lipolytica [S. cerevisiae has priority in the food industry due to its higher safety. In previous work, S. pastorianus had higher RNA and cell yield when compared to other S. cerevisiae such as wine yeast and baker’s yeast. However, there was no targeted use of S. pastorianus for high RNA yield strain breeding. In this study, a mutant S. pastorianus G03H8 with high RNA yield was developed via ARTP mutagenesis. However, it still needed to be cultured on solid medium with a selectable property, which prolonged the selection period. A more efficient screening method or a higher RNA yield in S. pastorianus are the goals of scientific research, such as high-throughput screening methods [The sources of ribonucleic acid are relatively wide; the industrial production method of ribonucleic acid is mainly the yeast fermentation method at present. revisiae  and Candpolytica  are main methods . In addi methods , metabol methods , etc.Bacillus subtilis EA-CB0575 by 17.2 times and 1.9 times, respectively, under the optimal carbon source and inorganic salt culture conditions. In other respects, the pH, which was controlled to be constant during the fermentation process, was more favorable for the strain’s growth compared to no controlling [High-cell-density culture is a widely used operation model in various industries that provides a higher product yield. It can become more effective by considering multiple factors in process design and implementation, such as microbial strains, their metabolic characteristics and nutritional requirements. The factors affecting high-density culture include types of nutrients, culture conditions, culture methods, and culture systems, etc. Posada-Uribe et al.  increasetrolling . The chotrolling . Fed-battrolling .In this study, a high-density fermentation strategy for molasses feeding was developed by comparing batch fermentations. Molasses contains various nutrients such as abundant sugars, amino acids, organic acids, inorganic compounds, and vitamins . This miS. pastorianus mutant with high RNA yield, was obtained via ARTP mutagenesis. Compared with the parental strain G03, the fermentation speed of G03H8 was faster. However, it had little effect on the assimilation ability of different carbon and nitrogen sources and environmental tolerance. Genome sequencing showed that the mutation direction was concentrated in ribosome biogenesis, yeast meiosis, RNA transport, MAPK signaling pathway, tryptophan metabolism, carbon metabolism, and longevity regulating pathway. After response surface model optimization, the highest RNA content of mutant strain G03H8 increased by 40% compared with the parental strain G03. The RNA production and DCW of G03H8 reached 3.58 g/L and 60.58 g/L in fed-batch with molasses flowing. These findings showed that G03H8 had the potential to greatly reduce the cost of RNA production and shorten the fermentation cycle and provided valuable insight into how to increase the RNA content in S. pastorianus strains for industrial production. The optimum culture composition obtained in these experiments and high-cell-density fermentation of molasses feeding provided a basis for further study using large-scale fermentation for RNA production.G03H8, a


Nature,1 Aguilar et al. describe the small molecule X1 that targets the long non-coding RNA Xist. Upon X1 binding, Xist changes its structure, which prevents its role in X-chromosome inactivation (XCI).In a recent study published in 3 However, protein coding sequences merely constitute ~1.5% of the human genome and only a small fraction of these proteins are currently targetable.2 In contrast, 90% of the human genome is transcribed into RNA in different forms, which makes RNAs compelling targets for small molecule drugs.3 Modulating mRNAs with small molecules could be an alternative route to change the levels of the corresponding protein products and their activities, especially for currently un-druggable proteins. Considering the diverse functions carried out by different classes of RNAs in cells, altering RNAs would provide a broad range of potential drug targets in the human genome. While modulating RNAs with small molecules carries a number of advantages, identifying RNA-targeting small molecules has proven difficult; previous compounds had been identified indirectly by phenotypic screens.3 Systematic approaches to screen for RNA-targeting compounds had been lacking.The vast majority of small molecule screens in drug discovery focus on targeting proteins.Xist , and performed a large-scale unbiased compound screen for the 431 nt-long GC-rich RepA domain of the non-coding RNA ist Fig. . Xist isXist spreading during XCI via its interactions with epigenetic regulators, including polycomb repressive complex 2 (PRC2) and the RNA recognition motif of SPEN.4 In vitro interaction assays indicated that compound X1 selectively competed for binding of RepA to both PRC2 and SPEN with varying inhibitory effects. RNA immunoprecipitation (RIP) studies on mouse embryonic stem cells (ESCs) confirmed that X1 maintained its inhibitory properties in cells and selectively disrupted PRC2 and SPEN interactions with Xist. Strikingly, the RIP assay in ESCs also recapitulated the differences in PRC2 and SPEN inhibition by X1 observed in vitro. Additionally, deleting RepA in fibroblasts entirely abrogated the inhibitory effects of X1, validating that X1 effects were mediated by RepA in cells.RepA facilitates Xist RepA domain had a functional impact on XCI. They observed defects on embryoid body outgrowth of female ESCs upon exposure to compound X1. This effect is Xist-dependent as XY male and XO female cells did not show the growth defects. XCI is characterized by PRC1-mediated monoubiquitylation of lysine 119 on histone H2A (H2AK119ub), followed by enrichment of repressive histone marks, such as tri-methylation of lysine 27 on histone 3 (H3K27me3), via PRC2 activity on inactive X chromosomes (Xi).5 Immunostaining assays confirmed enrichment of those epigenetic markers on Xi, which were significantly abolished by treatment with X1 without affecting Xist expression itself. CHIP-seq analyses showed that the inhibition of PRC2 and H3K27me3 accumulation observed upon X1 treatment were specific to Xi and not observed on active X chromosome (Xa) or autosome. In addition, allele-specific RNA-sequencing revealed that X1 administration prompted increased expression of Xi genes with only minor autosomal effects, suggesting minimal X1 off-target effects to modulate XCI. Notably, this X1 effect is reversible as retrieving X1 treatment restored embryoid body growth and Xi silencing.Ultimately, Aguilar and colleagues tested whether X1 interaction with the To investigate further the molecular mechanistic of X1 inhibition, Aguilar et al. analyzed RepA with and without X1 by small-angle-X-ray scattering. Overall, these analyses showed minor changes in RepA size and shape distribution and suggested that RepA underwent conformational changes upon interaction with X1 without a significant loss of its RNA structure. Further modeling of the RepA 3D structure revealed a distinct RepA structure and reduced conformational heterogeneity upon X1 addition, when compared to its free form. Aguilar et al. proposed that this X1-induced RepA structure reduces RepA affinity for its associated protein interactors, leading to X chromosome activation Fig. .Xist RNA is an untypical target with its large size and high likelihood of being mostly unstructured. However, this work has demonstrated that the ALIS approach can overcome this challenge and could potentially be extended to any type of RNA, especially when structure information is not available. It would still be intriguing to evaluate the versatility of this design to target RNA with different physical properties, for instance small or highly structured RNAs. This work highlights an exciting possibility to utilize RNA-targeting compound to modulate currently undruggable proteins and cellular pathways beyond their protein-driven functions.Altogether, the described work pioneers and exemplifies a direct RNA-binding screen design to identify small molecules that bind to RNA with the potential to exhibit phenotypic effects. In light of the recent increases in efforts to screen for RNA binding small molecules, this work provides a unique blueprint for such a screen. Adjustment to the ALIS design, which was originally developed to target proteins, could improve the identification of RNA-binding molecules and maximize the benefit to attain possibly higher output of identified small molecules in the future than currently presented in this study. In regard to ligandability,


