Creating three-dimensional (3D) representations of the world from two-dimensional retinal images is fundamental to visually guided behaviors including reaching and grasping. A critical component of this process is determining the 3D orientation of objects. Previous studies have shown that neurons in the caudal intraparietal area (CIP) of the macaque monkey represent 3D planar surface orientation . Here we compare the responses of neurons in areas V3A  and CIP to 3D-oriented planar surfaces. We then examine whether activity in these areas correlates with perception during a fine slant discrimination task in which the monkeys report if the top of a surface is slanted toward or away from them. Although we find that V3A and CIP neurons show similar sensitivity to planar surface orientation, significant choice-related activity during the slant discrimination task is rare in V3A but prominent in CIP. These results implicate both V3A and CIP in the representation of 3D surface orientation, and suggest a functional dissociation between the areas based on slant-related choice signals. Surface orientation perception is fundamental to visually guided behaviors such as reaching, grasping, and navigation. Previous studies implicate the caudal intraparietal area (CIP) in the representation of 3D surface orientation. Here we show that responses to 3D-oriented planar surfaces are similar in CIP and V3A, which precedes CIP in the cortical hierarchy. However, we also find a qualitative distinction between the two areas: only CIP neurons show robust choice-related activity during a fine visual orientation discrimination task.Perception of three-dimensional (3D) surface orientation is essential for many visually guided behaviors. Electrophysiological studies have identified 3D orientation-selective neurons in multiple brain regions of nonhuman primates . In partHere we measured the responses of V3A and CIP neurons to 3D surface orientation, as well as their functional correlations with behavior during a fine slant discrimination task. First, 3D surface orientation tuning was measured during a fixation task. The two areas were found to contain similar proportions of selective neurons, as well as similar degrees of selectivity. Second, neuronal activity was recorded while the monkeys viewed planar surfaces at different slants and reported the slant direction in a two-alternative forced-choice task. Receiver operating characteristic (ROC) analysis was used to quantify neuronal sensitivity and to assess choice-related activity . In contMacaca mulatta), denoted as monkeys N, P, and Z, weighing 4-5 kg at the start of the study. As previously described, the monkeys were chronically implanted with a lightweight plastic ring for head restraint, a recording grid, and scleral eye coils for monitoring binocular eye movements , neuronal threshold , surface orientation discrimination index , squared choice partial correlation , and squared slant partial correlation . A lack of effects of the labyrinthectomy on visual discrimination is not surprising given that the monkeys were head-fixed during the experiments and that previous studies found that visual heading discrimination performance is largely normal within days following a bilateral labyrinthectomy  were inserted into the cortex through a transdural guide tube using a hydraulic microdrive to record extracellular action potentials. Neuronal voltage signals were amplified, filtered (1 Hz to 10 kHz), and displayed on an oscilloscope to isolate single units using a window discriminator (BAK Electronics). Raw voltage signals were digitized at a rate of 25 kHz using a Power 1401 data acquisition system (Cambridge Electronic Design), and single units were sorted off-line as needed . In some experiments, action potentials were displayed and isolated using the SortClient software (Plexon).r = 0.621, p = 0.002), and the linear fit y = 0.47x + 1.8 was similar to previous measurements, y = 0.33x + 1.78 (y = 0.38x + 2.8 (p = 0.02).The CARET software was used to segment visual areas in MRI scans of monkeys N and P . Two MRIx + 1.78  and y = 8x + 2.8 , as obta8x + 2.8 . We compBehavioral control was conducted with custom Spike2 scripts. The monkeys sat in a primate chair ∼32.5 cm from a liquid crystal display (LCD) on which stimuli were displayed . An aperture constructed from a black nonreflective material was affixed to the screen such that the monkey could only see stimuli within a 30-cm-diameter (System 1) or 18-cm-diameter (System 2) circular aperture. The same material extended between the LCD and the monkey, occluding the view of the surrounding room. The OpenGL graphics library was used to program visual stimuli that were generated using an OpenGL accelerator board . The fixation point (yellow in color) was presented directly in front of the monkey at eye level and screen distance. Fixation was enforced using 2° vergence and 1° version windows. Due to eye coil failures in monkey P, the binocular eye movements of this animal were monitored in all experiments using an infrared optical eye tracker (ISCAN).p = 0.44); neuronal threshold ; behavioral threshold ; point of subjective equality ; squared choice partial correlation ; and squared slant partial correlation .Surface orientation tuning was measured as previously described ,b. BriefSlant was varied between 0° and 60° in 20° steps, and tilt was varied between 0° and 315° in 45° steps. All stimuli were centered on the fixation point and covered the same retinotopic area. Stereoscopic cues were created by rendering the stimuli as red-green anaglyphs. Each trial began with the monkey fixating a point on a blank screen for 300 ms. Fixation was maintained while a checkerboard stimulus was presented for 1000 ms, followed by 50 ms of fixation with a blank screen. There was a 1000 ms blank screen intertrial interval. Stimuli were presented in pseudo-random order. Surface orientation selectivity was assessed for all cells held for at least three repetitions of each stimulus. At most, seven repetitions of each stimulus were recorded. For each selective neuron (see Results), a one-way ANOVA was performed to determine whether there was significant slant tuning along the 90°/270° tilt axis A,B. NeurThe slant discrimination task was always performed along the 90°/270° tilt axis. To simplify the description of surface orientation, we do not refer to tilt for the slant discrimination task but instead denote planes with a tilt of 90° (top of the plane closer to the monkey) as having a negative slant, and planes with a tilt of 270° (top of the plane further from the monkey) as having a positive slant . As illuDuring pilot work, we observed that local orientation cues in checkerboard stimuli could be used to perform the task without having to judge slant. To avoid this potential confound, the discrimination task was performed using RDS planes with uniform dot density on the screen . In CIP,Slant was varied between ±20° with the intermediate slants tailored to each monkey’s performance. For monkeys N and Z, slants of ±20°, 10°, 5°, 2.5°, 1.25°, and 0° were used. For monkey P, slants of ±20°, 9°, 4.05°, 1.83°, 0.82°, and 0° were used. Neurons were recorded while the monkey performed the task for a minimum of 10 repetitions of each stimulus. Sufficient repetitions were recorded for 65 CIP and 23 V3A neurons.Rmax and Rmin are the maximum and minimum responses, respectively. SSE denotes the sum squared error around the mean responses, N is the total number of trials, and M is the number of tested slant–tilt combinations (M = 25). Neurons with strong response modulation relative to their variability have SODI values closer to 1, whereas neurons with weak response modulation have SODI values closer to 0.Analyses were performed in MATLAB (MathWorks). Unless otherwise noted, analyses were performed on firing rates or spike counts computed during the 1000 ms stimulus presentation period. The tuning strength of each neuron was evaluated using a SODI, motivated by previous studies , that waBehavioral performance in the slant discrimination task was quantified by plotting the proportion of top-far choices as a function of stimulus slant. The resulting psychometric function was fit with a cumulative Gaussian using the Psignifit toolbox . The poiNeuronal sensitivity was measured by using ROC analysis to assess the ability of an ideal observer to discriminate non-zero from zero slants  . To consval task , but theTo quantify the relationship between neuronal response and choice, CPs were computed using ROC analysis. For each slant, neuronal responses were grouped according to the choice. “Preferred” choices corresponded to those made in favor of the preferred slant of the neuron, as determined from the 3D surface orientation tuning profile measured during fixation. “Nonpreferred” choices corresponded to those made in the opposite direction. CP was computed by performing ROC analysis on the preferred and nonpreferred choice distributions for the (ambiguous) 0° slant stimulus. To achieve greater statistical power, a grand CP was computed by performing ROC analysis after normalizing the neuronal responses for each stimulus slant and combining the normalized data into two composite distributions corresponding to preferred versus nonpreferred choices . Only strFS.C , choice (C), and neuronal spike count (F). From these correlations, we computed a slant partial correlation, rFS.C  that quaC .Surface orientation tuning was measured for 427 CIP and 72 V3A neurons during a fixation task in which a checkerboard plane was presented at 25 slant–tilt combinations A. Of thep < 0.05) was fit with a Bingham function . In particular, there was a bias toward representing smaller slants  or threshold . Although not significant, there was a slight tendency for the P.S.E. to be negative at −2.25 cm , suggesting that the slightly negative P.S.E. at −2.25 cm was not due to a systematic vergence error. These data strongly suggest that the monkeys performed the task by assessing the slant of the plane rather than by judging local stimulus depth relative to the plane of fixation.A control experiment was conducted to confirm that the animals did not perform the slant discrimination task based on local absolute disparity cues signaling that the upper (lower) half of the plane was in front of (behind) the LCD. Each monkey performed the slant discrimination task for nine sessions with the stimuli centered at three depths (0 and ±2.25 cm) from the display A,B. Psyc−2.25 cm D. Howevep < 0.05) along the 90°/270° tilt axis used in the slant discrimination task  were significantly tuned for slant . We further confirmed that neuronal thresholds were similar between monkeys. The median CIP thresholds were 35.16° (monkey N) and 26.04° (monkey P), and not significantly different . Likewise, the median V3A thresholds were 31.30° (monkey Z) and 23.73° (monkey P), and were not significantly different . These results indicate that CIP and V3A neurons are similarly sensitive to changes in slant.Firing rate distributions for three pairs of slants  are shown in Neurometric functions can be directly compared with psychometric functions measured in the same recording session A,B. SimuDuring the slant discrimination task, variability was observed in both the neuronal firing rates and choices elicited by stimuli of the same slant. This variability is evident in histograms of the responses of the example CIP neuron to a slant of 0°, grouped by choice A. This sCP analysis was used to quantify the relationship between neuronal response and choice . We compt test: t = 3.89, p = 2.45 × 10−4). For the V3A neuron in t test: t = 0.64, p = 0.53). Second, to achieve greater statistical power, we calculated a “grand CP” by including responses to all slants for which the monkey made at least three choices toward each response target. For this analysis, responses to each slant were normalized using the balanced z-score method (p = 0.001). The grand CP for the V3A neuron in p = 0.36). Across the neural populations, the grand CP was highly correlated with the CP measured for the 0° slant stimulus . The analyses that follow are based on grand CPs.After sorting responses by choice, we used ROC analysis to compute the probability that an ideal observer could predict the choice of the monkey based on the responses of the neuron . The CP was calculated in two ways. First, we only considered responses to the ambiguous 0° slant stimulus. For the CIP neuron in e method . For thet test, p = 1 × 10−15). The mean CIP CP was also significantly different from chance for each monkey . In total, 51% of CIP neurons (33 of 65) had CPs that were significantly different from