Elevated triglyceride-rich lipoproteins (TGRL) in circulation is a risk factor for atherosclerosis. TGRL from subjects consuming a high saturated fat test meal elicited a variable inflammatory response in TNFα-stimulated endothelial cells (EC) that correlated strongly with the polyunsaturated fatty acid (PUFA) content. This study investigates how the relative abundance of oxygenated metabolites of PUFA, oxylipins, is altered in TGRL postprandially, and how these changes promote endothelial inflammation. Human aortic EC were stimulated with TNFα and treated with TGRL, isolated from subjects’ plasma at fasting and 3.5 hrs postprandial to a test meal high in saturated fat. Endothelial VCAM-1 surface expression stimulated by TNFα provided a readout for atherogenic inflammation. Concentrations of esterified and non-esterified fatty acids and oxylipins in TGRL were quantified by mass spectrometry. Dyslipidemic subjects produced TGRL that increased endothelial VCAM-1 expression by ≥35%, and exhibited impaired fasting lipogenesis activity and a shift in soluble epoxide hydrolase and lipoxygenase activity. Pro-atherogenic TGRL were enriched in eicosapentaenoic acid metabolites and depleted in esterified C18-PUFA-derived diols. Abundance of these metabolites was strongly predictive of VCAM-1 expression. We conclude the altered metabolism in dyslipidemic subjects produces TGRL with a unique oxylipin signature that promotes a pro-atherogenic endothelial phenotype. One-third of Americans are clinically categorized at higher risk for CVD in that they show a cluster of risk factors associated with high fat western diet and sedentary lifestyle defined as metabolic syndrome3. Among these risk factors is hypertriglyceridemia, which is characterized by elevated levels of serum triglycerides, circulating in ApoB-containing triglyceride-rich lipoprotein (TGRL). Elevated TGRL is a strong predictor for CVD7, and its uptake by the endothelium leads to inflammation that precedes and promotes atherosclerosis. Here we investigate how changes in the relative abundance of fatty acid metabolites circulating in TGRL link to its ability to prime an inflammatory response to a high fat meal.Disorders of metabolism are accompanied by an elevated systemic inflammatory state that accelerates the progression of atherosclerotic cardiovascular disease (CVD), the leading cause of death and disability in our society9. A high fat meal induces a postprandial inflammatory state, characterized by elevated levels of circulating TGRL and other inflammatory mediators, including cytokines (e.g. TNFα) that promote endothelial dysfunction accompanying atherosclerosis. A high-fat test meal challenge has been established as an effective tool to elicit inflammation of arterial endothelium and subsequent monocyte capture, changes not typically observed under fasting conditions. Therefore, a postprandial response may provide greater insight into the effects of metabolism on insipient inflammation than the fasting state11. We have investigated the capacity of TGRL produced by human subjects after consuming a high saturated fat meal, typical of western diet, to elicit a pro-atherogenic endothelial response.Atherosclerosis has been proposed to be a postprandial process that is tied to transient elevation of postprandial triglycerides12. VCAM-1 expression correlates with coronary lesion severity in atherosclerotic patients, and is causative in mouse models of atherosclerosis14. We showed that TGRL isolated from subjects after a standardized fast food breakfast meal high in saturated fat, can prime either a pro- or anti-atherogenic state, defined as a net up- or down-regulation of VCAM-1 expression in TNFα-stimulated human aortic endothelial cells (HAEC)18. In these studies, the level of VCAM-1 expression was strongly associated with the relative capacity to induce recruitment of monocytes from healthy subjects assessed in TNFα-inflamed HAEC16. The capacity for TGRL to alter VCAM-1 expression correlated closely with an individual’s metabolic status as reflected by their level of postprandial triglycerides and abdominal obesity. However, TGRL obtained from the same individuals when fasting did not prime inflamed HAEC for increased VCAM-1 expression15. Unknown is how the composition of TGRL is altered postprandially following the high fat meal to elicit a distinct endothelial inflammatory response by individuals exhibiting metabolic risk factors.Among the earliest inflammatory responses by the endothelium that precedes atherosclerosis is the acute upregulation of cell adhesion molecules (CAMs), notably vascular cell adhesion molecule (VCAM-1), the predominant receptor for monocyte recruitment from the circulation8. The fatty acid composition of circulating TGRL thus reflects both the content of a meal and an individual’s overall metabolic state, including the presence of diabetes or dyslipidemia19. We previously reported that pro- and anti-atherogenic TGRL were composed of distinct fatty acid profiles, and that pro-atherogenic TGRL in particular was enriched in non-esterified polyunsaturated fatty acids (PUFA)16. Though overall less abundant in TGRL, PUFA and their oxygenated metabolites, oxylipins, are potent mediators of inflammation20. PUFA are metabolized by three main enzymatic pathways to produce an array of oxylipins: cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450s (CYP). COX-derived prostaglandins are generally considered pro-inflammatory. While LOX-derived mid-chain alcohols, leukotrienes, and ketones can be either pro- or anti-inflammatory , CYP-derived epoxides are considered anti-inflammatory. However, epoxides are readily hydrolyzed by soluble epoxide hydrolase (sEH) to form diols, which are reported to promote or have a neutral effect on inflammation20. Fatty acids and oxylipins are found in both esterified (bound) and non-esterified (free) pools in TGRL, and can be released by the action of lipases or upon receptor-mediated uptake by endothelial cells (EC)21.Dietary fatty acids of all saturation levels are packaged into and circulated by TGRL, which consist of chylomicrons and VLDL, synthesized in the gut and liver respectively11. Metabolic syndrome is associated with a disruption of the oxylipin pattern across all lipoprotein classes that was putatively pro-inflammatory, and was mitigated by prescription omega-3 PUFA intervention22. Moreover, there is evidence that oxylipin metabolism can directly affect atherosclerosis. For example, inhibition of sEH reduced atherosclerosis in ApoE null mice on a high fat diet, which correlated with enrichment in the plasma epoxide to diol ratio23. We address whether oxylipins circulating in TGRL act to exacerbate endothelial inflammatory responses promoting atherosclerosis.Although 90% of oxylipins are esterified in lipoproteins, their exact origins are unclear, and the significance of their transport within lipoproteins is largely unknown. It has been demonstrated that diet and metabolism affect oxylipin abundance and distribution among lipoprotein pools. For example, plasma oxylipins were enriched in proportion to the fatty acid composition of a high-fat mealThe objective of the current study was to identify, using an unbiased metabolomics approach, novel fatty acid constituents that can significantly predict the pro- or anti-atherogenic postprandial response after uptake and processing of an individual’s TGRL by endothelium. We hypothesized that metabolic dysregulation in dyslipidemic individuals is associated with a unique fasting and postprandial TGRL oxylipin signature in response to a high fat test meal. We report that the composition of sEH-derived diols and LOX-derived alcohols in postprandial TGRL strongly discriminated the response to the meal between pro- and anti-atherogenic subjects. A postprandial oxylipin signature was identified in pro-atherogenic subjects, characterized by enrichment in eicosapentaenoic acid (EPA) metabolites and depletion of esterified sEH-derived diols. Moreover, fasting indices of lipogenesis activity and the abundance of sEH-derived diols in TGRL were the best predictors of enhanced VCAM-1 expression.2) and waist circumference (24.5–43.0 in), and ranged from normal to hypertriglyceridemic (fasting triglycerides 30–279 mg/dl). We measured the capacity of postprandial TGRL isolated from each subject’s plasma to alter TNFα-stimulated CAM surface expression in HAEC in an ex vivo assay. An increase in VCAM-1 surface expression from stimulation with a low dose of TNFα (EC50 = 0.3 ng/ml) correlated most strongly with the postprandial spike in triglyceride levels   Fig. , inset. ng) Fig. , inset.Fpro-atherogenic (pro-) response, eliciting the greatest increase in VCAM-1 surface expression (47.4 ± 22.5%) and expressing a ΔTG > 120 mg/dl. Another five subjects were selected whose TGRL produced an anti-atherogenic (anti-) response, eliciting the greatest decrease in VCAM-1 surface expression (40.2 ± 19.5%) and expressing a ΔTG < 120 mg/dl   was performed. Postprandial levels of each constituent were normalized by fasting levels for each individual  to adjust for baseline metabolic variation. Oxylipins yielded the strongest discrimination between the pro- and anti-TGRL samples (Q32) Fig. . In cont32) Fig. . LOX-derTo further evaluate the involvement of these 22 oxylipins in mediating a differential response to the meal, they were grouped into 4 clusters based on similar patterns of expression. Non-esterified diols and alcohols of C18-PUFA LA and ALA in cluster 1, represented by 9-HODE, were enriched to a greater extent postprandially in pro-atherogenic TGRL. Oxylipins in clusters 2 and 3, represented by non-esterified 5,6-DiHETrE and 19,20-DiHDoPE, respectively, consisted primarily of diols and alcohols of C20- and C22-PUFA, which were highly expressed in the fasting state in anti-atherogenic TGRL. These same metabolites were depleted in the postprandial state of anti-atherogenic subjects  in non-esterified sEH activity index, i.e. the diol to epoxide ratio summed over all parent fatty acids measured in the non-esterified pool, strongly correlated with induction in VCAM-1 surface expression  score was selected as the best predictor of the postprandial VCAM-1 surface expression  was composed of the two cluster components depicted by the equation in Fig. ex vivo VCAM-1 surface expression was Cluster 1, which consisted predominantly of EPA and esterified EPA-derived metabolites . This is in accordance with the elevated EPA-specific LOX activity observed postprandially in pro-atherogenic subjects. Cluster 2, comprised predominantly of C18-derived esterified diols , negatively correlated with VCAM-1 expression. These findings are consistent with the observations from the PLS-DA model.We next investigated the extent to which the abundance of specific oxylipins and fatty acids composing postprandial TGRL were predictive of the elicited changes in fasting subjects. As above the AIC score was used to select a model that best fit measured VCAM-1 expression data . Cluster 1 consisted