Caspase 7. Also, the effect on in vitro cellular samples was assessed (for reference genes and Caspase 7). This method can screen for the presence of AuNPs in RNA samples, which were isolated from biological material in contact with the nanomaterials, i.e., during exposure and risk assessment studies. This is an important quality control procedure to be implemented when quantifying the expression of a GOI from samples that have been in contact with various ENMs. It is recommended to further examine 18S, PPIA and TBP since these were the most reliable for detecting shifts in the difference curves, irrespective of the source of the RNA, or, the point at which the different AuNPs interacted with the assay.Genetic molecular studies used to understand potential risks of engineered nanomaterials (ENMs) are incomplete. Intracellular residual ENMs present in biological samples may cause assay interference. This report applies the high-resolution melt (HRM) feature of RT-qPCR to detect shifts caused by the presence of gold nanoparticles (AuNPs). A universal RNA standard (untreated control) sample was spiked with known amounts of AuNPs and reverse transcribed, where 10 reference genes were amplified. The amplification plots, dissociation assay (melt) profiles, electrophoretic profiles and HRM difference curves were analysed and detected interference caused by AuNPs, which differed according to the amount of AuNP present (i.e. semi-quantitative). Whether or not the assay interference was specific to the reverse transcription or the PCR amplification step was tested. The study was extended to a target gene-of-interest (GOI),  Gene expression studies commonly use Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) due to the sensitivity of the technique, as well as, the high diversity where different target genes-of-interest (GOI) can be studied. However, it relies on the normalisation of the expression data between samples, e.g. use of reference genes as internal controls –3. ReferThe high-resolution melt (HRM) feature of RT-qPCR analysis is an application of amplicon melting analyses, e.g., analysis of the melt curves of DNA fragments or PCR amplicons produced via amplification. Due to the improved qPCR instrumentation, as well as, the latest saturating DNA-binding dyes, this combination allows for the identification of small variations in nucleic acid sequences, e.g., via a controlled melting of double-stranded PCR amplicons. The improvements are related to the instrument calibration methods, which enable the rapid analysis of the resulting data sets by using HRM-compatible software, e.g. the discrimination of DNA sequences based on their composition, length, GC content, or, strand complementation . HRM expwww.bio-rad.com).Routine qPCR experiments rely on dissociations assays (melt peaks) in order to ensure primer specificity, where the data is typically collected over a temperature range of 65–95°C in 0.5°C increments. However, for HRM experiments, the data are generally collected at narrower temperature increments, i.e., 0.2°C. This increased density of data points that are collected can assist with melt curve profile generation, which then improves subsequent sequence discrimination. The analysis software specific for HRM identifies areas of stable pre- and post-melt fluorescence intensity from the HRM melt curve. These signals are then automatically normalized to relative values of “1.0” and “0.” In this way, differences in background fluorescence are eliminated, which increases the ability to detect subtle melt curve profile changes. Ultimately, both the melting temperature shifts, as well as, the curve shape can be used to identify sequence differences (in vitro Carboxyl-PEG (PCOOH) AuNP-treated samples. The results obtained included amplification plots, dissociation assay (melt) profiles, electrophoretic profiles and HRM difference curves, which were used to identify a concentration-dependent alteration in the amplification of the nucleic acid template. Thus, specific analyses of the HRM profiles detected a form of interference caused by AuNPs being present during the assay. This method may, therefore, be used to screen for the presence of AuNPs in RNA samples, which were isolated from biological material that had been in contact with the nanomaterials.This study is a continuation of our previous work, where we wanted to investigate the effect of gold nanoparticles (AuNPs) on the application of the high-resolution melt (HRM) feature of RT-qPCR to detect shifts in nucleic acid samples. Herein, a universal RNA standard was used as an untreated/control sample and spiked with known amounts of citrate-stabilised gold nanoparticles (AuNPs), in addition to the analyses of in vivo stability and to avoid uptake by the reticular endothelial system [The aim of this study was to determine if HRM profiles of common reference genes could detect the (known) presence of AuNPs in a sample. This is an important aspect of quality control of starting material used for ENM-related toxicity studies. A non-cytotoxic concentration of citrate-stabilised 1 nM AuNPs was used since these are the proposed conditions for all planned gene expression work yet to be performed . A univel system , the RNAReference genes, after RNA standard samples had been spiked with non-functionalized, but citrate-stabilised AuNPs at the cDNA reverse transcription step.Reference genes, after RNA standard samples had been spiked with non-functionalized, but citrate-stabilised AuNPs at the PCR amplification step.Target genes (GOI), after RNA standard samples had been spiked with non-functionalized, but citrate-stabilised AuNPs at the reverse transcription step.Reference genes, after BEAS-2B cells had been treated with PCOOH AuNPs.Target genes (GOI), after BEAS-2B cells had been treated with PCOOH AuNPs.4.3H2O aqueous solution [4.3H2O) and trisodium citrate (Na3C6H5O7.2H2O) were purchased from Sigma Aldrich (USA) and used without further purification.The AuNPs were fully characterised as previously described , 12. Thesolution , 14. The2)11–(EG)n–functional group, where EG is ethylene glycol (C2H6O2).The PCOOH AuNPs were saturated with the relevant PEG ligands complexed with carboxyl functional groups as previously described . Brieflyin silico analysis was performed in order to predict conformational changes under experimental conditions  software . The target type was selected as “DNA” since the samples would be RNA that had been reverse transcribed into cDNA using the random hexamer and oligo-dT primers. The determining factor for the selection was based on the structure with a Gibbs free energy within acceptable limits. In addition, Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/) was used to design the GOI primer, i.e. Caspase 7 (Casp7) primer.A literature search narrowed the list of putative human reference genes down to 10 candidates before initiating any wet-bench experiments . Thereaf4 cells/cm2 in a 75 cm2 flask and allowed to proliferate for 24 h before treatment. Total RNA was isolated using the RNeasy plus mini kit , according to the manufacturer’s instructions. In addition, the QIAshredder spin columns  were used to homogenize the samples. Since it takes time to both treat and process samples over the course of a time study, the RNAprotect stabilizing solution was used for all samples in order to minimise variations during the incubation and storage time periods. Following trypsinization and harvesting of the cells, RNAprotect solution was added to intact cells to stabilize the RNA. The RNA lysis buffer with guanidine thiocyanate was added and vortexed to lyse the cells. The cell lysate was passed through a QIAshredder column to aid homogenization. Thereafter, this eluent was passed through a gDNA Eliminator column to remove genomic DNA. Ethanol was added and the sample loaded onto an RNeasy MinElute column, where RNA binds to the column and contaminants were washed away during subsequent wash steps with the RNA wash buffer and the RNA ethanol-based buffer. Finally, RNA was eluted with RNase-free water. Each experiment was performed on a fresh isolation of RNA from BEAS-2B cells from a different passage number, i.e. completely separate experiments, where each time a new cDNA pool was reverse transcribed and amplified.For the preliminary study, total RNA was also isolated from a treated bronchial epithelial human cell line, BEAS-2B, as previously reported . BrieflyFor the main parts of this study, a universal human reference total RNA standard was purchased for qPCR  and used . This qPCR Human Reference Total RNA was composed of total RNA from 10 human cell lines, with quantities of RNA from the individual cell lines optimized to maximize representation of gene transcripts present in low, medium, and high abundance. The cell line derivations included an adenocarcinoma (mammary gland), hepatoblastoma (liver), adenocarcinoma (cervix), embryonal carcinoma (testis), glioblastoma (brain), melanoma (skin), liposarcoma, histiocytic lymphoma , lymphoblastic leukaemia (T lymphoblast) and plasmacytoma . According to the manufacturer, this reference RNA was carefully screened by spectrophotometry, MOPS agarose gel electrophoresis and analysis using the Agilent 2100 Bioanalyzer. In addition, the RNA was manufactured in large batch-lots in order to eliminate inconsistencies over long-term experiments, and, was treated with DNAse. Each experiment was performed on a fresh aliquot of the same universal RNA standard, where each time a new cDNA pool was reverse transcribed and amplified  in a final PCR volume of 40 μl was 0.72 nM, 1.44 nM and 2.2 nM, respectively. It should be noted that 1 nM AuNPs was determined to be a non-cytotoxic concentration by cell impedance analyses . These vNanoparticles that are coated with specific polymers display improved biocompatibility and stability, as well as decrease the cytotoxicity . AuNPs aA preliminary study was initially performed to test the primers’ ability to transcribe and amplify RNA from a human cell line using a SYBR Green-based RT-qPCR system . The BEAAfter testing of the primers in the preliminary study, a universal RNA standard from Stratagene  was used to verify the EvaGreen-based qPCR assay used in this study. The RNA was spiked at either the reverse transcription step , or, at the DNA amplification step . After treatment, 1 μg of total RNA was extracted as previously reported . The firhttps://www.bio-rad.com/en-us/sku/1845025-precision-melt-analysis-software?ID=1845025). Within part 4 and 5 of this study, the resulting cDNA from the RNA that was obtained after the bronchial epithelial human BEAS-2B cell line had been treated in vitro with 1 nM PCOOH AuNPs for 0.5 h, 1 h, 2 h and 24 h, was subsequently amplified in the same manner as described above.Within parts 1, 2 and 3 of this study, the resulting cDNA from the RNA standard was amplified using specific primers (indicated in The resulting amplicons obtained from the spiked RNA (part 1 of the assessment) and spiked cDNA (part 2) were separated on a 1% agarose gel see  Files. T™ Software . The pre-melt  and post-melt  signals of all samples are set to uniform, relative values from 100% to 0%. The differences in melting curve shape are further analysed by subtracting the curves from a