chance . For the majority of CIP neurons with significant CPs (26 of 33), firing rates increased when the monkey made a choice in favor of the preferred slant (CPs > 0.50). However, 7 CIP CPs were significantly <0.50, indicating that they fired more when the monkey made a choice in favor of the nonpreferred slant. In contrast to CIP, the mean V3A CP was 0.49, which was not significantly different from 0.50 . Neither monkey had a mean V3A CP that was significantly different from chance . Permutation tests revealed that only one V3A neuron had a CP that was significantly different from chance. As a control, we confirmed that there was no significant difference in CP associated with whether the neurons preferred positive or negative slants. The mean CIP CP was 0.55 ± 0.03 SEM (N = 30) for neurons preferring positive slants and 0.59 ± 0.03 SEM (N = 35) for those preferring negative slants . The mean V3A CP was 0.46 ± 0.03 SEM (N = 10) for neurons preferring positive slants and 0.50 ± 0.03 SEM (N = 13) for those preferring negative slants . Comparing choice-related activity across the two areas, we found that the mean CIP CP was significantly greater than the mean V3A CP . These findings indicate that the CIP, but not V3A, neurons displayed strong choice-related activity during the slant discrimination task.Histograms of grand CP for CIP and V3A are shown in r = −0.44, p = 3 × 10−4; r = −0.20, p = 0.36), and the V3A CPs clustered around 0.50 regardless of neuronal threshold (p = 0.03) between neuronal threshold and brain area, indicating a significant difference in the strength of the relationship between CP and neuronal threshold in CIP and V3A.We further found that the CIP neurons showed a significant negative correlation between neuronal threshold and CP . We therefore calculated CPs and neuronal thresholds after removing the dependence (linear trend) on vertical eye position, vertical eye velocity, and vergence from the neuronal responses. After removing the effect of vertical eye position, there was a small but significant reduction in CP . The CP measurements before and after correction were highly correlated , and the mean value remained significantly greater than chance after correction . Removal of the effect of vertical eye position had no significant effect on the median neuronal threshold . For vertical eye velocity, there was a small but significant effect on the mean CP  and the median neuronal threshold . The CP measurements before and after correction were highly correlated , and remained significantly greater than chance after correction . Neuronal thresholds were also highly correlated before and after correction . For vergence, there was no significant effect on mean CP (p = 0.58) or median neuronal threshold (p = 0.48). Thus, variations in eye position, eye velocity, and vergence had little effect on CIP CPs and neuronal thresholds.As a control, we confirmed that trial-by-trial variation in vertical eye position, vertical eye velocity, and vergence during the stimulus presentation had no appreciable effect on CIP CPs and neuronal thresholds . For eacDuring the slant discrimination task, both the stimulus and the choice may contribute to neuronal activity. The contributions of stimulus and choice to the activity of example CIP and V3A neurons is shown in p < 0.05), and the magnitude  of the slant partial correlations in CIP  and V3A  were not significantly different . The ranges of slant partial correlations in CIP (r = −0.51 to 0.47) and V3A (r = −0.48 to 0.46) were also similar. Correspondingly, the variance of the slant partial correlations was not significantly different between the areas .To dissociate the contributions of stimulus and choice to the responses of each neuron, partial correlations were computed between slant, choice, and spike counts (over the 1000 ms stimulus presentation period) using all trials. This analysis estimates how much variance in the responses can be accounted for by stimulus and choice while controlling for the fact that these variables are correlated. Similar percentages of CIP  and V3A  neurons had significant slant partial correlations (p = 0.003). The range of choice partial correlations was also greater in CIP (r = −0.55 to 0.49) than V3A (r = −0.15 to 0.20). Correspondingly, the variance of the choice partial correlations was significantly different between the areas . These findings confirm that choice had a greater effect on CIP than V3A activity.Although the slant partial correlations in CIP and V3A were similar, the choice partial correlations differed substantially. A greater percentage of neurons had significant choice partial correlations in CIP  than V3A , and the magnitude of the choice partial correlations in CIP  was significantly greater than in V3A . In contrast, the CIP neuron in p = 0.001). For comparison, a V3A neuron that preferred negative slants and top-near choices is shown in p = 0.29).In CIP, the relative signs of the slant and choice partial correlations were largely predictive of CP. The CIP neuron in N = 40), which was significantly >0.50 .The relationships among slant partial correlation, choice partial correlation, and CP are summarized for CIP and V3A in N = 25, Wilcoxon signed rank test, p = 0.95). Note, however, that neurons with the lowest CPs (darker blue points) are largely found in quadrants II and IV.There was also a substantial number of neurons for which slant and choice had opposite effects on firing rate (quadrants II and IV). Cells in quadrant II (top left) are those for which firing rate increased for negative slants and top-far choices. Cells in quadrant IV (bottom right) are those for which firing rate increased for positive slants and top-near choices. Assuming that CP was computed based on the true sign of the slant preference , neurons in quadrants II and IV should have CPs <0.50. This was not immediately evident: 12 of 25 CIP neurons (48%) in these quadrants had CPs <0.50, and the mean CP = 0.50 ± 0.02 SEM was not significantly different from 0.50 , suggesting that slant and choice can have independent effects on neuronal responses (see Discussion).To further test whether CPs are related to the relative signs of the slant and choice partial correlations, we fit a 95% confidence ellipse to the data from all CIP neurons with CPs >0.50 (green dashed ellipse) and a 95% confidence ellipse to those with CPs < 0.50 (blue solid ellipse), as shown in N = 9, Wilcoxon signed rank test, p = 0.09), but the mean CP for quadrants II and IV (0.44 ± 0.02) was significantly <0.50 . This suggests that there was some tendency for the relative signs of the slant and choice partial correlations to predict CP in V3A. However, this trend was weak compared to CIP, as demonstrated by the 95% confidence ellipses for CPs > 0.50 and CPs < 0.50 in V3A. For both ellipses, the major axis is oriented approximately along the slant partial correlation axis , reflecting that the V3A responses were substantially more dependent on slant than choice.In V3A, the mean (±SEM) CP for quadrants I and III (0.55 ± 0.03) was not significantly >0.50 (N = 65 neurons) was significantly >0.50 was 350 ms (bin center) after stimulus onset . The CP plateaued at ∼400 ms after stimulus onset and maintained this approximate level until the last time bin before stimulus offset (950 ms), at which point there was a further increase in CP, which may reflect additional choice-related activity and/or directionally selective saccade-related activity. The mean V3A CP was not significantly different from 0.50 in any time bin , but was slightly <0.50 throughout most of the stimulus duration. For comparison, mean CIP and V3A neuronal thresholds are shown in Last, we examined the time course of CPs, neuronal thresholds, and partial correlations in CIP and V3A by computing these quantities within a series of 200 ms bins shifted every 50 ms. Average CP time courses are shown in r = 0.93, p = 2.4 × 10−10, N = 22; V3A, r = 0.90, p = 1.9 × 10−8, N = 22). In CIP, the time course of the squared slant partial correlation peaked at ∼250–300 ms (bin centers), whereas the squared choice partial correlation increased later during the stimulus epoch (red curve). It was not until 450 ms (bin center) after stimulus onset that the squared choice partial correlation became significantly different from its initial value , further emphasizing that choice-related activity in CIP is substantially delayed relative to stimulus-related activity. Similar to the CP time course, the squared choice partial correlation plateaued until about the time of stimulus offset, at which point it increased further. In contrast, for V3A, the squared choice partial correlation remained close to zero throughout the stimulus duration, further reflecting that there was little to no choice-related activity in V3A. The squared choice partial correlation for V3A did, however, increase significantly above its initial value after stimulus offset, at the 1100 and 1150 ms time bins . Given that the V3A CP was not significantly different from 0.50 in these same time bins , and, given that a previous study reported saccade-related activity in V3A , squared SPC, and squared CPC are shown for CIP and V3A in y in V3A , the incWe investigated correlations between 3D surface orientation perception and neuronal activity in areas V3A and CIP of the macaque monkey. Our results show that surface orientation is similarly discriminable based on V3A and CIP responses, and that neurons in the two areas are similarly sensitive to small slant variations. Together with anatomical data , these rThe present results strongly agree with previous reports of 3D orientation selectivity in CIP , and areAlthough we found similar stimulus response properties in V3A and CIP, there was a stark difference in their choice-related activity. More than half of the CIP neurons had significant CPs, whereas only one V3A neuron had a significant CP. This difference suggests that CIP activity is functionally coupled with perceptual slant decisions, whereas V3A activity is not. However, given the small number of V3A neurons recorded in this study, this result should be considered preliminary. Additionally, the relationship between CIP activity and choice is not necessarily causal. Significant CPs could arise in a bottom-up manner , but thePrevious work has shown that the 3D orientation tuning of CIP neurons is largely invariant to changes in the mean depth of the stimuli relative to the fixation plane, as well as the defining visual  cue, suggesting that CIP neurons are sensitive to depth gradients . In the To dissociate the contributions of stimulus slant and choice to CIP and V3A responses, we computed partial correlations between these variables and the spike counts of individual neurons. In both areas, we found strong correlations between the stimulus and spike count. In contrast, correlations between choice and spike count were generally strong in CIP, but weak in V3A. This analysis validates the main CP finding; namely, that there was strong choice-related activity in CIP but not V3A. These results are reminiscent of a previous study, which found that V2, but not V1, neurons show significant choice-related activity during a disparity discrimination task , despiteThe pattern of slant and choice partial correlations observed in CIP may reflect a substantial top-down contribution to CPs. In a feedforward (bottom-up) scheme, it would be expected that stimulus and choice partial correlations would have the same sign, such that greater activity from a neuron constitutes evidence in favor of its preferred stimulus. In contrast, our CIP data show no significant relationship between slant and choice partial correlations A. In othIt is also possible that some of the choice-related activity that we observed in CIP was due to directionally selective saccade-related activity. However, the time courses of CP and squared choice partial correlation suggest that saccade-related activity may be limited to the time period between stimulus offset and saccade execution. First, choice-related activity became significant ∼400 ms after stimulus onset (>800 ms before the median choice time). The choice-related activity then plateaued at an elevated value until about the time of stimulus offset. Second, there was a sharp increase in choice-related activity starting around stimulus offset, which may reflect a choice signal and/or directionally selective saccade-related activity. Together, these observations suggest that by restricting our analyses of choice activity to the stimulus presentation period, we largely isolated choice-related (rather than saccade-related) signals.Last, we consider the relative timing of slant and choice signals. The time courses of slant-related signals in CIP and V3A were highly correlated with population-level spike density functions . In CIP,