predominantly of non-esterified diols suggesting elevated levels of sEH activity that contributes to the non-esterified oxylipin pool in fasting anti-atherogenic subjects. Clusters 2 and 3 were comprised of various lipogenesis indices and Σn-6/Σn-3, which are emerging biomarkers for dyslipidemia28. Both the fasting esterified and non-esterified ELOVL-6 indices  inversely correlated with VCAM-1 expression. Esterified Δ5D and SCD-1 and non-esterified levels of Σn-6/Σn-3 and ELOVL-2 were also inversely correlated to the measured VCAM-1 expression. Together, these data support the utility of these lipogenesis indices as potential biomarkers that predict a subject’s susceptibility to CVD. Moreover, the regression analysis reveals that fasting indicators of metabolism reflected in the composition of TGRL strongly predict the inflammatory postprandial response.Given the strong correlation of postprandial VCAM-1 expression with fasting markers of dyslipidemia, we evaluated whether the composition of TGRL in fasting subjects was also predictive of the postprandial VCAM-1 response. A second multiple linear regression model was generated iteratively using 18 clusters of oxylipins and fatty acids, identified by their similar pattern of expression in TGRL from To demonstrate that the differences in concentrations of individual oxylipins identified by our analysis of TGRL could directly affect endothelial inflammation, we delivered several of them as methyl esters to stimulated HAEC and measured the dose response in VCAM-1 expression. The methyl ester form is readily taken up by cells and is analogous to the non-esterified or free form measured in TGRL. In this analysis we selected representative non-esterified oxylipins that contributed significantly to distinguishing pro- from anti-atherogenic TGRL Fig. , and str15. Our platform provided a means to directly link changes in the fatty acid and oxylipin composition of an individual’s TGRL with its inflammatory capacity measured ex-vivo for the first time. By characterizing TGRL before and after administration of a test meal representative of a high fat western diet, we have established a gauge for an individual’s acute postprandial inflammatory response18. Postprandial TGRL mediated a pro- or anti-inflammatory response unique to each subject that was marked by an increase or decrease in endothelial VCAM-1 receptors. The magnitude of this inflammatory response was strongly influenced by an individual’s fasting metabolic status, in that it correlated strongly with plasma markers of metabolic dysregulation and dyslipidemia. In response to the high fat meal challenge, TGRL characterized as pro- or anti-atherogenic revealed different oxylipin signatures, a key distinguishing feature of which was a shift in the relative abundance of non-esterified (free) diols. Pro-inflammatory TGRL was enriched in EPA and EPA-derived oxylipins and depleted in esterified (bound) C18-PUFA-derived diols in response to the meal, reflecting a putative shift in sEH and LOX activity in those subjects. A remarkable finding was the ability to model the measured postprandial VCAM-1 response using the relative abundance of free diols and lipogenesis indices measured in the fasting state. We conclude that specific changes in the composition of TGRL elicited by a high fat meal reflect an individual’s in vivo lipoprotein metabolism and mitigate the TGRL-induced ex vivo inflammatory response.Herein we quantify the effects of metabolic stress associated with the progression of atherosclerosis, through direct measurement of relevant inflammatory outcomes in a monolayer of arterial endothelial cells under conditions that mimic a postprandial response16. Though PUFA are not the most abundant type of fatty acid in TGRL, by virtue of their double bonds they are readily metabolized to form oxylipins, which are known to elicit potent effects on inflammation, depending upon the metabolizing enzyme, the position of the double bond, and the parent fatty acid. Moreover, the fatty acids and oxylipins are present in both esterified and non-esterified forms in lipoproteins, which could lead to differences in their processing and effects on inflammation21. Esterified fatty acids and oxylipins in lipoproteins are released into tissues after their uptake and lipolysis21. We previously reported that the inflammatory response to postprandial TGRL was largely dependent upon their receptor-mediated uptake by HAEC18. In contrast, the non-esterified fatty acids constitute only a small proportion of the total fatty acid constituents in TGRL. However, free oxylipins and fatty acids in TGRL are more readily available upon uptake and thus may be significant mediators of endothelial inflammation. Our data points to the enrichment of non-esterified PUFA and oxylipins in pro-atherogenic TGRL, consistent with our previous analysis16. We proposed that the oxylipin metabolites constituting TGRL are bioactive and can in part account for the relative atherogenicity of these particles by altering endothelial inflammation.In quantifying the abundance of fatty acids of various saturation levels in TGRL we found that pro-atherogenic TGRL was enriched in long chain PUFA, consistent with our previous report10. sEH inhibition is associated with anti-inflammatory and vascular relaxation properties mediated by elevated epoxides such as EETs and EpOMEs29. For example, 11,12-EpETrE reduced TNFɑ-induced VCAM-1 surface expression in HAEC30. Treatment with sEH inhibitors has also been reported to lower systemic inflammation in a mouse model of septic shock31, and to reduce atherosclerosis in ApoE null mice32. Another study observed an increase in sEH-derived diols and LOX-derived HODEs in the atherosclerotic plaques of rabbits33. In the current study, we observed enhanced sEH and LOX activity in dyslipidemic subjects after the high fat meal challenge. However, a higher relative abundance of non-esterified diols of C20- and C22-PUFA which were depleted postprandially was a defining feature of fasting anti-atherogenic TGRL. Our analysis consistently revealed a shift in the levels of diols in TGRL as markers of an endothelial inflammatory response. However, additional studies are needed to determine whether these diols themselves mediate the inflammatory response in HAEC, or rather reflect a shift in metabolism away from an overall anti-inflammatory state that involves cross-talk with other pathways.We found that an increase in the abundance of certain vicinal -diols, including non-esterified 9,10-DiHOME, 12,13-DiHOME, and esterified 19,20-DiHDoPE, were among the strongest discriminators of a pro-inflammatory response to postprandial TGRL. Diols are downstream products of epoxides enzymatically converted by sEH20. As we did not directly measure the hepatic activity, the elevated LOX index may also reflect auto-oxidation within subjects. However, in monitoring markers of auto-oxidation within our samples, we found them to be expressed at low levels, and to not differ significantly between the pro- and anti-atherogenic groups. The postprandial abundance of esterified EPA and EPA-derived oxylipins  were positive predictors for TGRL-induced VCAM-1 expression by HAEC. Additional studies are needed to determine whether the observed postprandial enrichment of EPA-derived oxylipins that mark pro-TGRL are themselves proactive mediators of inflammation.This study further revealed enrichment in C18-derived midchain alcohols in the TGRL of dyslipidemic subjects. Moreover, a significant positive correlation was observed between the calculated postprandial shift in EPA-specific LOX activity and VCAM-1 expression. Although midchain alcohols are products of LOX metabolism, they can also be derived from auto-oxidation processes associated with oxidative stressin vivo are exposed to a suite of fatty acids, oxylipins, and other metabolites present in lipoproteins which could have opposing effects on inflammation, characterizing the integrated response to the native oxylipin profile and evaluating correlative trends with inflammatory response ex-vivo provides physiological relevance. Nevertheless, herein we demonstrate that individual oxylipins identified by our analysis of TGRL could directly affect endothelial inflammation  inversely correlated with the measured endothelial VCAM-1 expression. The liver metabolizes LA and ALA derived from the diet to produce downstream long chain PUFAs using ELOVL and desaturase enzymes. These enzymes also catalyze the production of MUFAs from SFA, and elongate SFAs. Impairment or hyperactivity of these enzymes is associated with altered lipid metabolism and disease states such as type 2 diabetes and hypertriglyceridemia, both risk factors for CVD36. We observed an overall reduction in ELOVL-6 activity in response to the high fat meal challenge, particularly in pro-atherogenic subjects, which implies impairment of elongation or saturation of palmitic acid. Elevated levels of palmitic acid are strongly associated with a sedentary lifestyle and hypertriglyceridemia37. In a recent ten-year longitudinal study, ELOVL-6 and Δ5D activity were significantly reduced in obese subjects who developed dyslipidemia28. Other reports correlated impaired Δ5D activity to high BMI, CRP, and elevated triglyceride and CVD related mortality38. Consistent with these studies, we report that Δ5D and ELOVL-6 activity inversely correlated with endothelial VCAM-1 surface expression, a direct measure of inflammation and marker of atherosclerosis14. These findings support the utility of measures of lipogenesis and lipid metabolism  in identifying subjects in a state of metabolic dysregulation associated with greater cardiovascular risk.Since subjects consumed an identical high fat meal, differences in the postprandial response, especially as they correlate with markers of 18. This mechanism distinguishes VCAM-1 expression from ICAM-1, which is independent of both IRF-1 and ER stress. By extension the varying oxylipin constituents transported in TGRL may directly induce downstream activation or inhibition of ER stress and subsequent increase or decrease in VCAM-1 surface expression. It is established that an excessive load of fatty acids can induce a state of ER stress in hepatocytes, macrophages and adipocytes39. ER is the hub of lipogenesis activity in cells and ER stress correlates strongly with obesity and CVD. Inhibition or knock-down of sEH can reduce high fat diet or pharmacologically induced ER stress in mouse models41. Individual oxylipins including 9-HODEs and 12/15-HETEs induced ER stress in macrophages and adipocytes, respectively43. 