reference curve . This enables the software to cluster samples automatically into groups that have similar melting curves. Samples with heterozygous SNPs can then be easily distinguished from the wild type or homozygotes by the different shapes of their melting curves.The dissociation assay (or melt peaks) data generated was analysed using the CFX ManagerThere were no human participants in this laboratory study and an ethics waiver was obtained for the use of an established cell line from the Human Research Ethics Committee  at The University of the Witwatersrand (Ref: W-CJ-150504-2).in vitro with PCOOH AuNPs. The study, thus, consisted of five parts, i.e., HRM analyses of:This report highlights the application of the HRM feature of RT-qPCR to detect shifts that are caused by the presence of residual intracellular AuNPs in nucleic acid samples. The same universal standard RNA was reverse transcribed into cDNA, but the reaction was spiked with various amounts of citrate-stabilised AuNPs and at different points within the assay. Therefore, the same PCR product was formed, where the only difference was the amount of AuNPs present in the reaction. The CFX Manager software generated data that was used to screen the gene expression profiles for differences, e.g., changes in the dissociation assay (melt peaks) of the different products formed. The gel electrophoresis results confirmed the RT-qPCR melt peak results, per reference gene tested. Thereafter, the experiment was repeated with RNA that was obtained from BEAS-2B cells that had been treated Reference genes, after RNA samples had been spiked with non-functionalized, but citrate-stabilised AuNPs, at the cDNA reverse transcription step see Figs –6.Reference genes, after RNA samples had been spiked with non-functionalized, but citrate-stabilised AuNPs, at the PCR amplification step , after RNA samples had been spiked with non-functionalized, but citrate-stabilised AuNPs, at the reverse transcription step see Figs  and 8A.Reference genes, after BEAS-2B cells had been treated with PCOOH AuNPs see Figs  and 6B.Target genes (GOI), after BEAS-2B cells had been treated with PCOOH AuNPs see Figs  and 8B.Human 18S ribosomal RNA (18S) reference gene. Similarly, peptidylprolyl isomerase A (cyclophilin A) (PPIA). Thereafter, TATA-box binding protein (TBP). The novel aspect of this work arises from the use of the Precision Melt Analysis™ software to identify the HRM dissociation assay differences caused by both citrate-stabilised and PCOOH functionalised AuNPs. The difference curves, with a characteristic HRM profile, are indicated for 18S  in in vitro cellular treatment . Therefore, since the starting amount of RNA did not change, the AuNPs influenced the conversion of RNA into cDNA, i.e., the reverse transcription (RT) efficiency. In addition, the plateau heights of the Amplification plot also varied. Therefore, the AuNPs influenced the amount of fluorescent dye that was detected, which is relative to the amount of double stranded DNA (dsDNA) present in the reaction . The amplification plots for the 18S gene showed the most changes in relation to the amount of AuNPs. Further analysis of the melt peak of the 18S reference gene . This could be verified by sequencing the numerous PCR products, but it would be very time consuming and costly to do this continuously. It should also be noted that the non-template control (NTC) for 18S only amplified after 35 cycles, which was most probably due to a primer dimer and was not due to contamination, i.e., it passed the internal QC check for the assay. Similarly, the PCOOH AuNP treated samples obtained from BEAS-2B cells were referenced against the 0 h untreated (control) cluster  presence of the AuNP could not be detected by dissociation of the dsDNA  presence of the AuNP by simply evaluating the melt peak of the TBP reference gene  BEAS-2B treated samples. These PCOOH AuNP treated samples were referenced against the 0 h untreated (control) cluster  in the amplification plots, see Figs ™ software was therefore used to generate difference curves that have a characteristic HRM profile for the reference genes, see Figs in vitro treatment, where the control of 0 h exposure time appeared as a flat line for reference genes 18S and PPIA (but not for TBP-2), where one could distinguish a difference between the control and the other treatments of 0.5 h, 1 h, 2 h and 24 h, respectively. These differences imply that assay interference would have occurred during the reverse transcription of the RNA into cDNA, i.e., both the AuNPs appeared to influence the transcription specifically.The Precision Melt AnalysisA comparison was made between the primer used for the TATA-box binding protein see . The priCaspase 7 (Casp7), is shown in Casp7 to the reference gene (YWAHZ). The spiked samples  with a non-cytotoxic concentration of PCOOH AuNPs? (3) Can the same assay method be used to detect changes in target GOI when the cells are treated? For this reason, the study consisted of five parts, i.e., HRM analyses of reference genes using a universal RNA standard (which was either spiked with citrate-stabilised AuNPs) at the cDNA reverse transcription, or, PCR amplification step), as well as using RNA obtained from human cells treated in vitro (cellular) with PCOOH AuNPs. Lastly, the Caspase 7 target gene was also analysed with both citrate-stabilised AuNPs, as well as after BEAS-2B cells had been treated in vitro (cellular) with PCOOH AuNPs.It is important to screen for residual AuNPs remaining in a sample, in order to prevent misrepresentation of gene expression data from toxicology studies, which will be used for exposure assessment and Risk assessments related to ENMs. Thus, the aim of this study was to determine if HRM profiles of common reference genes could detect the (known) presence of AuNPs in a sample. Different AuNPs were used, at different parts of the study, to observe different variables  of a cycle is unacceptable because it indicates the presence of nucleotide insertions/deletions (in/dels) or even single nucleotide polymorphisms (SNPs). The same amount of RNA template was used, where only the amount of citrate stabilised AuNP added to the reaction changed. Hence, a false measurement was recorded that would normally imply less template being available in that sample.The CFX Manager software generated data that was used to screen the reference gene expression for any differences, e.g., changes in the dissociation assay (melt peaks) of the different PCR products (amplicons) formed, or, changes in the amplification plots. There were very few changes observed in the temperature at which the amplicon melted, after treatment with various amounts of AuNPs. Hence, the unchanged melt peak of the DNA indicated that the same amplicons were formed each time, irrespective of the amount of AuNPs present in the reaction. Although there were no changes in the dissociation assay (melt peaks), there were changes in the quantitative cycle value in the amplification plots, i.e. the Csee Figs  and 5A. q, the clustering feature of the Precision Melt Analysis™ software was used as a novel approach to further identify AuNP-related changes in the qPCR assay, i.e., AuNP-related interference. Shifts in the HRM profiles were identified by an in-depth analysis of the difference curves, where readings were taken every 0.2°C values  citrate stabilized AuNPs induced results, versus, the in vitro PCOOH AuNP treated results, per gene indicated. These figures show a clear difference between the control (either 0% or 0 h) and the treated samples , where reference genes 18S and PPIA showed good correlation irrespective of which AuNP was used. However, the TBP reference gene produced a positive/increased control profile instead of the previously observed flat baseline. Although a clear stepwise difference relative to increased amounts of AuNPs was not observed, the control was still distinct from the treated samples. YWHAZ was previously validated [in vitro acellular, cell-free) citrate stabilized AuNPs induced results to the in vitro PCOOH AuNP treated results for the GOI. Again, a positive/increased control profile was observed, but the treated samples were still distinctly separate within the difference curve.Both the universal RNA standard (spiked with citrate-stabilised AuNPs) and isolated RNA (obtained after treatment of BEAS-2B cells with PCOOH AuNPs) were used to assess shifts in the HRM profiles. To summarise, Figs alidated . Fig 8 cMost of the literature regarding the toxicological impact of ENMs has reported on acute stress responses such as viability, oxidative stress or apoptosis –24. ApopCasp7 gene was screened as a potential GIO for our analyses of the toxicity of AuNP treatments, both in vitro acellular cell-free and in vitro cellular. YWHAZ was shown to be a stable reference gene under these experimental conditions [Casp7 is the GOI (and not a reference gene in this study), the results are displayed differently, where the normalised expression (ΔΔCq) of Casp7 to the YWAHZ reference gene is shown instead of showing the electrophoresis results, for example. The same universal RNA standard was reverse transcribed, where the only difference was the amount of citrate-stabilised AuNPs present  treated samples were also analysed in a similar manner and referenced against YWHAZ to obtain the normalised expression (ΔΔCq), where it was seen that the 1 h exposure time produced a slightly elevated gene expression  cluster (in vitro cellular exposure. The trend that was clearly seen between spiked in vitro acellular treatments (as previously observed and discussed for the reference genes in sections 4.1.1 to 4.1.3), was not observed to the same extent for the GIO, where only the untreated control sample could be easily distinguished from the treated samples that were clustered together in The HRM Difference curve of assessed . All spiassessed . It was  cluster . AlthougThe novel approach described herein uses HRM profiles and specific reference genes for visualising alterations of the thermal dissociation behaviour of dsDNA, which are caused by the (known) presence of AuNPs, to detect if these AuNPs interfere with the RT-qPCR assay. Normally qPCR experiments rely on the traditional use of Dissociation assay melt peaks to distinguish PCR products. However, melt peaks could not distinguish the known presence of AuNPs in this study, i.e. where the same RNA/cDNA sample was reverse transcribed, amplified and the same PCR product was produced, but the only difference was the amount of AuNPs present in the sample. Hence, the study progressed to analyse HRM profiles because any HRM shifts observed after a RT-qPCR run would normally indicate a change in the genetic sequence composition of the PCR product (e.g. mutations and in/dels). Thus, by using this knowledge, one could detect any assay interference caused by the presence of AuNPs by simply spiking the same RNA/cDNA sample with different mounts of AuNPs and observing the changes in the HRM profiles. The difference curves for the HRM profiles in this current study were able to detect alterations of the thermal dissociation behaviour of dsDNA. Thus, any differences observed were due to the AuNPs, which appeared to influence the transcription of RNA into cDNA to a