Membrane association is a hallmark of the genome replication of positive-strand RNA viruses [(+)RNA viruses]. All well-studied (+)RNA viruses remodel host membranes and lipid metabolism through orchestrated virus-host interactions to create a suitable microenvironment to survive and thrive in host cells. Recent research has shown that host lipids, as major components of cellular membranes, play key roles in the replication of multiple (+)RNA viruses. This review focuses on how (+)RNA viruses manipulate host lipid synthesis and metabolism to facilitate their genomic RNA replication, and how interference with the cellular lipid metabolism affects viral replication. Lipids are a diverse group of amphipathic or non-polar molecules essential for all cellular life forms. In eukaryotic cells, ~5% of genes are dedicated to the biosynthesis of thousands of lipid species  catalyzes the conversion of saturated FAs (SFAs) to unsaturated FAs (UFAs), is perhaps the best surveillance system of eukaryotic lipid saturation and membrane fluidity RNA viruses] are the most numerous of seven viral genetic classes and cause diseases in humans, animals, and plants. Despite genomic and structural diversity among various (+)RNA viruses, they share common features in genome replication RNA viruses remodel cellular membranes to form membrane-bound viral replication complexes (VRCs) or mini-organelles RNA virus replication by providing a physical scaffold for the assembly of replication machinery, including viral and host factors. Also, VRCs protect against host defense mechanisms including innate immunity sensors by shielding replication complexes containing double-strand RNA (dsRNA) replication intermediates. As major components of cellular membranes and signaling molecules, lipids have been demonstrated to be essential factors in many steps of the (+)RNA virus life cycle , SLs, and sterols function as structural constituents of membranes and are distributed throughout intracellular membranes  Albert . InteresThe membranes associated with viral replication are rearranged into distinct structures, which can be in the form of small spherules with necks, double-membrane vesicles, membranous webs, and reticular layers. Although these morphologies are distinct, they all require the bending of membranes. Membrane bending and deformation are essential to many cellular processes  and cells have evolved multiple mechanisms to induce membrane curvature. Three ways that cells induce membrane deformation include: local enrichment of specific lipids, protein scaffolding, and binding protein insertion/interaction P3 are inverted conical lipids that can lead to membranes with positive curvature. PC, PS, and SM can all form lipid bilayers, however, PE, PIs, and sterols cannot form lipid bilayers by themselves. In addition to the head groups, acyl chains can also play a role in membrane bending. The presence of a double-bond(s) in an acyl chain produces a “kink” that affects lipid packing, leading to changes in membrane shape and fluidity  and the last two are catalyzed by OPI3-encoded phospholipid methyltransferase Opi3p (overproducer of inositol 3) RNA viruses, such as brome mosaic virus (BMV)  alone is able to induce membrane rearrangements and PC synthesis, suggesting viral replication is not required to stimulate PC accumulation  at the inner mitochondrial membrane  and carnation Italian ringspot virus (CIRV), which both belong to the family Drosophila cells, where the Kennedy pathway predominates and the methyltransferase enzyme(s) required to convert PE to PC has not been documented. This work brings up interesting questions regarding lipid-type specificity vs. overall membrane composition as discussed in Conclusions, Cautions, and Future Directions.The NoV work regarding the role of PC was performed in yeast cells, where PC is primarily produced from PE via the CDP-DAG pathway, while the FHV work was performed in PE is a cone-shaped lipid with a relatively small, polar head group Burger, . Due to PI is synthesized by combining the phosphatidyl portion from the CDP-DAG pathway with inositol . ACBD3 interacts with both PI4KIIIβ and AiV viral proteins to form a viral protein/ACBD3/ PI4KIIIβ complex to produce PI4P  is formed by esterifying the -OH group at the 4-position of the inositol ring with a phosphate group . This es2) is another PI derivative that is modulated by poliovirus P2 Pincer (BAAPP) within its N-terminal amphipathic helix P2 is the product of PI4P catalyzed by phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) P2 and NS5A P2 and 3CD Pde novo synthesis from glycerol-3-phosphate , diacylglycerol kinase (DGK), and enzymes involved in the PAH1-encoded phosphatidate phosphatase . DAG is primarily converted to TAG and stored in LDs in the absence of free choline  cells  alteration affects HCV VRC formation in lipin1-deficient cells  to produce 3-ketodihydrosphingosine  are another class of membrane structural lipids. They are enriched in plasma membrane , which are present in DRM with the highest level compared to other SM species RNA viruses is best demonstrated in HCV. It has been reported that HCV increases SL levels in infected host cells  genome replication. WNV infection significantly increases levels of a number of SL species, such as SM, dihydroceramide, and ceramide , human rhinovirus (HRV), and echoviruses, along with an inhibited cholesterol esterification and depleted LDs in enterovirus-replicating cells  from ER membranes and PI4P from Golgi membranes. These OSBPs simultaneously shuttle cholesterols to the Golgi and PI4P to ER membranes RNA viruses may recruit sterols for their genomic RNA replication via different mechanisms. For instance, replication proteins p33 and p92 of TBSV bind directly to sterols What are the possible roles of enriched sterols in (+)RNA viruses replication? The direct binding of sterols to viral replication proteins, such as TBSV p33 and p92  are basic building blocks for the majority of cellular lipids, including PLs, SLs, and neutral lipids . They arFAs have been reported to play crucial roles in the replication of multiple (+)RNA viruses. Applying pharmacological inhibitors to disrupt FA synthase (FASN) inhibits the replication of DENV  of the hydrocarbon tails. The saturation degree of FAs affects genomic replication of (+)RNA viruses, likely through modulating membrane flexibility. For example, certain saturated and monounsaturated FAs promote HCV replication but some polyunsaturated FAs inhibit HCV replication, suggesting that balanced FA composition is needed for efficient HCV replication  serve as essential depots for storage of lipids  and energy. The LD core is mainly composed of StEs and TAG, which is surrounded by a phospholipid monolayer predominately constituted of PC and PE  Tauchi-. The surAUP1 blocked DENV virion production but not replication, which differs from what had been previously reported RNA viruses to acquire lipids for membrane or energy production to support their replication. DENV stimulates lipophagy, a specialized autophagy process targeting LDs for degradation and to mobilize the stored lipids as free FAs , lipids have been demonstrated to play key roles in almost every stage of the viral life cycle. In this review, we focused on crucial roles that lipids play in genomic replication of (+)RNA viruses. As summarized in Many research groups have identified some specific lipid species that are required for the replication of corresponding viruses . For exaCurrently, the majority of reports hypothesize the potential roles of lipids in the assembly of VRCs. However, how lipids are organized in VRCs and their roles in the assembly and maintenance of VRCs still needs to be further investigated. Fluorescent imaging has been used to observe lipids in relation to other cellular components and viral replication sites. However, conventional fluorescent microscopy techniques, such as confocal microscopy, pose limitations due to low spatial resolution, and the inability to image subcellular components in full detail. Super-resolution microscopy has the ability to precisely localize single fluorescent molecules with a precision of ~10–20 nm, depending on the sample and the instrument. This could provide a novel way of imaging lipids and subcellular organelles, leading to a better understanding of the trafficking and recruitment of certain lipids in (+)RNA virus replication. In addition, super-resolution microscopy has the potential to be used in live-cells, providing insight into the formation of VRCs upon viral infection. The precision of super-resolution microscopy allows for a more detailed picture of the localization and movement of lipids, providing further insight into their role in the replication of (+)RNA viruses.In conclusion, despite the complexity of lipid metabolism and virus-host interactions, knowledge of how viruses modulate host lipid synthesis pathways and remodel cellular membranes to facilitate replication, will undoubtedly unmask many new directions in cell biology, and accelerate the process of developing antiviral strategies.ZZ, GH, and NF collected references and drafted the manuscript. ZZ made all figures. ZZ, NF, BK, GB, GR, and XW revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Feeding management in modern dairy farms is becoming increasingly important economically and technologically as well as concern for the comfort and welfare of dairy cows. Automatic feeding systems enable more frequent delivery of fresh feed to dairy cows compared to conventional feeding systems and, in combination with automatic milking systems, provide cows more freedom to determine individual critical behavioral activities . This study examined the effects of feed delivery frequency on behavior of lactating dairy cows in an automatic feeding and milking system. The results highlight a potential negative effect of high frequency (11 times per day) feed delivery compared to low frequency (six times per day) on lying behavior. This indication can improve feeding management and the productivity and welfare of lactating dairy cows.Management systems in modern dairy farms is an important issue in relation to animal comfort and welfare. The objective of this study was to determine the effect of feed delivery frequency on the behavior patterns, visits to an automatic milking system (AMS) and on milk production of lactating dairy cows. The study was conducted on a commercial dairy farm with automatic feeding and milking systems. Feeding treatments consisted of two different frequencies, high feed delivery frequency (11 deliveries per day) and low feed delivery frequency (six deliveries per day). Lying behavior of 20 dairy cows was electronically monitored. The results obtained showed that 11 deliveries per day feed delivery frequency decreases the number of long-duration lying bouts, which may indicate that a very high feeding frequency disturbs the cows during their resting periods and thus influences both animal comfort and milk production. High feeding frequency may disturb the duration of lying bouts and alter the pattern of lying behavior throughout the day, affecting mainly the lying time during the 60 min before and following the provision of fresh feed. Delivering feed at a low frequency allow cows to distribute more evenly their lying time over the course of the day and improve their utilization of an AMS. Feeding system in modern dairy farms is an important issue in relation to animal welfare; moreover, the choice between the different types available on the market implies economic and technological consideration. The cost of feed needed for high-yielding herds makes efficient utilization of it essential for profitable farms. Feeding a total mixed ration (TMR) diet is now a preferred practice and has affected the popularity of mechanized feeding systems, mostly represented by conventional manually operated mixer-feeder wagons. The delivery of feed stimulates dairy cows to feed ,2. DelivMany researchers have examined the effect of feeding frequency on the performance of dairy cows, mainly on dry matter intake (DMI) and milk production ,6. FeediMäntysaari et al.  found thAutomatic milking systems (AMS) provide individual cows the freedom to choose their own milking times. Motivation to eat is a stronger incentive for attracting cows to a milking unit than is the motivation to be milked ,23. OostTo date, the majority of studies that have examined the effect of feeding frequency on cow behavior and performance have