Here, we describe the efficient DNA-templated synthesis and reverse transcription of libraries entirely composed of serum nuclease resistant alternative nucleic acid chemistries validated in nucleic acid therapeutics; locked nucleic acid (LNA), 2′-O-methyl-RNA (2′OMe-RNA), or mixtures of the two. We evaluate yield and diversity of synthesised libraries and measure the aggregate error rate of a selection cycle. We find that in addition to pure 2′-O-methyl-RNA and LNA, several 2′OMe-RNA/LNA blends seem suitable and promising for discovery of biostable functional nucleic acids for biomedical applications.Functional nucleic acids can be evolved  O-methyl-RNA (2′OMe-RNA), or mixtures of the two.Blends of engineered polymerases enable efficient DNA-templated synthesis and reverse transcription of diverse-sequence oligonucleotide libraries composed of locked nucleic acid (LNA), 2′- Instead, substitution of one or two of the four RNA or DNA nucleotides with analogues such as 2′-fluoro- and 2′-aminopyrimidines has been used to prepare partially-modified libraries for “modSELEX”,12,13 yielding mixed-chemistry aptamers with improved biostability. However, such mixed chemistry aptamers  remain vulnerable to nuclease degradation due to the presence of unmodified segments.For applications 14,15 selections involving complete substitution of all four nucleoside triphosphates ((d)NTPs) with biostable artificial analogues (xNTPs) have been reported.16–25 This has historically proven challenging due to the high substrate specificity of DNA and RNA polymerases, which – in most cases – must be evolved or engineered26–28 to permit DNA-templated XNA polymerisation at full substitution for library synthesis. Although elegant DNA-tagging approaches have been devised to avoid the requirement for an XNA reverse transcriptase (RT),21,29,30 where available it may be beneficial to utilise engineered RTs31 for XNA-templated cDNA synthesis, enabling efficient amplification and preparation of templates for subsequent rounds of selection.By comparison, comparatively few examples of “X-SELEX”O-methyl-RNA (2′OMe-RNA), a natural post-transcriptional modification found in ribosomal, tRNA and mRNA, and 2′-O,4′-C-methylene-β-d-ribo- or ‘locked’ nucleic acid (LNA)32,33 , which reduce the entropic penalty incurred by basepairing. Both 2′OMe-RNA and LNA have been shown to improve target strand invasion and specificity of antisense oligonucleotides, siRNAs and CRISPR/Cas systems, as well as boosting activity of DNAzymes and aptamers in physiological conditions, and are generally well tolerated in vivo.11,36 Furthermore, 2′OMe-RNA and LNA phosphoramidites are commercially available, enabling scalable chemical synthesis once functional sequences have been identified and optimised. Systems enabling the selection of biostable functional oligonucleotides using such chemistries therefore continue to be key technological goals for the field.Among the different nucleic acid analogues, 2′-NA)32,33  are of p37 where libraries were synthesised using a mutant T7 RNA polymerase 38 required inclusion of unmodified GTP as well as ‘forcing’ conditions (high xNTP concentrations and Mn2+) in order to achieve synthesis of N30 libraries. Furthermore, selection seemingly required this chimeric library to be supplemented with chemically-synthesised 2′OMe-RNA.37 Reverse transcription of 2′OMe-RNA was possible with Thermoscript (an MMLV RT variant), although inefficient (∼10% cDNA yields on 2′OMe-RNA templates), and the system had a high overall SELEX cycle error rate (51% of amplified cDNAs carried an error).37 Subsequently, improvements in 2′OMe-RNA synthesis and RT were achieved using laboratory-evolved mutants of T7,39–41 the Stoffel fragment of Taq DNA polymerase20 and KOD polymerase,25 although these, too, required Mn2+ and extended incubation times. Most recently, engineering a two-residue ‘steric gate’ in the Tgo DNA polymerase in order to reduce predicted clashes with bulky 2′-modified nucleotides yielded an efficient 2′OMe-RNA synthetase, “pol2M” and its variant “pol3M”.42 Likewise, an efficient 2′OMe-RNA reverse transcriptase was engineered using Tgo by directed evolution, “RT-C8”.31 In the case of LNA, a variety of polymerases have been explored for synthesis and RT,43 including engineered variants of the polymerases Tgo 17 and KOD,25,44 although LNA in selections has thus far been limited to primer regions45–47 or one LNA nucleotide.25An early example of selections for aptamers composed of 2′OMe-RNA49 Here, we expand the range of mixed-chemistry synthetic genetic systems using two XNA polymerase blends to efficiently synthesise and reverse transcribe mixtures of LNA and/or 2′OMe-RNA.Combinations of different XNAs in fully synthetic genetic systems , a natural terminal modification of eukaryotic mRNA involved in the regulation of transcript stability.58 m6Am provides a methyl side-chain that could contribute to formation of hydrophobic paratopes in aptamers evolved to target proteins of interest by analogy to nature's use of m6A RNA modifications as sites for recognition by epitranscriptome regulatory proteins.59Beyond sugar modifications, the addition of a variety of side chains to nucleobases, and the creation of alternative basepairs, has proven to be a successful strategy for the expansion of chemical diversity and function of aptamer and DNAzyme reagents.via cDNA) of longer, diverse-sequence (N40) XNA libraries and assess yields, library diversity and replication fidelity following a complete X-SELEX cycle.Typically, synthesis and reverse transcription of XNAs is assayed by primer extension reactions with short, defined DNA templates and incorporation of a limited number of nucleotide analogues. However, to thoroughly evaluate the suitability of our systems for X-SELEX – in particular for more sophisticated directed evolution experiments such as aptamer selections against challenging targets – we examine synthesis and replication  in the absence of Mn2+ . We initially screened all single- (each LNA-NTP into 2′OMe-NTP mixes) and double-nucleotide combinations  polymers from 2′OMe-RNA primers ; 2′OMe-A, -C, -G, LNA-T (78%); 2′OMe-A, -G, LNA-5mC, -T (80%); LNA-A, -T, 2′OMe-C, -G (81%) and all-LNA (94%). Of the combinations explored, only an LNA purine/2′OMe-RNA pyrimidine mix was inefficiently synthesised ised ESI, Fig. S1.17 LNA × DNA template (as well as LNA × LNA inter- and intra-molecular) hybridisation can be only incompletely denatured by 8 M urea (and boiling in formamide loading buffer) during polyacrylamide gel electrophoresis (Urea-PAGE), resulting in low mobility species , was capable of efficient synthesis of cDNA templated by N40 libraries composed of all six 2′OMe-RNA and/or LNA combinations ,17 had been optimised for LNA RT. However, the mixed-chemistry libraries prepared using one or two LNA nucleoside triphosphates (with others 2′OMe-NTPs) were well tolerated  (6A-containing mix (25% cDNA yield)  , as was A yield) . SpecifiA yield) . AlthougA yield)  Fig. S1,i.e. X-SELEX library quality) is maintained by the mixed-chemistry synthetic genetic systems. For these experiments, we chose to examine the complete replication cycle – i.e. the sum of synthesis, reverse transcription and cDNA amplification – rather than deconvolute the contributions of the individual polymerases as this aggregate measurement is a closer proxy of a full round of X-SELEX, and the performance of each polymerase has been described elsewhere.17,18,31,42 An unbiased defined sequence (“Temp25”)  ESI, Fig. S4,equences , were thequences . RT reac40 oligo was found to have slightly higher A content as well was used ; a spikewas used  Fig. S5 was used  Fig. S4.was used  Fig. S1 −3) and the mixed-chemistry systems (16.1–26.6 × 10−3) .The fidelity of a complete cycle of synthesis and replication using the pol3M/pol6G12[I521L] and RT-C8/RT521L blends (and a blend of Taq and the proof-reading polymerase Deep Vent for DNA amplification) was found to be similar between the pure 2-OMe-RNA (21.7 × 10 × 10−3)  Table S2−3 and 7.05 × 10−3, respectively) (17 (5.74 × 10−3), and lower than the analogous KOD variant system25 (14.0 × 10−3)), we also verified the fidelity of the pure LNA system by cloning and Sanger sequencing amplicons from the first step ‘out-nest’ PCR (rather than generating Illumina sequencing libraries) and obtained a similar total error (∼7.5 × 10−3)   Table S20−3) ESI, Fig. S6.40 sequences . Indeed, we find that the highest incidences of errors occur at AA(A) positions in the 2′OMe-RNA/LNA mixes ixes ESI, Table S325 . Howeve library  Fig. S4c42 and our data suggest that systems in which 2′OMe-NTPs are substituted by one (LNA-T), two  or all four LNA-NTPs offer further improvements. We provide a key proof-of-concept of a system bearing both sugar and nucleobase modifications, suggesting that selections for biostable functional oligos with expanded base chemistry are feasible using commercially available nucleotides , paving the way for development of XNA selection systems with more elaborate side chains.Using