12(S)-HETE was shown to directly enhance the monocyte adhesion in HAEC44. Further, pharmacological inhibition of LOX, COX, CYP, and sEH enzymes have consistently demonstrated that perturbing oxylipin metabolism can alter systemic inflammation. For example, knock-out of 12/15 LOX in Apo-E null mice resulted in a 95% reduction in monocyte arrest within the aorta45. These observations motivate a need for further investigation to establish a causative role for enrichment in individual oxylipins within TGRL in modulating ER stress responses that promote endothelial inflammation.We have previously reported that the upregulation of TGRL-induced VCAM-1 is in part signaled via the endoplasmic reticulum (ER) stress response that promotes increased activity of the transcription factor interferon regulatory factor 1 (IRF-1)Herein we develop regression models to predict the relative atherogenicity of an individual’s postprandial TGRL based on the oxylipins present in these lipoproteins. The models were generated from a cohort of subjects whose TGRL elicited the greatest modulation in VCAM-1 expression. Validation of these models in a larger independent cohort of subjects that includes patients with established metabolic syndrome and coronary artery disease, would ultimately demonstrate the predictive potential for gauging cardiovascular risk and disease progression over time. This approach could also prove useful in quantifying the effects of dietary intervention, statins, or other lipid-lowering therapies, by providing direct real-time measures of an individual’s lipid metabolism and inflammatory status.18. The meal is high in saturated fat and calories, and the cholesterol levels are above recommended intake for healthy diets. However, the meal is representative of a fast food breakfast meal not uncommon in the Western diet. Since this study identifies differences in the fatty acid composition of TGRL (particularly PUFA and PUFA-derived oxylipins) that correlate strongly with the nature of an inflammatory response, we envision future controlled dietary intervention studies that vary the fatty acid composition of the meal (e.g. replacing SFA with PUFA), or supplement with n-3 PUFA, and quantify the postprandial response.The aim of this study was to shed light on the variable inflammatory response in individuals elicited by an identical meal rather than to compare the response to meals of different fatty acid composition. Thus, we chose a test meal that we have used to elicit a variable postprandial endothelial inflammatory response in multiple studies15. One possible explanation is that the fasting state is primarily anti-inflammatory among most subjects, though it can reflect a different baseline on which a postprandial insult superposes to amplify differences in the inflammatory response of an individual. Consistent with this is the observation that the postprandial increase in subjects’ plasma markers CRP and sVCAM predicted the magnitude of the inflammatory response to TGRL measured ex vivo. Thus, this technique has the potential to be employed to discriminate patient populations who do not present elevated levels of conventional clinical risk factors but are still at high risk for atherosclerotic CVD. It is also noteworthy that hypertriglyceridemic subjects exhibit lower clearance of TGRL particles and higher accumulation of lipoproteins. In this regard, the differences we have observed in the oxylipin composition between pro- and anti-TGRL may exacerbate an inflammatory response in endothelium exposed to these particles for a prolonged period.Previous experience shows that the differential VCAM-1 expression observed in the postprandial state is not induced by fasting TGRLIn conclusion, we have identified a characteristic oxylipin signature in postprandial TGRL that distinguishes an acute pro- versus anti-inflammatory endothelial response in subjects consuming an identical high fat meal. Dyslipidemic subjects were uniquely characterized by a shift in sEH and LOX activity, and produced pro-atherogenic TGRL, postprandially enriched in EPA-metabolites and depleted in C18-PUFA-derived esterified diols. In contrast, normolipidemic subjects exhibited higher indices of fasting lipogenesis activity, and produced anti-atherogenic TGRL enriched in esterified C18-PUFA-derived diols, but depleted in non-esterified diols after the high fat meal challenge. These results implicate a role for the fatty acids and oxylipins constituting TGRL in mediating a pro- or anti-atherogenic endothelial phenotype. We further conclude that lipoproteins such as TGRL can serve as a depot for fatty acids and oxylipins, and propose that these bioactive fatty acid metabolites transported in TGRL could act remotely to directly modulate an endothelial inflammatory response.Human subjects (N = 39) were recruited according to an Institutional Review Board approved protocol at the University of California, Davis in accordance with the Helsinki principles and institutional guidelines and regulations. Informed consent was obtained from all research participants. The subjects varied from normal lipidemic to dyslipidemic, but did not have hypertension or elevated fasting glucose, and thus were not overtly characterized as having metabolic syndrome (Supplementary Table Plasma was spiked with 1x antibiotic/ antimycotic (GIBCO). Density-based ultracentrifugation  was used to isolate TGRL particles from plasma (ρ < 1.0063 g/ml) using a Beckman XL-90 ultracentrifuge. ApoB content was quantified using an ELISA kit (ALERCHEK) that measures both ApoB48 and ApoB100. In the fasting state TGRL consists of mainly ApoB100-containing very low-density lipoproteins (VLDL), whereas the postprandial TGRL is a mixture of VLDL and ApoB48-containing chylomicrons. ApoB concentrations were used to normalize the TGRL quantity for inflammatory characterization of HAEC and for mass spectrometry analysis. TGRL samples were characterized within 7 days of isolation or were aliquoted and frozen for future use. All samples were immediately flushed with nitrogen gas upon collection to prevent auto-oxidation. The extent of oxidation in the samples was monitored by tracking concentrations of 9-HETE, total TriHOME and F2-isoprostanes, which did not differ significantly between experimental groups.50 for CAM upregulation, R&D). HAEC were dissociated from the tissue culture plate using 1 mM EDTA , and stained with phycoerythrin-conjugated VCAM-1 antibody  and Alexafluor 488-conjugated intercellular adhesion molecule-1 (ICAM-1) antibody . VCAM-1 and ICAM-1 surface expression were measured using flow cytometry (Attune NXT). To account for day-to-day cell variability in HAEC response, CAM expression is reported as % change from TNFα stimulation alone. Based on previous studies, TGRL eliciting an increase in VCAM-1 expression ≥10% are characterized as pro-atherogenic, and those that decrease VCAM-1 by ≥10% as anti-atherogenic, as these changes correlate strongly with mononuclear cell recruitment.Human aortic endothelial cells (HAEC), passage 6–8 , were cultured with endothelial cell growth media-2  supplemented with 1x antibiotic/ antimycotic (GIBCO). Cells were seeded on tissue culture plates  and proliferated to form a 85–95% confluent monolayer. HAEC were then incubated with postprandial TGRL (10 mg/dl ApoB) for 4 hrs in the presence of TNFα , detected with a 5973 A mass selective detector (Agilent Technologies) using electron impact ionization and selected ion monitoring, and quantified against authentic standards. Results were corrected for the recoveries of analytical surrogates as previously described47. Non-esterified oxylipins were isolated directly from TGRL solution in PBS using solid phase extraction. Esterified oxylipins were transformed into oxylipin free acids by base hydrolysis, and isolated by subsequent hydrophilic/lipophilic interaction solid phase extraction. Non-esterified and alkaline stable esterified oxylipins were analyzed by UPLC-MS/MS using negative mode electrospray ionization and multi-reaction monitoring on a Sciex 4000 QTRAP47. Concentrations were calibrated using analytical standards as previously reported48.Non-esterified fatty acids (NEFA) and oxylipins, along with total fatty acids (TFA) and alkaline stable oxylipins were quantified using mass spectrometry: ultra-performance liquid chromatography mass spectrometry (UPLC-MS/MS) for oxylipins, and gas chromatography-mass spectrometry (GC-MS) for FA. For simplicity, since the non-esterified pools account for a minor component of the total pool, the total fatty acid and total alkaline stable oxylipin pools are referred to as esterified lipids for the remainder of this manuscript. The abundance of oxylipins was reported as pmol/mg ApoB and fatty acids as nmol/mg ApoB. Lipids were isolated using a liquid-liquid extraction with cyclohexane/isopropanol/ammonium acetate after enrichment with a suite of deuterated and rare compounds used as analytical surrogatesOxylipins were synthesized in methyl esterified forms dissolved in ethanol by the Hammock Lab (UC Davis). They were delivered to TNFα (0.3 ng/ml) stimulated HAEC at doses ranging from 0.1–100 nM for 4hrs. Change in VCAM-1 surface expression in HAEC from vehicle control was quantified by flow cytometry.Fatty acid and oxylipin data were categorized into non-esterified and esterified fatty acid pools and metabolites were grouped by their parent fatty acids. An activity index for each parent fatty acid was calculated based on product to substrate abundance ratios according to the following equations:An overall activity index was also estimated using the overall product to substrate ratio summed across all measured fatty acids. Analyses were then conducted on the postprandial to fasting ratio of these calculated enzyme activities.Indices of lipogenesis were calculated as a ratio of the measured abundance of fatty acid product to precursor in the fasting and postprandial plasma or TGRL: stearoyl-CoA desaturase-1 , SCD-C18 (C18:1n-9/C18:0), SCD-C16 (C16:1n-9/C16:0), delta 5 desaturase , elongation of very long fatty acid-6 , ELOVL-2 (C22:5n-3/C20:5n-3). The ratio of n-6 to n-3 PUFA was calculated across all measured PUFA (Σn-6/Σn-3).All statistical analyses were performed using JMP Pro 13 software. Fatty acid and oxylipin abundance data were Johnson transformed and checked for normality using the Shapiro-Wilk test. Outliers were assessed based on an interquartile range calculated per metabolite. Outliers were excluded if interquartile range <Q1 – (3 x interquartile range) or >Q3 + (3 x interquartile range). Missing values were imputed via a multivariate approach. Undetectable levels of metabolites were substituted by 0.5 x the lowest detected expression level measured for that specific metabolite.To assess differences in FA and oxylipin composition in TGRL as a function of the meal, TGRL-induced inflammatory phenotype, and interaction between the two factors, a two-factor analysis of variance (ANOVA) was performed. P-values were corrected for multiple testing using a Benjamini-Hochberg false discovery rate (FDR) approach. Likewise, a two-factor ANOVA and FDR was used to assess differences in plasma inflammatory markers.The Pearson method was used to correlate the change in VCAM-1 surface expression with clinical biomarkers in the full subject cohort (N = 39), since normality was satisfied. In the smaller set of subjects chosen for metabolomics analysis (n = 10), a non-parametric Spearman rank