taken place in conventional feeding systems in combination with parlor-milking systems. The range of the feeding frequencies in these studies has been between 1× and 5×, and with cows being milked in a conventional parlor at frequencies of 2× and 3×. However, an AFS enables more frequent delivery of fresh feed compared to conventional feeding systems, and in combination with an AMS, provides cows more freedom to determine individual critical behavioral activities .The objective of this study was to determine whether the frequency of feed delivery in an AFS affects: (a) the behavior patterns  of dairy cows, (b) visits to an AMS during the day , and (c) milk yields. We hypothesized that under AFS and AMS, an increased frequency of feed delivery would negatively influence lying behavior. We further hypothesized that the frequency of feed delivery would affect the pattern of lying behavior, particularly after fresh feed delivery and the utilization of AMS.The study was conducted between December and January on a commercial dairy farm located in Friesland (The Netherlands) where animals were milked with an AMS  and fed by an AFS .Two sensors with integrated data loggers, located in the middle of the barn, were used to measure and record the air temperature and relative humidity . The average temperatures of the two periods during the experiments were 5.2 °C and 7.9 °C, respectively.The barn housed 93 lactating Holstein Friesian cows, of which 21 were primiparous and 72 were multiparous ; mean ± SD), and featured a loose-housing layout with a total of 141 cubicles with rubber mats covered with sawdust. The manger, positioned sideways in the barn, had 61 feeding spaces . Twenty Treatments consisted of two different frequencies of MR distributions applied to all 93 cows. Each treatment lasted 7 d, in which a 3 d adjustment period  was follThe two treatments were (1) a feed delivery frequency of 11 deliveries per day (11×)  and (2) a feed delivery frequency of six deliveries per day (6×) . In each treatment, feed was pushed up at 13:45 and 15:45 by the AFS . In thisx-axis of the data logger was perpendicular to the ground. The degree of vertical tilt of the x- and z-axis was used to determine the lying behavior of the animal [Lying behavior patterns of the cows were automatically recorded using two types of electronic data loggers. HOBO Pendant G Data Loggers  were utilized to measure the leg orientation at 1 min intervals and allowed all the lying behavior data to be collected electronically . The deve animal .IceTag Activity Sensors  were used to sample acceleration with a frequency of 8 Hz, and to determine the percentage of time the cows spent lying for each recorded second . IceTag Data collected by the data loggers were used to calculate lying times (h/d), bout frequency (n/d), and bout duration (min/bout) for each cow and each day of the treatment. Pre-feeding and post-feeding lying times (min) were calculated as the average lying time per cow during the 60 min before and following provision of each fresh feed delivery (feeder wagon starts automatic feed distribution). These two 60 min periods were identified in an explorative analysis as the times when lying behavior of cows was especially influenced by feed delivery . The preMilking-related data for all 93 cows, including time of entrance to and of exit from the AMS, yield and type of visit per visit, were automatically collected by the AMS and stored as “log files”. AMS log files included classifications for three types of cow visits: milkings , refusals , and failures . The milking duration (time in AMS) was calculated as the difference between the times that a cow entered and exited the AMS. The log files were preprocessed with the support of MS-Excel 2007, and the mean milk yield (kg/d), milking frequency (n/d), milking duration (min/m), refusal frequency (n/d), and visit frequency (n/d) on a per cow basis were calculated for each day of each feed delivery treatment. The average hourly number of milkings and AMS refusals per cow were calculated for each feed delivery treatment.® software . Before analyses, all data were screened for normality using the UNIVARIATE procedure of SAS® software. Only lying behavioral residuals were transformed using logarithmic transformations (log10(x)) to achieve normal distributions. Lying behavioral data , AMS utilization , and milk yield were analyzed as repeated measures using the MIXED Procedure of SAS® software for testing the effects of feeding frequency treatment (11× and 6× feed delivery), period, and the interaction between feeding frequency and period. The cows monitored were considered replicates as their behavior can be considered independent. In fact, synchronized behavior has not been observed as the daily average percentage of animals simultaneously exhibiting the same posture (lying or standing) was 50.8% and lower than the threshold percentage of 70% for 90% of time [® software. The replicate was considered a random effect and cow within time was a subject effect. The fixed effects of type of treatment (11× and 6× feed delivery), period (Period 1 vs. Period 2), and their interaction (Treatment × Period) were included in the model, fitting a Kenward-Roger method to calculate the denominator degrees of freedom to approximate the F-tests in the mixed models.Descriptive statistics were used to characterize the distribution of the variables in the study using the MEANS and FREQ procedures of SAS of time . Least s® software with the LSMEANS options. Five subsets of bout duration were used: the first contained all short lying bouts with duration <50 min; the second contained only lying bouts with a duration between 50 min and 100 min; the third subset contained the lying bouts with a duration between 100 min and 150 min; the fourth contained the bouts with a duration between 150 and 200 min; and the last subset contained all long bouts with a duration >200 min.The effect of feeding frequency on distribution of lying bouts duration were analyzed using the GLM procedure of SASThe effect of the feeding frequency on the diurnal AMS visiting pattern  was assessed using a model including the following fixed factors: treatment (11× and 6× feed delivery), hour (1 to 24) and treatment × hour interaction.p < 0.05 and p < 0.01 . A tendency was declared when p < 0.10.In the statistical analyses, significance was declared when p = 0.01) and increased post-feeding lying time . Furthermore, we observed effect of period on the length of time cows spent lying per day. In period two cows increased their lying time of about one hour (from 11.6 h to 12.8 h per day).The frequency of feed delivery had no significant correlation with total daily lying time, lying bout frequency and bout duration . HoweverThe effect of feed delivery is also visible in When the cows were fed 11 times a day, the percentage of cows lying was below 30% twice; compared to only once when they were fed 6×. The differences in lying behavior between treatments were particularly pronounced at times of peak feeding behavior  after each delivery; lying activity during the period immediately after the distribution of fresh feed decreased an average of 30% when cows were fed at low frequency (6×). In fact, when the cows were fed 6×, fewer cows were lying during the time period immediately following the provision of fresh feed, as highlighted by post-feeding lying time. Cows fed 6× showed a decrease in percentage of lying behavior after each feeding, whereas the 11× feed delivery did not show this trend. The peak for 11× lying behavior appears to be higher than any peak for 6×, and lying behavior of cows fed 6× appeared to spend more time (<30%) than 11×, even if 11× cows reach below 30% more times. Also, the lying pattern appears to be similar among the treatment, especially during the late evening or night and early morning.p < 0.01), but significantly more lying bouts with a duration between 100 min and 150 min (p < 0.05).During period 2, bout frequency was increased respect the period 1 . Also, tp < 0.10) to increase the number of milkings during the morning, between 08:00 and 10:00, caused a significant increase at 13:00 (p < 0.05), and resulted in a significantly lower number of milkings at 14:00 (p < 0.01) compared to the low feeding frequency (6×). The high feeding frequency led to significantly more refusals per cow per hour only at 14:00 (p < 0.05) compared to the low feed frequency.We did find that the feeding frequency affected the diurnal AMS visiting pattern. To better analyze the effect the total visits have been divided into milkings and refusals . The nump = 0.01, The low feeding frequency showed a significant effect to result in a higher milk yield  and the lying time during the 60 min before and following the provision of fresh feed. These patterns could reflect the periods of higher feeding activity throughout the day of the cows, which were related to feed delivery times. Cows spent 12% more time lying during the hour before feed delivery and 13% less time lying in the hour following feed delivery when provided feed 6× compared with feed 11×. This displacement in lying time during the 60 min before and following the provision of fresh feed indicates that the delivery of fresh feed is a much stronger stimulus to get cows to feed, especially when cows are fed with high frequency. However, these results demonstrate that the cows are able to adjust and change their behavior against variation of feeding frequency and evenly distribute their lying time over the course of the day, on basis of feeding frequency delivery. Also, in the studies by DeVries and von Keyserlingk  and DeVrIn this study, we observed that the feeding frequency affected the distribution of lying bouts durations. The feeding frequency affected the longer lying bouts (those lasting between 100 min to 200 min), but not the shorter bouts (those less than 100 min). The possible explanation may be that cows fed 6× are motivated to feed less frequently and thus have more time to lie down continuously between deliveries of fresh feed, as demonstrated by the fact that lying has priority over feeding [Feeding frequency had no effect on the milking, refusal and visit frequency. However, during several hours of the day, the feeding frequency significantly affected the milking and refusal patterns. Our results for the effect of the feeding system on the daily number of milkings and refusals are consistent with those of Oostra et al.  and BellIn the present study, the lower feeding frequency increase significantly milk yield. In the study by Phillips and Rind , increasAutomatic milking systems in combination with AFS provide cows more freedom to determine individual moments of lying, feeding, and milking. Compared to conventional dairy systems, where synchronization of behavior does still occur (particularly around times of milking and feed delivery), in AFS and AMS behavioral activity is distributed over a 24 h period. This may explain the subdued effects of feeding frequency found in this study in comparison with other studies that have examined the effect of feeding frequency in conventional feeding systems in combination with parlor-milking production systems.Neither lying behavior nor utilization of AMS was affected by decreasing the number of daily feed deliveries via AFS in this study. In our study, high feeding frequency (11× feeding treatment) showed significant differences in the pre-feeding and post-feeding lying time, and in the duration of the longer lying bouts, which supports a conclusion that very high feeding frequencies disturb lying behavior throughout the day, affecting mainly the lying time coincident with the provision of fresh feed. The feeding frequency also had a significant effect on the milking and refusal patterns throughout the day. In conclusion, results obtained, may indicate that a very high feeding frequency disturbs the cows during their resting periods and this may influence both animal comfort and milk production. High feeding frequencies should be avoided to allow cows to distribute both their lying time and their utilization of AMS more evenly over the course of the day. So, it is necessary to find a feeding frequency that is high enough to distribute the feeding time of cows over the course of the day, yet not so high that it disturbs the pattern of lying behavior and long-duration lying bouts. Further studies concerning feeding places, feeding and aggressive behaviors, and individual feed intake in farms with AFS would be useful to help explain some of the other associations we observed and to improve feeding management and the productivity and comfort of lactating dairy cows.