higher degree than the PCR amplification efficiency. This novel approach is a putative diagnostic tool, where this term “diagnostic tool” refers to the process of detecting the presence of both citrate-stabilised as well as PCOOH AuNPs, within RNA used for RT-qPCR, by analysing shifts in the HRM profiles. Following the same approach as reported herein see , one canThere have been many reports of gene expression studies for other types of genes, as induced by other types of ENMs see . Howeverhttps://www.bio-rad.com). Another similar work included a presentation by Prado and colleagues at the qPCR-NGS 2013 Symposium held in Germany who noted that the amplification plot of SYBR Green was affected by the addition of increasing concentrations of Fe3O4 NPs. It should also be emphasised that the dye used by Prado and colleagues was also SYBR green, i.e., the “relocating” dye that has the potential to generate false readings [The only study found to be similar in design to that reported herein was by Haber and colleagues who used a SYBR Green I detection system and performed HRM analyses, screening at every 0.1°C change . Howeverreadings .18S, Act-b, GUS, PPIA and TBP (see Act-b), whereas Bai and colleagues were referring to amplicon sizes with a range from 588 to 3529 bp. In other words, our method used short amplicons that were useful in detecting subtle affects (via an EvaGreen saturated dye), as caused by low amounts of ENM, where this is important for determining assay interference of gene expression data obtained from toxicology studies.The report by Bai and colleagues on the other hand supported the idea that the effects of AuNPs on PCR were attributed to interactions with the PCR components, and they disproved the theory that ENMs inhibit non-specific amplification by false priming . Instead TBP see . Again, Ultimately, the reason why validating assays and developing diagnostic methods are important is because gene expression studies are used to predict long-term nanoparticle exposures. In order to conduct these predictions, one requires data from various exposure studies, where these studies must employ validated methods. A specific example of this concept is made evident via the report of one low-dose exposure of nanoparticles that induced long-term changes in human cells . There hin vitro cellular treatment of BEAS-2B cells with PCOOH AuNPs) were used to assess the amplification of 10 reference genes, which was monitored using the EvaGreen dye, i.e. a saturating dye. The study consisted of five parts in order to encompass multiple scenarios and analyse all of the variables. The Amplification plots did show slight changes. Changes were also observed for the Cq values, from the same RNA/cDNA sample, where the only difference was the amount of AuNPs present. The traditional use of Dissociation assay melt peaks in qPCR cannot distinguish effects from the known presence of AuNPs. The difference curves for the HRM profiles, however, were able to detect alterations of the thermal dissociation behaviour of dsDNA, where results were clustered to the 0% AuNP sample or the 0 h untreated (control), using the specific software. Thus, any differences observed were due to the AuNPs, which appeared to influence the transcription of RNA into cDNA to a higher degree than the PCR amplification efficiency. A target GOI, Casp7, was also tested, where differences in the normalised gene expression were observed, but these were not statistically significant. Lastly, it was recommended to perform HRM RT-qPCR with AuNP-spiked samples (as positive control standards), so that one can determine the error compensation required for experiments assessing gene expression as a result of AuNP treatment or exposure in toxicity studies. The Supplementary files show the high degree of reproducibility that was obtained when spiked samples were screened. This novel approach, which uses HRM profiles and specific reference genes for visualising alterations of the thermal dissociation behaviour of dsDNA as caused by the presence of AuNPs, is a putative diagnostic tool. Specifically, one can screen samples for possible AuNP interference of the reference gene, whilst using the same sample, in the same assay, in order to quantify the expression of a particular target or GOI. This diagnostic tool has a broad range of applicability and can be further developed in future studies of nucleic acid samples.Both the universal RNA standard (spiked with citrate-stabilised AuNPs) and isolated RNA (obtained after S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S6 File(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Mental health issues, including major depressive disorder, which can lead to suicidal behavior, are considered by the World Health Organization as a major threat to global health. Alterations in neurotransmitter signaling, e.g., serotonin and glutamate, or inflammatory response have been linked to both MDD and suicide. Phosphodiesterase 8A (PDE8A) gene expression is significantly decreased in the temporal cortex of major depressive disorder (MDD) patients. PDE8A specifically hydrolyzes adenosine 3′,5′-cyclic monophosphate (cAMP), which is a key second messenger involved in inflammation, cognition, and chronic antidepressant treatment. Moreover, alterations of RNA editing in PDE8A mRNA has been described in the brain of depressed suicide decedents. Here, we investigated PDE8A A-to-I RNA editing-related modifications in whole blood of depressed patients and suicide attempters compared to age-matched and sex-matched healthy controls. We report significant alterations of RNA editing of PDE8A in the blood of depressed patients and suicide attempters with major depression, for which the suicide attempt took place during the last month before sample collection. The reported RNA editing modifications in whole blood were similar to the changes observed in the brain of suicide decedents. Furthermore, analysis and combinations of different edited isoforms allowed us to discriminate between suicide attempters and control groups. Altogether, our results identify PDE8A as an immune response-related marker whose RNA editing modifications translate from brain to blood, suggesting that monitoring RNA editing in PDE8A in blood samples could help to evaluate depressive state and suicide risk. About 90% of suicide completers suffered from mental disorders; half of them met the current criteria for major depressive disorder2. Single genes variants might not explain the full risk of developing suicidal behavior3, genome-wide association studies for MDD failed to identify causative variants4. However, changes at the molecular level are detectable in brains of depressive patients and suicide completers. Modifications in serotonin receptor and transporter expression, both of which resulting in altered serotonergic signaling, were the first to be described5. Additional molecular changes in MDD include other neurotransmitters pathways, e.g., glutamate and gamma-aminobutyric acid6. Alterations of inflammatory response have also been linked to both MDD and suicide (for review see ref. 7). A close interaction between immune system activation and changes in brain circuits related to mood and behavior has been described8. Deterioration of the normal structure and function of microglia, possibly caused by inflammatory signals, can lead to depression9. It is now admitted that depressed individuals display dysregulation of immune system (for review see ref. 10). Very recently, an association between pro-inflammatory cytokines and brain activation has been identified in patients with MDD and vulnerability to suicide11. A significant increase in depressive symptoms was observed in subjects injected with Lipopolysaccharide from Salmonella abortus, suggesting a direct link between immune response and mood alteration12. Occurrence of severe depression with suicidal ideation in patients undergoing interferon (IFN) treatment is well documented (for review see ref. 13), suggesting strong connections between inflammation and depression. Inflammation can modify dopaminergic, serotoninergic, and glutamatergic systems (for review see ref. 14).On the opposite, type I interferon signaling was recently shown to be altered in blood cells of patients suffering from severe recurrent depression15.Suicidal behavior and suicide are a major public health issue worldwide; suicide is one of the primary causes of death, with an estimated 1 million deaths every year16). Significant differences in the methylation patterns associated with astrocytic alterations have been described in depression and suicide17. In the central nervous system (CNS), RNA editing is an essential cellular mechanism for diversifying the transcriptome. Introducing codon changes in mRNAs encoding key proteins for nervous system activity, including glutamate and serotonin receptors, has been shown to modulate their activity18. Major changes in RNA editing patterns have been correlated with neuronal disease pathogenesis19. Adenosine Deaminase Acting on RNA (ADARs), the enzymes responsible for A-to-I editing of RNAs, have been implicated in immune regulation20. Interestingly, the p150 isoform of ADAR1 (ADAR1a) is induced by a variety of inflammatory mediators, including TNF-alpha and IFN-alpha21. Upregulated expression of ADAR1 was also observed in the dorsolateral prefrontal cortex of major depressive suicide victims22. We have previously shown that region-specific alterations of A-to-I RNA editing of 5HT2CR occur in different areas of the prefrontal cortex in suicide decedents23. More recently, region-specific RNA editing alterations of an immune response marker, phosphodiesterase 8A (PDE8A), were described in the cortex of suicides with major depression24. Cyclic nucleotide phosphodiesterases are a family of enzymes which selectively hydrolyze cAMP, cGMP or both, and thus affect G-protein-coupled signal transduction (for review see ref. 25). The pre-mRNA of PDE8A has previously been shown to be post transcriptionally edited by ADARs26 in T cells from systemic lupus erythematosus (SLE) patients and in T cells activated with type I interferon27. There is a high prevalence of depression in SLE patients28.Transcriptomic and epi-transcriptomic studies have recently identified peripheral markers of suicide risk . The samplings were performed under the expertise of Prof. Philippe Courtet, in charge of the Psychiatry collection of the Centre de Resources Biologiques at Montpellier Hospital.Participants (MDD = DEP + SA), were recruited from September 2016 to January 2019 among the outpatients of the Department of Emergency Psychiatry and Post-Acute Care (CHRU of Montpellier). Age-matched, race-matched, and sex-matched control subjects were recruited from a list of volunteers from the Clinical Investigation Center (CHRU of Montpellier). All participants, aged between 18 and 65 years, understood and signed a written informed consent before entering the study.n = 99), which includes individuals with no history of any major DSM-IV axis I disorder and of suicidal behavior/act; ii) depressed patients :depressed patients without any records of suicidal act lifetime and iii/ Suicide attempters : depressed patients with a history of at least one suicide attempt (defined as