method was used to assess correlations between CAM expression and subject characteristics, plasma lipid, plasma cytokines or TGRL constituents.To identify the shift in fatty acid and oxylipin constituents differentiating pro- and anti-atherogenic TGRL, a partial least squares discriminant analysis (PLS-DA) was performed. Postprandial abundance data were adjusted to the fasting values  for each subject (n = 10). This approach reduced the multivariate data for projection on the 2 coordinate axes resulting in the maximum separation between the pro- and anti-TGRL groups based on fatty acid and oxylipin abundance. The scores plot depicts the separation between the samples characterized for their relative atherogenicity, while the loadings plot superposes the metabolites that contribute to the maximum variability between the two groups. Variable importance in projection (VIP) scores the relative contribution of each metabolite to the separation visualized in the scores plot. Metabolites with a VIP score of over 1.1 were considered to contribute significantly to the separation. The leave-one-out method was used to cross-validate the model. Next, the 22 oxylipins identified as the significant contributors to the PLS-DA model were clustered by expression pattern using the JMP 13 Pro cluster variable algorithm. The fasting to postprandial change of the most representative metabolite in the cluster, i.e. that for which the expression correlated most strongly to the principal component of the cluster, was graphed in order to visualize the change in abundance of the metabolite in response to the meal.49 that was an input into the iterative regression model. To satisfy the linearity requirement, VCAM-1 expression was Johnson transformed50. The regression model fit the response variable , using stepwise addition of cluster components as independent input variables (xi). The Akaike’s information criteria (AIC) and Beyesian information criteria (BIC) scores were used in model optimization with a goal of minimizing the number of independent terms. The addition of a term was considered to result in a stronger model if it improved the p-value and R2 while also reducing the AIC score by 2 or more52. The resulting model can be expressed by the following equation, where b0, b1, b2, b3, b4, …, bx are coefficients corresponding to each input variable (cluster):A stepwise linear regression modeling approach was used to fit metabolite expression data to measured change in VCAM-1 surface expression in HAEC. Metabolites were first clustered to account for collinearity and maintain independence between the input variables in the model. A cluster variable algorithm in JMP Pro 13 was used to group metabolites based on the correlation of their expression levels across all subjects, using the fatty acids, oxylipins, and lipogenesis indices as inputs. This resulted in 19 clusters for the postprandial case, and 18 clusters for the fasting case. Each cluster was reduced to a representative cluster componentSupplementary InfoRaw data used for the analysis


DMPK (DM1) and CNBP (DM2) genes results in the synthesis of CUG and CCUG repeat expansion  RNAs, respectively. These CUGexp and CCUGexp RNAs are toxic since they promote the assembly of ribonucleoprotein (RNP) complexes or RNA foci, leading to sequestration of Muscleblind-like (MBNL) proteins in the nucleus and global dysregulation of the processing, localization and stability of MBNL target RNAs. STR expansion RNAs also form phase-separated gel-like droplets both in vitro and in transiently transfected cells, implicating RNA-RNA multivalent interactions as drivers of RNA foci formation. Importantly, the nucleation and growth of these nuclear foci and transcript misprocessing are reversible processes and thus amenable to therapeutic intervention. In this review, we provide an overview of potential DM1 and DM2 pathomechanisms, followed by a discussion of MBNL functions in RNA processing and how multivalent interactions between expanded STR RNAs and RNA-binding proteins (RBPs) promote RNA foci assembly.Short tandem repeat (STR) or microsatellite, expansions underlie more than 50 hereditary neurological, neuromuscular and other diseases, including myotonic dystrophy types 1 (DM1) and 2 (DM2). Current disease models for DM1 and DM2 propose a common pathomechanism, whereby the transcription of mutant  Repetitive DNA sequence elements compose 50–70% of the human genome ,2. TheseC9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD), the C9orf72 intron 1 GGGGCC expansion (GGGGCCexp) results in alternative first exon selection, intron 1 retention, altered sense and antisense transcription, sequestration of multiple repeat-binding proteins  and RAN translation of dipeptide repeats (DPRs) .,66.65,66e family ,68 whilee family . Third, s of DM1 ,71,72.exp mutation does not inhibit CNBP transcription . I. Iexp anelopment . MBNL loy (FECD) ,143 and y (FECD) . Finallydiseases ,146,147.CNBP is expressed at much higher levels than DMPK  using a fluorescent probe complementary to the expanded repeats . Later, exp repeats  and a single ZnF pair is sufficient to bind to RNA  . . exp rep repeats A   or block., DDX5)  foci for., DDX5) ,165. It DMPK and CNBP expression. For instance, DMPK is expressed at a significantly higher level during fetal muscle development than in mature tissues and expression varies significantly between tissues [The dynamic nature of RBP and expanded RNA interactions in foci has been reported for MBNL and RBFOX paralogs for various length STR RNAs ,162,168. tissues .exp and CCUGexp expressed at endogenous levels promote RNA foci formation. For instance, DMPK transcripts are present at one to a few dozen molecules per cell in DM1 patient-derived myoblasts so RNA foci must be assembled from only a few transcripts [Early studies assessed the effects of RNA-MBNL interactions in the formation of RNA foci , howevernscripts ,173.exp and CCUGexp RNAs provide a scaffold for multivalent homotypic and heterotypic interactions that allow the formation of higher order assemblies. In CDM, with high mutant DMPK loads and relatively low MBNL expression, foci might be formed primarily through multivalent extra- and intramolecular interactions that are more prone to RNA gelation. In contrast, in DM1 mature skeletal muscles, CTG expansion size progressively increases while DMPK expression decreases and MBNL protein levels are relatively high.Recently, a four-phase model was proposed whereby RNA-RNA, RNA-RBP and RBP-RBP interactions must reach an assembly threshold . Based oDMPK CTGexp and CNBP CCTGexp mutations in DM1 and DM2, respectively, have resulted in key insights into the developmental regulation of RNA alternative splicing and polyadenylation in the nucleus as well as RNA localization in the cytoplasm [Studies designed to elucidate the downstream pathways altered by the ytoplasm ,119. Earytoplasm  while anytoplasm  and bothytoplasm ,176. Nev


Experiments involve through‐bond correlations of nucleobases and the phosphodiester backbone of RNA for chemical shift assignment and make information on hydrogen bonding uniquely accessible. Previously unobservable NMR resonances of amino groups in RNA nucleobases are now detected in experiments involving conformational exchange‐resistant double‐quantum 1H coherences, detected by 13C NMR spectroscopy. Furthermore, 13C and 15N chemical shifts provide valuable information on conformations. All the covered aspects point to the advantages of low‐γ nuclei detection experiments in RNA.Ribonucleic acid oligonucleotides (RNAs) play pivotal roles in cellular function (riboswitches), chemical biology applications (SELEX‐derived aptamers), cell biology and biomedical applications (transcriptomics). Furthermore, a growing number of RNA forms  but also RNA modifications are identified, showing the ever increasing functional diversity of RNAs. To describe and understand this functional diversity, structural studies of RNA are increasingly important. However, they are often more challenging than protein structural studies as RNAs are substantially more dynamic and their function is often linked to their structural transitions between alternative conformations. NMR is a prime technique to characterize these structural dynamics with atomic resolution. To extend the NMR size limitation and to characterize large RNAs and their complexes above 200 nucleotides, new NMR techniques have been developed. This Minireview reports on the development of NMR methods that utilize detection on low‐γ nuclei . The multitude of current approaches to achieve maximal signal‐to‐noise ratio have been recently summarized.Since the development of multidimensional NMR spectroscopy and the availability of isotope‐labeled RNAs, NMR spectroscopy has contributed more than 40 % of all RNA structures in databases. Considerable challenges, however, remain for the structure determination particularly of large RNAs and their complexes by biomolecular NMR spectroscopy. Often, the maximum concentration, at which RNA and RNA–protein complexes can be prepared for NMR studies, does not exceed 50 μ1H) have for long been the experimental gold standard in the field of biomolecular NMR spectroscopy. Since 1957,meta‐stable secondary structures controlling gene‐regulation during transcription1H‐isotope and its highest sensitivity of all NMR‐active and stable nuclei.Multidimensional NMR pulse sequences that rely on excitation and detection of the most sensitive nuclei protons . In combination with the traditional proton‐detected experiments, they will open new possibilities in the structural description of RNA dynamics and function at atomic resolution.However, also proton‐detected experiments exhibit difficulties for NMR studies of RNAs. Chemical‐shift dispersion is limited for all their resonances with the exception of imino protons. Further, the proton‐density within the nucleobases of RNA is low and many nucleobase‐protons are susceptible to conformational and solvent exchange. Thus, methods to circumvent these problems are needed and we here summarize recent developments in heteronuclear‐detected experiments  chemical exchange, and 4) relaxation—can be circumvented in heteronuclear‐detected NMR experiments. The disadvantage due to low‐γ detection can be minimized thanks to new cryogenic probes with inner NMR coils optimized for 13C‐, 19F‐, or 15N‐detection.13C nuclei in RNA have chemical shifts from δ=65 to 170 ppm. If the chemical shifts of the heteronuclei are detected during direct acquisition, high resolution due to long FID sampling can be achieved without lengthening the experiments, as relaxation delays can be shortened.All four difficulties—1) resonance overlap, 2) low 1H‐excitation or 1H‐detection. Information on quarternary carbon or nitrogen atoms then becomes feasible. Additionally, in heteronuclear‐detected experiments, the introduction of deuterium‐labeled nucleotides in large RNAs exploits their favorable relaxation properties in deuterium‐decoupled spectraThe heteroaromatic nucleobases