Self/foreign discrimination by the innate immune system depends on receptors that identify molecular patterns as associated to pathogens. Among others, this group includes endosomal Toll-like receptors, among which Toll-like receptors (TLR) 3, 7, 8, and 13 recognize and discriminate mammalian from microbial, potentially pathogen-associated, RNA. One of the discriminatory principles is the recognition of endogenous RNA modifications. Previous work has identified a couple of RNA modifications that impede activation of TLR signaling when incorporated in synthetic RNA molecules. Of note, work that is more recent has now shown that RNA modifications in their naturally occurring context can have immune-modulatory functions: Gm, a naturally occurring ribose-methylation within tRNA resulted in a lack of TLR7 stimulation and within a defined sequence context acted as antagonist. Additional RNA modifications with immune-modulatory functions have now been identified and recent work also indicates that RNA modifications within the context of whole prokaryotic or eukaryotic cells are indeed used for immune-modulation. This review will discuss new findings and developments in the field of immune-modulatory RNA modifications. Detection of pathogens by the innate immune system relies on a small subset of germline-encoded pattern recognition receptors (in contrast to re-arranged receptors of the adaptive immune system) that recognize structurally conserved microbial molecules. Beside cell wall components of bacteria and fungi, nucleic acids (NAs) have been identified as potent stimuli initiating a robust immune response. As both ‘self’ and ‘non-self’ nucleic acids are composed of the same basic building blocks, the ability to identify the origin of DNA and RNA is a crucial necessity for the innate immune system. Beside cytosolic receptors including amongst others retinoic acid inducible gene I (RIG-I), melanoma differentiation antigen 5 (MDA-5), absent in melanoma 2 (AIM2) and cyclic GMP-AMP synthase (cGAS), NAs are recognized by the Toll-like receptors (TLRs) 3, TLR7, TLR8, TLR9, and TLR13 which reside in the endosome. Numerous excellent reviews  have addThree main principles have been described that allow the innate immune system to avoid recognition of host (‘self’) NAs but allow stimulation by microbial (‘foreign’) NAs: First, discrimination can be based on spatial restriction of NA-sensing receptors to specific subcellular compartments. Thus, TLRs reside in the endolysosome to which self-RNA/DNA has no or only limited access. In contrast, microbial RNA/DNA can get access after endosomal uptake and degradation of microbes specifically in phagocytes. Second, nucleotide composition, naturally occurring chemical modifications, and distinct sequence motifs affect primary, secondary and tertiary structures of RNA and DNA and determine NA recognition. As such, 5’-triphosphorylated cytosolic RNA that arises during RNA-virus replication activates cytosolic RIG-I whereas eukaryotic “self” RNA avoids recognition through capping ,7,8. FaiEndosomal NA-sensing TLRs are synthesized in the endoplasmic reticulum (ER) and shuttle via the Golgi apparatus to endosomes by the help of uncoordinated 93 homolog B1 (UNC93B1) . EctodomDNA containing unmethylated CpG dinucleotide motifs which are common in bacterial DNA are recognized by TLR9 . Interes6 methylation at A2085 of Staphylococcus aureus 23S rRNA (corresponding to A2058 of E. coli 23S rRNA) which also induces macrolide resistance of certain bacteria abolishes TLR13 stimulation [Interestingly, recognition of ssRNA differs between mice and humans, as TLR8 has limited function in mice and TLR13 is not present in humans ,29. Of nmulation . The latRecognition of ssRNA by human TLR7 and TLR8 has been shown since years to be important to mount antiviral responses. However, whether RNA recognition also plays a role in bacterial infection remained underexplored. Of note, several studies in recent years now elucidated the importance of bacterial RNA recognition for innate immune response ,32,33,34Recognition of foreign RNA in the cytosol is generally considered as hallmark of viral infection and replication . Yet, evHowever, RNA recognition by the innate immune system is not always beneficial. Sensing of self NA can trigger IFN and proinflammatory cytokine-associated severe autoimmune diseases like systemic lupus erythematosus (SLE) or psoriasis ,48,49. ELimitation of endogenous DNA and RNA recognition by spatial restriction of NA-sensing TLRs to the endolysosome, degradation, and discrimination by sequence motifs and chemical modifications are secure strategies to impede self-recognition.Why are TLRs recognizing nucleic acids restricted to the endosome, whereas other NA-sensing PRRs are localized in the cytosol? First of all, host DNA is not expected to be freely available in the cytosol. However, accumulation of endogenous DNA derived from viral retroelements is impeded by the cytosolic DNase three-prime repair exonuclease 1 (TREX1 or DNase III). Thereby, degradation of DNA within the cytosol negatively regulates cGAS-STING signaling and prevents IFN production . HoweverIn contrast, recognition of self-RNA is especially avoided by RNA modifications. More than 150 different, naturally occurring posttranscriptional RNA modifications are meanwhile identified . EukaryoN(1)-methylpseudouridine into mRNA, either alone or in combination with m5C, was shown to further decrease stimulation of innate immunity, especially TLR3 as compared to pseudouridine [N(1)-methylpseudouridine modified mRNA vaccine induced better T-follicular helper cell responses and germinal B cell activation—yet it remained unclear whether this was due to effects on mRNA stability or reduced innate immune stimulation [Recently, incorporation of ouridine . As mRNAmulation .Besides RNA modifications affecting stimulation of TLRs, the same principle also holds true for other innate immune sensors. As a brief overview, the following findings have been made in the field. Polyadenylation in eukaryotic RNA limited induction of IL-12 from human monocyte-derived DCs, which was high upon activation by bacterial . ConfirmAdar1 mutant mice die by embryonic day E12.5 and show aberrant interferon production. Yet, mice mutant for Adar1 and Mavs, the latter being the main signal adaptor for RIG-I and MDA5, survived thus showing that the phenotype probably is linked to recognition of unmodified dsRNA by cytosolic innate sensors. The rescuing effect on interferon production was dependent on the editing activity [Of utmost importance, cytosolic sensing of self-RNA by RIG-I or MDA-5 is prevented by specific modifications impeding RNA recognition. A to I conversion by the enzyme adenosine deaminases acting on RNA (ADAR) is one important strategy to make host RNA invisible for the innate immune system ,70. Thisactivity . In the activity . The datactivity .O-ribose methylation of the first (cap 1) and often the second (cap 2) nucleotide which abolishes interaction with RIG-I and MDA5 [O-methylation avoiding recognition by the innate immune system [N1-2’-O-methylated RNA and thus contributes to self/foreign discrimination [O-methylation of the viral mRNA cap in West Nile Virus is important to restrict the actions of antiviral IFN-induced proteins with tetratricopeptide repeats (IFIT) resulting in loss of pathogenicity in a mutant absent of 2’-O-methyltransferase activity [O-ribose methylation of the cap, NS5 protein of flaviviruses might also act as methyltransferase for internal adenosines thus extending the amount of modified nucleotides and thereby eventually reducing immunostimulation [O-methylation it was also shown that MDA5 activation is impeded by 2′-O-methylation in viral mRNA: Coronavirus mutants lacking 2′-O-methyltransferase activity showed increased type I interferon production and were also more susceptible to IFN actions [N-6-methyladenosine (m6A) and pseudouridine within a polyU/UC RNA domain of hepatitis C virus were tested for cytosolic RIG-I stimulation and showed to reduce RIG-I signaling. Similar modifying effects were also seen for 2FdU, 2FdC, 5mC, 5moC, and 5hmC. However, while m6A modified RNA bound RIG-I poorly, the mode of action was different for pseudouridine: this had high affinity for RIG-I but failed to induce a conformational change necessary for activation [Moreover, RNA transcribed in the nucleus is modified with a 5’ cap. This is characterized by an inverted 7-methylguanosine linked the first transcribed nucleotide (5′ cap) and 2’-and MDA5 . In conte system . Structuactivity ,78. Thusmulation . Besides actions . Moreovetivation .O-methylation of guanosine within certain tRNA isoacceptors was described to prevent RNA from TLR7 and TLR8 activation. This cis-silencing (which means the modified RNA itself is not recognized) properties of ribose-methylation are used to modify siRNA to avoid immune recognition [O-methylation is known to act as a dominant inhibitor on TLR7 and TLR8, which means 2’-O-methylation of only a proportion of RNAs is sufficient to abrogate immune stimulation of the entire RNA preparation. Mechanistically, it was shown that 2’-O-methylated RNA competed with unmodified RNA for TLR7 binding [O-methylated RNA might displace non-methylated stimulatory RNA from its receptor without inducing the change in TLR7 conformation that is necessary for activation. Supporting the same argumentation, 2’-O-methylated RNA was shown by AlphaScreen™ to bind with higher affinity to TLR7 compared to unmodified control, which might explain the trans-silencing effect (that is otherwise immune stimulatory RNAs are not recognized in the presence of 2’-O-methylated RNA) properties of RNA 2’-O-methylation) [Preferential recognition of RNA is based on sequence composition and RNA modifications. Several studies investigated the immunosilencing capacity of ribose-methylation within ssRNA. In this regard, a single 2’-ognition ,92,93. M binding . At the ylation) .O-methylation of G18 within tRNAs is described to stabilize their L-shaped three-dimensional structure. In this regard, extremophilic bacteria tend to exhibit more excessively modified tRNA [O-methyltransferase trmH, which is responsible for 2’-O-methylation of conserved guanosines at position 18 (Gm18) in the D-loop of tRNAs, is one example for diversity of enzymes. Based on the substrate specificity, trmH enzymes can be assigned to two classes: type I trmH methylates all tRNA species at G18, whereas type II trmH only modifies a subset of tRNA isoacceptors. Homologues of trmH are characterized in lower as well as higher eukaryotes. For instance, in Saccharomyces cerevisiae the tRNA-2’-O-methyltransferase is called trm3 and in humans it is termed TARBP1 [O-methylation is one of the most abundant posttranscriptional modifications within eukaryotic ribosomal RNA. Up to one hundred 2′-O-methylation sites within yeast or human rRNA are known and modified by nuclear protein 1 (NOP1) or fibrillarin, respectively [O-methylation is supporting ribosomal structures and therefore seems to be essential for stability and function [In its physiological sequence context, 2’-ied tRNA ,96. Mostied tRNA . The bacd TARBP1 ,99,100. ectively ,102. Thoectively ,104. rRNfunction . Of notefunction . In geneO-methylation of siRNA was used to improve stability of synthetic siRNAs in the serum [O-methylation of siRNA to inhibit immune stimulation, a common undesired side-effect of siRNA application [O-methylated RNA in human as well as murine plasmacytoid DCs and monocytes were confirmed then in a number of studies [O-methylation induced less type I IFN.Initially, 2’-he serum . Howeverlication : The aut studies ,108,109.O-methylated RNA as TLR7 antagonist [O-methylated RNA in trans, which means TLR7 signaling was impeded when only one strand of the siRNA duplex was modified. The data argued for a “dominant” inhibitory effect of 2’O-methylated RNA over otherwise immune-stimulatory RNA. To examine whether inhibition requires annealing of modified and unmodified RNA, 2’-O-methylated RNA was encapsulated together with non-complementary immune stimulatory RNA. Of note, co-delivery of non-complementary modified and unmodified RNA at a 1:1 ratio efficiently abrogated immune stimulation of PBMCs. Yet, early data with 2’O-methylation made only use of synthetic RNA.As another key finding, Robbins et al. identified 2’-tagonist . InteresO-methylation however is also naturally occurring at significantly higher abundance in eukaryotic as compared to prokaryotic  RNA [2’-ial) RNA ,111 and O-methylation in its physiological context was described for the first time within tRNAs. In 2012, Jöckel et al. and Gehrig et al. described the immune silencing effect of 2’-O-methylation of guanosine at position 18 (Gm18) of tRNAs. Gm18 within E. coli tRNA was sufficient to abolish immune stimulation of certain isoacceptors as well as total tRNA fractions [O-methylation within a self-RNA is able to interfere with TLR stimulation thus hinting towards a physiological function in self/foreign discrimination. Interestingly, also in in this study the suppressive activity, when 2’O-methylation was placed in a different than the immune stimulatory RNA strand, was confirmed [Immune modulatory potential of 2’-ractions ,85. It wonfirmed . O-methylation, 2’-O-fluoro modification has also been shown to limit inflammatory responses by therapeutic RNAs [O-methylated guanosine, adenosine and uridine but ribose-methylation of cytosine failed to inhibit TLR7 or TLR8 response [O-methylation-dependent immune inhibition was not elucidated. Thus, Kaiser et al. performed a permutation study of 2’-O-methylated nucleotides also considering the nucleobase downstream of the modification [cis. However, a sequence motif inhibiting TLR8 stimulation was pending. Furthermore, Kaiser et al. only focused on TLR inhibition by 2’-O-methylation in cis but did not verify if identified immune inhibitory sequences act as TLR7/8 antagonists in trans. Co-transfection experiments with immune inhibitory 2’-O-methylated oligoribonucleotides (ORNs) and immune stimulatory bacterial RNA performed by Schmitt et al. characterized the two dominant inhibitory tri-nucleotide motifs (DmRC) motif  and (DmDN) motif  inhibiting TLR8 and TLR7 in trans, respectively [O-methylated nucleotide was most stringent for TLR8 inhibition. Previous findings of Jung et al. suggested that a single 2’-O-methylation within a synthetic 18S rRNA derived sequence can abolish TLR7 activity while retaining TLR8 stimulation [cis without inhibiting TLR7 in trans. Interestingly, those modifications did not exhibit a discernible common term of chemical structures. Steric shielding of RNA was introduced by several kinds of modifications like small molecules, sugar derivates or fluorescent dyes. Besides 2’-tic RNAs . Severalresponse ,82. Howefication . The autectively . Interesmulation . This obmulation . Indeed,mulation . Those kO-methylated RNA has been acknowledged for many years. Interestingly, the native human tRNALys3 was described as non-stimulatory on the innate immune system without exhibiting previously described 2’-O-methylation at position 18. Co-transfection experiments with otherwise immune stimulatory bacterial RNA elucidated that tRNALys3 itself is indeed non-stimulatory without inhibiting TLR7 response against stimulatory RNA. A double-methylation of uridine to 2’-O-methyl thymidine at position 54 of human tRNA was identified as sufficient to partially silence immune recognition of RNA without dominant inhibition of TLR7 [Lys3 contribute to its immune silencing effect. Moreover, previously described shielding effects of bulky RNA modifications which were also found in the anticodon of human tRNALys3 might reinforce immune silencing effects of the RNA. Dominant inhibition of TLR7 by 2’- of TLR7 . HoweverO-methythymidine closed a gap in the understanding from inert to immune silencing to antagonistic RNA modifications based on 2’-O-methylation. Increasing structure of the nucleobase seems to shift immune stimulatory properties of a pyrimidine base to immune inhibitory features of a purine base , a diseases which is associated with increased levels of IFN-α [Mutations in nucleic acid modifying enzymes are associated with severe diseases including cancer, asthma and mitochondrial dysfunctions . Moreovesability . Reducedsability . Besidesof IFN-α ,118. ThuO-methylations in ribosomal RNAs are introduced by fibrillarin in humans [O-methylation of rRNA [O-methylation within rRNA might affect RNA sensing by TLR7 and TLR8 and therefore could facilitate self-NA recognition and type I IFN secretion. The above described studies investigating the effect of 2’-O-methylation with regard to its immune silencing properties were mostly performed with isolated RNA species. Most often certain tRNA isoacceptors or whole tRNA preparations were used to elucidate the relevance of ribose methylation for immune recognition. However, the relevance of Gm18 within pro-and eukaryotic tRNAs in a more physiological context like during infections with certain bacteria or yeast has not been elucidated so far. Although eukaryotic rRNA is more heavily modified concerning 2’-O-methylation compared to prokaryotic rRNA, studies on the immune modifying relevance of ribose methylation are still pending. Knock-out of Nop1 in yeast (necessary for rRNA 2’O-methylation) is described as lethal [O-methylation within human rRNA facilitates on one hand the investigation of isolated rRNA with respect to immune stimulation and on the other hand, knock-down experiments of fibrillarin in human immune cells like pDCs or monocytes might answer the question if self-recognition of host RNA is facilitated by inappropriate ribose methylation.2’-n humans . Fibrilln humans . Ribosom of rRNA . Of note of rRNA ,121,122. of rRNA ,124. Hows lethal , howevers lethal . DecreasThe ability to posttranscriptionally modify certain nucleotides within DNA and RNA recently has been shown to have an additional function beside of structural needs and epigenetics which is marking NAs as self or non-self to allow immune recognition. Modified nucleotides not only reliably prevent self-NA recognition under homeostatic conditions but also modulate innate immune response. The broad repertoire of NA modifications allows a receptor specific inhibition of RNA or DNA recognition. However, the importance of modifications to regulate immune activation is a double-edged sword needing to balance adequate immune activation upon infection and self-tolerance. Misused RNA modifications by certain pathogens may cause immune evasion while inappropriate processing of self NAs can cause autoimmune diseases. Thus, the understanding of complex NA modification pathways and the impact of nucleotide modifications on the innate immune system will remain a challenging task that now enters the stage of studies under physiological and pathophysiological conditions.