blends of previously engineered XNA polymerases, we have established a series of synthetic genetic systems based on mixtures of 2′OMe-RNA and LNA. We report the efficient synthesis and (indirect) replication of diverse-sequence oligonucleotide analogue libraries without major biases in incorporation or reverse transcription. All systems show similar efficiency and fidelity as an all-2′OMe-RNA system, which we have already successfully used to select 2′OMezymes,in vivo.63,64 The use of oligo libraries fully resistant to serum nucleases minimises or abolishes the need to apply extensive post-selection modifications to prepare resulting functional oligonucleotides for in vivo applications (which can negatively affect activity), and enhances the prospects for selections in more realistic biological settings: in live cell or organoid culture or even in whole animals.65 Excitingly, such approaches may offer the prospect of selections for aptamers capable of cell- or organ-specific delivery, and could enable direct screening for modulators of biological phenotype, a crucial approach in the immunoglobulin antibody discovery pipeline,66 but which has yet to be implemented for oligonucleotide aptamers and catalysts.The establishment of a variety of XNA systems based on highly biostable chemistries offers promising platform technologies for the development of a wide range of biostable functional oligonucleotides for diagnostic and therapeutic applications A. I. T. conceived and directed the project. J. R. D. H. acquired, curated and analysed data with A. I. T. and G. D. Polymerases were supplied by N. F., G. H. and P. H. A. I. T. and J. R. D. H. wrote the manuscript with contributions from the other authors.There are no conflicts to declare.CB-003-D2CB00035K-s001


Cardiovascular disease (CVD) is a significant public health issue due to its high prevalence and considerable contribution to the global disease burden. Recent studies suggest that genetic factors, including noncoding RNAs, have an important role in the progression of CVD. Noncoding RNA plays a critical role in genetic programming and gene regulation during development. Ferroptosis is a form of iron-dependent regulated cell death (RCD), which is mainly caused by increased lipid hydroperoxide and redox imbalance. Ferroptosis is essentially different from other forms of RCD in morphology and mechanism, such as apoptosis, autophagic cell death, pyroptosis, and necroptosis. Much evidence suggested ferroptosis is involved in the development of various CVDs, especially in cardiac ischemia/reperfusion injury, heart failure, and aortic dissection. Here, we review the latest findings based on noncoding RNA regulation of ferroptosis and its involvement in the pathogenesis of CVD and related treatments, aimed at providing insights into the impact of noncoding RNA regulation of ferroptosis for CVD. From theroxides –5. Curreeroxides –9.Noncoding RNA (ncRNA) is the RNA molecules transcribed from genomes that do not encode proteins. ncRNA is not only important to transcriptional and posttranscriptional levels but also plays a significant role in epigenetic regulation of gene expression. Recent studies have revealed that about 90% of the genes in eukaryotic genomes are transcriptional genes. Interestingly, only 1-2% of these transcribed genes encode proteins. Most genes are transcribed as ncRNAs.There are two subtypes of ncRNA, including basic structural and regulatory ncRNA. The former plays a role as a housekeeping gene during the translation and splicing, which includes ribosomal RNA, transfer RNA, and small nuclear RNA. From an epigenetic point of view, regulatory ncRNA matters more, because it involves the modification of other RNA. The regulatory ncRNA contains microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), and long noncoding RNA (lncRNA). At present, a large number of studies have demonstrated ncRNAs as cardinal regulators to regulate a series of genetic process including transcription and translation –12.With the in-depth research, a large number of human diseases have been considered to correlate with gene expression under the regulation of ncRNA , includiHere, we conducted a comprehensive review on whether ncRNAs affect ferroptosis in cardiovascular disease and critically analyzed the mechanism of action between ncRNAs and ferroptosis in cardiovascular diseases. We aim to (i) provide insights into the ncRNA-regulated ferroptosis, (ii) emphasize that ncRNAs are involved in the regulation of ferroptosis in cardiovascular diseases, and (iii) explore the possibility of using ncRNAs as targets to interfere with ferroptosis in cardiovascular diseases.At present, studies have revealed that programmed cell apoptosis is closely related to the occurrence and development of cardiovascular disease . Dixon e2+ reacts with ceruloplasmin to convert to Fe3+, which then binds to transferrin (TF) to form a complex. The complex is endocytosed into cells by membrane protein TF receptor 1 (TFR1) [3+ is stored in unstable iron pools (LIP) and ferritin is reduced to Fe2+ for the synthesis of iron-dependent enzymes [The iron metabolism pathway is the key to ferroptosis. Fe1 (TFR1) , 24. Int enzymes , 26 and  enzymes . Unstablc− play an important role in iron death. GSH is one of the key reductants in vivo and enables to maintain the redox state of cells. System xc−, as a cystine-glutamate exchanger, is composed of two proteins, including 12 channel transmembrane protein transporter solute vector family 7 member 11 (SLC7A11) and single-channel transmembrane regulatory protein solute vector family 3 members 2 (SLC3A2). Cystine and glutamate are exchanged in equal proportions by the system xc−. GSH is synthesized by cysteine, glutamate, and glycine in two steps.Glutathione (GSH) and system xWith GSH, GPX4 eliminates lipid peroxides by reducing the presence of complex phospholipid hydroperoxides (PL-OOH) in cell membranes, thus preventing ferroptosis , 30. TheRecently, many studies have indicated that ferroptosis also has an important effect on cardiovascular disease. Fang et al. found that the mechanism of doxorubicin (DOX) induced cardiomyopathy by upregulating heme oxygenase-1 (Hmox1) through Nrf2, which degrades heme, increases free iron in cardiomyocytes, leads to lipid oxidation, and then damages mitochondrial structure and function . This stcis-/trans-regulatory elements, introns, pseudogenes, repetitive sequences, and telomeres. At present, ncRNA is mainly classified according to its length and function. In terms of length, it is divided into small RNA (18-200 nt) and long ncRNA (>200 nt); small RNA includes ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and piwi-interacting RNA (piRNA). Functionally, they are divided into housekeeping ncRNAs such as rRNA and tRNA, and regulatory transcripts such as miRNA, long noncoding RNA (lncRNAs), and circular RNA (circRNA). It is found that ncRNA plays a functional role in remote tissues, which also means that ncRNA plays a part in a variety of biological functions through various mechanisms. miRNA is essential in the regulation of human diseases, especially in cancer progression [cis- and trans-genes through histone adornment or destroys the miRNA regulation [Most human RNA transcripts do not encode proteins, so they are called ncRNAs, which were used to be regarded as “junk” DNA . In the gression , 13. Whegression . The fungulation , 40. Witgulation .+, thereby enhancing ferroptosis [c−31 [Some ncRNA can target the mRNA directly and regulate relevant proteins, including GSH, ferroportin, and mitoferrin. Ding et al. found that miR-182-5p and miR-378a-3p negatively regulate the expression of GPX4 and SLC7A11 by directly binding the 3′UTR (untranslated region) of GPX4 and SLC7A11 mRNA to promote ferroptosis . In the roptosis . In radiroptosis . In addiis [c−31 . miR-541is [c−31 .On the other hand, some ncRNA induced methylation modification and posttranscriptional regulation of related protein, or promote ferroptosis indirectly via the activation of the upstream protein. Zhang et al. found that miR-522 inhibits ferroptosis in gastric cancer by inhibiting the activity of arachidonic acid lipoxygenase 15 (ALOX15) . ClinicamiRNA is an endogenous noncoding single-stranded small RNA molecule, which is composed of 21-23 bases. The role of miRNAs is to hybridize with part of the UTR of mRNA, causing mRNA to be cleaved or inhibiting mRNA translation. Research has demonstrated that a miRNA can regulate different target genes. Besides, several different miRNAs can jointly regulate a target gene .in vitro [miRNAs play an important regulatory role in the pathophysiology of CVD . Recentlin vitro . Studiesin vitro . At the in vitro . Additioin vitro . Similarin vitro . Also, min vitro .cis- or trans-regulation of chromosome structure, transcription, splicing, mRNA transport, stability, and translation, thus extensively affecting many cellular processes. At present, studies have shown that lncRNAs play a vital role in biological functions [β cell-specific transcription factor network. PLUTO of a lncRNA encodes a key β cell transcription factor by affecting chromatin structure and PDX1 transcription. It was also found that the levels of PLUTO and PDX1 were decreased in the islets of donors with type 2 diabetes or impaired glucose tolerance [Long noncoding RNAs (lncRNAs) are transcripts that are longer than 200 nucleotides but do not encode protein . lncRNAsunctions . For exaunctions . Previouolerance .