represent a cyclized chain of C−N fragments. This particular feature can be exploited for the direct magnetization transfer in NMR experiments in (multiple) INEPT steps without being dependent on 13C or especially 15N can bring potential advantages to extend molecular size limitation as line widths increase slower with molecular size when compared to 1H. On a technical side, further advantages of heteronuclear‐detection are their insensitivity to certain experimental conditions including pH value, temperature or salt concentration and the non‐necessity of water suppression.Moving towards slower relaxing nuclei including 13C‐detected experiments. In particular, the sizeable homonuclear 1JCC couplings lead to splittings, as they are larger than the carbon line widths. However, decoupling schemes like IP/APThe loss in sensitivity due to the lower gyromagnetic ratio is fundamental and remains, however, the major disadvantage in heteronuclear‐detected NMR experiments. Further, in uniformly isotope‐labeled samples, homonuclear J couplings decrease the chemical shift resolution in 213C‐ or 15N‐detection are even better suited for recording heteronuclear‐detected experiments. As opposed to the so‐called inverse probes, which are used as standard probes in biomolecular NMR, they connect the channels of those heteronuclei to the inner coil of the probe enhancing the sensitivity for 13C‐ or 15N‐detection due to a larger filling factor. When working with fluorinated nucleotides, a two channel probe for 19F‐detection with simultaneous 1H‐decoupling is needed as JHF scalar couplings tend to be large.Due to the reduced sensitivity of low‐γ‐detected NMR experiments, probes with cryogenically cooled detection coils and preamplifiers, so called cryogenic probes, are needed as they increase sensitivity about a factor of 3–4 when compared to room temperature probes.13C‐ and/or 15N‐isotope labeled samples are indispensable.13C‐ and/or 15N‐labeled rNTPs yields milligram quantities of RNA and is well established. For the characterization of larger RNAs, however, selective labeling schemes or the incorporation of modified nucleotides are often necessary.In biomacromolecular NMR of RNA, T7 RNA polymerase during in vitro transcription allows incorporation of several modifications including rNTPs modified with fluorine or amino groups at the 2′‐position.Using mutants of the 13C‐pyrimidine,13C‐methoxy nucleoside,13C5′‐sugar labeled nucleoside15N‐imino/amido nucleosideT4 RNA ligase 1 that is able to incorporate nucleoside 3′,5′‐bisphosphates with modifications at the sugar‐, phosphate‐, or base‐site.T4 RNA ligase 2 in an ATP‐dependent reaction. With this method, shown in Figure Solid‐phase synthesis is one of the most commonly used methods for preparation of RNAs carrying a wide range of modifications.3The first reports utilizing carbon‐direct detection in protein NMR occured directly after the introduction of cryogenic probes.3.1endo or C3′‐endo) can be determined.Due to limited chemical shift resolution, complete assignment of protons in particular for ribose protons is sparse in the BMRB database.31P chemical shifts in the 5′‐ and 3′‐site CC‐TOCSY experiment is furthermore not only applicable to RNA but to any kind of ribose‐containing molecules as shown by Fontana et al. for carbohydrates.A sequential assignment from nucleotide to nucleotide can then be achieved in the (H)CPC‐experiment that correlates the C4′ resonance with the 3.213C direct‐detected experiments can also significantly contribute to complete resonance assignment of the nucleobases including their quarternary carbon or tertiary nitrogen atoms. There are two alternative approaches to assign these nuclei: a first suite of experiments exploits 1H‐excited and 13C‐detected experiments and the second suite exploits 13C‐excited and ‐detected experiments.J‐coupling constants are sufficiently large. For U, C, and G nucleobases, a near‐to‐complete resonance assignment can be achieved  couplings between C6N1, C2N1, C2N3, and C4N3 , C4, C5, and C6 atoms in purines are assigned ‐ and J‐coupling constants can be measured. 1J scalar couplings are determined, for example, in the H6C6C5‐experiment or in H5C5C4(C5)‐experiment =67 or 1J=55 Hz.With the chemical shift assignments of 3.41H‐detected experiments, because the imino proton reporter signals are broadened beyond detection by exchange with the hydrogen atoms from the solvent water ‐coupling on the proton exchange rate. If the proton is in slow exchange with solvent water, the 1J‐coupling can evolve under an unscaled coupling of ≈90 Hz. If the proton is, however, in fast exchange with solvent water, the scalar coupling is decoupled through scalar relaxation of the second kind.2O‐imino chemical exchange process. As a consequence, 1J is no longer observable. The spin filter has no effect for nucleotides with fast exchanging imino protons, whereas it inverts the 15N coherences for imino sites of nucleobases involved in stable interactions  coherences are excited in the indirect dimension. This magnetization is transferred to the neighboring carbon atom, where it is detected H‐HDQC experiment a C−H correlated spectrum is obtained, in which the 1H‐double quantum signals resonate at the mean proton chemical shift  values of 55 and 1J of 75 Hz. Virtual decoupling schemes coined IPAP sequences remove these couplings that cannot else be decoupled for example, by homodecoupling. Thus, either two experiments are recorded with optimized values that match the respective J‐coupling or one experiment with an IPAP filter tuned to the averaged coupling of 65 Hz is recorded. The C(N)H‐HDQC represents a 13C‐detected experiment that allows detection of NMR signals that completely evade observation in proton‐based experiments.Technical details require recording two independent experiments. During detection, the signal of the C6 in adenosines and the C4 in cytidines are doublets due to 13C‐direct detection in tailored experiments allows also observation of NOE cross peaks from amino groups. The “amino”‐NOESY experiment  can be extracted.Carbon‐chemical shifts of the ribose atoms are used for the determination of the ribose conformation in the RNA′s backbone. Based on an empirical calculation of the so‐called ‘canonical coordinates’ the ring pucker and the conformation of the exocyclic angle , terminal (Watson–Crick base pairing and one site π‐stacking), and disordered. All of the average chemical shifts of the atoms of the different nucleobases were referenced to the average chemical shift of the helical region and a clear trend can be observed for several carbon atoms in different structural elements Figure  A.35 Als415N‐direct‐detected multidimensional NMR experiments for RNA has only recently been introduced.15N‐detected HN‐correlation experiments were applied to RNAs of increasing molecular size. This study was motivated by the development of 15N‐detection TROSY experiments for the analysis of proteins.15N‐dimension, whereas the 1H‐dimension only covers less than δ≈2 ppm.15N‐resonances increases much slower with molecular size  than their proton counterparts. , several experiments have been developed that detect magnetization of either non‐native nuclei (19F) or on rather exotic nuclei.Besides the well‐established nuclei for heteronuclear‐detection schemes . It has previously been shown that fluorine modifications do not necessarily disturb the structure of the RNA, with the exception of fluorine labels at the 2′‐position.19F experiments is that in biomolecular samples no background signals arise. However, 19F labelling comes also with two major disadvantages. The large chemical shift anisotropy (CSA) renders detection of 19F in large RNAs difficult. Further, the incorporated nucleotides are only 19F but not 13C labeled, so that only correlation experiments with proton‐nuclei can be recorded by exploiting the heteronuclear overhauser effect. Recently, these problems have been overcome by the introduction of nucleotides containing a pair of 13C–19F labels in the heteroaromatic nucleobases that allow recording of 19F–13C TROSY spectra.Although naturally occurring RNA nucleotides do not contain +, Li+, and K+ are NMR‐active. NMR has extensively been conducted for DNA for which particular relaxation measurements have contributed towards understanding the nature of the interaction for Na+ and Li+ with double‐stranded DNA.23Na, 39 K, 87Rb, and 205Tl NMR spectroscopy.+ in the interaction with G‐quadruplexes in which different binding sites for the G‐quartets can be detectedAs a negatively charged biopolymer, interactions with cations are crucial for RNA/DNA folding and function.613C‐ and 15N‐nuclei will become even stronger at higher magnetic fields that are now on the horizon.The development of low‐γ‐detection schemes in NMR spectroscopy has been an active field over the last 15 years. Now, these possibilities are also exploited for NMR spectroscopy of RNA. Given the increasingly recognized biological relevance of RNA and the power of NMR spectroscopy to characterize its functional dynamics, the application of low‐γ‐detection schemes now allows forwarding NMR spectroscopy to larger RNA molecules. In order to reach this goal, development of advanced NMR methods runs hand‐in‐hand with improved methods in RNA sample preparation. The low‐γ‐detection schemes are further very compatible with solid‐state NMR experiments for RNA, as pioneered in the group of Carlomagno.The authors declare no conflict of interest.Robbin Schnieders, born in 1991, studied chemistry at the University of Frankfurt and finished studying in 2016 with her master's degree. Since then she has been working on her PhD in the group of Harald Schwalbe and is focused on the development of NMR spectroscopic methods for the characterization of RNAs.Sara Keyhani, born in 1988, studied chemistry at the Goethe University in Frankfurt. She finished her PhD thesis in the group of Harald Schwalbe and holds, since then, a postdoctoral position in this group. Her research focusses on the development of new methods for the synthesis of RNAs and their non‐natural analogues for advanced biophysical studies.Harald Schwalbe, born 1966, studied chemistry at the University of Frankfurt and obtained his PhD in 1993 with Christian Griesinger. He was postdoctoral fellow with Chris Dobson in Oxford from 1993–1995. After work on his habilitation till 1999, he became assistant professor at MIT from 1999–2001. In 2002, he accepted the offer to become full professor at Goethe University in Frankfurt. His research focussed on NMR spectroscopy of protein, RNA, and DNA molecules and their complexes. In particular, his group has made important contributions to NMR studies of RNA structure, dynamics, and folding.Boris Fürtig, born 1978, studied biochemistry at the University of Frankfurt and obtained his PhD in 2007 with Harald Schwalbe. He was postdoctoral fellow with Renée Schroeder at the MFPL in Vienna and returned 2011 to Frankfurt University. His work focusses on the NMR‐based characterization of RNA dynamics.