any act carried out with some intent to die) during the last month before sample collection. All patients met the MDD criteria in Diagnostic and Statistical Manual of Mental disorders IV (DSM-IV) using the Mini-International Neuropsychiatric Interview. During the standardized interview, psychiatrists managed the French version of the Montgomery-Åsberg Depression Rating Scale (MADRS)29 and the 30-item Inventory of Depressive Symptomatology, Clinician Rated (IDS-C30)30 to score the depression. Depression severity levels, i.e., low, moderate, and severe were defined by 7 ≤ MADRS ≤ 19 and/or 12 ≤ IDSC-30 ≤ 23, 20 ≤ MADRS ≤ 34 and/or 24≤IDSC-30 ≤ 36, MADRS ≥ 35 and/or IDSC-30 ≥ 37, respectively. The severity of the most severe suicide attempt was evaluated with the Risk Rescue Rating Scale (RRRS)31, which measures the medical danger of the attempt (risk factors) and the probability of being rescued (rescue factors), and the 19-item Suicide Intent Scale (SIS), which evaluate the intent to die.The study included three groups: i) healthy controls , according to the manufacturer’s protocol. During sample preparation and RNA extraction, standard precautions were taken to avoid RNA degradation by RNAses. Total RNA concentrations and quality levels were determined with Qubit Fluorometer (Invitrogen) and LabChip  instruments, respectively. Only samples with RIN number >7 were taken into consideration for further analysis.Reverse transcription was carried out on 12.5 ng/µL of RNA per sample using the Takara kit . The resulting cDNA was combined with TaqMan universal PCR Master Mix (Applied Biosystems) and with the following specific gene probes: ADAR1-1 (ADAR1a-p150) (Hs01020780), ADAR1-5 (ADAR1-b-p110) (Hs01017596), ADAR2 (Hs00210562), PDE8A (Hs00400174), GAPDH (Hs02758991), HPRT1 (Hs02800695), TBP (HS00427620) and PGK1 (Hs99999906) (Life Technologies) in 20 µL final volume. Quantitative PCRs were performed in 384-well plates on LightCycler 480 Real Time PCR instrument (Roche). The analysis was performed using a second derivative absolute quantification, normalized by the geometric mean of four housekeeping genes .32. Experiments were performed five times independently for each sample.For NGS library preparation, we chose a targeted approach to selectively sequence the region of interest within intron 9 of the PDE8A gene . For each position, the script computes the percentage of reads that have a “G” [Number of “G” reads / (Number of “G” reads + Number of “A” reads) * 100]. The genomic location “A” reference with percentage in “G” reads > 0.1 are automatically detected by the script and are considered as “A-to-I edition site”  and demultiplexed as fastq file. The procedure for sequencing quality evaluation, trimming, and alignment has been described previously33. RNA editing values are usually presented as means ± standard error of the mean (SEM). A differential analysis was carried out with the non-parametric Wilcoxon rank sum test and the Welch’s t-test. With the multiple testing methodologies, it is important to adjust the p-value of each editing isoforms to control the false discovery rate (FDR). The Benjamini and Hochberg (BH) procedure34 was applied on all statistical tests with the “multtest package” and an adjusted p-value below 0.05 was considered as statistically significant. Relative proportion of editing levels was normally distributed and consequently no normalization was applied.All statistics and figures were computed with the “R/Bioconductor” statistical open source software35. Random forest require the use of a training set used to build the model and a test set to validate it. We have shared our data set: 2/3 of the dataset (n = 125) are used for the learning phase and 1/3 are used for the test phase (n = 50). This sharing has been randomized and respect the initial proportion of the various statutes in each sample. To estimate RF parameters, we used the 10-fold cross-validation method. For these approaches, we used the “randomForest package 4.6-14” of the R software version 3.5.3 with mtry = 2, ntree = 1000 as parameters for the model. Results of best RF model selected are represent by ROC curve and diagnostic performance on test phase.A random forest (RF) approach was applied to assess the RNA editing isoform combinations of PDE8A mRNA transcript. The concept of aggregating the results of many decision trees has resulted in a stable algorithm, robust to noisy datar2 = 0.90, p < 0.0005) between the two evaluations . We identified a significant decrease of editing rates at sites B, C, and E between healthy controls and depressed patients , suggesting that differential editing in intron 9 has no effect on mRNA levels. When we measured expression levels of the ADAR deaminases, we could not identified any significant modification in expression of ADAR1a , ADAR1b  or ADAR2 when we compared depressed patients or suicide attempters vs healthy controls (data not shown). As MDD has no effect on enzyme expression in the blood, these data suggest that the changes observed in editing rates are more likely due to modifications in ADAR activity or recruitment during depressive episodes.To evaluate their potentials as diagnostic test to predict suicide risk, all significant RNA Editing isoforms and sites were combined using random Forest (RF) machine learning approach. The diagnostic performance of RF model to separate suicide attempters from controls displayed an AUC ROC = 0.870  Fig. , a speci36. All patients included in the study were treated with different medications. Treatments were classified into five categories, the distribution of the five classes of treatments among patients being not statistically different between DEP and SA groups. Treatments might however represent a possible bias in the measurement of RNA editing. Some antipsychotic or antidepressant drugs, e.g., aripiprazole, sertindole, or nortriptyline tested in a cellular mode (SHSH-5Y cells) have been shown to modify the RNA editing profile of the 5HT2CR and to induce an editing profile similar to the one obtained after IFN-alpha treatment37. Indeed, IFN-alpha is known to induce ADAR1-1(p150) expression and increase editing of 5HT2CR in SHSH-5Y cells37. A recent work shown that PDE8A RNA editing is altered in both HCV patients following interferon alpha treatment and in IFN-alpha-treated SHSY-5Y neuroblastoma cells32. In the 225 bp studied in intron 9 of PDE8A, we identified significant alterations of PDE8A RNA editing in both depressed patients and suicide attempters. Editing levels were significantly decreased in all depressed patients, regardless of previous suicide attempt. Of note, suicide attempters had a trend to even lower editing levels when compared to DEP without history of suicide, though the decrease did not reach statistical significance. This decrease could be attributed to both pathology and pharmacology. As the five classes of medications did not cluster in a subgroup of patients, it is likely that the effect of treatments on RNA editing is uniformly distributed between patient groups.In this study, we used targeted Next-Generation Sequencing to study the PDE8A RNA editing profile in whole blood from controls, depressed patients with no history of suicide attempt and suicide attempters. We observed a marked association in substance abuse disorders in depressed patients, which is particularly common and well documented in depression24. Moreover, PDE8A RNA editing modifications were shown to be related to depression status in patients with chronic hepatitis C virus (HCV) infection undergoing antiviral combination therapy with IFN-α and ribavirin32. If we cannot exclude a possible effect of treatments on PDE8A editing profile, it is likely that the decrease observed in PDE8A RNA editing reflects the changes due to the pathological status of patients. Activation of the immune system might also be involved in the differences observed here between DEP and SA, as illustrated by a trend in the increase of CRP in SA vs. DEP. Editing levels for sites B, C D, E, and F were strongly and positively correlated, suggesting that the modifications in editing levels occurs concomitantly within the whole editing island of PDE8A mRNA, the result being a global decrease in editing activity in patients’ blood. PDE8A isoform B, which is by far the major isoform observed in blood, was significantly decreased in patients, while at the same time an increase in the non edited isoform was observed, suggesting a modification in either ADAR expression, activity, or subcellular localization in depression. Moreover, the identified PDE8A RNA editing modifications strongly discriminated depressed suicide attempters form healthy controls.We have previously described alterations in both 5HT2CR and PDE8A RNA editing in the brain of unmedicated suicide decedents24. In the blood of MDD patients, the increase in the non-edited isoform is in contrast with what we observed in the brain. In the brain, modifications in both the nature of isoforms and their relative abundance compensate each other, leading to no change in non edited isoform. In blood, we observed a global decrease in RNA editing activity in patients. In this case, the change in editing activity in PDE8A was not due to differential ADAR expression levels between groups, as measured by RT-qPCR. Previous studies suggested that ADARs mRNA and protein expression levels are not consistently associated with RNA editing levels. During brain development, editing levels of neurotransmitter receptors such as 5HTR2CR and GABAAR (Gabra3) increase, despite constant protein expression of ADAR proteins38. A recent report of A-to-I RNA editing signatures within the Drosophila brain identified editing differences between nine different neuronal populations, which were site-specific and not driven by changes in ADAR expression, suggesting a complex, targeted regulation of editing levels in key transcripts39. Indeed, spatial distribution of ADAR enzymes, and their interaction with partner proteins, which can act as inhibitor or activators, might influence ADARs’ editing activity40. As gene expression experiments did not show any modification in either PDE8A or ADARs editing enzymes, it is likely that the decrease might result from decreased ADARs activity and/or modified subcellular localization in patients.In a previous work, PDE8A RNA editing profile measured in two brain regions showed that the non-edited isoform was quite stable between controls and suicide decedents41. A recent survey of RNA editing in six different tissues confirmed that brain contained the highest number of tissue-specific RNA editing events42. Here, the PDE8A RNA editing levels for site B measured in blood were similar to those measured in brain, while editing levels for the four other sites considered were 5-fold less edited than in the brain. RNA editing is known to be strongly tissue-dependent; levels of RNA editing events might also be different in different cell types from the same tissue. It has been shown recently that the landscape of global RNA editing differs between brain and blood43. Characterization of RNA editing sites in human monocytes showed that RNA editing in blood cells was enriched in intronic region of repetitive elements, most of the sites being edited at lower