κB and AKT signals, leading to increased expression of adhesion molecules [Recently, studies have found that lncRNAs can be used as biomarkers for many cardiovascular diseases. Ye et al. indicated that the myocardial infarction-associated transcript (MIAT)/miR-149-5p/CD47 pathway of macrophages plays an important role in the formation of atherosclerotic plaques. The decrease in MIAT level can alleviate the progression of atherosclerosis, stabilize plaque, and promote the clearance of apoptotic cells by macrophages. MIAT is a tiny ribonucleic acid sponge and regulates the expression of the antiphagocytic molecule CD47 through the sponge miR-149-5p . Studiesolecules . Sun et olecules .Wang et al. showed that lncRNA-LINC00336 as an oncogene promotes tumor cell proliferation, inhibits ferroptosis, and induces tumorigenesis in an ELAV1-dependent manner . StudiesκB in colorectal cancer cells [In back splicing, the downstream (3′) splice donor site reversely accesses the upstream (5′) splice acceptor site, resulting in the formation of covalently linked circRNAs, lack of 5′ caps and 3′ poly-A tails. This makes circRNAs resistant to exonuclease RNase R digestion, so circRNAs are more stable than linear ribonucleic acid. Initially, circRNAs were considered waste products from abnormal splicing events. Only testis-specific circular ribonucleic acid of the sex determining region Y (Sry) gene may be functional . In receer cells . Holdt eer cells .With the continuous deepening of circRNA and ferroptosis research, whether circRNA can impact CVD by regulating ferroptosis has become a focus of attention. Li et al. found that circRNA1615 is inhibited ferroptosis by miRNA152-3p/LRP6 axis regulated cell autophagy in cardiomyocytes of myocardial infarction. Moreover, a previous study showed that Ferrostatin-1, a ferroptosis inhibitor, has an important function in protecting myocardial infarction and reperfusion injury via suppressing ferroptosis . LRP6 haEndothelial cell death is associated with vascular diseases such as arteriosclerosis and tissue ischemia. Erastin was utilized by Hong et al. to trigger ferroptosis in HUVEC cells. They discovered that miR-17–92 protects HUVEC cells against ferroptosis by inhibiting the A20-ACSL4 axis . Wu et aThe related mechanism of of drugs treating CVD is shown in In recent years, research on non-coding RNA and ferroptosis has continued to progress. A large number of studies have shown that both are involved in the occurrence and development of a variety of cardiovascular diseases. Noncoding RNA is widely expressed in the cardiovascular system and plays an important role in the occurrence and development of cardiovascular diseases (98). Specially, ncRNA crosslinked ferroptosis and CVDs. Ferroptosis is triggered by lipid oxidative stress and iron excess, and ncRNAs can regulate the expression of genes associated with different ferroptosis-related events, including iron homeostasis (FTH1) , cell pr


Green Human Resource Management (HRM) supports promoting and incorporating sustainable development with regard to their resources. Managers and customers actively utilize the limited resources efficiently and effectively to accomplish environment-friendly goals and objectives. The study focuses on investigating the moderating role of diffidence between Green HRM, among eco-friendly behavior and Employee Performance of frontline employees of the hospitality sector. Two hundred ten individuals particapted in the research from hospitality sector with regard to examine green HRM policies of employees along with their influence on consumer buying behvaiour. Frontline employees incorporated the data on HRM performance, eco-friendly behavior, and diffidence. Besides, consumers gave their opinion on consumer buying behavior. The study’s findings revealed that Green HRM, aka Green HRM, directly impacts consumer behavior. In addition to this diffidence moderates the relationship between Green HRM and employee performance and employee eco-friendly behavior. Besides, future studies can explore the clothing and banking sector as the current study was conducted in fast food sector. Studies have indicated that organizations are adopting those practices, which are more eco-friendly . This isOrganizations continue to convert their conventional HRM practices to Green HRM practices to enhance their environmental performance through human capital development . In addiFirst, recent studies have observed the impact of green HRM; at the organizational and individual levels  and on eSecond, the research argued that green HRM affects employees, shareholders, contractors, communities, and consumers . PreviouThird, for the last few years, the conservation of the natural environment has been an important issue, and for this purpose, nearly every organization is switching toward environmental protection practices . PreviouForth previous studies have explored that employee involvement  has a poThis research aims to fill the given gaps: First, to examine Green HRM’s impact on consumer buying behavior. Second, to observe the mediating role of employee eco-friendly behavior between Green HRM and consumer buying behavior, Third, to evidence the mediating role of employee performance between Green HRM and consumer buying behavior. Fourth, to know the moderating effect of employee diffidence on the relationships between Green HRM and employee eco-friendly behavior and employee performance.This study offers both theoretical and practical contributions. Theoretically, this research adds that Green HRM is a crucial aspect that influences the consumer buying behavior and as well as the employee behavioral outcome like employee eco-friendly behavior and employee performance. Moreover, it also contributes to the current literature that diffidence moderates the social interaction between employees and consumers. Practically, organizations should start focusing on Green HRM practices to positively impact consumer buying behavior to enhance the organization’s reputation. Moreover, organizations should focus on Green HRM to develop eco-friendly employee attitudes that can motivate and support employees in reducing their workplace diffidence.Social Identity theory defines as the people enjoy belonging to groups that have great reputations because it reinforces their sense of self-worth . The socSocial exchange theory elaborated the conduct between two or more parties in terms of conducting social interaction for the sole objective of exchanging goods. Besides, further illustrates that actions are contingent in terms of rewarding others in accordance with their behavior . For thaGreen HRM is defined as the process of making green employees with the use of green HR policy and practices, and this is for the benefit of individuals, community, business, society, and the whole planet . EnvironH1:Green HRM has a positive impact on Consumer Buying Behavior.One of the main purposes of green HRM is to develop environmental responsibility and sensitivity in employees to become aware of how their behaviors (in-role and extra-role) affect the environment . The resH2:Green HRM has a positive impact on Employee’s Eco-friendly behavior.Employee job performance is an important part of every organization . An orgaH3:Green HRM has a positive impact on Employee Performance.As employees are shifting toward green practices, consumers are also becoming keenly aware of green issues such as a threat to natural resources, global warming, and pollution; thus, they consider these issues when making green purchasing . The empH4:Employee’s Eco-friendly Behavior has a positive impact on Consumer Buying Behavior.Consumers are the most salient groups where frontline employees interact . ConsumePositive interaction between employees and customers increases customer loyalty . Liao anH5:Employee Job Performance has a positive impact on Consumer Buying Behavior.Organizations have an HRM department for hiring and selection, training and development, reward, compensation, and performance improvements of their employees . The praH6:Employee Eco-friendly Behavior will mediate the relationship between Green HRM and Consumer Buying Behavior.The shift of responsibility toward the society