Protein recoding by RNA editing is required for normal health and evolutionary adaptation. However, de novo induction of RNA editing in response to environmental factors is an uncommon phenomenon. While APOBEC3A edits many mRNAs in monocytes and macrophages in response to hypoxia and interferons, the physiological significance of such editing is unclear.Here, we show that the related cytidine deaminase, APOBEC3G, induces site-specific C-to-U RNA editing in natural killer cells, lymphoma cell lines, and, to a lesser extent, CD8-positive T cells upon cellular crowding and hypoxia. In contrast to expectations from its anti-HIV-1 function, the highest expression of APOBEC3G is shown to be in cytotoxic lymphocytes. RNA-seq analysis of natural killer cells subjected to cellular crowding and hypoxia reveals widespread C-to-U mRNA editing that is enriched for genes involved in mRNA translation and ribosome function. APOBEC3G promotes Warburg-like metabolic remodeling in HuT78 T cells under similar conditions. Hypoxia-induced RNA editing by APOBEC3G can be mimicked by the inhibition of mitochondrial respiration and occurs independently of HIF-1α.APOBEC3G is an endogenous RNA editing enzyme in primary natural killer cells and lymphoma cell lines. This RNA editing is induced by cellular crowding and mitochondrial respiratory inhibition to promote adaptation to hypoxic stress.The online version of this article (10.1186/s13059-019-1651-1) contains supplementary material, which is available to authorized users. RNA editing is an evolutionarily conserved post-transcriptional modification that can result in amino acid recoding and altered protein function . ProteinThe more widespread ADAR-mediated A>I RNA editing, mostly occurs (~ 98%) in non-coding repetitive regions , likely In mammals, C>U RNA editing by cytidine deamination is infrequent in baseline transcript sequences under normal physiological conditions. An exception is APOBEC1-mediated RNA editing, which is mainly involved in the production of short isoform of the ApoB protein in intestinal cells . The relC nucleotides both in its ssDNA and RNA substrates, whereas other A3 enzymes prefer TC nucleotides  caused a significant reduction in A3G mRNA and protein expression and were used for further studies.A3G knockdown in Hut78 cells was performed at the RPCCC gene modulation shared resource. For A3G knockdown, GIPZ human A3G shRNAs with the following clone ID’s were used: V2LHS_80856, V2LHS_80785, V2LHS_80786 (KD1a and KD1b), V3LHS_400156 (KD2), and V3LHS_303306 (Dharmacon). Lentiviruses were produced by cotransfection of 293T cells with A3G shRNA (or pGIPZ non-silencing control) along with psPAX2 and pMD2.G packaging plasmids, using the LipoD293 reagent (1:2.5 DNA to lipoD293 ratio) (SignaGen Laboratories) as per the manufacturer’s instructions. Culture supernatants were collected 48 and 72 h after transfection and cleared by filtration through 0.45 μm cellulose acetate syringe filter. For shRNA expression, 1 × 10Total RNA was isolated and reverse transcribed to generate cDNAs as described earlier . DNA priA3G, A3F, and A3C gene expression, reactions using LightCycler™ 480 Probes Master and SYBR™ Green I dye were performed on a LightCycler™ 480 System (Roche). Quantification cycle (Cq) values were calculated by the instrument software using the maximum second derivative method, and the mean Cq value of duplicate PCR reactions was used for analysis.For quantitative PCR to assess Whole cell lysates were prepared and immunoblot was performed as described previously , 34. APO6 cells in 100 μl per well) were seeded in 96-well round-bottom plates and incubated covered in the culture medium for 22 h in a 37 °C humidified hypoxia chamber (1% O2) or 37 °C humidified culture chamber (21% O2). Cellular dehydrogenase activity was determined using a WST-8 viability stain-based colorimetric assay . Plates were read at 450 nm on an Epoch2 microplate reader (Biotek) using the Gen5 software (Biotek). Proliferation in KD2 and its control Hut78 cell line is measured by [3H]-thymidine (1 μCi per well) incorporation for 18 h with T cells in 96-well plate. After the initial cultures with cellular crowding (1 million cells per 0.1 ml volume in 96-well plate), 100,000 viable cells per well are used for the incorporation assay. Results are expressed as net counts per minute (cpm).Control and KD1a HuT78 cells . These ratios were then normalized to the corresponding CTRL and KD T0 (unstressed cells) ratios within each experimental group, which are set to 1 . A3G expression levels and mean editing levels in different cell types Fig.  were firo 1 Fig. . The comhttp://weblogo.berkeley.edu/ with default parameters [Gene expression meta-analysis of A3G is performed on two online platforms: (1) BIOGPS , searcharameters .Additional file 1:Figure S1-S12. Supplementary figures. (PPTX 1499 kb)Additional file 2:Table S1. C>U RNA editing events after the first filter (FDR < 0.05). These events were present at least 5% frequency in any sample and overrepresented in the hypoxia or normoxia group. (XLSX 286 kb)Additional file 3:Table S2. C>U RNA editing events after the second filter (− 1 C or T). These events have C or T at − 1 nucleotide position. (XLSX 222 kb)Additional file 4:Table S3. C>U RNA editing events after the third filter (stem-loop). These events are located at the 3′-end of loops in putative stem-loop structures within exons or UTRs. (XLSX 59 kb)Additional file 5:Table S4. C>U RNA editing events shared between NK cells and 293T/APOBEC3G overexpression system. (XLSX 22 kb)Additional file 6:Table S5. C>U RNA editing events shared between NK cells and 293T/APOBEC3A, and NK cells, 293T/APOBEC3A and 293T/APOBEC3G overexpression systems. (XLSX 14 kb)Additional file 7:Table S6. A>I RNA editing events in RADAR database that are induced by hypoxia in NK cells. (XLSX 11 kb)Additional file 8:Table S7. Evolutionary conservation analysis of all non-synonymous C>U RNA editing sites. (XLSX 18 kb)Additional file 9:Table S8. Gene expression levels in normoxic and hypoxic NK cells. (XLSX 3002 kb)Additional file 10:Table S9. Oligonucleotide primer sequences used for PCR amplification and Sanger sequencing. (XLSX 10 kb)


RNA flow between organisms has been documented within and among different kingdoms of life. Recently, we demonstrated horizontal RNA transfer between honeybees involving secretion and ingestion of worker and royal jellies. However, how the jelly facilitates transfer of RNA is still unknown. Here, we show that worker and royal jellies harbor robust RNA-binding activity. We report that a highly abundant jelly component, major royal jelly protein 3 (MRJP-3), acts as an extracellular non-sequence-specific RNA-aggregating factor. Multivalent RNA binding stimulates higher-order assembly of MRJP-3 into extracellular ribonucleoprotein granules that protect RNA from degradation and enhance RNA bioavailability. These findings reveal that honeybees have evolved a secreted dietary RNA-binding factor to concentrate, stabilize, and share RNA among individuals. Our work identifies high-order ribonucleoprotein assemblies with functions outside cells and organisms.  •Worker and royal jellies harbor robust RNA-binding activity•Major royal jelly protein 3 (MRJP-3) is the dietary RNA-binding factor•RNA stimulates higher-order assembly of MRJP-3 into extracellular RNP granules•MRJP-3 granules concentrate, stabilize, and enhance environmental RNA bioavailability Honeybees share RNA between individuals through secretion and ingestion of worker and royal jellies. Maori et al. identify a jelly factor, MRJP-3, that forms extracellular ribonucleoprotein granules that concentrate, protect, and enhance RNA uptake. They propose that MRJP-3 facilitates RNA transfer that drives social immunity and signaling among bees. Distinct biochemical activities are often found in specialized subcellular compartments. These compartments are demarcated either by a membrane barrier or through the process of phase transition, which drives macromolecular condensation and formation of membrane-less organelles . MultivaProtein-coding and non-coding RNA can spread among cells and tissues of an organism. Such mobile RNA has been documented in plants and animals . FurtherApis mellifera) plays a key role in agriculture, pollinating a large number of crops that feed humans and farm animals. In recent years, elevated honeybee losses have become a major global concern. Bee colony losses have been linked to various biotic stressors, including the mite Varroa destructor, Israeli acute paralysis virus (IAPV), and other viruses (The honeybee ( viruses . Previou viruses . Field t viruses . Followi viruses . On the Apis mellifera genome (MRJP-1 to MRJP-9), but their physiological functions are not well understood. MRJP-3 is a polymorphic protein that represents 10%–15% of the total RJ protein content  aqueous solution of proteins, sugars, lipids, vitamins, salts, free amino acids, and RNA . Major r content . The pol content . MRJP-3  content , and pur in vivo .Here we show that MRJP-3, an abundant jelly ingredient, is a secreted non-sequence-specific RNA-binding protein and that multivalent RNA binding mediates the transition of MRJP-3 into extracellular RNP (eRNP) granules that concentrate, stabilize, and enhance environmental RNA uptake.