frequency43. RNA editing might have crucial implications in non-brain tissues. As an example, a non-synonymous RNA editing event in the coding region of Filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure and is associated with cardiovascular diseases44. Contrary to the recoding editing event in Filamin A, most edited sites are located in repetitive regions, and 90% occur in Alu elements45. They are usually found as adjacent inverted Alu elements, which form a stem-loop structure triggering ADARs binding46. It has been suggested that Alu elements function as editing inducer elements (EIEs), which recruit ADARs and contribute to efficient editing at specific sites47. The region studied here does not belong to an Alu element but rather to a non-repeated intronic element. As intron 9 of PDE8A also form a long stem-loop structure24, editing sites located in 3′ to site B, namely sites C, D, E, and F could also possibly serve as inducer elements for editing at site B.A-to-I RNA editing is typically more frequent in brain than in other tissues, both in terms of number of editing sites and editing levels48. Brain tissue from suicide decedents showed increased activation of microglia, which could release pro-inflammatory and anti-inflammatory mediators such as cytokines and chemokines upon activation (for review see ref. 49). The association between inflammation and depression also translates in blood. Common genetic variants, including polymorphisms in the genes IL-1β, IL-6, or CRP are involved in both immune activation and depression50. Many studies revealed that patients suffering from depression have increased circulating pro-inflammatory cytokines, such as TNF-α, IL-1β, or IL-6, suggesting that a neuroimmune axis interfacing the immune system and CNS might be involved in the control mood and behavior51. A genome-wide pharmacogenetic study in MDD patients identified single nucleotide polymorphisms in IL-11 and IL-6, which could predict antidepressant response52. A discovery approach to identify gene expression changes in suicidality identified a panel of 76 biomarkers, a subcategory of which have biological roles in immune and inflammatory response53. Recent data suggest that peripherally derived chemokines and cytokines can directly act in the brain, and that some immune cells can actually access the brain54. The chronic low-grade inflammation observed during depressive episodes can activate the hypothalamic-pituitary-adrenal (HPA) axis, resulting in an increase in cortisol levels55. Moreover, inflammation induced dysregulation of the tryptophan-kynurenine pathway, which contributes to the neurodegeneration observed in major depression and suicidality56.Inflammation is known as a pathogenic factor, possibly explaining a part of physiopathology of depression40. PDE8A is an immune response marker, RNA editing of which has been observed in brain and blood. RNA editing is altered in cancer, autoimmune, and neurological disorders (for review see ref. 57). A recent study reported an overall increased RNA editing in SLE58. In that study, patients could even be discriminated by the presence of the IFN-inducible genes’ expression signature. Interferons are crucial regulators of the immune system and are known to trigger the protective defenses of the immune system (for review see ref. 59). Importantly, Type I interferon can upregulate ADAR1-1 (ADAR1p150), and hence increase general RNA editing activity60. Treatment of a human glioblastoma cell line with IFN-alpha induced a 5-fold increase in ADAR1a, which was accompanied by significant changes in the editing of serotonin 2C receptor (5HT2CR) mRNA61.Genome-wide analysis of RNA edited sites in human blood recently revealed a strong enrichment in genes involved in immune system and interferon signaling25). Different classes of antidepressants have been shown to increase cAMP pathway in both hippocampus and cortex62. PDE8A is expressed in different brain region, including BA9 and BA24, where it participates in serotonin signaling. PDE8A mRNA is twofold decreased in the temporal cortex of MDD patients63. Consistent with our data, Orlowski et al. showed decreased RNA editing in T lymphocytes from SLE patients and in T cells activated with IFN-alpha27, suggesting that increased ADAR expression following interferon response might not correlate with ADARs editing activity40. Interestingly, serotonin receptors are present on immune cells and serotonin has immune-regulatory effects (for review see ref. 64). Serotonin modulated the release of pro-inflammatory cytokines from monocytes, including IL-1β and IL-6, when stimulated by lipopolysaccharide65. Therefore, PDEs are very likely to play an important role in immune cells during inflammation. Noteworthy, PDE8A has been shown in vitro to physically interact with endogenous IκB proteins, and binding to IκB increased enzymatic activity of PDE8A66. Therefore, the transcription factor NF-κB, which is a pivotal mediator of inflammatory response might also participate directly to the regulation of second messenger levels, as cAMP-induced signals might interfere with NF-κB and modulate its actions67.Phosphodiesterases (PDEs) are the only enzymes hydrolyzing adenosine and guanosine 3′,5′-cyclic monophosphates , which are key modulators of signal transduction that mediate the cells’ response to a broad variety of hormones and neurotransmitters (for review see ref. In conclusion, the data presented in this study identified PDE8A as a peripheral immune response-related marker previously documented in the brain. The observation of altered PDE8A RNA editing in the blood in depressed patients, combined with the identification of a combination of isoforms, that allowed discrimination between healthy controls and suicide attempters raises the possibility of providing a molecular signature, whose modifications in blood mirror those occurring in the brain. This suggests that both the central nervous system and the immune system act on the control of mood and behavior. A role of RNA editing in the non-coding intron of PDE8A in both brain and blood might suggest that this non coding intron might have functional role during or after splicing, yet to be identified. The finding that PDE8A RNA editing translated from brain to blood strengthen the association between inflammation and depression, and could pave the way for predictive blood-based biomarkers to evaluate depressive symptoms.Supplementary_MaterialSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5


In the last decade, an increasing number of studies have demonstrated that non-coding RNA (ncRNAs) cooperate in the gene regulatory networks with other biomolecules, including coding RNAs, DNAs and proteins. Among them, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are involved in transcriptional and translation regulation at different levels. Intriguingly, ncRNAs can be packed in vesicles, released in the extracellular space, and finally internalized by receiving cells, thus affecting gene expression also at distance. This review focuses on the mechanisms through which the ncRNAs can be selectively packaged into extracellular vesicles (EVs). EVs are a family of membrane-coated vesicles with different proteomic and lipidomic profile, as well as different size.In their study, Pan and Johnstone firstly described extracellular vesicles containing the transferrin receptor during reticulocytes maturation . In the Due to their biogenesis, we can identify two different types of EVs: exosomes and microvesicles (MV) . The lasThe different biogenesis of the EVs also affects the composition of the vesicles’ membrane. The invagination process of ILs determines a specific lipidic and protein composition of the exosome’s membranes, which gives them a distinctive molecular profile. Exosomes’ membranes contain ESCRT components and associated proteins, as TSG101 and Alix. Moreover, the participation of tetraspanins in exosomes formation determines an abundance of these proteins in the exo-membranes .MV as shed directly from the plasma membrane, hold the annexin A1  and are Finally, EVs are characterized by their size; generally, microvesicles range from 200 to 2000 nm while exosomes from 30 to 150/200 nm. Recently the ISEV society established that it is better to prefer other methods of classification because many vesicles are quite similar in size range ,15.Many studies have focused on the biological effects of EVs in cell-cell communication both between neighboring cells and between cells in distant body districts. Biological fluids carry the EVs released in the extracellular space until they find a landing place where dump their content through the fusion of plasma membranes or endocytosis . RecentlMuch of the recent interest in EVs was triggered by the discovery of their involvement in horizontal transfer of secreted extracellular RNA (exRNA). From the first evidence of functional mRNA in EVs, a huge number of studies revealed a significant assortment of ncRNAs in EVs. The deep sequencing RNA technique allows demonstrating a selective enrichment of small ncRNAs in human-derived extracellular vesicles, isolated from different cytotypes ,19. AmonAs pointed out by  althoughStill controversial is the presence of miRNA precursors: their identification, together with Dicer and Argonaute 2, let scientists to suppose that complete silencing machinery could be transferred by EVs ,23. HoweOther conflicting observations in studying exRNAs are due to the different isolation strategies. For example, ultracentrifugation, the gold standard method for EVs purification, biases RNAs data. It in fact does not allow to separate vesicles from free ribonucleoproteins or lipoproteins both associated with RNAs .To overcome this limitation, Lässer group developed a method combining size exclusion chromatography with a density cushion; this allows to isolate and characterize EVs from blood with minimal contamination by plasma proteins and lipoprotein particles .Meanwhile, Jeppesen et al. used high-resolution density gradient fractionation to separate EVs from non-vesicular material, then, with a direct immunoaffinity capture (DIC) targeting classical exosomal tetraspanins, exosomes were specifically isolated from other types of EVs . NucleicNew isolation techniques have allowed more accurate identification of the RNA families transported through the EVs, further confirming the role of vesicles in the horizontal transfer of this nucleic acid.Many studies demonstrated the functional role of the RNA-cargo that, selectively packaged inside EVs, are transferred to target cells. Firstly, the transfer of exosomal mRNAs and miRNAs was reported by Valadi et al. in 2007, which revealed a “novel mechanism of genetic exchange between cells” . Since tStudies on tumor cells revealed as exosome-mediated RNA transfer may control tumor growth, affect its microenvironment, and promote metastases.It is well known that a central event in tumor progression is the induction of angiogenesis. Focusing on this pathway it has been demonstrated that glioblastoma, chronic myelogenous leukemia, and breast cancer derived EVs can reprogram endothelial cells through horizontal transfer of their miRNAs cargo ,28.GS1-600G8.5 [The involvement of cancer derived EVs in facilitating