and environment has been the concern of organizations, scholars, and government . It gaveH7:Employee Performance will mediate the relationship between Green HRM and Consumer Buying behavior.Interpersonal communication is essential for an organization because a better relationship can be developed between employees and consumers, better expressing and receiving consumer needs . It has H8 (a):Diffidence moderates the relationship between Green HRM and Employee Ecofriendly Behavior.H8 (b):Diffidence moderates the relationship between Green HRM and Employee Performance.Data for this study was obtained from the frontline employees and customers of different fast-food chains. Frontline employees and customers visiting fast-food chains were selected for this study because globally, the hospitality sector is considered agents working to promote sustainability . This reDuring service encounter, the consumer interacts with first-line employees (and observe his performance) which ultimately affects consumer behavior. Instance, Data was collected from 210 respondents by adopting a questionnaire technique. A questionnaire has two parts: the first was for the frontline employees, and the second was for the customers. Both parts of the questionnaire comprised three sections. The first section briefly described the purpose of the data collection and assurance of confidentiality of all responses. The second section included information on the respondents’ demographics to verify the study respondents’ diversity. The third section consisted of predefined, closed-ended questions about the research. Researcher applied convenience data collection technique due to the time constraints and pandemic restrictions.Data was collected from the hospitality sector especially from restaurants. The main focus of the study was the fast-food sector within the hospitality sector. Customers and frontline employees were contacted and informed about the study’s objectives. Favorable support was obtained to administer, the questionnaires and all management confirmed that their organizations had established programs involving employees and their green behavior. A contact person from every fast-food chain was identified to provide customers who agreed to assist with data collection from each brand. The questionnaire was distributed and collected directly from the frontline employees and customers during their visit to that particular brand. Complete anonymity and secrecy of the respondents were assured as the management and customers had no access to the completed responses. The researchers ensured that the frontline employees and customers filled their respective parts of the questionnaire separately at separate timings to maintain secrecy. After the data collection, the researchers paired each questionnaire of frontline employees with their customers by asking for the customer’s feedback about their facilitated frontline employee in both parts of the questionnaire. Sixty questionnaires were distributed at each fast-food brand, the total number of 300 questionnaires was distributed. Data of 210 respondents was the appropriate sample size because Out of 360 frontline employees, only 255 participated in this survey from all six fast-food brands because of their visits and personal availability. Removing 45 incomplete questionnaires, 210 were useable for the study representing a response rate of 58.33%. The detail of these respondents can be seen in Green HRM, employee eco-friendly behavior, diffidence, and employee performance was rated by the frontline members of different fast food brands Green HRM and Eco-friendly behavior were measured with six and seven items scale, respectively . DiffideThe consumers of different fast-foods’ industry rated consumer buying behavior and it was measured using the two-item scale adapted from Data set of 210 responses were entered in Statistical Package for Social Sciences (SPSS). Initial screening was done through normality, reliability, and validity. Normality was observed with Skewness and Kurtosis. Findings approved that all the items are within the normal range of + 3 . ReliabiResearcher deleted some of the items to develop a good model fit and for the reduction of the cross loading. The development of the good model fit indices is the requirement to develop a good reliability and validity analysis . So thisDiscriminant validity was examined through the correlation between the variables of the study, and the square root of AVE should be greater than its peer values . All theBiases were observed through common method variance using Herman’s single factor test as proposed by Hypotheses testing was estimated using the partial-structural equation modeling technique in AMOS. Initially, the structural model fit indexes were observed. All the values of model fit were found in acceptable range that is CMIN/DF = 16.018, GFI = 0.964, AGFI = 0.644, CFI = 0.915, NFI = 0.912, TLI = 0.490 and RMSEA = 0.268. Furthermore, the approved conceptual model can be viewed in Researcher testified the mediation analysis by applying the amos plugin (My specific indirect effects estimands) this plugin eases the researcher to conclude mediation analysis directly without using bootstrapings.The moderation was checked through Partial-SEM in AMOS by following the path of researcher . It was Based on those study responses of the respondents, all hypotheses were accepted. The conceptual framework of this study contributed theoretically and empirically for academicians and was found to be practical for management.This research added to the scant amount of existing literature for academicians. The findings of this study offer a significant contribution to the literature. Firstly, it contributes to the green HRM literature by adding supportable evidence of the relationship between organizations’ green HRM and consumer buying behavior. Secondly, this study also contributed to the literature of the diffidence that employee’s diffidence influences social interaction between employee and consumer, affecting the employee’s behavior and performance. Employee shyness, loneliness, lack of confidence, etc., have been studied in the past, but this is the first study that has focused on the combination of these factors i-e diffidence. Thirdly, it also contributes to the literature to the indirect effect of Green HRM on consumer buying behavior that has never been investigated in terms of mediators such as employee eco-friendly performance and behavior. The study provides an important understanding of the relationship between Green HRM and employee green behavior and performance by hypothesizing the moderating effect of employee diffidence.Green HRM is the first step organizations should embrace involving environmental sustainability. The firms should focus on their green HRM practices and provide green training, initiatives, and green rewards to employees to get them engaged in green practices. Moreover, such practices can influence consumers’ buying behavior and, in return, can influence and increase the organization’s environmental environment social image. Employee Eco-friendly is the extra-role behavior of employees. If the organizations want to encourage eco-friendly behavior at the individual level, they can do so by implementing HRM activities, practices, and policies directed toward the environment. Employee performance is measured by HRM performance evaluation. Organizations can improve their performance management system by aligning their performance management system with environmental management objectives which can also act as performance indicators during evaluation by setting such eco-objectives for the employees to achieve those and improve their performance. Diffidence or shyness of employees can be reduced by motivating them for eco-friendly behavior. The lower the diffidence, the higher employees will exhibit eco-friendly behavior. Organizations should help, inspire, and give support to the employees and give training to them on how they can minimize their diffidence in social interactions and gatherings in the workplace.Results suggest that managerial practitioners can achieve their sustainable goals more efficiently and effectively by following this study’s findings. This study presented an approved hierarchical model for consumer behavior. This model observed that sustainable consumer behavior can be manifested through the employment of Green HRM, eco-friendly employee behavior, and employee performance. Green HRM positively and significantly contributed to employee eco-friendly behavior and employee performance. Ecofriendly behavior and performance of employees simultaneously support consumer behavior. In addition to this, it has also been observed that Green HRM makes a direct and indirect effect (through employee eco-friendly behavior and employee performance) upon consumer behavior. Diffidence has been proven to be a barrier for employees to perform their green motives, and especially involving their eco-friendly behavior and performance.This study has several limitations that will help guide future research in this area. First, this study considered information from respondents in a hospitality industry in a developing country. Although the cities are diverse in terms of demographics, behavior toward sustainable models can vary. Future studies are encouraged to apply the same model using different countries in different demographics so that intra-city and intra-country comparison can be estimated to determine the model’s acceptability. Second, this study collected the responses of employees and consumers of fast-food and observed their behavior for green policies of fast-food sector. Future researchers can observe the same model for educational institutions, banks, and other services sectors. Third, this study addressed diffidence as only one barrier regarding employees and Green HRM. Future studies should consider other barriers such as communication skills, short-term focus, resistance to change, etc.