∗). On day 5, queens were removed to initiate queen rearing and RJ secretion  and screened the unbound and eluted fractions for dsRNA-binding activity by EMSA see . By usinees only A.Figure Kd of 3.5 μM (Purified MRJP-3 bound to both dsRNA and single-stranded RNA (ssRNA) A. As obsf 3.5 μM E. Finallf 3.5 μM F. We theKd values of 4 nM, 300 nM, and 25 μM for ssRNA. We observed two distinct binding events for dsRNA and identified Kd values of 11 nM and 150 nM. The tight interactions at low protein concentrations suggest that the MRJP-3 monomer has high affinity to both unstructured and duplexed RNA. The defined Kd values also indicate that MRJP-3 binds ssRNA and dsRNA in different self-associated states: before, during, and after the completion of oligomerization. Because the Kd for self-association was 3.5 μM and the MRJP-3 concentration in RJ is ∼26 times more than the highest Kd value measured with RNA, RNA is expected to be bound to the fully self-associated MRJP-3 oligomers in native conditions.MRJP-3 binds ssRNA and dsRNA of different length and sequence, indicating a non-sequence-specific mode of binding A. HoweveQ17060; aa 424–523) has a pI of 10.10, implying that this region is positively charged in the acidic RJ environment (pH 3.5–4.5). To test if the positively charged TRR plays a role in RNA binding, we produced recombinant MRJP-3 that lacks the TRR and demonstrated, using EMSA, that the prion-like TRR is required for the RNA-binding activity  and RNA   B. ssRNA∗nditions C. When sApis mellifera) and exogenous  RJ RNA  RJ RNA D. MRJP-3) RJ RNA D. We obs) RJ RNA D. MRJP-3) RJ RNA .We previously demonstrated that bees transmit biologically active dsRNA, and diverse complementary viral RNA fragments occur in worker and royal jellies . ConsistEnvironmental RNA persistence requires RNA stabilization. MRJP-3 RNPs could potentially function to protect RJ RNA from factors such as nucleases. To test this, we mixed ssRNA with MRJP-3 or MRJP-1 and then introduced RNase-A. MRJP-3-bound RNA is protected from RNase-A degradation, whereas MRJP-1, which lacks the TRR domain, neither bound RNA nor protected it from degradation A. This eC. elegans nematode system. C. elegans is susceptible to environmental RNAi through ingestion  phenotype , dsRNA-unc-22 alone, dsRNA-unc-22 mixed with MRJP-1, or MRJP-3 bound to a control dsRNA (dsRNA-Fluc)  C. AnimalC. elegans, we designed an imaging experiment to detect dsRNA uptake shortly after ingestion. Animals were soaked for 2 h with labeled dsRNA alone, and in the presence of MRJP-3 or MRJP-1, followed by fixation and imaging  and Eric Miska (eam29@cam.ac.uk).Further information and requests for resources and reagents should be directed to the lead contacts, Eyal Maori . Next, the hives were transferred into two net-houses separating between dsRNA treated, and untreated hives. The bees were free to fly within the net-houses and to forage for water from buckets. The first 14 days were an adaptation period, during which the colonies were fed on demand with candy, and pollen supplement patties (10 g each) which were placed on top of the combs and replaced once a week. An established colony was determined by at least two constructed combs and egg-laying activity of the queen; only these hives were included in the experiment. During the experiments, established colonies (two per treatment) were fed on pollen supplement patties (10 g each), and had an unlimited water supply.C. elegans Bristol N2 strain was grown and maintained as previously described  agar plates and transferred using a platinum wire under a dissecting microscope (Leica M50). Alternatively, pieces of one plate were chunked and placed facing down on a new plate.escribed . The nem∗, 4 ng/μl final concentration) were provided on days: 1, 2, 4 and 5 (two hives per treatment). On day-5, queens were removed to initiate queen rearing and RJ secretion. On day-9, 3rd-4th instar larvae were carefully removed from queen brood cells with a fine paintbrush, and the larvae were checked to be intact without any physical damage. RJ was harvested from such queen brood cells and samples from each hive were pooled and stored at −80°C.50 mL 10% (v/w) sucrose solutions with or without Alexa Fluor-488 labeled dsRNA . 10μM sections were cut by a cryostat , put on slides and left at room temperature to dry. Slides were fixed for 15 min in 4% PFA (in PBS) and washed twice for 5min with 1xPBS. Immunostaining was performed by the Dako Autostainer Link 48 with the Envision Flex kit system (Dako) according to the manufacturer’s instructions using 1:250 diluted Alexa Fluor-488 antibody . More specifically, sections were incubated for 10 min with peroxidase-blocking reagent, 60 min with 1:250 diluted primary polyclonal rabbit anti-Alexa Fluor-488, 30 min with the EnVision FLEX/HRP Detection Reagent, 5 min with EnVision FLEX DAB+ Chromogen/EnVision FLEX Substrate Buffer mix, and 5 min with EnVision FLEX Hematoxylin. The slides were then dehydrated  followed by 2 times 5 min wash in xylene and then mounted.rd-5th instar queen larvae. Worker jelly was collected from brood cells with 4th-5th instar worker larvae by washing cells with nuclease-free water to resuspend the low jelly quantity available. Prior to jelly harvest, larvae were carefully removed and checked for any physical damage. Commercial RJ was sourced from Well Bee-ing UK and was used for MRJP-3 isolation.Royal and worker jellies, which were directly applied in experiments (raw or soluble fraction), were produced in collaboration with Springwell Apiaries, UK. RJ was harvested from brood cells containing 3in vitro transcription using Megascript kit (Ambion) according to the manufacturer’s instructions including DNase-I treatment. Transcription DNA templates, carrying a single (for ssRNA synthesis) or double opposite T7 promoters (for dsRNA synthesis), were generated by PCR or gene synthesis. HPLC-purified RNA oligos (up to 50 nt) were ordered from Integrated DNA Technologies (IDT). List of nucleic acid sequences, their corresponding NCBI accession number and source is shared in dsRNA and ssRNA that are longer than 50 nt were synthesized by in vitro transcription reaction using Alexa Fluor-488 labeled UTP (Thermo Scientific). 2 μl of 1mM labeled UTP was added to a standard 20 μl in vitro transcription reaction of the Megascript kit (Ambion). Ulysis kit (Thermo Scientific) was used according to the manufacturer’s instructions to label RNA oligos (50 nt and shorter) with Alexa Fluor-488 as well as the Alexa Fluor-647 labeled dsRNA.Long ssRNA/dsRNA (> 50 nt) was labeled by Due to the large size of RNA-MRJP-3 complexes, non-denaturing agarose gel was used for the EMSA assays. Prior to the electrophoresis, RNA and protein samples  were mixed and incubated at room temperature for 0.5-3 hours, and then mixed with 1x loading buffer . Samples were loaded in Ethidium Bromide containing 0.8% non-denaturing agarose gel and run in 1xTAE buffer for 45 min in 150 mA.cf. for 10 min at room temperature and the aqueous fraction was collected. The centrifugation and aqueous fraction separation were performed three additional times until a pure soluble fraction was extracted.Soluble RJ fraction was prepared by diluting raw RJ with “RJ buffer” (v/v) that was formulated based on RJ ash content : 77 mM K1 mL of 25% (v/v) RJ was split into 2 equal aliquots. RNA was purified separately from each aliquot by phenol/chloroform/isoamyl alcohol extraction and pooled together.Two-step primer-specific RT-PCR was performed by following standard SuperScript-III (invitrogen) and KAPA HiFi Hotstart ready mix PCR (Kapa Biosystems) protocols. Reverse transcription was performed by applying 0.5 ng total RJ RNA template, two dsRNA-specific primers  was predicted by PONDR VSL2 (http://www.pondr.com/) and IUPred (http://iupred.enzim.hu/), which apply different approaches for disorder prediction (Intrinsic disorder in MRJP-3 (UniProt ID ediction . The defQ17060) was blasted against the available non-redundant protein sequence database (nr). The outcome was analyzed for the presence of the yellow-related protein and TRR (amino acids 1-419 and 424-523 respectively). Next, the NCBI taxonomy database was applied to generate a common tree , centrifuged at 16,000 rMRJP-3 was purified using cation exchange FPLC. The RJ sample was loaded onto a Source 15S column , equilibrated with MES binding buffer (2 mL per min) followed by a 6-column volume wash with MES binding buffer. Elution was performed with a linear 38 column volume salt gradient, from 25 mM MES pH 6.0 / 0.15M NaCl (Buffer-A) to 25 mM MES pH 6.0 / 1.0 M NaCl (Buffer-B). The cation exchange included overall 10 unbound and 58 eluted fractions. 20 fractions (approximately 5 mL per fraction) were collected over the first third of the elution gradient and their protein concentration was measured by Nanodrop spectrophotometer. MRJP-3 eluted over the first 25% gradient. Fractions containing the protein peak were run on 4%–12% gradient SDS-PAGE gel and those with the most concentrated and pure MRJP-3 were pooled and dialyzed into CHAP binding buffer (5 mM phosphate pH6.8 / 0.15 M NaCl). MRJP-3 was further purified on a Ceramic Hydroxyaptatite (CHAP) column . MRJP-3 sample was passed through the column (2 mL per min) followed by washing with 6 column volumes of CHAP buffer. Elution was performed with a linear 25 column volume gradient, from 5 mM phosphate pH 6.8 / 0.15 M NaCl (Buffer-C) to 500 mM phosphate pH 6.8 / 0.15 M NaCl (Buffer-D). The hydroxyaptatite chromatography included overall 12 unbound and 70 eluted fractions. MRJP-3 eluted over the first 30% gradient and 20 fractions (approximately 3 mL per fraction) were collected. Fractions with the most concentrated and pure MRJP-3  were pooled and dialysed into RJ Buffer. MRJP-3 concentration was determined by absorbance at 280 nm value using a 1 mg/ml/cm extinction coefficient of 0.8.0c and purified protein identities were validated by a MALDI/MS peptide mass fingerprinting, performed by the Cambridge Centre for Proteomics.Starting material was unbound protein in the run-through from the Source 15S column (in MES binding buffer) under the conditions for purifying MRJP-3. MRJP-1 was purified using anion exchange FPLC performed on Source 15Q Tricorn 10/100 column . The unbound protein sample was passed through the column (2 mL per min) followed by washing with 6 column volumes of MES buffer. Elution was performed with a linear 35-column volume gradient, from Buffer-A to Buffer-B. The anion exchange included overall 12 unbound and 54 eluted fractions. 30 fractions (approximately 5 mL per fraction) were collected over the first half of the elution gradient. Fractions containing the protein peak were run on 4%–12% gradient SDS-PAGE gel and those with the most concentrated and pure MRJP-1 were pooled and dialyzed into CHAP binding buffer. MRJP-1 was further purified on a CHAP column . The MRJP-1 sample was passed through the column (2 mL per min) followed by washing with 6 column volumes of CHAP buffer. Elution was performed with a linear 25-column volume gradient, from Buffer-C to Buffer-D. The hydroxyaptatite chromatography included overall 9 unbound and 53 eluted fractions. 