brain infiltration is worth mentioning. Lu and collaborators have recently demonstrated that exosomes derived by highly brain metastatic breast cancer cells are able to destroy the blood–brain barrier through its lncRNA -600G8.5 ,30.miR100-5p as the mediator of this process [Exosomal miRNAs have a role also in tumor drug resistance. Mao et al. support this hypothesis demonstrating that Adriamycin-resistant breast cancer cells deliver specific miRNAs through exosomes thus promoting drug resistance in neighboring cells . Quin an process .H19 induces doxorubicin resistance in breast cancer [lncH19; these exosomes increase the chemoresistance of doxorubicin once internalized by sensitive cells. Moreover, downregulation of H19 in sensitive cells ablated this effect thus confirming the direct role of the long non-coding RNA [miR-9 and miR-155, carried respectively by breast cancer derived exosomes and pancreatic cancer derived microvesicles, are able to reprogram fibroblast to cancer associated fibroblast (CAF) phenotype thus promoting tumor progression [miR-105 and -miR-122 reprogram CAFs metabolism to sustain tumor growth [miR196a-5p and miR-501-3p were significantly downregulated in exosomes isolated from the urine of prostate cancer patients [let7-b and miR-18a, isolated from plasma of multiple myeloma patients, were associated with overall survival [In addition, exosome-transported lncRNAs may participate in drug resistance induction. A recent paper by Wang et al. proved that the exosome-mediated transfer of lncRNA t cancer . The autding RNA . EVs relgression ,35. Whilr growth ,37. Morer growth . In addipatients , while lsurvival . As expemiR-210 inhibits cardiomyocyte apoptosis by targeting Ephrin A3 (cell surface GPI-bound ligand for Eph receptors) and PTP1b (protein-tyrosine phosphatase 1b). Moreover, EVs derived miR132 stimulates angiogenesis acting on RasGAP-p120, Ras GTPase activating protein p120 [miR21 exert similar effects preventing cell apoptosis by targeting PDCD4 (Programmed Cell Death 4) [Barile and colleagues revealed the cardioprotective role of the different miRNAs transported by EVs derived from cardiac progenitor cells (CPC). They demonstrated that ein p120 . FurtherDeath 4) . SimilarDeath 4) .miR-21a [Regarding differentiation, it has been demonstrated that exosomes released from Neural stem/progenitor cells (NPCs) have an important role in neurogenesis; Ma and collaborators demonstrated that mouse cortical NPCs, isolated from fetal brain, promote neuronal differentiation through exosomal miR-21a .miR-99b has been demonstrated to control in vivo fibroblast growth factor 21 (FGF21) production [Adipose tissue is an excellent resource for circulating exosomal miRNAs that have a role in regulating liver gene expression, as well as affecting obesity or diabetes. Adipose-derived exosomal oduction . While eoduction .All these experiments suggest that exosomes may be a vehicle of therapeutic non-coding RNAs in physiological and pathological conditions as well as in the field of regenerative therapy  Table 1Table 1. Recent evidence highlighted the main role of RNA-binding proteins in RNA sorting and loading in EVs. Santangelo et al. have identified the RNA binding protein SYNCRIP  as a component of the hepatocyte exosomal miRNA sorting machinery. They showed that SYNCRIP knockdown impairs the internalization of miRNAs in exosomes . SubsequmiR-122, a well-known prognostic biomarker for metastasis in breast cancer patients [miR-122 vHD enrichment in breast cancer cell lines [Recently, Temoche-Diaz and collaborators showed that the metastatic breast cancer cell line MDA-MB-231 releases two sub-populations of EVs, called vesicular low density (vLD) and vesicular high density (vHD) EVs. Mass spectrometry analysis revealed that CD63, known as an exosomal marker, was enriched in the vHD subpopulation, as well as other endosome-associated proteins. MiRNA analysis identified five miRNAs that were enriched in vHD vesicles and not in vLD ones, thus suggesting that there is a specific mechanism of miRNA sorting for the vHD subpopulation . Interespatients ,51 was ill lines .LNMAT2) that promotes lymphangiogenesis. Interestingly, they found that LNMAT2 was loaded into exosomes by the heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1). The complex LNMAT2/hnRNPA2B1, once in receiving cells, can migrate into the nucleus and interact with PROX1 promoter. Here HNRNPA2B1 mediates H3 lysine 4 trimethylation (H3K4me3) and subsequent activation of PROX1 which, in turn, regulates endothelial cell differentiation and metastatic dissemination [RNA binding proteins not only promote RNA loading but also contribute to RNA function into target cells. Chen et al. studying bladder cancer metastatization identified an exosomal lncRNA, termed lymph node metastasis-associated transcript 2  is one of the proposed mechanisms. HNRNPA1, after sumoylation, recognizes the long non-coding RNA into EVs . While Hinto EVs .The ribonucleoprotein HNRNPA2B1, again, through GlcNAcylation, can regulate the internalization of specific miRNAs into EVs. Recently, Lee and collaborators demonstrated that Caveolin1 (CAV1), after its tyrosine phosphorylation (Y14), interacts with HNRNPA2B1, promoting HNRNPA2B1 O-GlcNAcylation on two serine domains. This modified complex directly controls the packaging of specific miRNAs into EVs . AnotherIn a recent work, Robinson and coworkers have identified HNRNPK as a CAV1-regulated microRNA binding protein. They observed that CAV1 drives HNRPNK localization to MVB, which brings the miRNA containing the AsUGnA motif. Moreover, they found that membrane-rafts take part in this transport . Export Again, HNRNPK participates in EVs loading and secretion of non-coding RNAs, through an alternative pathway mediated by LC3-conjugation machinery and called LDELS, LC3 dependent EV loading and secretion .Leidal et al. demonstrated that lipidated LC3-II is involved in the loading of specific proteins into intraluminal vesicles (ILVs) for their subsequent release as EVs. Interestingly the process is distinct from classical macroautophagy/autophagy because it requires components of the LC3 conjugation machinery, but not other ATGs involved in autophagosome formation. By proteomic analyses of LC3-conjugated protein obtained from secretome, the authors demonstrated that 33% of identified proteins have been previously associated with EVs. Moreover, after sucrose density gradient purification, they found that endogenous LC3-II co-fractionated with well-defined EV markers. This result further confirms the presence of LC3-II residues inside the lumen of EVs. Through different knock out models, the authors identified several RNA binding proteins that require LC3 to be internalized in EVs, among these the HNRNPK and scaffold-attachment Factor B (SAFB). Interestingly the deficiency in LC3-conjugation machinery affects the amounts of RNA loaded in EV. In particular, snoRNAs and miRNAs were reduced with an increase in tRNAs while no effects were found in large RNA levels .New evidence from Arabidopsis proteomic analysis revealed that also plant- derived EVs contain several RNA-binding proteins, as Argonaute 1 (AGO1) and RNA helicases (RHs). These proteins selectively associate with EV-enriched small RNAs, thus suggesting their involvement in specific loading of sRNAs into EVs also in plants .In conclusion, several RNA binding proteins, alone or in combination with other molecular interactors may control RNA sorting inside EV .It is well known that the ESCRT pathway is responsible for protein sorting into EVs ,63.Alix is an adaptor protein involved in EVs biogenesis and cargo sorting through an ESCRT dependent pathway . HoweverCargo sorting could also be driven via ESCRT-independent pathways, e.g., through the neutral sphingomyelinase, phospholipase or other lipids and associated protein such as tetraspanin ,10,60,68Janas et al. have proposed that the RNA loading into exosomes could be mediated by the direct interaction between RNAs and the lipid raft of MVB membrane . MoreovemiR-210 packaging in EVs that finally affect the initiation of metastatic process through the induction of angiogenesis [miR-100 while the oncomiR miR-10b was preferentially secreted in EVs derived from wild type KRAS cell line; these data demonstrate a key role for KRAS in orchestrating RNA trafficking. Interestingly nSMase2 is the mediator of this mechanism since its inhibition caused the miR-100 confinement in KRAS mutated cells [Other recent evidence suggests that neutral sphingomyelinase 2 (nSMase 2), via ceramide production, regulates EVs cargo loading. Kosaka and coworkers demonstrated that nSMase2 promotes the ogenesis . Cha et ed cells .Sphingomyelinase takes part also in the transport of viral RNA mediated by EVs. Zhou et al. demonstrated that ZIKA viruses, which infection induces severe neurological manifestations, use exosomes as mediators of viral transmission between neurons. The authors demonstrated that cortical neuronal cell-derived exosomes contain ZIKA virus RNAs and proteins and that the treatment with GW4869, nSMase-2/SMPD3 specific inhibitor, significantly reduced the loading of viral molecules. Interestingly, viral RNA positively participates in transmission promoting in neuronal cells the expression and activity of the nSMase-2 .PVT1) that, highly expressed in several cancers including pancreatic cancer, promotes exosome secretion [PVT1 promotes the docking of MVBs by altering RAB7 expression and localization. Moreover, PVT1 promotes the palmitoylation of the v-SNARE homolog YKT6 and its colocalization with vesicle-associated membrane protein 3 (VAMP3) thus determining the fusion of MVBs with the plasma membrane. The involvement of lncRNA in exosomes formation or release let also suppose a direct role of these molecules in transporting other smaller RNAs.Finally, it has to consider the possibility that the RNAs are not passively loaded into the exosomes but may themselves participate in vesicle formation. This is the case of lncRNA plasmacytoma variant translocation 1  protein, which binds specifically to the stem-loop-containing trans-activating response (TAR) element RNA. They added TAR directly to the 5′ end of a cargo mRNA and fused Tat peptide directly to the C-terminus of ARRDC1, the Arrestin domain containing protein 1 .Despite numerous attempts to engineer vesicles with specific RNAs, the major limitation is still the partial knowledge of the sorting and loading mechanisms in nature.Recent investigations have shown the central role of ncRNAs in gene regulation at different levels, including the ‘remote’ regulation mediated by EVs.Here we summarized the newest studies describing the RNA binding proteins and other interactors controlling the RNA sorting inside EVs. However, many aspects of RNA trafficking need to be further investigated. Moreover, it remains unclear why cells selectively load specific RNAs into subclasses of extracellular vesicles unless we still want to convince ourselves that it is only a strategy for discarding excess materials.Detailed knowledge of the loading mechanisms may point towards the wider use of EVs as diagnostic and prognostic tools. Moreover, it can provide essential indications for enhancing the use of EVs as drug delivery systems.