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by this study involving human participants was reviewed and approved by the Ethics Committee of the Department of Management Sciences, Riphah International University Islamabad, Faisalabad Campus, Faisalabad, Pakistan. The participants provided their written informed consent to participate in this study. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


As of December 31, 2020, there were 12 facilities located in Asia and Europe which were treating cancer patients with carbon ion radiotherapy (CIRT). Between June 1994 and December 2020, 37,548 patients were treated with CIRT worldwide. Fifteen of these patients were United States (U.S.) citizens. Using the Surveillance, Epidemiology, and End Results cancer statistics database, the Mayo Clinic in Rochester, MN has conservatively estimated that there are approximately 44,340 people diagnosed each year in the U.S. with malignancies that would benefit from treatment with CIRT. The absence of CIRT facilities in the U.S. not only limits access to CIRT for cancer care but also prevents inclusion of U.S. citizens in phase III clinical trials that will determine the comparative effectiveness and cost effectiveness of CIRT for a variety of malignancies for FDA approval and insurance coverage. Past and present phase III clinical trials have not been able to enroll U.S. citizens due to their unwillingness or inability to travel abroad for CIRT for an extended period. These barriers could be overcome with a limited number of CIRT facilities in the U.S. Carbon ion radiotherapy (CIRT) is a more physically targeted form of radiotherapy compared to photon (X-ray) or proton radiotherapy due to a sharper lateral beam penumbra and end of range Bragg Peak –4. One oA series of dose-escalating phase I/II and phase II clinical trials conducted at QST Hospital  in Chiba, Japan since 1994 have demonstrated that CIRT is safe and effective in the treatment of a number of recurrent, previously irradiated, photon-based radiation-resistant, and/or hypoxic malignancies including prostate cancer, salivary gland cancer, bone and soft tissue sarcoma, paranasal sinus mucosal melanoma, recurrent rectal cancer, non-small cell lung cancer, and liver cancer . These iThis original evaluation was conducted to determine the number of U.S. citizens who have traveled abroad to receive CIRT. In addition, we present a case series describing the types of malignancies which have been treated in U.S. patients, and patient outcomes including survival, tumor control, and toxicity.www.ptcog.ch) was used to identify all CIRT facilities in the world treating patients as of December 31, 2020. The CIRT facilities were contacted to determine if they have treated U.S. citizens who have traveled abroad for CIRT from the date their facility opened and treated their first patient through December 31, 2020. U.S. citizens who had traveled abroad were verified by passport information. U.S. citizens living abroad and non-U.S. citizens living within the U.S. were not included.The Particle Therapy Co-Operative Group (PTCOG) website  increasing the number of multinational randomized clinical trials, 2) leveraging the existing CIRT facilities to launch larger multinational trials directed at common cancers combined with high-level quality assurance; and 3) developing more compact and less expensive next-generation treatment systems integrated with radiobiologic research and preclinical testing (The absence of U.S. CIRT facilities not only limits access to CIRT for cancer care but also prevents inclusion of U.S. citizens in phase III clinical trials that will determine the comparative effectiveness and cost effectiveness of CIRT for a variety of malignancies for FDA approval and insurance coverage. Past and present phase III clinical trials have not been able to enroll U.S. citizens due to their unwillingness or inability to travel abroad for an extended period due to the barriers noted above. These barriers could be overcome with a limited number of CIRT facilities in the U.S. with perhaps one located in the Eastern, Midwestern, and Western U.S.via responsive, respected radiation oncologists with particle therapy experience and subspecialty expertise in malignancies with indications for CIRT who will communicate openly and in a timely manner with referring physicians, patients and their families; radiation therapists, certified medical dosimetrists and medical physicists with particle therapy expertise; educational infrastructure to train the next generation of CIRT providers, research infrastructure to conduct basic science and clinical research, and a comprehensive cancer and medical center to provide surgery, medical therapy, rehabilitative services, social services, psychologic support, chaplain services and spiritual support, and concierge services to help patients and family members with transportation, food and housing, entertainment, and insurance coverage.The ideal CIRT facilities in the U.S. would have existing patient volumes to match the capacity of the facility, allow easy and rapid access to CIRT Fifteen U.S. citizens have been treated with CIRT. Substantial barriers to traveling abroad for CIRT exist. A limited number of CIRT facilities in the U.S. would improve access to CIRT and enhance accrual to phase III clinical trials. U.S. patients deserve more practical access to life saving cancer treatments which are available elsewhere in the world.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was reviewed and approved by QST Hospital. Written informed consent for participation was not required for this study in accordance with national legislation in the United States and institutional requirements of Mayo Clinic.All authors have made substantial contributions to conception and design, acquisition of data, analysis, and interpretation of data; and have been involved in drafting the manuscript or revising it critically for important intellectual content; and given final approval of the version to be published. Each author has participated sufficiently in the work to take public responsibility for appropriate portions of the content; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Membraneless cytoplasmic condensates of mRNAs and proteins, known as RNA granules, play pivotal roles in the regulation of mRNA fate. Their maintenance fine-tunes time and location of protein expression, affecting many cellular processes, which require complex protein distribution. Here, we report that RNA granules—monitored by DEAD-Box helicase 6 (DDX6)—disassemble during neuronal maturation both in cell culture and in vivo. This process requires neuronal function, as synaptic inhibition results in reversible granule assembly. Importantly, granule assembly is dependent on the RNA-binding protein Staufen2, known for its role in RNA localization. Altering the levels of free cytoplasmic mRNA reveals that RNA availability facilitates DDX6 granule formation. Specifically depleting RNA from DDX6 granules confirms RNA as an important driver of granule formation. Moreover, RNA is required for DDX6 granule assembly upon synaptic inhibition. Together, this data demonstrates how RNA supply favors RNA granule assembly, which not only impacts subcellular RNA localization but also translation-dependent synaptic plasticity, learning, and memory. RNA granules are important regulators of RNA metabolism. Here the authors report that RNA granules containing RNA helicase DDX6 disassemble during neuronal maturation. The nervous system sticks out as it displays by far the highest transcriptomic diversity5. Hence, to enable spatiotemporally controlled protein expression, RNA-binding