In order to validate the putative trans acting networks, we used various biochemical and molecular techniques which confirmed predictions of the RNA network approach. A combination of mutagenesis and reverse genetics systems revealed that the RNA–RNA interacting sites identified are indeed responsible for segment assortment and complex formation, which are essential criteria for genome packaging. This paves the way for their exploitation as novel types of drug target, either to inhibit assembly, or for designing defective interfering particles containing an incomplete set of genomic segments.Viruses with segmented genomes, including pathogens such as influenza virus, Rotavirus and Bluetongue virus (BTV), face the collective challenge of packaging their genetic material in terms of the correct number and types of segments. Here we develop a novel network approach to predict RNA–RNA interactions between different genomic segments. Experimental data on RNA complex formation in the multi-segmented BTV genome are used to establish proof-of-concept of this technique. In particular, we show that  Reoviridae, have multi-segmented genome. These viruses are transmitted by arthropods  causing disease in animals and plants, often with a high economic impact on agriculture and animal health. Although Bluetongue virus  and the related epizootic haemorrhagic disease virus (EHDV) of deer, African horse sickness virus (AHSV) are all transmitted by biting midges , BTV is the most common throughout the world including Europe and UK , a genus in the family e and UK . Conseque and UK .BTV is structurally highly complex with a genome of 10 double-stranded RNA segments enclosed by a multi-layered protein capsid. It shares a virus family relationship with several other veterinary and medically important viruses (e.g. Rotaviruses). The BTV RNA segments vary in sizes (3.95–0.8 kb) and sequence, but each shares common short 5′ and 3′ untranslated regions (UTRs) of variable length, including highly conserved hexanucleotides at both ends . Recent trans interactions between segments, given the probabilities of cis interactions for each individual one. Predicted interaction sites were validated by mutagenesis, electrophoresis mobility shift assays (EMSAs) and virus recovery using reverse genetics. Our data describe the essential drivers of segment assortment in BTV as multiple sites dispersed along the full length of the segments but not localized in a single site of each segments as described previously  base pairing probabilities of <75% was compiled using RNAplex  assays and beads binding RNA–RNA interaction assays suggest that smaller segments are more critical for BTV genome assembly  targeting region (nt 699–737), which is critical for virus replication and RNA complex formation (trans interactions with a low mean free energy (MFE) of formation  and with mostly contiguous base-pairing were identified via RNAplex . Due to the dynamic nature of the interaction network, both steps need to be recomputed iteratively in the growing complex, because interaction probabilities recalibrate across the entire segment once a new interaction has been formed. To identify putative interaction sites between different segments in the complex with incoming segments (or groups of segments), sites of existing trans interactions in the complex need to be factored into the sampling of the conformations of RNA segments that already form part of the complex. For this, the predicted trans interaction sites are used as constraints on the folds of the interacting segments, i.e. interaction probabilities are sampled based on the understanding that a site already engaged in an interaction is no longer available to interact. This recalibrates all interaction probabilities, and in some cases makes interactions highly favourable that previously only have had a low probability, thus imposing a sequential order on interactions at different stages of complex formation. This dynamic and iterative sampling of the secondary structures of the segments in the growing complex is thus an essential step, and indeed is increasingly important with complex size. It was applied here to identify trans interactions at three stages of complex formation: Formation of the initial S7+S8+S9 complex (Stage 1), association of the latter with S10 (Stage 2), followed by association with S6 (Stage 3). The numbers of interactions and the geometries of the Stage 2 and 3 complexes rendered with Gephi  are shown in Figure The 10 individual RNA segments of BTV are recruited and packaged in sequential order, mediated by specific multi-site RNA interactions ,9. As inassembly ,9,16, weassembly , we idenAmong others, Figure It also demonstrates the importance of the S6–S10 contacts at Stage 3. Both their number Figure  and theiin vitro RNA–RNA interaction assay  did not form retarded bands when co-transcribed together with the other four segments  or at nts 479-490 . The interaction network implies that both are unlikely to occur, the former, due to competition with a pre-existing contact (S7-S9), and the latter, due to the distance of the putative interaction sites in the network, consistent with the absence of any complex in Figure s Figure . Major cs Figure , right aTo confirm that RNA complex formation is essential for virus replication, the predicted highest affinity SAS site on S6 (#20) was mutated in the viral genome using a BTV reverse genetics system. As S6 encodes NS1, an essential viral protein, the silent mutation was designed, i.e. aiming for maximum nucleotide changes without altering the amino acid sequence. In this case, the modified S6 (#20) construct was created via 10 nucleotide changes Figure  and RNA To determine whether virus recovery failed due to the failure of RNA packaging, which requires RNA–RNA interactions, we co-transcribed either the mutated S10 (#20) or mutated S6 (#20) together with the four (S6–S9 or S7–S10) wt segments. As predicted, the RNA shift mobility assay showed disruption of RNA complex formation and disappearance of certain retarded RNA bands. For mutated S10, only band A and C were visible Figure , lane 7,Several recent studies have revealed the essential function of the smallest RNA segment S10 in packaging and RNA–RNA interactions . Of the in vitro mutagenesis data are also consistent with the predicted wt interaction network. Mutagenesis of #20 on S10 enables S6 to form a new contact with S9 , significantly changing the interaction network and thus accounting for the occurrence of a weak band A. In order to verify this, we also mutated this site on S9, co-transcribed it with other four wt segments and analysed it by native gel, which revealed the loss of all bands as expected  and S9 , thus making it impossible to interpret experimental results based on the wt interaction network. We therefore recomputed the interaction network for this case, as well as for S6–S9 WT + S10 (#20–#24) Figure , lane10 Of the five interaction sites between S6 and S10, the site #20 is the strongest by free energy. Mutagenesis in this region of S6 or S10 perturbed complex formation, confirming the importance of this contact. We investigated if it is possible to recover complex formation by introducing complementary mutations in S6 and S10 at this region. Both S6 and S10 were mutated at this site such that the sequences at #20 are complementary between these two segments, but differed from wt sequence . Co-trancis or trans interactions anywhere on the segment. In particular, as discussed above, a trace of band A was visible after mutagenesis of S10, most likely due to a stabilizing effect on this band through a new bond formed between S6 and S9 that does not displace any other bond. Simultaneous mutation of #20 on S6 and S10 does not result in conflicting constraints and bond formation, and it is therefore not surprising that the same signature, i.e. band A, was seen following simultaneous mutagenesis of S6 and S10, thus providing an explanation based on the complex interaction network for why recovery was not possible.It is noteworthy that the complementarity between S6 and S10 in the #20 region was not sufficient to drive RNA complex formation, implying that not just the contact itself but also the sequence of specific nucleotides involved in this contact, are important. This is exactly what we expect from our analysis. As we have shown above, any mutations do not only affect the precise region mutated, but indeed the entire segment and its probabilities of forming Since it was not possible to compensate for the mutation at the #20 interacting site, we selected a weaker interaction site between these two RNA segments. Mutation at #24 on S6 could still generate three of the four signature bands Figure , lane 8,https://doi.org/10.1101/236620). Here, we have introduced a bespoke network approach to predict the possible RNA–RNA interactions between different segments, and have provided proof-of-principle of its predictive power using molecular assays and virus recovery. In particular, we identified vital trans interaction sites in the formation of RNA complexes among the five smaller segments of BTV, i.e. S6–S10. Data from in vitro RNA–RNA interaction assays, substantiated by isolating the assembled five segments as one single band via a sucrose gradient ultracentrifugation as in Lourenco and Roy  in the complex. Indeed, the major consequences of such mutations are expected from the model, because these mutations have dramatic consequences for the structure and topology of the interaction network (as e.g. in Figure As discussed above, the only band out of the four, band C, is the correct complex for further genome packaging, and other three bands might represent different stages in the sequential complex formation in different conformations. Alternatively, these complexes might represent conformations, in which alternative, transient contacts are made between the segments that are later replaced by those characteristic of the final complex. However, how this preferred complex is selectively packaged is yet to be revealed. Recent data suggest that in the absence of VP6, i.e. the RNA binding helicase protein of BTV, the RNA complex fails to be packaged into the assembling capsid. Further, the interaction of capsid proteins VP3 and VP6 is essential for genomic RNA recruitment ,24. Thesin vitro assembly assays and protein-free RNA–RNA interaction assays that genomic RNA segments recognise each other (trans interactions that are crucial for segment assortment and formation of an RNA complex for packaging into the protein container. The RNA sites involved in key trans interactions are reminiscent of, yet distinct from, the packaging signals discovered recently in different viral families (It had previously been demonstrated based on ch other , but thefamilies . They cofamilies ,21,22,29Taken together, the network approach introduced here can identify a number of inter segment contacts crucial for segment assortment and RNA complex formation, a prerequisite for packaging. We expect that this method, in concert with experiment, will enable new avenues for anti-viral intervention against viruses that either inhibit or misdirect RNA segment assortment.Supplementary DataClick here for additional data file.