Immune checkpoint inhibitors (ICIs) targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein (PD-1), and its ligand PDL-1, are finding increasing application in the treatment of malignant neoplasms. The widespread clinical use of these drugs, however, resulted in the discovery of side effects. The occurrence of celiac disease (CD) after ICIs therapy has been reported in the literature, but its incidence remains unknown and the role of ICIs in its onset is not yet clear. In this review, we examine the published data on this topic in order to better understand and define this entity from a histological point of view. We performed an electronic literature search to identify original reports in which CD or pathological CD-like conditions were documented histologically in patients treated with ICIs. We identified ten papers. A total of twenty-five patients were included in these publications, eleven of them receiving a serologic and histological diagnosis of CD, and four a histological diagnosis of CD-like conditions, in which pathogenesis appears to be multifactorial. ICIs can cause a CD-like enteropathy and biopsies with clinical integration are crucial to diagnose this condition. CD rarely has been observed during treatment with ICIs and its morphological aspects are similar to ICIs-CD enteropathy. Moreover, the onset of ICIs-CD may have a distinct immune mechanism compared to classical CD. Thus, the pathologists must make a histological diagnosis of CD with caution and only in adequate clinical and serological context. Biological therapy development in inflammatory bowel disease (IBD) has allowed for the identification of several novel compounds that can interfere with distinct immunological pathways ,2. AmongThe Food and Drug Administration has approved several monoclonal antibodies blocking CTLA-4 (ipilimumab), PD-1 , and PDL-1  for treatment of solid tumours, such as melanomas , non-smaThe increasing implementation of these drugs in different settings has underlined the possibility of side effects arising in treated patients. Their interference with the immune system appears to favour the onset of autoimmune manifestations, defined as immune-related adverse events (IRAEs) ,17. IRAEGiardia, Cryptosporidium, Cyclospora), and drug enteropathies  have rarely been associated with severe villous blunting, showing only a more or less pronounced increase of intraepithelial lymphocytes [Celiac disease (CD) is an immune-mediated inflammatory disorder of the small intestine developing in genetically susceptible individuals after exposure to gluten, characterized by malabsorption resulting from mucosal injury after ingestion of wheat gluten or related rye and barley proteins . CD can phocytes ,43,44,45phocytes , to indiphocytes . In somephocytes . In contphocytes ,49.In this review, we summarize CD and CD-like histological features in patients with ICIs therapy reported in the literature and comment on the possible immune-related pathogenic mechanisms in order to better understand the relationship between ICIs and the development of CD and CD-like alterations, including the histological diagnostic approach.A systematic review was conducted following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. A search of PubMed and Web of Sciences (WoS) databases was performed up to 30 September 2021 using the terms: immune checkpoint, immune checkpoint inhibitor, imatinib, pembrolizumab, nivolumab, atezolizumab, avelumab, and durvalumab in combination with each of the following: celiac, coeliac, celiac-like, sprue-like, sprue like, duodenitis, enteropathy. Only articles in English were selected.All original reports in which the development of CD or CD-like conditions were documented histologically in patients of any age being treated with ICIs were considered for inclusion. Only articles in English were selected. Case reports, letters to the editor, and original articles were also included.Based on our predefined inclusion criteria, the literature search identified ten papers in the period 2013–2021. While two of them were original papers, the remaining ones were either case reports or letters to the editor ,56,57,58Among the reported treatments, pembrolizumab appears to be the most frequent one associated with CD, even after months of treatment initiation. All ICIs-CD cases reported so far have shown typical CD histological findings with variable villous atrophy and intraepithelial lymphocytosis. The clinical presentation of affected patients was mostly characterized by diarrhoea, frequently associated with weight loss. Interestingly, patients diagnosed with ICIs-CD benefited from a gluten-free diet (GFD). In two cases, steroid treatment was also implemented. In addition, four case reports of ICIs-related CD-like conditions have been described, but CD serologic testing was negative and, of note, only one of them showed clinical benefits from the introduction of a GFD. We recently observed the case of a 45-year-old man treated with ipilimumab for follicular lymphoma. Four weeks after initiating the therapy, the patient developed non bloody diarrhoea and weight loss. The duodenal biopsies showed a picture of an ICIs-related CD-like condition characterized by a moderate-severe atrophy of villi and intraepithelial lymphocytosis. After a GFD, the duodenal biopsies were performed again, and we observed improvement in villous architecture but persisting intraepithelial lymphocytosis .Helicobacter pylori infection, the histological presentation of duodenitis was similar to the classical CD but, in our opinion, duodenitis is a misnomer and the ICIs-related injury of the duodenal mucosa should more correctly be defined as CD-like enteropathy. In detail, the histological clue of ICIs-related duodenal injury is represented by a marked villous atrophy (from moderate to severe) with a mechanism similar to olmesartan, and increased intraepithelial lymphocytic infiltration; when compared to classical CD, ICIs-related CD-like conditions show an increased presence in the lamina propria of CD3+ and CD8+ T lymphocytes, with a reduced CD4:CD8 ratio [In recent years, the continuously wider implementation of ICIs in oncology has increased the occurrence of IRAEs. Regarding events arising in the gastrointestinal tract, CD and CD-like conditions have rarely been described and their real incidence among ICIs-related IRAEs is unknown, with only a very small number of cases reported to date. On the other hand, ICIs-related duodenitis and enteritis appear more frequent. The involvement of the upper gastrointestinal tract is more common among patients treated with anti-PD1/PDL-1 agents compared to patients in treatment with anti-CTLA4 agents, in which the endoscopic and histological injuries are detected more often in the stomach than in the duodenum . The mosD8 ratio .Thus, ICIs can cause atrophic lesions of the intestinal mucosa with CD-like features that are difficult to differentiate from classical CD. Subtle histological change such as patchy intraepithelial lymphocytosis  or more About etiology in CD-like lesions, Freeman et al.  suggesteConcerning ICIs-CD, since 2013, only eleven cases have been reported with histological documentation in the literature ,55,56,57These histological and immunological differences, along with clinical improvement after GFD and a reported increased serological value of autoantibodies in ICIs-CD, suggest that the onset of this entity may have a distinct immune mechanism which in turn is triggered by the implementation of the same drugs. Conversely, though ICIs-related CD-like enteropathy share the same histological and immunophenotypic characteristics with ICIs-CD, the lack of clinical improvement after GFD and the negativity for IgA TTGA suggest a different immune mechanism. Moreover, since ICIs-CD improved with GFD, similarly to the classic CD, it is likely that gluten becomes a trigger for these forms as well, upon treatment initiation . FinallyWhile it is well known that ICIs may trigger gastrointestinal inflammatory responses, the underlying mechanisms are poorly understood. The pathogenesis appears to be multifactorial and may involve the activation of effector T cells, increasing memory T cells, and lymphocytes in the intestinal mucosa, which may interact with the resident microbiota, infectious triggers, and drugs . In addiFinally, ICIs can induce intraepithelial lymphocytosis with villous atrophy in the intestinal mucosa. These lesions can be the expression of a drug-induced damage or of a latent CD. In fact, ICIs do not cause CD but promote the appearance of a latent CD through the loss of immunological tolerance .The limitations of our conclusions are due to the few papers, case reports, and case series that are reported in the literature and an absence of controlled studies. Furthermore, the papers published are lacking relevant data (serology and genetics) useful for validating a correct diagnosis of CD.ICIs can induce enteropathy and mucosal sampling for histological evaluation is mandatory. In the case of morphological lesions resembling CD, serological and genetic testing should be performed for differentiating true CD from drug-induced villous atrophy. This diagnostic path must be as more accurate as possible, including both IgA TTGA and EMA as well as HLA-Q2 and DQ8 typing. Moreover, in this setting, the diagnosis of CD should be made with caution and after a thorough evaluation because the therapeutic approach to ICIs-CD (GFD) differs from ICIs-related CD-like conditions (corticosteroids).