proteins (RBPs) play crucial roles by guiding mRNAs through the different stages of their life cycle in the cell6. RBPs assemble with their target mRNAs into RNA granules or ribonucleoprotein particles (RNPs)7. Since their original discovery9, many different types of RNA granules have been identified and described, e.g. processing bodies (P-bodies), stress granules, and transport granules among others12. Importantly, the content of these RNPs is dynamically regulated by both the demand and supply of RNAs15. While the demand crucially depends on translational activity, the supply of RNAs is additionally determined by transcription, splicing, translational repression, and eventually decay. Consequently, RNPs are able to dynamically assemble and disassemble in order to react to changing cellular demands. This behavior has been nicely visualized by time-lapse fluorescence microscopy17. In addition to their assembly dynamics, RNPs are very heterogenous in protein and RNA composition18. Therefore, it is tempting to postulate that a network of RBPs and eventually RNA granules serves as a dynamic buffer system to allow homeostatic RNA metabolism inside the cell6. This RNA-based mechanism is a prerequisite for cells to respond to different stimuli in order to remodel the neuronal proteome. Thereby, RNPs balance the RNA demand and supply that is needed for adequate protein synthesis, which ultimately enables cellular plasticity. Disrupting the balance of RNA homeostasis can have important consequences, such as the development of neurodegenerative diseases, which in some cases are hallmarked by pathological deposits of RNA granule components19. Here, the concept of liquid-liquid phase separation has an important impact on our understanding of how RNA and RBPs form RNA condensates inside cells20. Interestingly, RNA recruitment affects the size and composition of RNA-protein condensates in cells21. While the relevance of RNA availability for RNA granule assembly22 has already been studied in vitro and under diseased or stress conditions, its physiological impact is much less understood. Therefore, we aimed to investigate the assembly and disassembly of RNA granules under physiological conditions in cells. We chose the RNA helicase DDX6 , which has been shown to mediate translational suppression of specific mRNAs in P-bodies24. Notably, DDX6 is not only found in P-bodies, but also in other types of RNA granules, including stress granules and Staufen2 (Stau2) containing transport RNPs18. The yeast homolog Dhh1 regulates RNP assembly and turnover25. Together, this underlines the importance of DDX6 in gene expression regulation26. Here, we report that DDX6 granule formation is regulated by RNA supply in the neuronal cytosol, as well as neuronal maturation and synaptic activity.Eukaryotic cells constantly adapt their gene expression in response to specific cellular demands. This happens primarily at the transcriptional level, however, a number of additional mechanisms allow for subsequent post-transcriptional regulation. Post-transcriptional gene regulation crucially contributes to many important cellular processes, e.g. development, differentiation, and maturation of cells in tissues, but also to many pathological alterations. Prominent examples are cancer and neurodegeneration among many others27, we speculated that these alterations might influence RNA granule properties as well, and studied DDX6 granule formation at different stages of maturation28. We observed cells with larger and smaller DDX6 granules at all observed time points during maturation, though at different proportions. To quantify this variability in the cell population, we manually counted > 100 cells per time point and biological replicate by eye and categorized cells into two groups: cells containing either preferentially large or small granules. Examples of this quantification are provided in Suppl. Fig. F3,8 = 0.0044). This occurred in a similar fashion during the maturation of cultured cortical neurons   vs. 29 DIV), but an increase in their number (p = 0.0081), further corroborating RNA granule reorganization . The observed disassembly was accompanied by decreased DDX6 protein levels, both in hippocampal  and investigated its impact on DDX6 granule formation. Interestingly, the inhibition of synaptic activity in mature neurons resulted in the reassembly of DDX6 into larger granules . Detailed single particle analysis revealed a significant increase in average granule size and a decrease in the number of granules in the cell body . Importantly, these newly formed assemblies were not stress granules, nor did this treatment induce stress granules . Additionally, DDX6 disassembly could be rapidly induced by synaptic activation of NMDA receptors, independent of the prior treatment. To gain further insight into how granules are disassembled upon NMDA treatment, we transiently expressed GFP-DDX6 in hippocampal neurons and performed live imaging during NMDA application and subsequent NMDA wash-off  of its targets and is an essential RNA transport protein responsible for localizing a significant fraction of the neuronal transcriptome within different cellular compartments36. A DEAD box RNA helicase such as DDX6 may play a role in the regulation of such complex targets. Moreover, Stau2 can transiently interact with other types of RNA granules in living neurons17 suggesting that it serves as a dynamic RNA distributor and regulator in cells. To investigate whether Stau2 indeed associates with DDX6 in the cell body of neurons, we performed co-immunostainings . However, in the absence of Stau2 neuronal inhibition failed to induce DDX6 assembly. This was confirmed by single particle analysis, showing that a significant increase in DDX6 granule size  and a reduction in granule number , harringtonine (HRN), and cycloheximide (CHX). Though all three compounds inhibit translation, they differ in their underlying mechanism, as PMY dissociates translating ribosomes and releases the bound transcript, HRN blocks initiating ribosomes and thereby induces ribosome runoff, whereas CHX results in stalling and stabilization of ribosomes on mRNAs43. Thus, all three inhibitors have a different impact on the pool of mRNAs that are available for granule formation; while PMY and, in a translation speed dependent manner, HRN eventually increase the pool, making mRNAs abundant in the cytoplasm and available for other interactors, CHX restricts the number of assembly competent RNAs. We found that PMY treatment resulted in fast DDX6 granule assembly . Additionally, HRN treatment caused a gradual assembly upon prolonged incubation , possibly due to the induction of ribosome runoff that depends on translational speed  and decrease in granule number  in both cases. Conversely, neurons treated with low concentrations of CHX showed a moderate decrease in the neuronal population containing large DDX6 granules , opposing the assembly promoting effect of neuronal inhibition. Again, these assemblies did not represent stress granules, nor did the treatments induce stress granules on their own , spatially distinct to DDX6 granules  and confirming our overexpressed reporter phenocopied endogenous DDX6 . Interestingly, the smaller granules formed by GFP-DDX6-RNase1 did not significantly increase in size after neuronal inhibition , which was C-terminally tagged with the endonuclease RNase1 Fig. , to effeion Fig. . This stion Fig. .Fig. 5RN50. Hippocampal neurons were transduced using a lentivirus expressing a short-hairpin RNA targeting DDX6 mRNA (shDDX6) resulting in a ~ 40% reduction of DDX6 protein and DDX6 granule size, mimicking the reduction in protein levels and granule size observed during neuronal maturation  embryos of timed pregnant Sprague-Dawley rats (Charles River Laboratories) were isolated, cells dissociated, and plated on Poly-l-Lysine coated coverslips and cultured in NMEM + B27 medium (Invitrogen). Experiments were performed with cultured neurons between 8–29 days in vitro (DIV). Neurons were transiently transfected by calcium phosphate co-precipitation as previously described65. A total amount of 3 µg DNA was used for single or co-transfected plasmids. All animals were used according to the German Welfare for Experimental Animals .Primary rat hippocampal and cortical neuronal cell cultures were generated as previously described67. In brief, brains were removed, postfixed in 4% PFA overnight, washed 3x with PBS and placed in cold 30% sucrose in PBS till they sunk down (~2 days). Samples were embedded in OCT (Tissue-Tek) and cryopreserved. Sagittal cryosections (40 µm thick) were permeabilized with PBS-0.1% Triton X-100 (PBT) and then blocked with 1% BSA in PBT. Primary polyclonal goat anti-DDX6 (Abnova) antibody was incubated o/n at 4 °C. Secondary calf anti-goat Alexa488 or Alexa555 conjugated antibodies (Dianova) were