CircRNAs are a class of noncoding RNA species with a circular configuration that is formed by either typical spliceosome-mediated or lariat-type splicing. The expression of circRNAs is usually abnormal in many cancers. Several circRNAs have been demonstrated to play important roles in carcinogenesis. In this review, we will first provide an introduction of circRNAs biogenesis, especially the regulation of circRNA by RNA-binding proteins, then we will focus on the recent findings of circRNA molecular mechanisms and functions in cancer development. Finally, some open questions are also discussed.  Discovered more than three decades ago , circRNAHere, we summarize the expanding findings on circRNA and provide an up-to-date account of their biogenesis, regulatory mechanisms, and cellular functions in carcinogenesis.In eukaryotes, linear mRNA is formed by processing exons of pre-mRNAs through alternative splicing. Unlike linear RNAs, circRNAs were originally thought to be aberrant RNA splicing products . BecauseWith the optimization of next-generation sequencing technology, several circRNA subtypes have been discovered in recent years. There are four main subtypes of circRNAs: exonic circRNAs (ecircRNAs), which are mainly derived from single or several exons; on the other side, circular intronic RNAs (ciRNAs) contain only introns; exonic-intronic circRNAs (EIciRNAs), which consist of both introns and exons; and tRNA intronic circRNAs (tricRNAs) are formed by splicing pre-tRNA intron . CurrentArchaea and Drosophila . I. I10]. Iosophila ,15,16. TPreviously, analysis of the surrounding introns of circRNAs in human fibroblasts has indicated they are usually longer and harbor complementary ALU repeats . A recenRNA-binding proteins, which usually contain RNA-binding motifs, play a central role in transcriptional regulation of genes . BecauseQKI, KH domains containing RNA binding proteins, is the first identified RBP involved in circRNA formation during the epithelial to mesenchymal transition. It enhances circRNA formation by binding to its consensus target motif, single-stranded RNA (ssRNA), in introns that flank the circRNA-forming exons. Consequently, insertion of synthetic QKI binding sites into introns was sufficient to produce circRNAs [NF90/NF110) could couple circRNA biogenesis and function during viral infection. They found the nuclear export of NF90/NF110 to the cytoplasm correlated with decreased circRNA expression, while NF90/NF110 bound to viral mRNAs to stimulate an antiviral immune response [ESRP1) is an essential splicing factor during pluripotency [NANOG and OCT4 regulate ESRP1 expression in hESCs, which leads to the promotion of circBIRC6 generation. Consequently, circBIRC6 works as a ‘molecular reservoir’ of miR-34a and miR-145, collectively contributing to pluripotency maintenance [circRNAs . Based oresponse . Epithelipotency . A recenntenance . ADAR1) to inhibit circRNA formation [ADAR1 binds double-stranded RNA to mediate adenosine-to-inosine (A-to-I) RNA editing [ADAR1 expression level globally. Approximately 3.5% of detected RNA editing sites are associated with potential clinical relevance, many of which are in noncoding regions [ADAR1 depletion can up-regulate the formation of circRNAs [Different RBPs may play different, or even opposite, roles in the back-splicing process. For example, all the above three RBPs can promote the production of circRNAs, whereas the RNA-editing enzyme (that edits adenosine to inosine) acts on RNA enzyme 1  are indeed produced from transcribed exons of distinct genes and affected by the translocations. Furthermore, they also found circular EWSR1/FLI1 fusion in SK-NEP-1 sarcoma cells and EML4/ALK1 in H3122 lung cancer cells. However, a recent search of MiOncoCirc data, which included 17 cancer sequencing datasets, did not detect any f-circRNAs resulting from chromosomal translocations and deletions. Instead, they discovered read-through circRNA (rt-circRNA), a novel class of circular transcripts that span exons originating from two adjacent genes on the same strand. They found some of these rt-circRNA reads were commonly found across different cancer types, and they were even detected in normal tissues or in samples with a normal copy number of the parent genes, which indicated the prevalence of rt-circRNAs in cancer samples [Oncogenic gene fusion is commonly found in cancer samples and plays a role in carcinogenesis. Guarnerio et al. hypothesized chromosome rearrangement will result in the juxtaposition at a close enough proximity to favor new events of back splicing, which would promote the generation of aberrant circRNAs . To veriThe functional mechanism of circRNAs are diverse in human cancer including acting as miRNA sponges, interacting with protein, regulating gene splicing or transcription, translating proteins, or peptide and epigenetic regulation . AccordicircFECR1, a FLI1 exonic circular RNA, interacts with the FLI1 promoter and recruits TET1 demethylase to induce DNA demethylation in the CpG islands. Concurrently, circFECR1 also binds to and downregulates DNMT1, the critical enzyme that maintains DNA demethylation during DNA replication. Thus, circFECR1 promotes tumor cell invasion by coordinately regulating TET1 and DNMT1 in breast cancers [Accumulated reports observed that circRNA can, in a complicated way, regulate its parental gene in terms of epigenetic control, splicing, transcription, or translation. For example, Chen et al. reported that  cancers .If the circRNA contains the same exon as the parental gene, the processing of circRNA will compete with the splicing of pre-mRNA. Previous studies have shown that back-splicing can compete with pre-mRNA splicing in circRNA biogenesis, resulting in low levels of linear mRNA that contain exon inclusions. In general, the more an exon is circularized, the less it presents in the processed mRNA ,11. ANKRD52) [CircRNA can also affect the transcription of its parental gene. For example, intron-retained circRNAs, such as EIciRNA are located in the nucleus. It is reported that EIciRNAs can bind to U1 snRNPs through RNA–RNA interactions, while U1 forms a complex with RNA polymerase II by binding to TFIIH. EIciRNAs regulate RNA polymerase II activity and promote parental gene transcription . SimilarANKRD52) . circYap was found to bind with Yap mRNA and the translation initiation associated proteins eIF4G and PABP. The complex containing overexpressed circYap abolishes the interaction of PABP on the poly (A) tail and eIF4G on the 5′-cap of the Yap mRNA, which functionally leads to the suppression of Yap translation initiation. Individually blocking the binding sites of circYap on Yap mRNA, or respectively mutating the binding sites for PABP and eIF4G, de-represses Yap translation [Besides, some circRNAs have been demonstrated to regulate gene transcription via both RNA polymerase II complex and translation-related proteins. Recently, nslation . CDR1as consists of a single exon and 63 conserved binding sites for miR-7, can suppress miR-7’s activity, and can upregulate the expression of miR-7 targets such as SNCA, EGFR, and IRS2 [circTADA2A was repressed in a large cohort of triple-negative breast cancer (TNBC) patients compared to adjacent normal tissues. Through bioinformatics analyses, miR-203a-3p and miR-302c-3p binding sites were identified in circTADA2A, which was confirmed in subsequent wet experiments. Functional analysis indicated circTADA2A-E6 binds with miR-203a-3p, thus affecting its downstream gene SOCS3 to suppress cell migration, invasion, and clonogenicity. Furthermore, their down-regulation was correlated with survival time in TNBC patients. We conclude that circTADA2A-E6 is a tumor-suppressor circRNA and could be utilized as a promising prognostic biomarkers and therapeutic target for TNBCs [circAGFG1 was significantly up-regulated in TNBC, and it promoted TNBC progression through the circAGFG1/miR-195-5p/CCNE1 axis [circHIPK3 targets miR-7 to promote colorectal cancer growth and metastasis, while it also targets miR-558 to suppress the expression of HPSE and inhibits migration, invasion, and angiogenesis of bladder cancer cells in vitro [CircRNAs are stable in cells because they do not have a 5′end and a poly-A tail, which prevent ribonuclease degradation. Furthermore, there are multiple miRNA response elements on the circular sequences. Thus, circRNA is natural miRNA sponge. or TNBCs . InteresNE1 axis . The samin vitro ,35.Ccnb1 could bind with H2AX in p53 mutant cells and suppress mutant p53 in tumor progression. This study found that circ-Ccnb1 could interact with both Ccnb1 and Cdk1 proteins, thus counteracting the effects of p53 mutations in breast cancer [AMPK activation is usually associated with upregulated expression of circACC1 [CircACC1 functions to stabilize and promote the enzymatic activity of the AMPK holoenzyme by binding with the regulatory β and γ subunits. Argonaute 2 (AGO2) is a core component of the miRNA-induced silencing complex. Recently, Chen et al. identified that circAGO2, one intronic circRNA generated from the AGO2 gene, binds with the HuR protein to facilitate HuR-repressed functions of AGO2–miRNA complexes in gastric cancer [CircFOXO3, which is highly expressed in noncancer cells, could arrest the function of CDK2 and block cell cycle progression via formation of a circFOXO3–p21–CDK2 ternary complex [Some circRNAs that harbor binding sites for RNA-binding proteins may serve as protein sponges or decoys in addition to acting as miRNA sponges. For instance, the circular RNA t cancer . In colocircACC1 . CircACCc cancer . CircRNA complex . Undoubt complex .circCTNNB1 produces a novel, 370 amino acid CTNNB1  isoform that uses the start codon as the linear β-catenin mRNA transcript, and translation is terminated at a new stop codon created by circularization. They found that this novel isoform can stabilize full-length β-catenin by antagonizing GSK3β-induced β-catenin phosphorylation and degradation, leading to activation of the Wnt pathway [lncRNA-PINT can be translated into a small peptide to suppress glioblastoma cell proliferation; this action is mediated by trapping PAF1c to inhibit translational elongation of oncogenes [GFP) [circRNAs were initially defined as a distinct class of endogenous noncoding RNA that could not translate proteins. Recently, strong evidence from several research groups has shown that circRNAs can encode proteins. Liang et al. reported that  pathway . In addincogenes . Howeveres [GFP) . And anoes [GFP) . Based oes [GFP) .In recent years, considerable interest has focused on circRNA in cancers. In this review, we outline current understandings on its biogenesis, molecular mechanism, and function, but there are also some open questions. Firstly, controls of circRNA and its parental gene are thought to be intensely coupled together ,30,31. HcircQTL SNPs might influence circRNA formation by altering the canonical splicing site or the reverse complementary sequence match [circQTL SNPs are highly linked to genome-wide association study signals in different cancer types. Besides, genetic or epigenetic factors contributing to circRNA regulation have not been fully explored in cancers. For example, genomic aberrations include both chromosome translocation and short nucleotide variants . Canonicce match . It woulFinally, circRNAs possess several characteristics that are well-suitable to serve as biomarkers in cancers. Some circRNAs have tissue-specific expressions and correlate with clinical indicators ,9,32. Th