Arabidopsis gene FLC, repression involves alternative processing of FLC antisense transcripts linked to delivery of a local chromatin environment that determines FLC transcription initiation and elongation rate. Here, we show that AGO1, a factor known predominantly for its role in posttranscriptional gene silencing, is involved in this cotranscriptional repression mechanism. Conserved cotranscriptional regulators, the THO/TREX complex and NTC components of the activated spliceosome, physically associate with AGO1. Our analysis suggests that alternative interactions of cotranscriptional regulators with the RNA Pol II–spliceosome link RNA processing and chromatin modification to quantitatively regulate transcriptional output.RNA processing generally occurs as transcripts are being produced and the concomitant cotranscriptional processes are interconnected with chromatin regulation. These cotranscriptional mechanisms quantitatively influence transcriptional output. At the  Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3′-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins—the spliceosome NineTeen Complex (NTC) and related proteins (NTR)—and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The  Xist)–mediated X chromosome inactivation in female mammals  plays a central role in determining reproductive strategy—overwintering or rapid cycling—with considerable consequence on reproductive success in different climates , SET-domain proteins, SWR1 chromatin-remodeling complex, and H2B monoubiquitination mediators , RNA 3′-end processing and splicing factors, and chromatin modifiers. These repressors were grouped into the autonomous pathway that regulates FLC through an RNA-mediated chromatin-silencing mechanism  in RNAi (Arabidopsis AGO1 may play a much wider role in gene regulation.Argonaute proteins are well-known effectors in widely conserved RNA interference (RNAi) pathways. AGO proteins loaded with sRNAs form the core of RNA-induced silencing complexes that function in posttranscriptional gene silencing, either through cleavage of target RNA or translational inhibition, both of which mainly occur in the cytoplasm . AGO pro16 in RNAi , but rec in RNAi –28. ThesCOOLAIR-mediated FLC repression mechanism. We find that AGO1 physically associates with the FLD/SDG26/LD complex. Genetic analysis confirmed the AGO1 involvement in FCA-mediated FLC repression, and we show AGO1 associates with antisense RNA COOLAIR and influences COOLAIR splicing. Proteomics analyses revealed a close relationship between AGO1, the THO/TREX and NTC/NTR complexes, and RNA Polymerase II (Pol II). We show the THO/TREX complex functions antagonistically with AGO1 on COOLAIR processing and thus FLC regulation. The detailed dissection of the FLC/COOLAIR transcriptional unit has enabled an understanding that differential assembly of cotranscriptional machineries with RNA Pol II results in changes in RNA processing that feeds back to alter local chromatin environment and thus influence subsequent transcriptional output.Here, we report AGO1 as a player in the SI Appendix, Fig. S1). Additionally, unique peptides of AGO1 were detected in cross-linked nuclear immunoprecipitation of SDG26-GFP . The interaction between AGO1 and SDG26 was further confirmed by coimmunoprecipitation (co-IP) using nuclear extracts from a stable transgenic line expressing SDG26 fused to a FLAG-HA TAP tag (SDG26-TAP)  A. The phago1-25; this is slightly late-flowering (FLC expression (ago1 fca and ago1 fld double mutants did not show any additive effect on FLC expression (ago1 was combined with FRIGIDA (FRI), a strong activator of FLC  carrying FRI and overexpressing FCAγ that we have used to successfully screen components required for FCA in FLC regulation . These data collectively demonstrate AGO1 is required for FCA-mediated FLC repression. AGO1 harbors an Asp-Glu-Asp-His/Asp (DEDH/D) catalytic tetrad, which is commonly used for slicing target messenger RNA (mRNA) in Arabidopsis , suggesting that slicer activity or an intact slicer domain is required for AGO1-mediated FLC repression.Since a complete loss of AGO1 function is sterile we exploited a weak allele lowering  and confpression B, similapression C; however of FLC D. This igulation , 30, 31.ed to C2 E. Neithebidopsis , 32. We  ago1-25 . The derArabidopsis genome to positively regulate gene expression, especially in response to hormone and stress stimuli  sequencing data in this report did not suggest FLC chromatin is enriched for AGO1. Instead, the physical association and the genetic requirement of AGO1 in the autonomous pathway of FLC regulation prompts us to investigate a direct role of AGO1 in this antisense RNA-mediated chromatin silencing pathway. We performed RNA immunoprecipitation (RIP) in a transgenic line expressing hemagglutinin (HA)–tagged AGO1. Intriguingly, AGO1 associates with specific segments of COOLAIR, especially at a region spanning the exon–intron junction in the proximally polyadenylated COOLAIR , the subtle difference likely due to ago1-25’s being a weak allele. Nevertheless, the slight AGO1 stabilization of the FCA condensates may increase dwell time over the locus sufficiently to promote splicing of the proximal intron linked to 3′-end processing to promote proximal COOLAIR formation.FCA forms liquid-like nuclear condensates in vivo that concentrate 3′-end processing factors to promote efficient polyadenylation . Thus, wCOOLAIR promotes the resolution of a COOLAIR induced R-loop, which links to histone demethylation-induced chromatin silencing of FLC -qPCR analysis showed the R-loop over the 3′ end of FLC indeed increases in ago1-25 . Therefore, we conclude AGO1 is involved in FCA-mediated FLC silencing independently of DCL3.AGO1 canonically functions with miRNA and small interfering RNA (siRNA), which are mainly produced through DICER-LIKE (DCL) proteins in hat line . IntroduSI Appendix, Fig. S6A). We immunopurified HPR1/THO1, a component of the THO/TREX complex, from a transgenic line expressing HPR1 tagged with green fluorescent protein (GFP) . We took advantage of the FLC/COOLAIR transcriptional unit to further understand the functional relevance of the physical associations between AGO1 and these conserved cotranscriptional regulators.To understand the function of AGO1 in RNA processing, we investigated the proteins associated with AGO1 in the nucleus. A previous mass spectrometry analysis of nuclear AGO1 coimmunoprecipitated proteins identified SWI/SNF complex as an interactor that promotes transcription in response to stimuli . Other fin (GFP) . We wereCOOLAIR-induced R-loop, we first tested the possibility that the THO/TREX complex may prevent COOLAIR-induced R-loop formation. Using DRIP-qPCR analysis, we did not observe an obvious R-loop level change in the THO/TREX complex mutants hpr1-5 and tex1-4 . We did, however, find a reduction of FLC expression in these mutants . Our data show AGO1 and the THO/TREX complex function antagonistically on COOLAIR processing and FLC regulation. In Arabidopsis, the isolation of loss-of-function mutations of the NTC/NTR complex has revealed differential regulation by different components even on the same target FLC. For example, a mutation on PRP8 causes elevated FLC level  and DCL1 is mainly required for producing miRNA (FLC is not a miRNA target), redundant DICER activity could produce sRNA. Therefore, we searched sRNA sequence databases (http://ipf.sustech.edu.cn/pub/asrd/) . This location was intriguing given the observed change in COOLAIR splicing in ago1. We therefore analyzed RNAs coimmunoprecipitated with AGO1 and found sRNA fragments complementary to COOLAIR over the proximal intron/exon junction region , where AGO1 was enriched on COOLAIR   and foun COOLAIR B. A prev COOLAIR . The pri COOLAIR , 23, 48.ago1-25  medium plate without glucose. Plates were stratified at 4 °C for 3 d before transferred to long-day conditions .UBC. Primers are described in SI Appendix, Table S1.Ten-day-old seedlings were harvested, and RNA was extracted. TURBO DNase (Ambion) was used to remove DNA contamination before reverse transcription. RNA was reverse-transcribed by SuperScript IV Reverse Transcriptase (Invitrogen) using gene-specific primers. qPCR analysis was performed, and data were normalized to Total protein extracts from 10-d-old seedlings were prepared and immunoblot was performed as described previously . Anti-FC2, 0.5% Triton X-100, 5 mM dithiothreitol [DTT], and 1× protease inhibitor mixture [Roche]) in the presence of 10 U/mL RNase inhibitor . After resuspending the pellet in 1 mL Nuclear Lysis Buffer , 40 U/mL DNase (Invitrogen) was added for incubation for 10 min at 37 °C. The lysate was incubated 1 h at 4 °C after adding 10 μL 10% sodium dodecyl sulfate (SDS) and 5 M NaCl. The lysate was then sonicated with Diagenode Bioruptor 10 times, 30 s on/30 s off at high setting. Soluble chromatin extracts were collected after centrifugation. GFP-trap magnetic agarose beads (ChromoTek) were prewashed with low-salt wash buffer and blocked with 0.1% bovine serum albumin and 1 mg/mL transfer RNA (tRNA) in low-salt wash buffer for 1 h before adding them into IP solution. After 2 h incubation at 4 °C, the beads were washed through high-salt washing buffer and low-salt washing buffer. The beads were suspended in RNA elution buffer  at 95 °C for 15 min then were reverse cross-linked at 65 °C for 1 h in the presence of Protease K and RNase inhibitor. RNA was extracted using TRIzol and subjected to reverse transcription using gene-specific primers. qPCR was performed and data were presented as IP/1% input. UBC was used as a negative control. Primers are listed in SI Appendix, Table S1.For HPR1-GFP RIP, 3 g of 14-d-old seedlings were cross-linked in 1% formaldehyde. Nuclei were enriched using Honda buffer  and suspended in RIPA buffer . Nuclear extracts were sonicated and incubated with Benzonase for 15 min. Four micrograms of AGO1 antibody  was added into the soluble extracts incubated for 2 h before adding 25 μL Dynabeads Protein A (Invitrogen) for another 1.5 h. Beads were washed three times before eluted in 1x LDS buffer.SI Appendix, Table S1.DRIP was performed as previously described . BrieflySI Appendix, Table S1. Data were normalized to 1% of input and presented as the ratio of H3K4me1 to H3.Histone ChIP was performed as previously described with modifications . ProteinNuclei from 2 g of 10-d-old seedlings were prepared as described before in RIP. The nuclei pellet was weighed and resuspended in an equal volume of resuspension buffer . After washing twice with two volumes of UREA wash buffer  via pipetting up and down, the chromatin pellet was spun down, which then was rinsed gently with TES. The RNA was extracted using TRIzol.ago1-25 and homozygous FCA-mTurquoise2 were sown and the 7-d-old seedlings were segregated into two populations. One population which developed normally was referred to genotype carrying wild-type AGO1 and the other with developmental defects (unexpanded dark green cotyledons) was referred to genotype carrying ago1-25 homozygous mutation. Root tips were imaged as described previously  plus the MaxQuant contaminants database (245 entries). The quantitative LFQ results from MaxQuant with default parameters were used together with search results from an in-house Mascot Server 2.4.1  on the same databases. The Mascot search results were imported into Scaffold 4.11 (https://www.proteomesoftware.com/) using identification probabilities of 99% for proteins and 95% for peptides.Fine powder from 14-d-old seedling was dissolved in IP buffer . Extracts were immunoprecipitated with GFP-trap magnetic agarose beads (ChromoTek). The affinity-purified protein samples were separated on gel. The gel slices were washed with 50 mM TEAB buffer, pH 8 (Sigma) and incubated with 10 mM DTT for 30 min at 65 °C followed by incubation with 30 mM iodoacetamide at room temperature (both in 50 mM TEAB). After washing and dehydration with acetonitrile, the gels were soaked with 50 mM TEAB containing 10 ng/µL Sequencing Grade Trypsin (Promega) and incubated at 50 °C for 8 h. The eluted peptide solution was dried down, and the peptides were dissolved in 0.1%TFA/3% acetonitrile. Aliquots were analyzed by nanoLC-MS/MS on an Orbitrap Fusion Tribrid mass spectrometer coupled to an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific). Recalibrated peaklists were generated with MaxQuant 1.6.0.16 REF in LFQ mode using the TAIR10_pep_20101214 COOLAIR sequence across the proximal COOLAIR transcript) and the adaptor as the reverse primer. miR159 was included as a positive control. qPCR was performed using the Mir-XTM miRNA qRT-PCR SYBR Kit (Takara) on LightCycler480 II (ROCHE) and qPCR data were normalized to tRNA. Primers are listed in SI Appendix, Table S1.One gram of Col-0 and HA-AGO1 seedlings were ground into fine powder and subjected to immunoprecipitation using anti-HA magnetic beads . The beads were then extracted with TRIzol agent (Invitrogen) to retrieve the RNAs bound to AGO1. RNAs were reverse-transcribed using a Mir-XTM miRNA First Strand Synthesis Kit (Takara) according to the manufacturer’s instructions. The complementary DNA was amplified using sRNA sequences as forward primers (designed multiple primers complementary to Supplementary FileSupplementary File