incubated for 2 h at RT. Slides were mounted with Aqua-Poly/Mount (Polysciences).Eight day postnatal and 10 month old male black 6 mice  were anesthetized and intracardially perfused with PBS (Invitrogen) followed by 4% paraformaldehyde (PFA) and processed as published2+ imaging was obtained from Addgene (#40753)68.The DDX6, RNase1, and the first 99 nucleotides of TOMM20 sequences were obtained by PCR amplification from rat cDNA without a functional stop codon. The pEGFP-DDX6 plasmid was generated by placing the DDX6 sequence in frame with GFP in the pEGFP-C1 vector (Clontech). The pEGFP-DDX6-RNase1 plasmid was obtained by placing the RNase1 sequence in frame into the pEGFP-DDX6 vector. A pTOMM20(1-99)-TagRFP plasmid was generated by placing the pTOMM20(1-99) sequence in frame with TagRFP in the pTagRFP-C vector (Evrogen). pTagRFP-RNase1 and pTOMM20(1-99)-TagRFP-RNase1 plasmids were obtained by cloning the RNase1 sequence in frame with pTagRFP-C (Evrogen) and pTOMM20(1-99)-TagRFP vectors. The pGP-CMV-GCaMP6s plasmid for Ca30. In brief, cells were incubated with 100 µM 6-cyano-7-nitroquinoxaline-2,3-dione , 50 µM 2-amino-5-phosponopentanoic acid  and 1 µM tetrodotoxin  in NMEM + B27 medium overnight at 37 °C, unless otherwise stated. Vehicle treated cells were incubated with an equivalent amount of DMSO. Wash off experiments were performed by a short wash in pre-warmed HBSS and subsequent 15 min recovery in NMEM + B27 medium at 37 °C. Stimulation by NMDA was done by a quick wash with pre-warmed PBS and 15 min incubation with 100 µM NMDA in NMEM + B27. Cycloheximide  was incubated for 4 h, puromycin  was incubated for 15 minutes and harringtonine  was incubated for 10, 20 or 30 minutes in NMEM + B27 medium before fixation.To inhibit neuronal activity, cells were treated as published34 and shDDX6 lentiviral particles were obtained from HEK293T cells  co-transfected with the plasmids psPAX2, pcDNA3.1-VSV-G and either pFu3a-H1-shNTC-pCaMKIIα-tagBFP, pFu3a-H1-sh-Stau2-2-pCaMKIIα-tagBFP, pFu3a-H1-shNTC-pCaMKIIα-tagRFP or pFu3a-H1-shDDX6-pCaMKIIα-tagRFP, respectively, using calcium phosphate co-precipitation. The sequence of shDDX6 was GATCCCCTTTATCTGGTAGGGATATCTTCAAGAGAGATATCCCTACCAGATAAATTTTTA. Supernatants were filtered , concentrated by ultracentrifugation , and resuspended in Opti-MEM™ (Life Technologies)69. Hippocampal neurons were transduced for 4 days before fixation.Control shNTC, shStau2-234. The following primary antibodies were used: polyclonal rabbit anti-Rck 1:500 (MBL), polyclonal goat anti-DDX6 1:500 (Abnova), polyclonal mouse anti-DCP1a 1:500 (Abnova), polyclonal rabbit anti-G3BP1 1:500 (Proteintech), monoclonal mouse anti-Stau2 1:50018, monoclonal mouse anti-CYT C 1:200  and mouse monoclonal anti-Homer1 1:500 (Synaptic Systems); and detected by the following secondary antibodies: donkey anti-rabbit or donkey anti-mouse AlexaFluor488, AlexaFluor555 or AlexaFluor647 conjugated antibodies 1:1000 (Life Technologies).Neurons were fixed for 10 min with 4% PFA and immunostained as describedImaging of sagittal brain sections was performed at the core facility bioimaging of the Biomedical Center with an inverted Leica SP8 microscope, equipped with a Plan-Apochromat 63x objective, a laser for 552 nm excitation, hybrid photo detectors (HyDs), and a convectional photomultiplier tube . Image pixel size was 361 nm. Z-stacks were acquired at 700 nm interval. Imaging of fixed cells was performed on a Zeiss Z1 Axio Observer microscope, including a Plan-Apochromat 63x objective, a COLIBRI.2 LED light source and the Axiocam 506 mono camera. Live cell imaging was performed on a Zeiss Cell Observer spinning disk system, consisting of a Zeiss Z1 Axio Observer microscope including a Plan-Apochromat 63x objective, a Yokogawa CSU-X1 spinning disk unit with 4 laser lines  and an Evolve 512 Delta EMCCD Camera. A custom made EMBL environmental chamber (EMBLEM) was used for temperature control. Hippocampal neurons were imaged at 36.5 °C in HBSS (Life Technologies) supplemented with 20 mM HEPES buffer pH=7.3 (Sigma Aldrich). Time-lapse images were acquired at an approximate frame interval of either 30 or 0.5 sec. Z-stacks at 1 µm intervals spanning the entire cell body were acquired per time-point for live imaging of NMDA and wash-off treatments, and subsequently subjected to maximum intensity projection. Cells were selected for proper expression of plasmid as well as for cell morphology and cell viability.70 script, available upon request. In short, images were gamma adjusted, a Laplacian filter was applied and images were thresholded using the automatic MaxEntropy method, to generate a detection mask for the granular compartment. A second detection mask for an approximation of cytoplasmic intensity outside granules was generated by dilating the first mask by 2 pixels and subtracting the first mask, leaving only the dilated part outside granules. Both masks were used to measure the mean fluorescence in the original unprocessed images.Assessment of neuronal population with large or small granules , as indicated. The subscript of F values denotes the degrees of freedom. All statistical tests were performed on individual biological replicates.The R75. In brief, 4 million cortical neurons were treated either with 100 µg/mL cycloheximide (CHX) or, for ribosome runoff experiments, with 2 µg/mL harringtonine (HRN) for 10 minutes at 37 °C. Cells were washed with prewarmed HBSS supplemented with CHX or HRN and lysed in polysome lysis buffer  sodium deoxycholate supplemented with 100 µg/mL CHX and 2 mM dithiothreitol (DTT). Lysates were spun at 13,000 x g for 5 min at 4 °C. Supernatants were loaded onto a sucrose density gradient (18% (w/v) to 50% (w/v) sucrose in 100 mM KCl, 5 mM MgCl2, 20 mM Hepes pH 7.4). Gradients were centrifuged at 35,000 rpm  for 1.5 h at 4 °C. Gradients were fractionated in 10×500 µL fractions using an automated fractionator . Proteins were extracted using methanol/chloroform extraction76. Individual fractions were subjected to Western blot analysis using polyclonal rabbit anti-Rck 1:1000 (MBL) or polyclonal rabbit anti-RPL7A 1:1000 (Abcam) primary and the donkey anti-rabbit IRDye 680RD conjugated 1:10,000 (LI-COR) secondary antibodies.Polysome profiling was performed as described78. In brief, 16 million cortical neurons were lysed in homogenizing buffer  on ice. Homogenate was spun at 16,000 × g for 10 min at 4 °C . Supernatant S16 was then spun at 100,000 × g for 20 min at 4 °C . When required, samples were treated with RNase1 (Ambion) prior to centrifugation. P100 pellets were volume-even resuspended in RIPA buffer  sodium deoxycholate, 1 vol% NP-40, 0.1% (w/v) SDS, 1× complete protease inhibitor, Roche) at 37 °C. All fractions  were methanol/chloroform extracted as described76. Samples were analyzed by Western blot, using polyclonal goat anti-DDX6 1:1,000 (Abnova) and mouse anti-Gephyrin 1:1,000 (Synaptic Systems) primary and donkey anti-goat IRDye 680RD conjugated 1:10,000 and donkey anti-mouse IRDye 800CW conjugated 1:10,000 (LI-COR) secondary antibodies.Differential centrifugation was performed as describedg for 10 min at 4 °C. The resulting supernatant (S16) was added to the beads and incubated for 4 hours at 4 °C. Beads were washed with IP-buffer and then either used for RNA digestion or protein analysis. For RNA digestion, 4,7 µg of total rat brain RNA was incubated with the beads at 22 °C for 30 min. RNA samples were subsequently loaded on a 1% agarose gel. For Western blot analysis, beads were boiled in SDS-sample buffer and analyzed by immunoblotting using the mouse anti-GFP 1:500  as primary antibody and donkey anti-mouse IRDye 800CW 1:10000 (LI-COR) as secondary antibody.20 µL of slurry protein G sepharose beads  were washed with IP-buffer and coupled with 100 µg of monoclonal anti-GFP antibody diluted in IP-buffer overnight at 4 °C. Beads were blocked with 1.25 mg/mL BSA in IP-buffer overnight at 4 °C. pEGFP-DDX6 and pEGFP-DDX6-RNase1 plasmids were transiently transfected in HEK 293 T cells. For IP, cells were lysed in IP-buffer  and homogenized using ultra sonication. Lysate was spun at 16,000 × 75. In brief, protein samples were transferred on a nitrocellulose membrane and blocked in 2%(w/v) BSA. Proteins were detected using rabbit anti-Rck 1:1000 (MBL), goat anti-DDX6 1:1,000 (Abnova), mouse anti-ß-actin 1:5000 (Sigma-Aldrich), rabbit anti-RPL7A 1:1000 (Abcam), mouse anti-Gephyrin 1:1000 (Synaptic Systems) and mouse anti-GFP 1:500  primary antibodies and donkey anti-rabbit IRDye 680RD conjugated 1:10,000, donkey anti-mouse IRDye 800CW conjugated 1:10,000 and donkey anti-goat IRDye 680RD conjugated 1:10,000 (LI-COR) secondary antibodies. Primary antibody binding was detected using the LI-COR Odyssey IR scanner.Western blot analysis was performed as describedFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting Summary