20 fractions (approximately 4 mL per fraction) were collected over the first 40% of the elution gradient and MRJP-1 eluted over the first 30% gradient. Fractions with the most concentrated and pure MRJP-1  were pooled and dialysed into RJ Buffer. MRJP-1 concentration was determined by absorbance at 280 nm value using a 1 mg/ml/cm extinction coefficient of 1.2. MRJP-1 and MRJP-3 aliquots were stored in −80 Recombinant MRJP-3 expression was serviced from the Israel Structural Proteomics Centre, Weizmann Institute. Full-length MRJP-3 (1-544) and truncated MRJP-3 (1-424) were cloned into baculovirus expression vector pVL1393. T7-epitope and three amino-acid (aa) linker (SAG) followed by 6xHis were introduced into the gene following the authentic secretion signal (aa 1-20). Each expression vector construct was co-transfected with the ProGreen green fluorescent protein (GFP) linearized baculovirus DNA (AB vector) into Sf9 insect cells. Viruses were produced for each construct, and were used to infect Sf9 cells for protein expression. Infection efficiency was monitored by GFP fluorescence of infected cells. Cells were grown in ESF921 protein-free culture medium (Expression Systems). Three days post-infection, medium containing the secreted protein was collected concentrated and dialyzed against 50 mM Tris 8.0 / 100 mM NaCl. Protein was purified on HisTrap_FF_crude_5 mL column  followed by desalting column to exchange the buffer of the eluted protein. The protein was further purified by anion exchange at pH 8.0 on Tricorn Q 10/100 GL column  and eluted at 75 mM salt. The fractions containing the eluted protein were dialyzed against RJ buffer.The binding affinities were measured using the Monolith NT.115 . Both single- and double-stranded 22 nt RNA were fluorescently labeled with Alexa Fluor-488 using the Ulysis kit (Thermo Scientific) according to the manufacturer’s protocol. Labeling efficiency was determined to be 1:1 (RNA to dye) by measuring the absorbance at 260 and 488 nm. A 16 step dilution series of the unlabeled MRJP-3 was prepared and mixed with the labeled RNA at 1:1 ratio and loaded into capillaries. Measurements were performed at 25°C in RJ buffer containing 0.01% Tween 20. Data analyses were performed using Nanotemper Analysis software, v.1.2.101 and were plotted using Origin 7.0. All measurements were conducted as triplicates and the errors were presented as the standard error of the triplicates.MRJP-3 was concentrated to 100 μM and the concentrated MRJP-3 was injected onto a 16/100 Superdex 200 size exclusion column . Protein was eluted at 0.5 ml/min in RJ buffer. The oligomer formation was judged by the appearance of a peak with an earlier retention time. The size exclusion column was calibrated with Thyroglobulin (669 kDa), Ferritin (440 kDa), Aldolase (158 kDa), Conalbumin (75 kDa), Ovalbumin (43 kDa), Carbonic anhydrase (29 kDa), Ribonuclease (137 kDa) and Aprotinin (6.5 kDa). Data analyses were performed using Unicorn 7.0 and were plotted using Origin 7.0.C. elegans images were acquired using a UPlanSApo 20x objective with 2x magnification. Imaging settings (laser power and exposure) were set so that negative control images did not show signal. The same microscope settings were then used for all treatments. Sample mounting was not applied prior to imaging.Confocal imaging was performed using an inverted Olympus FV1000 microscope equipped with the FLUOVIEW 4.2 software. Nucleoprotein images were acquired using a 60 × UPlanSApo/1.35 oil objective with 1-2x magnification. Super-resolution SIM (structured illumination microscopy) images were acquired using a Deltavision OMX 3D-SIM System V3 BLAZE from Applied Precision  equipped with 3 sCMOS cameras, 405, 488, 592.5 nm diode laser illumination, an Olympus Plan Apo N 60x 1.42NA oil objective, and standard excitation and emission filter sets. Imaging of each channel was done sequentially using three angles and five phase shifts of the illumination pattern. The refractive index of the immersion oil (Cargille) was adjusted to 1.513 to minimize spherical aberrations. Sections were acquired at 0.125 μm z steps. Raw OMX data were reconstructed in SoftWoRx software version 6.5.2 . Reconstructions were carried out using channel specific Optical Transfer Functions (OTFs) and a Wiener filter of 0.002. OTFs were generated within the SoftWoRx software by imaging 100 nm beads (Life Technologies) using appropriate immersion oils to match the data. For figures, reconstructed data were further processed in FIJI software (open source) to remove negative values created by the reconstruction algorithm. Sample mounting was not applied prior to imaging.4-Biotin  was used to biotinylate the proteins as follows; 170 μl nuclease-free water was added to the EZ-Link NHS-PEG4-Biotin (‘biotin’) powder aliquot and mixed gently. 3 μl of biotin solution was added per 500 μl MRPP-3 or BSA solution, mixed well and incubated at room temperature for 30 min. Biotinylated proteins were then dialyzed in 1xPBS with Slide-A-lyzer MINI dialysis units 7 KDa (Thermo Scientific) for 2 hours followed by buffer change and additional dialysis for 4 hours.11.8 nmol MRJP-3 and BSA were dialysed in 1xPBS. EZ-Link NHS-PEG0c. Next, the RJ pH was adjusted to 5.0-5.5 by adding 0.23 mL of 500 mM Tris pH 8.8. Each RJ-biotinylated protein mixture was then split into two tubes containing 0.4 mL pre-washed Pierce Streptavidin magnetic beads  and rotated at room temperature for 3 hours. Beads were then placed in magnetic stands for 10 min followed by removal of the RJ solutions. Next, beads were washed with 0.5 mL 1xPBS for 10 min at room temperature. After three wash steps, 0.35 mL 1xPBS was added to each beads tube, followed by Phenol/Chloroform/Isoamyl alcohol RNA extraction.1 mL raw RJ was diluted with 1.5 mL nuclease-free water and split into two aliquots (1.25 mL per tube). 500 μl of biotinylated protein (MRJP-3 or BSA) was added to each RJ aliquot and rotated overnight at 4 Animals were grown until young adult stage and washed twice in M9 medium. The animals were then transferred into 10 μl treatment solutions placed on a paraffin film, which was sealed within an empty NGM plate and incubated for 2 hours at room temperature. Next, the animals were individually picked and washed three times in M9 medium before fixation in 4% formaldehyde with PBS for 30 min. The animals were then mounted in 1xPBS for imaging.unc-22 (ZK617.1) expressing RNAi bacterial feeding clones were kindly received from J. Ahringer’s laboratory, Cambridge University. Bacteria were grown in LB- Ampicillin for 16 hours. The dsRNA-unc-22 expressing bacteria (positive control for unc-22 RNAi phenotype) were seeded onto 50 mm NGM agar plates containing 1 mM IPTG and 25 g/ml Carbenicillin at a volume of 50 μl bacterial culture per plate and left to dry for 48 hours. Empty vector bacteria were seeded similarly, but in the absence of IPTG. For unc-22 dsRNA uptake assays, on day-1, 100 μl of treatment solutions were placed on the center of the empty vector bacterial loan and left to dry for 10 min. Next, 50 L1s synchronized by starvation arrest were spotted onto each plate in a drop of M9. On day-2, second 100 μl of treatment solutions were applied. On day-4, twitcher phenotype was scored at the adult stage. Each treatment had three biological repeats. 5.4 pmol (0.05 μM) dsRNA and 4 nmol (39.7 μM) MRJP-3 or 5 nmol (49.9 μM) MRJP-1 were applied in all dsRNA- and protein-containing treatments in RJ buffer.HT115 empty vector and dsRNA-cf. and passed through a 0.22 μM filter. Jellies proteins were identified by LC-MS/MS applied directly on the RJ and WJ solutions (serviced from Cambridge Centre for Proteomics)10% soluble RJ and WJ were dialyzed overnight in 1xPBS buffer, centrifuged at 16 K rTotal RJ RNA or pulled-down MRJP-3 bound RNA was first subjected to Tobacco Acid Pyrophosphatase  and Polynucleotide kinase  treatments according to the manufacturers’ instructions. Total stranded RNA library preparations were performed using the TrueSeq stranded total RNA sample preparation kit (Illumina) according to manufacturer’s instructions omitting the rRNA removal and fragmentation steps since input RNA was of low molecular weight and did not contain obvious rRNA contaminants. In brief, total RJ RNA (ca. 20 ng) or 25% of MRJP-3-bound purified RNA was diluted to 8.5 μl total volume in Elution buffer. After addition of 8.5 μl Elute/Prime/Fragment High Mix (containing the Reverse Transcription primers), RNA was denatured at 65°C for 5 minutes followed by rapid cooling on ice. All subsequent steps were performed following the manufacturer’s protocol with the following modification: PCR amplification of the MRJP-3 bound input cDNA samples was by using 20 cycles in total. All libraries were quantified by standard dsDNA High Sensitivity Qubit assay (Invitrogen) and sizing of libraries was controlled by running 1 μl sample each on a D1000 screen tape using a Tapestation 2200 system (Agilent). Sequencing was performed by a Hiseq 1500  instrument using a 100 bp paired-end read run.Apis mellifera (GCA_000002195.1) and known honeybee viruses using STAR v2.5.2b (NC_009025.1), Acute Bee Paralysis Virus , Kashmir Bee Virus , Deformed Wing Virus , Varroa Destructor Virus 1 , Black Queen Cell Virus , Sacbrood Virus , Chronic Paralysis Virus , Chronic Paralysis Virus , Bee Macula-like virus , Slow Bee Paralysis Virus , Lake sinai virus strain-1  and Lake sinai virus strain-2 . Gene expression was quantified using featureCounts v1.5.0-p2 from Subread  and two MRJP-3 bound RJ RNA libraries . RNA-seq reads were adaptor trimmed using cutadapt v1.11 to a minimum length of 10 nt . Trimmed v2.5.2b  with par Subread  on gene Varroa Destructor Virus-1 was computed strand-specifically using bedtools genomecov 2.27.1 (10(x+1) on counts x (and flipping the axis for the antisense strand).RNA-seq coverage of the v 2.27.1 . CoveragFor GO analysis, genes were considered as expressed if they had a read count after RNA-seq mapping and quantification of at least 2. We considered only GO terms with at least 5 annotated genes; GO terms were downloaded from Ensembl Metazoa Biomart. A null distribution was calculated from expressed genes using the gene lengths (as the mean length of a gene’s transcripts) as a bias term (via goseq 1.28 ). GO terA. mellifera genome were re-mapped against the “hairpin.fa” pre-miRNA reference from miRBase, release 21 as single-end reads, using STAR with parameters --outFilterMismatchNoverLmax 0.15 --outFilterMismatchNmax 1 --alignIntronMax 1 --scoreDelOpen -10000 --scoreInsOpen -10000 --outFilterMultimapNmax 100.To discover unannotated microRNAs, reads that mapped to the Apis mellifera genome were analyzed for the occurrence of putative honeybee dsRNA. Long RNA-seq data were split into first and last read of each fragment. Subsequently, we tested for pairwise overlaps of reads on the forward and reverse strand via bedtools intersect 2.27.1 . We furhttps://github.com/klmr/royal-jelly (https://doi.org/10.5281/zenodo.1542860). Other Software used in this work are all publicly available, with the links to them in the above tables. The raw imaging data, including images of gels and blots, have been deposited in Mendeley Data and can be accessed: https://doi.org/10.17632/5w7rbd8452.1. All the rest of the data are available in the manuscript or the supplementary materials.The RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI under accession number E-MTAB-6732. All in-house scripts have been deposited in Github and can be downloaded: