However, the redundant use of the 1 × 1 convolution in traditional light-weight networks, such as MobileNetV1, has increased the computing time. By utilizing the 1 × 1 convolution that plays a vital role in extracting local features more effectively, a new lightweight network, named PlaneNet, is introduced. PlaneNet can improve the accuracy and reduce the numbers of parameters and multiply-accumulate operations (Madds). Our model is evaluated on classification and semantic segmentation tasks. In the classification tasks, the CIFAR-10, Caltech-101, and ImageNet2012 datasets are used. In the semantic segmentation task, PlaneNet is tested on the VOC2012 datasets. The experimental results demonstrate that PlaneNet (74.48%) can obtain higher accuracy than MobileNetV3-Large (73.99%) and GhostNet (72.87%) and achieves state-of-the-art performance with fewer network parameters in both tasks. In addition, compared with the existing models, it has reached the practical application level on mobile devices. The code of PlaneNet on GitHub:  Convolution neural networks (CNNs) have achieved excellent performance in image classification, semantic segmentation, and object detection tasks. Due to the limitations of computer resources, the scale of CNNs is restricted. With improved computer performance, deeper CNNs, where the CNN structure becomes more complex with many parameters and floating-point arithmetic operations, are needed to improve the precision of computer vision tasks. For example, ResNet50 has approximately 25.6M parameters and requires 4.1B Madds to process an image of shape 224 × 224 .A deep convolutional neural network is composed of many convolutions, and its scale is often represented by two factors: the number of channels and the number of layers. By deepening CNNs, smaller convolutional kernels are generally used, and the calculation can be further reduced by pooling, such as with VGG networks . As a neIn recent years, with the rapid development of the Internet of things, the trend of deep neural networks is designing a lightweight network that simplifies the model while achieving comparable accuracy, which can reduce the demand for computer hardware resources. There are many efficient deep neural networks with fewer parameters and computations. For example, SqueezeNet  decreaseBased on these observations, we believe that the 1 × 1 convolution is crucial in a lightweight network. Using the 1 × 1 convolution more reasonably can effectively improve the accuracy of a model and reduce the negative effects of the numbers of parameters and floating-point operations caused by redundancy. Based on this principle, we make more effective use of the 1 × 1 convolution by adjusting the position of the 1 × 1 convolution and setting the stride in the deep separable convolution and propose our network, named PlaneNet. In summary, our contributions are as follows:1. We make more effective use of the 1 × 1 convolution, which improves the accuracy while reducing the unnecessary redundancy; and provide a practical reference for subsequent efficient network design.2. Plane local features are the key elements of image classification. Increasing the information interaction of plane regions and effectively extracting the local features of images can help models understand the heart of models more effectively. We proposed a novel lightweight network PlaneNet, whose dimension is increased by the separable convolution, and then the local features are extracted more effectively by the 1 × 1 convolution. Finally, the plane local features are extracted from the data with spatial dimension features by using the separable convolution.3. In our experiments, our model can achieve state-of-the-art performance in classification and semantic segmentation tasks.The organizational structure of the remainder of this article is as follows. We begin with ‘Materials & Methods’ that describes the principles and methods used by PlaneNet. In ‘Results’, we compare our model with other mainstream lightweight networks. In Section 4, we discuss the experimental results of our model and consider the advantages and disadvantages of the improved strategy of our model. ‘Conclusion’ contains the conclusions and future work.This section introduces the plane module taking the MobileNetV2 module as a representative for a detailed comparison. Then, the analysis of two critical factors and the complexities of the plane module is demonstrated. Finally, PlaneNet is developed.To prove the importance of the 1 × 1 convolution in the model, experiments are conducted in ‘Hyperparameter Strategy on CIFAR-10’. For example, the CIFAR-10 experiment, an extreme example, assumes that local plane characteristics are not considered and fully uses the 1 × 1 convolution kernels for the standard convolution operation. The result is calculated using 22.29% of the time. The remaining 1 × 1 convolution kernels form a set of complete traditional convolution networks with 90% accuracy; therefore, extracting plane local feature from the network has a crucial role.The essential functions of the 1 × 1 standard convolution are to extract the depth pixel features and change the number of channels, but the disadvantages are obvious. The number of parameters is still large, and the receptive field is tiny. The 1 × 1 standard convolution in MobileNetV1 accounts for 95% of the computation time and contains 75% of the parameters. Therefore, we need to seek a strategy that can consider both. Thus, the overall strategy guiding our designed module has two points:Strategy 1: Within a specific budget of computing resources, the network should extract as wide a range of regional features as possible to richer network features.Strategy 2: The network should reduce the redundant 1 × 1 standard convolutions so that the network, based on the effective use of depth extraction features, can also reduce unnecessary calculations to a certain extent.Deep convolutional neural networks usually consist of many different-sized convolutions, resulting in high computational costs. Since the SqueezeNet after the 3 ×3 convolution is replaced by the 1 × 1 convolution, the efficient network model design adopts the 1 × 1 convolution to extract local features. Then, different methods, such as the ShuffleNet group convolution and EspNetV2 that replace the point-by-point convolution with the grouping point convolution, are used to reduce the redundancy. All methods eased the partial feature extraction due to redundancy of the 1 × 1 convolution to a certain extent. Therefore, to more effectively use the 1 ×1 convolution, we propose the plane bottlenecks and use them to extract the features. As shown in k is the image size, and Dk is the size of the feature map. M is the number of input channels. N corresponds to the primary function used to change the number of channels in the output. The point-by-point convolution follows the separable convolution, that is, the simple 1 × 1 standard convolution . The funThe Madds of the ascending dimensional point-by-point convolution (both the input dimension and the output dimension) is calculated as follows: We can reduce the proportion of the calculation formula as This result is further verified in ‘Compared with MobileNetV2 on Caltech-101’. Therefore, this design almost meets the performance requirements of the MobileNetV2 module design, and the required computing resources are significantly reduced. It can handle cross-channel features without the lack of interaction of plane local features.Through our separable convolution and point-by-point convolution, we achieve the ascending dimension function of direct point-by-point convolution and introduce the information interaction between the local regions of the plane to increase its receptive field, which achieved by direct point-by-point convolution . Plane bρ in MobileNetV1. The coefficient of expansion is introduced to increase the dimensionality with the depthwise convolution. Note that the size of the convolution kernel at this time is not 1. The advantage of this is that strategy 2 is perfectly addressed. This strategy can replace the 1 × 1 convolution to increase the dimension and attain a large receptive field to extract local features  as shown in ɛ = 1, there are M Dw × Dw convolution kernels convolved with M channels and Df × Df × M, which is a standard depth separable convolution, output feature maps. When ɛ = N, there are N × M × Dw ×Dw convolution kernels, each group has N convolution kernels to convolve with each channel of the input image, and the output after convolution is Df  × Df  × N × M. N corresponds to the primary function used to change the number of channels in the production. Because of the average grouping concept, the value of N can only be positive integers; therefore, when N > 1, this is an excellent operation to increase the dimension of the input image. However, it is worth noting that although ɛ > 1 conducts the operation for increasing the size of the input image, there is still no step of addition or multiplication on the channel dimension, which is entirely different from the traditional convolution. The numbers of parameters and Madds of conventional convolution are, respectively, The numbers of separable convolution parameters and Madds are, respectively, It is evident that when Madds reaches the same dimension, the standard convolution operation (even a 1 × 1 convolution kernel) is N times that of the separable convolution.α, making the network more universal; different networks can be customized for various scenes. We denote the PlaneNet with width multiplier α as PlaneNet α ×. The width factor α can change the size and amount of calculation of plane bottlenecks. In general, more extensive networks can achieve much more powerful performance, but this is at the expense of speed. Therefore, how to measure this hyperparameter is discussed in detail in ‘Efficiency of plane bottlenecks’.Because the performance requirements for the network are not consistent in different situations, we use the width factor in MobileNetV1. In each plane bottleneck, the number of convolution kernels is multiplied by w = kh). Take the MobileNetV2 famous deep separable convolution whose convolution kernel size is k. The size of the plane bottlenecks of the convolution kernel of the first separable convolution is k1, and the size of the second separable convolution kernel is k2. Because the filters specified in most layers are the same as the input channels, we simplify the processing by using c, where padding= ’same’ and h and w are integer multiples of s for convenience.Here, we further analyze the number of parameters and Madds used in our plane bottlenecks. e.g., k = 3) because multiple smaller convolution kernels can replace a larger convolution kernel, and the same receptive field can be obtained. First, this can result in fewer parameters. Second, this can allow more nonlinear transformations, which increases the ability of the CNN to learn features. The value of ɛ is relatively small , at least 1∼2 orders of magnitude larger than that of ɛ and K. Therefore, the theoretical parameter ratio of the two is α fluctuates around 1 , so the parameter ratio between the two is 0.33 to 0.60. The setting of some of the hyperparameters in a lightweight network is insufficient to make us ignore k and ɛ, but this is sufficient to make plane bottlenecks take advantage of their merits. Section ‘PlaneNet on semantic segmentation’ shows the plane replacing MobileNetV2 with the rest of the hyperparameters and the same network structure setting. The parameter savings of the model are up to 34.27%.Generally, the value of k is relatively small (ɛ and α (much greater is defined as 103). According to the We use Madds to assess the computational efficiency. Ideally, the theoretical parameter ratio of the two is Based on the experiments in ‘Efficiency of plane bottlenecks’ and our plane bottleneck architectures, we can easily incorporate our plane bottlenecks into existing well-designed neural architectures, thus reducing the computing costs. Using the coefficient of expansion and the width multiplier, we can tweak the architecture to achieve different performance points and adjust the architecture according to the desired precision and performance trade-off.α = 1, 224 × 224), as shown in We present PlaneNet , and the performance of MobileNetV2 can be improved by adding a nonlinear activation function in the latter part of the network. Therefore, to trade-off the information loss and performance gains, our network uses the LeakyReLU activation function to activate the nonlinearity . The mosNote the variation rule of the expansion coefficient of PlaneNet. The expansion coefficient only changes when the stride is 2 because when the stride is 2, the image size is halved by subsampling the image twice, and the small expansion coefficient has a good effect when applied to small-sized data. The strong extraction of regional features characterizes PlaneNet, but in fact, the channel depth features cannot be ignored, so a deep convolution is applied to extract the channel features again.Settings: For all the experiments in this section, the back end of the training model is TensorFlow, and most of the training settings of each model are modeled following MobileNetV2. The initial learning rate is 0.045, and the learning rate decreases at a rate of 0.98 in each epoch. The SGD optimizer is used, the momentum of the SGD optimizer is set to 0.9, and an RTX 5000 ×2 is used for training. The data enhancement strategy of our training model is to randomly rotate an image within the range of 30 deg, shift an image by 20% at the horizontal position, shift an image by 20% at the upper and lower positions, cross-cut transform 20% of an image, randomly shrink an image by 20%, and perform the horizontal flipping operation on the picture at random.ɛ = 6 to the input dimensions unless otherwise specified.For the experiment in this section, we apply width coefficient α = 1 and expansion coefficient To evaluate our plane bottlenecks, we used the CIFAR-10 dataset and replaced all the concentric layers in the VGG16 model with our proposed plane bottlenecks substitute (the pooling layer was replaced with stride = 2 plane bottlenecks). The new models were referred to as Plane-VGG16. Our training strategy is modeled after the setup of MobileNetV2, including the optimizer’s momentum, learning rate, etc. In this paper, the effects of the hyperparameters k and s on plane bottlenecks are analyzed, and then we have compared plane bottlenecks with the most advanced light-weight network models.CIFAR-10 is a computer vision data set for pervasive object recognition that was collected by Hinton students Alex Krizhevsky and Ilya Sutskever. It contains 60,000 32 × 32 RGB color images in 10 categories. A total of 50,000 images are used as the training set, and 10,000 images are used as the testing set.α=1, ɛ=6} and correct and adjust the 9 sets of k1 and k2 combinations in {1, 3, 5}. The size of the plane bottlenecks of the convolution kernel of the first separable convolution is k1,d the size of the second separable convolution kernel is k2. We list the results on the CIFAR-10 verification set in 1 = k2 = 3 is much better than the small or large ones in the convolution kernel. This is because the 1 × 1 kernel cannot introduce the feature information of the neighborhood plane in the feature map, and the small convolution kernel uses too much local information instead of global information. The combination of larger cores, such as {{k1=3, k2=5}, { k1=5, k2=5}}, can lead to overfitting and the failure to extract detailed local features. Therefore, to improve the effectiveness and efficiency of the experiment, we adopted the combination of k1=k2=3 in the following experiment.In this section, we analyze two hyperparameters, including k for controlling the perception fields and s for the kernel size of the linear operations of the feature mappings . The impacts of these two parameters were tested on the VGG16 model architecture. First, we set {s = 2, α=1, ɛ=6, k1=k2=3} and the hyperparameter s is set in the range of {2, 3, 4, 5, 6}. S is directly related to the computational costs of the obtained network. As analyzed in ‘The importance analysis of 1 × 1 convolution’ the larger s is, the lower the number of parameters and the computational costs. As the results in s = 2, our algorithm is even slightly better than the original model, but the number of Madds is only 43.12% of that of VGG16, which shows the superiority of plane bottlenecks in this paper. The constant number of parameters and the rapidly decreasing number of Madds prove the correctness of our derivations in After studying the convolution kernel sizes that are used in the proposed plane bottlenecks summarized, our remaining hyperparameters are set to {Caltech-101 consists of images with objects belonging to 101 classes, plus one background clutter class. Each image is labeled with a single object. Each class contains approximately 40 to 800 images or a total of approximately 9k images. The images have variable sizes, with typical edge lengths of 200–300 px. To study the performance of the classification task, we removed the background clutter in images and ended up with 101 classes. Since the data size is not fixed, we specifically conducted experiments with three different input sizes, namely, 192 × 192, 224 × 224, and 256 × 256. In contrast to training large models, we use fewer regularization and data augmentation techniques because small models have less trouble with overfitting. However, to be fair, we have performed experiments using data enhancement.Next, we use plane bottlenecks to customize our MobileNetV2 algorithm. We replace plane bottlenecks with plane bottlenecks, denoted as Plane-MobileNetV2; and use the Caltech-101 data set for our experiments. The standard MobileNetV2 has approximately 3.4M parameters and 300M Madds. MobileNetV2 is based on TensorFlow open-source code. To better study the substantial differences between replacing bulk, we remove the classification layer when counting the numbers of parameters and Madds. To make a fair comparison, settings such as the optimizer and the learning rate remain the same as those set in ‘Hyperparameter Strategy on CIFAR-10’. α  and ɛ  were conducted. More detailed data are given in the appendix. Overall, the number of Madds of our Plane-MobileNetV2 is approximately 52.30%∼78.83% that of MobileNetV2 in the same setting, but our accuracy is higher than that of MobileNetV2 in most of the width and expansion coefficient settings. In comparison, our method achieves significantly better performance. More details are given in the appendix.To better show the difference, we present the results in From the appendix, it is interesting to note that regardless of whether we are analyzing the data-enhanced or strengthened experiment, some common phenomena can be found: for a small-sized input image, the expansion coefficient for 3 can reach almost the same accuracy as that for 5 or 6; and for a large-sized input image, the expansion coefficient is the highest because the accuracy is almost the highest. These two interesting phenomena have constructive significance to guide us to construct PlaneNet.To verify the performance of PlaneNet on the ImageNet datasets, we conducted experiments on the ImageNetTE and ImageNetWoof classification tasks . ImageNe1=k2=3 in all plane bottlenecks. We choose some lightweight networks with good performance and fast speed, including the MobileNet series  64-bit SoC @1.5 GHz while 2b is BCM2836 Cortex-A7 (ARM v7) 32-bit SoC @900MHz. RAM for 4b is 4G while 2b is 1G, and the results are shown in As shown in To verify PlaneNet’s generalization ability on other computer vision tasks, we used the PASCAL VOC 2012 dataset to conduct experiments on semantic segmentation tasks . PASCAL vs. 0.47B Madds). However, PlaneNet’s mIOU is the highest except for that of ResNet50 (74.48% vs. 79.92%).In this section, we use the ResNet50, MnasNet, GhostNet, and MobileNet series as feature extractors to test semantic segmentation tasks on the main framework based on PSPNet. The ratio of the input image spatial resolution to the backbone output resolution is set as 16, that is, the layer with the fifth convolution step of 2 is changed to 1, and the dilation rate of all separable convolution layers after this layer is set as 2. We conducted experiments on the PASCAL VOC 2012 dataset using additional annotated images. We segmented 90% of the data as the training set and 10% as the validation set. The training loss function is the Dice loss with CE and does not use auxiliary branches for auxiliary training. The Adam optimizer with 1 = 0.9 and 2 = 0.999 is adopted. The initial learning rate is 0.001, and the learning rate is multiplied by 0.1 every 500 epochs. Padding is used to crop the images to 473  × 473. The advantage is that the images will not be distorted. We applied general data enhancement strategies such as flipping. Finally, the mIOU evaluation index is calculated on the verification set. Similarly, for the semantic segmentation task, we also calculate the Frames Per Second (FPS) for every backbone based on PSPNet. The highest index indicates the stronger real-time performance of the model. We still used the same platform in the classification network, and we chose four graphics cards: an RTX1060, an RTX2060S, an RTX5000, and a Titan V. Then, AMD R5-3600 and Intel I7-8750H CPUs were selected to test the operating effectiveness of our model in different environments. The experimental results are shown in To reduce the computational costs and improve the accuracy of a deep neural network, a new plane bottleneck module is proposed to construct an efficient neural network structure. The basic plane module is divided into three parts. The first part increases the dimensions using separable convolution, which effectively reduces the number of 1 × 1 convolutions. Then, the local features are extracted through point-by-point convolution. Finally, the plane features are obtained through separable convolution. Experiments on classification tasks and semantic segmentation tasks show that this module is a plug-and-play module that can reduce the calculation costs of the original model and obtain better performance to a certain extent. Additionally, the PlaneNet constructed using the proposed new module is superior to the most advanced portable neural networks in both efficiency and accuracy. The original data are given in


We did so by beading the capsid and then also by replacing each of its bulbous spikes with a single bead. However, each coronavirus spike is a glycoprotein trimer, and each spike bulb is triangular. In this work, we replace each bulbous coronavirus spike with a bead triplet, where each bead of the triplet is charged identically. This paper, thus, explores the role of bulb triangularity on the rotational diffusivity, an effect not previously considered. We thus use energy minimization for the spreading of triangular bulbs over the spherical capsid. The latter both translates and twists the coronavirus spikes relative to one another, and we then next arrive at the rotational diffusivity of the coronavirus particle in suspension, from first principles. We learn that the triangularity of the coronavirus spike bulb decreases its rotational diffusivity. For a typical peplomer population of 74, bulb triangularity decreases the rotational diffusivity by Recently, the rotational diffusivity of the coronavirus particle in suspension was calculated, from first principles, using general rigid bead-rod theory [M. A. Kanso, Phys. Fluids  I.ab initio calculations from general rigid bead-rod theory on coronaviruses is that the peplomer arrangement is not known. However, we do know that the spikes are charge-rich.Thomson problem, we mean determination of how identically charged particles repel and then spread over a sphere by minimizing system potential energy. This minimum system electrostatic potential energy, when divided by the sphere area, is not to be confused with surface energy.Recently, we calculated the rotational diffusivity of the coronavirus particle in suspension as a function of peplomer population, from first principles, using general rigid bead-rod theory  whose center is the center of mass and whose principal moments of inertia match those of the virus particle. The MIE, thus, determines the orientability of the virus particle, and thus, the virus rotational diffusivity. We use Eqs. (3)–(13) in Ref. For this work, we chose general rigid bead-rod theory for its flexibility and accuracy Sec.  and 9. UEqs. (3)–3 in Ref.Eqs. (3)–3 in Ref.III.small amplitude, general rigid bead-rod theory yields [Eq. (32) of Ref. In this paper, we focus on small-amplitude oscillatory shear flow (SAOS). For this flow field, for the molecular definition of minus the imaginary parts of the shear stress responses to small-amplitude oscillatory shear flow as functions of frequency, following Ferry (Secs. 2.A.4.-2.A.6. of Ref. et al. (Sec. 4.4 of Ref. The polymer contributions to the complex viscosity,As IV.As shown by Kirchdoerfer,A.Let B.ϵ is the dielectric permittivity. Using the constrained minimization approach of Ref. Let each vertex of each triangle Our trimeric model amounts to a replacement for the Thompson problem,V.From From ab initio calculation of coronavirus transport properties. Specifically, we have determined the rotational diffusivity, the property governing the particle alignment for cell attachment (see Sec. I of Ref. Whereas much prior work on fluid physics related to the virus has attacked transmission,et al.Chaurasia pleomorphism, we must eventually consider this too. Whereas this work considered the detailed triangular structure of the peplomer head as triads of three point-charges, we could also consider uniformly charged triangular rigid peplomers constrained to a sphere. By uniformly charged triangular, we mean that the charge would be uniformly distributed over the edges of the triangle rather than point charges at its vertices. We leave this task for a later date.Since the coronavirus capsid can be ellipsoidal (Fig. 3. of Ref. One cognate transport problem is the transient translation and twist of coronavirus spikes rearranging freely under their own electrostatic repulsions, for instance, the transient following the extraction of a single spike. This paper is, of course, silent on this interesting problem, which we leave for another day.As in our previous work,


RNase-HII) transcript which means that overall unresolved DNA–RNA hybrids can ultimately weaken DNA and cause skin lesions. Since the genome is actively transcribed, cellular RNase-HII is essential for removing RNA from the DNA–RNA hybrid in controls of genome stability and epigenome shaping and can be used as a causal prognostic marker in patients with psoriasis.Functional long non-coding RNAs (lncRNAs) have been in the limelight in aging research because short telomeres are associated with higher levels of TERRA (Telomeric Repeat containing RNA). The genomic instability, which leads to short telomeres, is a mechanism observed in cell aging and in a class of cancer cells. Psoriasis, a skin disease, is a disorder of epidermal keratinocytes, with altered telomerase activity. Research on the fraction of nascent RNAs in hybrid with DNA offers avenues for new strategies. Skin and blood samples from patients were fractionated to obtain the RNA associated with DNA as a R-loop structure. The higher amount of TERRA levels attached with each chromosome end was found with psoriasis patients in blood and skin. In addition to telomeric TERRA, we evidenced accumulation of others non-coding RNA, such as non-telomeric TERRA and centromeric transcripts. Increased levels of non-coding RNAs attached to DNA correlates with a decreased in Ribonuclease HII ( Psoriasis is a chronic, recurrent inflammatory disease of the skin characterized by abnormal proliferation of keratinocytes, vascular hyperplasia, and infiltration of inflammatory cells into the dermis and epidermis ,2. It isKeratinocytes normally proliferate over a period of 40 days, but in patients with psoriasis their proliferation accelerates to only 4–8 days . For manTelomeres consist of the double strand TTAGGG repeating units of DNA sequences associated with six telomere-specific proteins called Shelterin complex, three of them—Telomere Repeat binding Factor 1 (TRF1), Telomere Repeat binding Factor 2 (TRF2), and Protection of telomere 1 (POT1)—recognize repeated motifs to protect each chromosome ends ,17,19. AThe rate of telomere shortening may be accelerated by inflammation and oxidative stress in vitro and participate in the aging process . Wu et aIt is proposed that TERRA regulates the structure of telomeric chromatin ,32, but RNase HII) transcripts in the skin of the psoriasis patients. RNase HII present in all organisms is required to cleave RNA primers of RNA–DNA hybrid during DNA replication [RNase HII knockout mice are not viable, and mutations in the human genes cause uncleaved RNA–DNA hybrids with Aicardi–Goutières Syndrome, a severe inheritable neurodevelopmental disorder [We propose a model where higher levels of lncRNAs held in hybrid with DNA, including the ends of chromosomes, indicate epigenetic changes in the genome and could link to predisposition to the diseases. We recently reported  in normalication ,39. RNasdisorder . OverallMoreover, higher levels of DNA interactions with RNAs (strengthens) reveal epigenetic marks that distinguish the epigenome of patients with psoriasis from the epigenome of unaffected individuals.Patients diagnosed with psoriasis and healthy volunteers admitted to the Skin and Venereal Dermatology unit of the Faculty of Medicine at Erciyes University were included in this study after approval by the university’s Human Ethics Committee: Erciyes University with decision no 17/002.Skin biopsy samples were taken from the area with (LA) and without lesions (NL) from patients with psoriasis, who agreed to participate in the study admitted to the Department of Dermatology of the Faculty of Medicine at Erciyes University. The research method of the patients included in the study was prospective, and they had a clinical diagnosis of psoriasis. After calculating the psoriasis area severity index (PASI)  value ofp > 0.05).A total of 35 people over the age of 18 years old were included in the study, including 20 patients diagnosed with chronic plaque-type psoriasis and 15 healthy volunteers who were consistent with age and gender as a control group. The psoriasis patient group consisted of 10 women and 10 men patients and the healthy group consisted of 8 women and 7 men. The mean age of the control groups was 42.6 and 39.5 years, respectively. There is no statistically significant difference between the groups in terms of age and sex  of the volunteers in the control group, with a 2 mm punch tool after local anesthesia was applied under the skin, and completely sterile conditions were provided. Tissue samples from patients and controls were immediately delivered to the Genome and Stem Cell Center (GenKok) Genome Unit. Half of the samples were used for DNA isolation and DNA–RNA hybrid determination to assess TL and hybrid TERRA amount, and the other half for RNA isolation to determine the total expression of TERRA. The isolated samples were stored at −80 °C until used.www.zymoresearch.com (accessed on 24 January 2022) ZR-Duet TM DNA–RNA MiniPrep Plus Cat No: D7003, Irvine, CA, USA)), from tissue samples. The concentrations and purity of DNA, RNA, and DNA–RNA hybrid samples were measured in the Biotech Biospec-Nanodevice.DNA, RNA, and DNA–RNA hybrid isolation was performed in accordance with the manufacturer’s protocol, using the ZR-Duet DNA–RNA MiniPrep Plus Kit  device using appropriate primers (Tel F:CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGT and Tel R:GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT) and standards . Telomer® 480 Real Time PCR  device using previously reported [In order to determine the TERRA expression level from total RNA and DNA–RNA hybrid samples obtained from the patient and control groups; cDNA synthesis was performed using the EvoScript Reverse Transcriptase cDNA synthesis  kit. The cDNA samples of the total RNA and DNA–RNA hybrids of the patient and control groups obtained were determined in the Roche Light Cyclerreported  primers le study .Rad51 and RNAse HII genes in total RNA of tissues and blood samples.RT-qPCR experiments were performed using the high-throughput Light Cycler 480 II Real-Time PCR system . In order to determine the genes expression levels of total RNA samples obtained from patient and control groups, cDNA synthesis was performed using the EvoScript Reverse Transcriptase cDNA synthesis . cDNAs were diluted with nuclease free water in 1:5 ratio. SYBR Green Master Mix  was used to determine mRNA expression levels of Rad51 F: GGCCATTAGCCCTTCACCAT and Rad 51 R: TCTGCAAGTGGGACTTTCCT; RNase HII F: GACCCTATTGGAGAGCGAGC and RNase HII R: TATTTGACCCGCCCAAGCAT) were mixed and dispensed into 96 multi-well plates. The total volume was completed to 20 µL by adding 4 µL of cDNA on it. Thermal cycling conditions consisted of an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of 94 °C for 20 s, 60 °C for 20 s, 72 °C for 45 sn and 95 °C for 15 s, and, finally, melting curve was performed at 67 °C for 01 sn (melting curve) and cooled at 40 °C for 30 s. Human beta-actin (ACTB F: CTCGCCTTTGCCGATCC and ACTB R: TCTCCATGTCGTCCCAGTTG) was used as the reference gene. Changes in gene expression were determined using the 2−ΔΔCt method of relative quantification in all groups.The reaction mix was prepared according to the manufacturer’s instructions: 10 µL of 2t-test was applied to compare the differences in telomere lengths and TERRA expression of lesional and non-lesional tissues among patients. A One-Way ANOVA test was used to multiply compare Telomere lengths and TERRA expressions between patients and the control group. The relationships between quantitative data were evaluated by Spearman correlation analysis. Data were treated using Graphpad Prism  software. P values were considered statistically significant as <0.05. One-way ANOVA analysis of peak annotation data were carried out using the SPSS Statistics for Windows version 19.0 . The mean of all the sample groups psoriasis patients and healthy controls data were compared using ANOVA followed by Duncan’s multiple comparison tests. The data were presented as means ± SD.The suitability of the data for normal distribution was evaluated by histogram, q-q graphs, and Shapiro–Wilk test, and Variance homogeneity with Levene’s test. A two-sample DNA-associated RNA molecules were purified from nine skin sample biopsies (three Control (C); three lesional (LA); three non-lesional (NL). After digestion of DNase, 10–100 ng of RNA were obtained.Plateform Génomique Institut de Biologie – IBENS (ENS) Paris  performed libraries of all samples corresponding to DNA-associated RNA molecules and small RNA high-throughput sequencing ). Totally, 14 million (m) paired-end reads were mapped to unique sites in the human genome (1L), 20m (2L), 8m (1N), 17m (2N), 7m (2C), 8m (3C), and 10m (5C) per sample. Average length of the reads was about 75 nt. All the primary sequence characteristics, for sample libraries of the DNA-bound RNA sequences of psoriasis skin samples are summarized in http://genome.ucsc.edu, accessed date: 24 January 2022) [FastQC version 0.11.7 was used to do some quality control on the RAW sequencing data. Illumina adapters sequences removed by cutadapt v1.16. Based on the FastQC results, trimming of bad-quality reads was performed using Trimmomatic version 0.36  . Then alry 2022) ,48.Genome-wide locations of TERRA repeats were found by aligning sequences with lengths of 24 and 48 nucleotides composed of four and copies copies of TERRA to the reference genome using bowtie2 using “−a” argument to report all alignment ,37.p-value over local region <0.0001, false discovery rate (FDR) threshold <0.001).Statistically significant peaks of expression were identified using HOMER . To find enriched peaks and regions associated with nearby genes and genomic features in the genome using Homer, “findPeaks –o auto” called out was used to perform peak calling and transcript identification analysis. Then to annotating regions in the genome “annotatePeaks.pl” called for performing peak annotation. FDR cutoff of 0.001 used for significant peaks identification. Annotated positions for different regions plotted. The percentage of significant peaks located in each genomic region is visualized. Annotated positions for exons, intergenic, intron, promoter-TSS, and TTS are based on the hg38 genomic features Peak annotation.t-test with Welch’s corrections. Data are expressed as the median with p-values < 0.05 considered to be statistically significant. The standard error of the mean (SEM) represented as mean ± SD.Because of the rheostat-type  distributions and the limited number of patients, non-parametric methods were used, and a median value comparison was performed by Wilcoxon rank analysis. Statistical analyses were performed with an unpaired https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE188763 (accessed on 24 January 2022). Enter token axyfuoyyjbyjlkt into the box.To review GEO accession GSE188763: Go to p > 0.05). The mean of Psoriasis Area and Severity Index (PASI) of the patient group was 15.6.Twenty patients with chronic plaque psoriasis were included in the study. Ten of these patients were women and 10 were men. The healthy group consisted of 15 people, 8 women (53.33%) and 7 men (46.67%). Three sample groups of lesional (LA) and non-lesional (NL) psoriasis and healthy control were analyzed. The mean age of the control groups was 42.6 and 39.5 years, respectively. There is no statistically significant difference between the groups in terms of age and sex (p < 0.05). There was no significant difference in TL between PSO NL skin samples from patients with psoriasis and healthy controls  , it is nqYq, and XpYp see The aqueous phase extracted total RNAs (free fraction) from, both LA  and NL  skin samples from patients with psoriasis expressed TERRAs  in the relative levels expression of TERRA maintained to the chromosomes 1q, 2q, 7p, 9p, 10q, 13q, 15q, 17p, 18p, XqYq, XpYp, and sub-telomeric regions (The DNA-bound RNA hybrid (DRNA) obtained from PSO LA and PSO NL skin biopsy samples, compared to the control group shows a significant difference  was performed. Recently, we reported that with a conventional sperm RNA extraction protocol, unlike the free RNA fraction, a fraction made up of DNA–RNA hybrid molecules is tightly associated with the genome ,50. The To prepare the RNAs for the RNA-seq, the cutaneous cells were treated with proteinase K and then the DNA and RNAs fractions were recovered by column separation. The DNA retained on the columns was treated with DNase, and the RNA was released and further purified. The RNA fraction recovered from DNA is referred to as fraction D or DRNA for details see reference herein ,50. The We assessed RNA sequences from human psoriasis skin cells of Lesions (LA), Normal (NL) and Control (C) individuals. LA and NL biopsies were taken from the same individuals. We first focused on abundantly expressed TERRA transcripts. Interestingly, DRNA fraction from human somatic cells shows telomeric TERRA hybrids. To view genome-wide TERRA loci throughout the different chromosomes, DNA–RNA hybrid density tracks along with TERRA loci (shown as a green bar) have been visualized on the ends of two arms of most of human chromosomes . TERRA lRNA-seq expression profiling of LA, NL, and control samples shows that TERRA levels signals are relatively higher in LA and NL than from controls .This observation also confirms the RT-qPCR results according to which the DRNA level of TERRA in the LA and NL samples groups is much higher than the control samples groups.Finally, Costa et al., also reported a novel gene family, indicating that an ancestral gene, originated as a rearranged portion of the primate DEAD/H-Box Helicase 11 (DDX11) gene, and propagated along with many sub-telomeric locations of human chromosomes . Sub-telMoreover, we also reported in our previous study that in addition to telomeric TERRA regions there are some non-telomeric regions in sperm DRNA fractions . Non-telAnnotated positions of exons, intergenic, intron, promoter-TSS (Transcription Start Site), and TTS (Transcription Termination Site) were based on the hg38 assembly genomic features which shows that most of the peaks in skin DRNA samples fall into enhancer regions (intergenic and intronic regions) compared to others. The general transcripts of the regions: exon, promoter-TSS, and TTS are perfectly comparable levels in all samples . Thus, mOn the other hand, in accordance with the RT-qPCR technique results, higher expression values are also observed in non-lesional samples from psoriasis patients by the report to the control. But, the lesional samples profiling is more complex. More analysis is required to compare other genomic regions, such as ribosomal, etc., that can be considered for further studies.Centromeric regions (see below) also accumulate more DNA-bound RNA compared to controls not only in lesion samples but also in the healthy samples from patients with psoriasis .It is accepted that RNAs are associated with centromeric chromatin. In humans, R-loops are detected at centromeres in mitosis; and an R-loop-driven signaling pathway promotes faithful chromosome segregation and genome stability . It is aWith deep-sequencing analysis, we detect signals at the centromeric regions of chromosomes 4, 5, 12, 13, 20, 21, and Y, of all DNA-bound RNA samples with highly significant enriched repeat sequences of CCATT/GGTAA . Most ofThese observations are in accordance with previous reports that the centromeric sequences have a repetitive nature  . The cenOur hypothesis was that the accumulation of DNA–RNA hybrids more in patients with psoriasis may lead to chromosomal instability, which can result in the development of lesions in patients with psoriasis. Evaluation of the skin genome associated transcripts here by deep sequencing revealed that the amount of non-coding RNAs such as TERRA, non-telomeric TERRA, from intron and transcripts from centromeres are retained with DNA and is generally increased in psoriasis samples compared to controls .Rad51 and of Ribonuclease HII (RNase HII) in patients with psoriasis. The results are shown in RNase HII is highly significant especially in the LA tissues of psoriasis patients, in contrast the levels of Rad51 transcripts do not seem to increase. According to these results, correlation analysis was done and the positive and moderate relation was detected between telomere length and RNase HII expression in PSO lesional tissue (p = 0.001 rho = 0.688). Taken together, these results indicate that an increase in DNA-associated lncRNAs levels could be due to the decreased levels of RNase HII transcription in skin tissues of patients with psoriasis. In addition, we determined the level of expression of the RAD51 and RNase HII transcripts in blood samples from PSO patients and controls. Patients with PSO have a lower expression level of RAD51 and RNase HII transcripts compared to healthy control in blood; but these results are not significant.Here we report an increase in the level of several lncRNAs associated with DNA, which means an increase in the non-physiological formation of the R-loop that could trigger genome fragility. DNA repair protein RAD51 and its partner Breast Cancer 2 (BRCA2) interact with TERRA and promote the association of TERRA with short telomeres . On the Here, in clinically defined psoriasis, we have revealed, with molecular tools and genomic knowledge, modification of the retention levels of lncRNAs on DNA. We propose causal epigenomic mechanisms that add to the already described clinical pathways of phenotype and cytokines. The aim of this study was to search epigenetic signals in patients with psoriasis. Our hypothesis is that altered epigenetic signals are transcripts such as TERRA due to shorter telomere observed in psoriasis lesion. In fact, our results show a higher amount of TERRA molecules attached to the telomeres (psoriasis genome). Thus, similar to TERRA, the RNAs mediated epigenetics signals must be in this disease strongly attached to the DNA (genome). To achieve our goals, we are measuring the level of lncRNAs such as TERRA associated with telomeres and others long non-coding RNAs (non-telomeric TERRA and centromeric) in the genome of patients with psoriasis. We also know that psoriasis cells develop short telomeres after lesions, and must, therefore, contain one or more epigenetic changes that make them susceptible to lesions under certain conditions. Our results indicate that, indeed, lncRNAs, such as TERRA, centromeric, and others, RNAs are retained in R-loop at higher levels on the genome of patients with psoriasis than controls. The results demonstrated more than expected, an increase in overall R-loops in the genome indicates changes in the activity for removal of the RNA fraction of DNA–RNA hybrids. Indeed, the cellular transcripts levels of RNase HII essential for the degradation of RNA templets are reduced in patients with psoriasis. Here, we propose a way to predict the severity and occurrence of the lesion, or possibly to be used as a source of causation and development of inflammation due to the endogenous epigenetic changes.As it is known, telomeres, which are ribonucleoprotein structures at the ends of linear chromosomes, prevent the natural ends of chromosomes from being recognized as DNA breaks and can lead to inappropriate repair activities, such as telomere dysfunction, homologous recombination (HR), and non-homologous junction. Due to incomplete DNA replication and nucleolytic degradation, telomeres shorten with each replication cycle, ultimately leading to cell death, known as replicative senescence or apoptosis .In psoriasis, keratinocytes proliferate faster than in normal people. This situation is caused by the formation of keratinized tissue in patients with psoriasis. In fact, it is due to the increased rate of proliferation and accelerated death of keratinocytes resulting from this rapid cell proliferation, the barrier skin deteriorates, and inflammation occurs in patients. This indicates that either certain localized factors cause a further shortening of the telomere length in the lesion tissue, or because the keratinocytes in the lesioned area continue to divide faster than in normal skin.In this study, we used analysis of TERRA retention profiles on DNA of the skin as marker. Our studies revealed that the increased level of RNA attachment to telomeres correlated with the state of patients with psoriasis. We evidenced that the amount of TERRA associated with DNA was higher in patients with psoriasis even in non-lesional tissues (skin and blood) than in healthy controls. Interestingly, the overall levels of TERRA are not much changed but regulation of TERRA levels depends on the location of TERRA in loci associated with chromosomes. This highlights alterations in pathways that protect actively transcribed regions from genotoxic stresses. Here, it is observed localized epigenetic regulatory changes with development of TL abnormalities implicated in disease progression. In particular, damaged skins progress to telomeric shortening. These results supported the hypothesis that increasing the level of TERRA in the R-loop structure compromises the long-term maintenance of TL and cell survival. These keratinocytes cells from skins of psoriasis patients do not even over-express TERRA overall compared to the control, but are more retained in the genome at the telomeres. The amount of TERRA retained in the chromosome is already high in patients with psoriasis, and how TL is controlled in normal tissues compared to scarred spots requires further study.It has been reported that the lesions promote and accelerate cell proliferation in culture experiments. The downstream signaling pathway of cell growth acceleration has not yet been clarified, but several reports have shown that short telomeres play a role in triggering cell replication and cancer ,16,24. HRNase HII transcripts. The detailed mechanisms by which skin cells negatively regulate RNase HII transcription to let more TERRA in telomeres remain unknown. Further studies are needed to clarify the relationship between TERRA and intercellular signaling. In addition, TERRAs are not the only lncRNA retained with DNA in patients with psoriasis, in fact, centromeric regions and other regions also exhibit higher levels of RNA retention on DNA. This suggests an alteration of the general pathways of resolution of hybrid DNA–RNA structures due to robust down-regulation of RNase HII.We observed a relationship between TERRA retention and disease progression, with decreased levels of RNase HII enzyme, which removes transcripts from the genome before DNA replication. The reduced levels of RNase HII transcripts in the skin of patients with psoriasis explain the higher levels of accumulation of lncRNA with DNA. At the same time, this indicates dysregulation of RNase HII transcripts, but we do not know if initially, this is due to a general transcription alteration. Down-regulation at the transcript level could well occur with deregulation of small non-coding RNAs at post-transcriptional levels or with a silencing event at the start of transcription. RNase HII transcription analysis in patients with psoriasis compared to control could evidence the controlling factors.Several factors are involved in the regulation of physiological R-loop formation and resolution for review see references . Our finThe possibility of cancer risk in patients with psoriasis is mentioned in the literature. Lymphomas and keratinocyte cancer have been reported to be at increased risk in patients with psoriasis ,62. HoweFinally, we observe also more retention of abundant non-telomeric TERRA transcripts on the DNA, which confirms overall changes in RNA retention on the genome in patients with psoriasis.Psoriasis is not yet a curable disease, and current treatments block intermediary molecules that play a role in the mechanism of inflammation or reduce the severity of the disease by suppressing or reducing the proliferation of keratinocytes. Psoriasis seriously and negatively affects the quality of life of patients. Psoriasis is consistently considered an inflammatory disease and, at this stage, the causality is uncertain. With our results, it appears that the epigenetic mark is already present in unaffected tissues of patients with psoriasis. It is the first time that it has been revealed with epigenetic signals in the form of lncRNAs marking the genome in patients with psoriasis. On the other hand, in psoriasis cytokine pathways are mainly described and are related to immune diseases. We are opening the question of causality in the development of inflammation due to epigenomic changes in the regulation of the factors involved in genome integrity such as RNaseHII.Thus, there can be epigenetic mechanisms at play, and it is clearly very important to develop new strategies for the treatment of this disease .In conclusion, the levels of TERRA and centromeric regions were higher in the RNA attached to the DNA of patients with psoriasis, regardless of clinical stage, thus making the complex lncRNA as a potential epigenetic DRNA marker and a therapeutic target.


We propose that the diversity and instability of messenger RNAs, transcribed by RNA polymerase II, have conditioned the appearance of regulatory mechanisms based on different gene promoter strength and mRNA stability. However, for the regulation of ribosomal RNA levels, which are very stable and transcribed mainly by RNA polymerase I from only one promoter, different mechanisms act based on gene copy variation, and a much simpler regulation of the synthesis rate.In eukaryotic cells, three nuclear RNA polymerases (RNA pols) carry out the transcription from DNA to RNA, and they all seem to have evolved from a single enzyme present in the common ancestor with archaea. The multiplicity of eukaryotic RNA pols allows each one to remain specialized in the synthesis of a subset of transcripts, which are different in the function, length, cell abundance, diversity, and promoter organization of the corresponding genes. We hypothesize that this specialization of RNA pols has conditioned the evolution of the regulatory mechanisms used to transcribe each gene subset to cope with environmental changes. We herein present the example of the homeostatic regulation of transcript levels  A key step in the central dogma of molecular biology is the transcription of pieces of DNA information into RNA molecules, which will, in some cases, be translated into proteins but will remain, in other cases, as functional non-coding RNAs (ncRNAs). In all living systems, the transcription of cellular genomes is carried out by cellular multisubunit DNA-dependent RNA polymerases (RNA pols). Eubacteria and archaea possess a single such enzyme, while eukaryotes carry out nuclear transcription with at least three RNA pols with functional specialization by each one transcribing different non-overlapping subsets of genes. Although all these enzymes have originated from a common ancestral enzyme, the increasing complexity of genomes, cells, and organisms has imposed the evolution of transcription machineries to more sophisticated systems in terms of composition, interactions, selection of target genes, and regulation. In this mini review, we summarize the presumed evolutionary origin and functional reasons that have led to the multiplicity of nuclear RNA pols in eukaryotes, and its consequences for their regulation and the homeostasis of their different RNA products. We finally focus on the different adaptation of transcription regulation by eukaryotic RNA pols to changes in cellular volume. Other eukaryotic RNA pols aspects have been extensively reviewed elsewhere .All RNA pols, from eubacteria to higher eukaryotes, share basic mechanistic functioning: use of a DNA template, processive translocation on the template during RNA synthesis, utilization of ribonucleoside triphosphate as substrates, Watson–Crick base pairing of the new added nucleotide with the complementary one in the template DNA, and formation of a new phosphodiester bound by a metal-dependent mechanism. To perform these basic functions, all RNA pols contain two largest subunits  with douRNA pol gained greater complexity in terms of acquiring new subunits following the split of the eubacterial and archaeal–eukaryotic branches from the universal tree of life . ArchaeaArchaeal and eukaryotic lineages diverged more than 2 billion years ago, with eukaryotes originating from an archaeal linage with already diverse eukaryotic signature proteins . Other iSaccharomyces cerevisiae, and it is thought that they are good models for other eukaryotic RNA pols. For this reason, we use the names of yeast RNA pols genes and subunits throughout this review  of the largest catalytic subunit (Rpb1) of RNA pol II is also one source for innovation in mRNA transcription regulation and a mark of the eukaryotic lineage  in all eukaryotes is remarkable and suggests their early evolutionary divergence. At the same time, the substantial conservation of the central RNA pol core since LUCA indicates that it performs essential processes required for gene expression that allows very little innovation. Therefore, in order to generate complex eukaryotes, most evolutionary innovation is expected to occur in periphery subunits, especially in RNA pol II, which specifies the cellular proteome that confers unique characteristics to different cell types through mRNA synthesis. Additionally, the unique  lineage . CTD con lineage . Of all  lineage . It can  lineage . RNA pol lineage .cis-acting elements and ancillary factors for termination  has similar principles in all three nuclear RNA pols, the specific features of their transcription modes are reflected in their subunit structures. RNA pol II targets a large set of differently regulated genes, which requires the capacity to interact with a bigger set of transcription initiation and elongation factors than the other two RNA pols. Perhaps this was accomplished by having less permanent subunits than the other two RNA pols, but by also having dissociable subunits (Rpb4/7) and independent initiation and elongation factors , while the equivalent factors in other polymerases form an intrinsic part (subunits) of the RNA pol complex. For example, RNA pol I has a single promoter to recognize but requires high-speed, efficient elongation to avoid collisions between polymerases in its highly crowded genes . This ismination . In shorgulation .Chromatin imposes a major limitation to transcription by three eukaryotic RNA pols preventing their direct targeting to gene promoters, which probably explains why all nuclear RNA pols are first engaged in pre-initiation complexes before starting transcription. Pre-initiation complexes minimally consist of the TATA box-binding protein (TBP), which is common to all three transcription systems, initiation factors TFIIB (RNA pol II) and Brf1 (RNA pol III), and the RNA pol II-specific TFIIE factor . MoreoveTranslation machinery (ribosomes and tRNAs) synthesis requires the tight coordination among all nuclear RNA pols because rRNAs are synthesized by RNA pols I and III and ribosomal proteins are made from mRNAs transcribed by RNA pol II. Hence their coordination at all times and in all growth regimes is clearly necessary. The existence of five common subunits and one universal initiation factor, TBP, in all three RNA pols may be used to establish common regulatory mechanisms for nuclear transcription. RP mRNAs are some of the most abundant mRNAs in actively growing cells , and, thS. cerevisiae: the regulation of global RNA pol I and II activities with respect to changes in cell volume.The different properties of eukaryotic RNA pols and their RNA products predict that the regulatory mechanisms used by each one to cope with changes will be different. We discuss here an example that we have recently studied in yeast Homeostasis is defined as the state of steady internal conditions maintained by living beings and includes the control of concentrations of cell molecules. The terms ribostasis and proteostasis refer to the modulation of RNA and protein levels, respectively, in response to changes in the environment. Proteins are mostly the final goal of gene expression and are in charge of catalytic and structural functions. For this reason, their homeostasis is very strictly controlled, and the total protein concentration remains quite constant . NonetheHomeostasis deals with the molecular concentration, and not with the number of molecules. Therefore, changes in the cell volume are expected to provoke adaptation mechanisms to maintain homeostasis. In yeast cells, and probably in other organisms, volume varies depending on the genotype, the cell cycle phase , aging , ploidy Schizosaccharomyces pombe (S. cerevisiae, nascent TR remains constant by controlling the expression of RNA pol II coding genes, while mRNA stability increases to maintain mRNA ribostasis (S. cerevisiae resulting in two cells with different volumes: a small daughter cell and a large mother cell. In this scenario, the strategy adopted by eukaryotes with symmetric cell division, such as S. pombe or fibroblasts, is not applicable, as it would result in a higher mRNA net synthesis rate in small daughter cells (Studies carried out in different model organisms have established differences between transcription regulatory responses to cell volume depending on the organism and the RNA pol studied and suggest the existence of a size-sensing mechanism that produces alterations in transcription . Changeses pombe , RNA poles pombe . Thus, fbostasis . This dier cells . Howeverer cells . This moer cells .Saccharomycotina clade (Why is there a different solution for an identical problem in RNA pols I and II? We hypothesize that the differences in their targets, the 35S gene, and protein-encoding genes conditioned the evolution of different regulatory mechanisms for RNA pol I and RNA pol II. As the rRNA TR needs to reach much higher levels than that of any of the RNA pol II genes, eukaryotic cells evolved a specialized faster polymerase with a single gene template with many repeated copies. RNA pol is able to form extremely dense head-to-tail “camel caravans” in which the A49 subunit from one molecule contacts directly with A43 from the neighboring molecule. Thus, the specialized dimer A49/A43 allows a higher RNA pol loading rate than in RNA pol II . On the na clade , which cna clade .To summarize, eukaryotes differentiate from prokaryotes not only because of a more complex intracellular organization with nuclear and organelle evolutionarily independent genomes but also because the unprecedented job division occurs between several distinct nuclear RNA pols. The specialization of each one in the synthesis of a specific subset of transcripts with different abundance, stability, and function has forced differences in transcription initiation, elongation, termination, and regulation strategies but has provided, at the same time, the versatility to make phenotypically different cells from the same genome as a requisite for multicellular organisms.JP-O conceived the manuscript. PA, MB-A, and JP-O wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407–Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR140 (1–140 nt) highlight that the Thr367–Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein–RNA interaction as a “fuzzy” complex.The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the  HOX Transcript Antisense Intergenic RNA (HOTAIR) [Epigenetic regulator proteins play key roles in cell proliferation and differentiation. The enhancer of zeste homologue 2 (EZH2), a part of the gene-regulating polycomb repressive complex 2 (PRC2), is responsible for the trimethylation of histone H3 lysine 27 (H3K27), resulting in the silencing of several genes, including tumor suppressors. This effectively fuels cancer proliferation . Recent (HOTAIR) . This ln(HOTAIR) ,14,15. T(HOTAIR) ,17.Since EZH2 does not contain a canonical RNA-binding element, the location of the RNA-binding site on EZH2 is debated in the literature. Long et al. reported on different key binding residues throughout EZH2, most of which are located in the N-terminal helical region, and they noted that residues from other PRC2 components may be involved in RNA binding as well . Based oRNA-binding intrinsically disordered protein regions (IDRs) have received increasing attention during the previous years, as more and more information surfaces on their abundance and biological relevance ,26. Whil1HN-detected approach. Proline residues, which are more abundant in IDPs than in folded proteins [N protons, further complicate assignment strategies, especially if Pro–Pro motifs are present. Moreover, proline cis/trans isomerization might give rise to minor components, and a certain equilibrium is established [Since disordered proteins are famously recalcitrant to crystallize, NMR spectroscopy is the only method  to give proteins , and lacablished .140 (1–140 nt) lncRNA, localizing binding regions on the EZH2 loop.In the light of all these, we aimed to understand how the disordered loop of EZH2 recognizes one of its most important RNA partners, HOTAIR, and whether phosphorylation modifies the interaction. To achieve these goals, we performed a detailed atomic-level characterization, and we studied the changes caused by the phosphomimetic mutation and characterized the interaction with the HOTAIR1HN detected method is limited due to the fast chemical exchange of amide protons with water [1H,15N-HSQC measurement at 310 K, pH = 7.2 delivered only ~30% of the expected signals  are also clearly visible, as shown in 1H-1H couplings . Amide 15N and carbonyl 13C chemical shifts can be obtained from the 3D HCAN, its complementary 3D HCA(CO)N, as well as from the 3D HCA(N)CO experiments. The information gained from both 1HN- and 1Hα-detected approaches under these conditions result in a nearly complete backbone assignment, including 100% of 13Cα and 15N chemical shifts, with some 1HN, 1Hα, and carbonyl 13C missing due to severe signal overlap. As, at low temperature, the water peak is positioned favorably downfield from the 1Hα region, the classical measurements can be used without further modification. However, at 310 K, the water peak situated at 4.62 ppm is overlapping with the 1Hα region, and a loss of ~15% of signals in the 3D experiments is observed. To overcome this effect, improvements are needed. Solutions to obtain a more efficient water peak removal and a better phase behavior are: the addition of a weak presaturation, a pair of low power 90° pulses with phases x, y before the composite pulse decoupling (CPD) block on 1H [To perform sequential assignment, the already reported eriments ,35 were ck on 1H , a purgeck on 1H , and theck on 1H , as well1HN and 1Hα dimensions is only ca. 1 ppm  chemical shifts were determined using two methods that consider the effect of both temperature and pH. The predictor developed by Kjaergaard et al. uses experimentally determined chemical shifts of short peptides [The signal dispersion of both a. 1 ppm a,b, india. 1 ppm b. This ipeptides ,39, whilpeptides . As the Results show that the two predictors give a similar picture , with SCT345D phosphomimetic mutant. This substitution introduces a negative charge, mimicking the Thr345-phosphorylated protein. Investigations for EZH2T345D were performed at 278 K and 310 K, using both 1HN- and 1Hα-detected assignment strategies. Comparison shows that 15N, 1HN, and 13Cα chemical shift differences between the wild type and T345D mutant are insignificant. The exception is the region in the vicinity of the mutation; the backbone chemical shifts of Pro346 suffer the highest deviations (BMRB Entry 51421). SCS values of 13Cα atoms calculated with the predictor of Kjaergaard et al. [It was previously documented that phosphorylation of Thr345 increases affinity towards RNA . As a fid et al. ,39 and Pd et al.   region, indicating that the nucleotide bases are not involved in H-bonds. The appearance of broad signals in the presence of Mg2+ confirms the presence of H-bonded nucleotide bases, although the majority of these signals appear over 11 ppm, suggesting the formation of Watson–Crick type base pairs instead of quadruplex structure  and charged residues (21% negatively and 17% positively charged) together with the depletion of aromatic (1%) and hydrophobic residues (13%) as well as cysteines (1%) suggests that this protein segment lacks a stable 3D fold ,45. Inde1HN-detected approach fails to deliver satisfactory results at physiological conditions, 1Hα-detected experiments, with further enhanced sensitivity and resolution, can represent a way of reaching the peak assignment in aqueous solutions with 5–10% D2O, at 310 K and pH = 7.0. This finding is of general validity for any isotopically labeled IDP.Regarding the applied experimental set-up, we showed that, while the Analysis of the determined chemical shifts experimentally proved the highly disordered behavior of the EZH2 loop. An α-helical tendency is observed in the Ser408–Ser412 region, and the high disorder is maintained over a large temperature range. This lack of transient structural elements is relatively uncommon even amongst IDPs. EZH2 maintains its highly disordered state even when bound to RNA, and the nascent helical region does not play any role in the interaction. As a counterexample, the disordered p53 transactivation domain possesses nascent helical regions, which undergo a disorder-to-order transition upon binding, while other regions remain fuzzy in the complex .cis-proline minors was also investigated. For most of the 10 proline residues, we found that the amount of minor cis-Pro peaks follows the empirical regularities established from the statistical analysis [cis conformation lower than 5% due to the presence of positively charged residues in the proline neighborhood at (i − 3 and i ± 1 positions relative to Pro); indeed, these minors could not be detected. Six proline residues  should give rise to over 5% cis conformer, and these do not contain positively charged amino acids at i + 3 and i ± 1 positions, but are abundant in polar and negatively charged residues in the proline neighborhood. Note that aromatic residues, that can increase cis-proline ratio, are not present in the proline proximity in the EZH2 loop. Amongst these six expected cis conformers, five were detected with such a ratio, while the minor form of Pro427 could not be detected.The appearance of analysis . Four prcis conformer. The significant downfield shift of the cis-Pro346 1Hα signal indicates a more pronounced turn-like behavior, suggesting that interactions of EZH2 may be affected or regulated via this phenomenon.Regarding the phosphomimetic T345D mutant, results revealed that the overall disordered nature of the EZH2 loop is not affected, and that only local changes occur. The neighboring Pro346 is mostly affected, especially its minor 140 interaction shows that, upon binding, EZH2 remains highly unstructured, thus suggesting that a fuzzy complex is formed. Fuzzy interactions are typical to IDP complexes and involve varying levels of flexibility within the bound structures [Investigation of the EZH2 loop–HOTAIRructures . The binructures  such as ructures . We also140 lncRNA. Given that the investigated region of EZH2 is localized on the surface of the PRC2 complex, it is ideally placed to serve as a first recognition point during the RNA-binding process. RNA molecules anchored to this region then have the opportunity to form high-affinity interactions with other, more difficult-to-access binding surfaces on the PRC2  and subcloned into pET22b (+) cloning vector. The T345D mutant was generated using site directed mutagenesis method . The pET-22b (+) vector containing the appropriate construct was transformed into competent E. coli BL21 * (DE3) pLysS cells and grown in LB medium containing 0.05 mg/mL carbenicillin overnight at 37 °C with shaking at 180 rpm. After inoculation with the starter cell culture into fresh LB medium containing 0.05 mg/mL carbenicillin, the cells were grown to OD600 = 0.8 and transferred one hour prior to induction to M9 minimal medium, complemented with 15N-labeled ammonium chloride and 13C-labeled glucose . Induction was done for 4 h at 37 °C by 0.5 mM IPTG, then cells were pelleted by centrifugation  and incubated at room temperature for 30 min in lysis buffer  pH 7.5 and EDTA-free SIGMAFAST Protease Inhibitor Cocktail Tablets, Merck KGaA, Darmstadt, Germany) with vigorous shaking. After sonication, the cell debris was removed by centrifugation . The supernatant was filtered through 0.2 μm nitrocellulose filter then purified over HisTrap HP  column on an AKTA Explorer system using a gradient elution of two buffers . Representative purification results are shown on The same methods of protein overexpression and purification were used for both protein constructs, EZH2140 (1–140 nt) DNA sequence cloned into pEX-A128 vector was purchased from Eurofins Genomics . After 2 h digestion with EcoRV restriction enzyme at 37 °C, the gel-purified, linearized DNA template was used to synthesize RNA by in vitro transcription carried out with New England BioLabs HiScribe™ T7 Quick High Yield RNA Synthesis Kit . After transcription, remaining DNA template was eliminated with DNaseI treatment. RNA sample purification was carried out using Macherey-Nagel NucleoSpin® RNA Clean-up XS Kit . The quality and intactness of the purified transcription product was analysed by native and formaldehyde agarose gel electrophoresis. Final RNA concentration was determined using Implen NanoPhotometer™ N60 Spectrophotometer . Purified RNA was stored at −80 °C until usage in the presence of RNAINH-RO Roche Protector RNase Inhibitor (20 U). Before usage the RNA sample was refolded by incubation at 75 °C for 5 min and then allowed to cool to room temperature.HOTAIR3, then 5% D2O and 1% DSS  were added. Final concentration of the protein was (0.5–1.0) mM in each NMR sample. For protein–RNA-binding studies, the 15N labelled wild type or the T345D mutant EZH2 loop was dissolved in assay buffer  containing 10 mM TCEP and 3 mM NaNffer see  containi1H, 176.03 MHz for 13C, 70.94 MHz for 15N) equipped with a Prodigy TCI H&F-C/N-D probehead . 1HN-detected experiments for backbone assignment at 278 K were: 2D 1H-15N correlations (Fast-HSQC [1Hα-detected experiments: 2D SHACA-HSQC [All NMR measurements were performed on a Bruker Avance III 700 MHz spectrometer (700.05 MHz for ast-HSQC  and SOFAast-HSQC ), BEST-tast-HSQC ,53 ACA-HSQC ,56,57, aACA-HSQC  were conACA-HSQC . Tempera


Our results demonstrate that all RNA mutants display different levels of decreased replication and translation initiation efficiency in vitro. The translation defect was correlated with significant reduced viral titer of mutant particles in comparison with the wild type. When inoculated in mice, mutant viruses were checked for inflammation and necrosis.In vitro and in vivo Findings strongly suggest that the most attenuated mutant strain could be considered a candidate for live-attenuated CVB3 vaccine.Coxsackievirus B3 (CVB3) is a principal causative agent of viral myocarditis, meningitis and pancreatitis. There is no vaccine available for clinical use. It has been demonstrated that the primary molecular determinant of virulence phenotype is located in the 5’ UTR of the viral genome. Translation initiation of CVB3 RNA is directed by the IRES element situated in the 5’UTR. In the present study, we analyse the effects of single point mutations introduced in different positions in the domain V of the IRES RNA of CVB3 wild type. We characterize  Picornaviridae family, a large and complex family of small RNA viruses.Enteroviruses infecting humans are assigned to four species, Enterovirus A to Enterovirus D. Coxsackieviruses B, six serotypes of which are known (CVB1 to CVB6) belong to the Enterovirus B species . During the 1960, Albert Sabin obtained attenuated strains of each poliovirus serotype, which were unable to productively infect and destroy neuronal cells . Each ofCVB3 virus replicates in murine host and induces diseases, which mimic those in humans. The mouse model is useful for the study of EV pathogenesis  methionine/ml , and 80 μM unlabeled amino acids . Added MgCl2 and KCl were optimized on the bases of the RRL used from 0.2 to 1.2 mM and 50 to 100 mM, respectively. Concentrations of HeLa cell extracts were varied from 0 to 20% (vol/vol), and that of H100 buffer  was varied from 33 to 13% (vol/vol) of the final reaction volume. Reactions were programmed with uncapped mRNAs at concentration of 10 μg/ml, and essay reaction mixtures were incubated for 1h at 30°C. Reactions were stopped by adding stop RNase solution and incubated for 10 min at room temperature before adding of blue solution . Translation products were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis by using gels containing 15% (wt/vol) polyacrylamide. Quantification of translation of the viral polyprotein before its cleavage was carried out by densitometry of autoradiograms by using NIH image software (Image J 1.34s), with multiple exposures of each radiogram to ensure that the linear response range of the film was respected.4 cells per well were inoculated with the CVB3 wild type or with one of produced mutants at the same multiplicity of infection of 10 TCID50%. Transfected plates were frozen at different times of post infection . Virus titers of each mutant and wild type were determined by Reed and Munch method on HeLa cells using 96 well plates.Virus single-step growth cycles in HeLa monolayer cells were performed at 37°C as described previously , 31. Twe5 TCID50 of either CVB3 viruses (mutants and wild type viruses) previously titrated on HeLa cell monolayers. Control mice (uninfected mice) obtained 0.3 ml of BME medium only. Mice controls were bred in the Biotechnology Institute of Monastir animal facility. All animal experiments complied with the recommendations of the Committee of Ethics of the institution in a protocol approved and supervised by the committee. The protocol was controlled and supervised by of a member of the committee. Animal experiences were conducted with making all efforts to minimize suffering animals during anesthesia.Female Swiss mice (3 to 4 weeks old) were purchased from the Pasteur Institute of Tunis, Tunisia. All mice were divided separately in 3 groups and rested until body weights were between 12 and 20 g per mouse. Mice were inoculated by oral route using a sterile polyethylene tube and syringe with 5 x 10Mice were sacrificed every day from 0 to 21 days post inoculation. Heart and pancreas tissues were fixed in formalin for paraffin embedding. Serial 3–5 μm thick sections of different organ tissues were stained with eosin and hematoxylin and examined for the presence of inflammatory lesions by light microscopy.P< 0.05) using Graphpad Prism 6 Version 6.02.Student’s t test was used to establish statistical significance of three (n = 3) replicated experiments  or RNA encoding wild type of CVB3 were transfected into HeLa cells, which are known to support productive CVB3 infection. Seventy two hours later, supernatants were stocked and infectious virus titers were determined by Reed and Muench method. Viral supernatant from cells transfected with the M3, M4, M5 and M6 RNAs yielded viruses with almost1 log decreased titers, suggesting that mutations introduced within the stem-loop V of the IRES of these mutants were attenuating. However, infectious viruses of M1 and M2 mutants were detected with titers comparable to that of wild type CVB3, suggesting that the virus mutants were not severely disabled in tissue culture .All mutant virus stocks were compared to wild type of CVB3 by the use of single-step growth cycles. Stocks of wild type of CVB3 and mutant viruses of similar titers, were used to infect HeLa cells at an MOI of 10  and samples were harvested at 3, 5, 7, 10, 15 and 20 h p.i. and then titred by Reed and Munch method. As demonstrated in However, M3, M4, M5 and M6 CVB3 mutants were severely handicapped in HeLa cell monolayers, as demonstrated by their single-step growth cycles showed in in vitro in a RRL supplemented with different increasing concentrations of S10 cytoplasmic extracts of HeLa cells (0 to 20%) and with a 10 μg/ml as RNA template in order to investigate the translation efficiencies of the different produced RNA mutants. The quantification of all polyproteins generated by each IRES RNA mutants was conducted by the measure of the intensity of bands obtained by SDS-PAGE  and M4 (20%) RNA mutants, a little translation was detected. Moreover, their RNA mutants showed low sensitivity to the addition of non-canonical factors, which prove that the 2 mutants were affected in their translation. M5 RNA mutant directed translation at low efficiency (62.67% compared with the level of wild type). In addition, M6 RNA mutant revealed a dramatic decrease in translation efficiency with the same conditions. M6 RNA mutant was translated only at 42.13% comparing with the wild type RNA .In order to determine whether the attenuated replication and translation of CVB3 mutants in HeLa cell corresponded to an attenuation of the virulent phenotype of the wild type CVB3 strain, mice we inoculated by oral route . Heart and pancreas tissues were evaluated for evidence of pathologic damage. Mice tissues stained with hematoxylin and eosin in thick sections were examined by light microscopy. Tissue damages of heart and pancreas due to virulent CVB3 replication has been demonstrated previously to be evident at 10 days post inoculation . ResultsThe IRES sequence belonging to the 5’UTR of PV has a modular structure in which especially stem-loop domain V regulate replication and translation of the viral genome . MutatioPartially attenuated CVB3 strain was obtained by long-term passage of a virulent CVB3 strain in host cell culture . A singl5’UTR of EV is a highly ordered structure among different species and serotypes. Because it is considered as the central of both transcription and translation of the viral genome, our main objective of this present study was the study of the relationship between the IRES stem-loop V of CVB3 and the degrees of replication and translation efficiencies. More previous study demonstrated that in poliovirus 5’UTR, a single nucleotide mutations defined by nt 472 to 481 is significantly and perturb RNA structures . These 5Our results were based on phenotype analyses of 6 CVB3 mutants whose genomes harbour one to six nucleotide substitutions  compared to the genome of wild type CVB3 strain. Secondary structure prediction of the region surrounding the IRES domain V (nucleotide position 470 to 530) of all mutants and the wild type, using mFOLD prediction software reveals that there is no alteration in the RNA structure in mutants comparing to the wild type. Properties growth of these six CVB3 mutants and wild type CVB3 were investigated by carrying out a one-step growth cycles study in HeLa cells. The M1 and M2 mutants displayed respectively a 1 to 1.5 log unit reduction of viral particles production in HeLa cells. The other 4 CVB3 mutants showed reduction of their replication. They displayed a 3 to 4.5 log unit reduction of infectious particles production in HeLa cells comparing with CVB3 wild type.in vitro in a cell-free system and to correlate it to the viral production capacities. The cellular factors necessary for EV translation are present in sufficient amounts in HeLa cell S10 extract but not in RRL used in our experience. Thus, the in vitro system is useful for the study of the cap independent translation and the identification of cellular factors necessary. Indeed, using this system, the M1 and M2 mutated RNA templates directed translation as efficiency as wild type RNA. However, a significant reduction in the translation efficiency of the M3, M4, M5 and M6 RNA was observed in in vitro translation experiments. Our results of in vitro translation suggest that the RNA of M1 and M2 CVB3 mutants interact with translation machinery present in HeLa cells with the same manner of that of the wild type RNA template. However, the four other mutants, especially the M4mutant present a pronounced level of defect in translation that is more important than those of the M1 and M2 mutants. Even if the nucleotide changes in domain V of the M3, M4, M5 and M6 RNAs may not influence the overall RNA structure of the IRES element, the altered nucleotide sequences of these four mutants stop the RNA interaction to proteins involved in translation initiation. This RNA-protein stopped interaction may then be responsible for the reduced levels of viral protein synthesis observed.A previous work conducted by Bhattacharyya and Das  observedin vitro translation in standard RRL lysate and in prolonged infection in mice model. Indeed, 10 days after oral inoculation, CVB3 wild type induce widespread inflammatory lesions in heart and pancreas in infected mice, whereas M5, M6 and especially M4 mutant viruses demonstrate no evidence of inflammatory disease neither in heart or pancreas of orally inoculated mice. Results of our in vivo study were comparable to those obtained for other well-characterized, attenuated strains of CVB3 [The molecular genetic information that determine the CVB3 pathogenic phenotype have been remained unsolved. Results of the present study support our originally hypothesis witch suppose that the CVB3 5’UTR of the genome comprises one or more primary genetic determinants witch influence strongly the biological properties of CVB3. Worldwide poliomyelitis vaccination effort to eradicate wild polioviruses moves to completion, it is important to evaluate alternative HEV other than poliovirus to fill this gap. Thus, it is important to explore methodology to attenuate HEV like CVB3 for the use as potential vaccine candidates. In the present study, we demonstrate that introduced mutations within the domain V of the IRES of a virulent strain of CVB3 leads to different avirulent mutant viruses. Our produced mutated viruses leads to different levels of attenuation in replication on cell cultures,  of CVB3 . AdditioS1 FigShown are murine heart (a) and pancreas (b) tissues stained with hematoxylin and eosin from mice inoculated with CVB3 M4 mutant, (c-d) heart and pancreas from mice inoculated with CVB3 M5 mutant and (e-f) heart and pancreas from mice inoculated with M6 virus.(DOCX)Click here for additional data file.


Enhanced replication of rubella virus (RuV) and replicons by de novo synthesized viral structural proteins has been previously described. Such enhancement can occur by viral capsid proteins (CP) alone in trans. It is not clear whether the CP in the virus particles, i.e., the exogenous CP, modulate viral genome replication. In this study, we found that exogenous RuV CP also enhanced viral genome replication, either when used to package replicons or when mixed with RNA during transfection. We demonstrated that CP does not affect the translation efficiency from genomic (gRNA) or subgenomic RNA (sgRNA), the intracellular distribution of the non-structural proteins (NSP), or sgRNA synthesis. Significantly active RNA replication was observed in transfections supplemented with recombinant CP (rCP), which was supported by accumulated genomic negative-strand RNA. rCP was found to restore replication of a few mutants in NSP but failed to fully restore replicons known to have defects in the positive-strand RNA synthesis. By monitoring the amount of RuV RNA following transfection, we found that all RuV replicon RNAs were well-retained in the presence of rCP within 24 h of post-transfection, compared to non-RuV RNA. These results suggest that the exogenous RuV CP increases efficiency of early viral genome replication by modulating the stage(s) prior to and/or at the initiation of negative-strand RNA synthesis, possibly through a general mechanism such as protecting viral RNA. Rubella virus (RuV) is a member of the Rubivirus genus within the Matonavirus family . RuV is Studies of RuV RNA replication were expedited after development of infectious cDNA clones and replicons ,10,11. EThe dissociation of the RuV genome from the nucleocapsids after entry is not fully understood. It was suggested that the phosphorylation of RuV CP in the virions is critical in facilitating the release of viral genome from the nucleocapsids during disassembly, probably because of the less stable interaction between the CP and RuV RNA ,18. WhilThe mechanism(s) by which RuV CP modulate genome replication is not fully understood. RuV CP have been found to colocalize with RuV NSP ,24 or viBased on the previous finding that GFP expression was only detected in RuV-infected cells transfected with RUBrep/GFP_ΔNotI , a repliWe hypothesized that the RuV SP in the pseudovirus particles were promoting the replication of RUBrep/GFP_ΔNotI after entering cells. To test this, RuV antigens, including UV-inactivated RuV virus  and virus-like particles , were added to the Lipofectamine-RUBrep/GFP_ΔNotI RNA mixture during transfection. By the end of transfection, the monolayers were subjected to three PBS washes to remove excess external materials. As shown in 3 pfu/mL) if the RNA was mixed with VLP , with and without RuV antigen, was also compared. When RuV antigen was added to a transfection with the pCH110, no significant increase was observed in the intracellular β-galactosidase activity (relative activity of 0.91 ± 0.28)  and SINV, from Vero cells transfected with viral RNA with and without RuV antigens was compared. The viral RNAs, PV RNA transcribed in vitro from infectious cDNA clones  or total ± 0.28) . Taken tPreviously, we have shown that de novo synthesized CP enhanced RuV genome replication . To confGFP expression was detectable if the rCP was added 4 h before or after the transfection of RUBrep/GFP_ΔNotI RNA. If the RNA and rCP were added 24 h apart, no GFP was detected, showing that the window of CP accompanying RuV RNA is also important (data not shown).® rabbit reticulocyte lysate with FluoroTect™ GreenLys labeling system  in the presence of rCP or rE2. The constructs included RUBrep/GFP (a full-length replicon), genomic mini-Xpress (g41-GFP or g1700-GFP), a subgenomic mini-Xpress (sg-GFP), and a control construct pCI-GFP, in which the GFP gene was expressed from the early promoter of human cytomegalovirus (CMV) (Promega)  . A speciPromega) . No diffPromega) , indicatThe effect of rCP on translation was also assessed in transfected cells with RuV mini-Xpress. The fluorescent proteins in g-GFP and sg-GFP transfected cells were detected within 4 h after adding the RNA. Supplementing rCP did not make a difference in the amount of GFP from the experiments with rE2 for up to 4 d post-transfection . The samOne proposed mechanism of intracellular CP-enhanced RuV replication was by chaperoning viral NSP to the endosomal membrane . This waAt 18 h, aggregates of the fluorescent punctate pattern were noticed. This shift of RFP distribution also occurred in cells with rE2, as well as in cells transfected with non-replicating replicons. Thus, this shift is apparently independent from the replication of replicon RNA   . Thus, tThe data presented above indicated that RuV rCP enhances RuV replication before or during viral RNA synthesis. To closely examine which stage of viral RNA synthesis rCP is most effective, replication of RuV replicons known to have specific defects during genome replication was assessed in the presence of rCP or rE2. These replicons included RUBrep/GFP (WT), RUBrep/GFP_ΔNotI (or RUBrep/GFP/NSP-Rfp), RUBrep/GFP-1301S, and RUBrep/GFP-RdRp*. RUBrep/GFP-1301S is an NSP cleavage mutant with amino acid 1301 (a glycine) changed to serine. This change completely abolished the processing of NSP polyprotein precursor (p200). As a result, the synthesis of the (+) gRNA, but not the (−) RNA, was greatly reduced .Replication of these replicons was first assessed by the dsRNA synthesis by IFA A. In celConsistent with the detection of dsRNA, (−) RNA was readily detected in the replication-competent replicons with rCP, while it was hardly seen with rE2 by Northern hybridization C. No (−)rCP exerted marginal impact on sgRNA synthesis: from the replication of RUBrep/GFP, the ratio of (+) RNA to sgRNA was 0.86 ± 0.01 with rCP, and 0.77 ± 0.01 with rE2 at 3 to 6 d post-transfection from four independent experiments. To confirm this observation, we compared the replication of rubella replicons with the intergenic region (IR), which contained the putative sgRNA promoter  replacedN-acetyl-transferase (PAC), of which the replication capability can be measured by the number of puromycin-resistant cells such that the dependence on rCP can be quantifiable , were mixed with Lipofectamine 2000 in Opti-MEM followed by inoculation onto Vero cells for 4 h in a 37 °C, COTransfection with RuV replicons encoding a puromycin-resistant gene was carried out using BHK cells in 96-well plates. Cells were trypsinized and seeded in 6-well plates at the end of transfection, grown in DMEM with 5% FBS, and subjected to puromycin selection (2 μg/mL) at 24 h post-transfection. The numbers of surviving cells were counted using a hemocytometer and the cell colonies were subjected to crystal violet staining at 6 to 7 d post-transfection.7G(5′)ppp(5′)G RNA cap analog (New England Biolabs) as described previously .The intracellular fluorescence was measured using a FluoroSkan Ascent Microplate fluorometer (Thermo Fisher Scientific). Cell monolayers were washed once with Opti-MEM and stained with 2.5 μg/mL Hoechst 33258 (Thermo Fisher Scientific) in a 37 °C, 5% COTo assess β-galactosidase in pCH110-transfected cells , cellular proteins from 5 d post-transfected cells were collected following three freeze-thaw cycles in PBS. The amount of intracellular β-galactosidase was determined from approximately one-third of the lysate using the Luminescent β-galactosidase detection kit II  and was analyzed by a TD-20/20 Luminometer .Quantification of RuV RNA by Northern hybridization was carried out by densitometry scanning of the chemiluminescent signal after exposures on X-ray films by UVP BioImaging Systems. The quantification was analyzed using LabWorks 4.0 Image Acquisition and Analysis software .−ΔΔCT). The ΔCt at a later time point n was used to compare with the ΔCt value at 0 h (ΔCt0) to obtain the difference, ΔΔCtn. The ΔΔCtn was plugged into the equation of 2−ΔΔCt to calculate the change in the amount of target RNA over a period of time. The relative ΔΔCt (rΔΔCtn = 2−ΔΔCt_rCP/2−ΔΔCt_rE2) was used to indicate the ratio of the amount of input RNA in the presence of rCP to the amount of RNA in the presence of rE2 at the specific time point. Quantification of PV RNA was performed using a real-time RT-PCR assay for the 3Dpol coding region, and PV titer was determined by standard plaque assay as described previously [Intracellular RNA retention was determined using the comparative CT method (ΔΔCt). In vitro transcripts were treated with two cycles of DNase I (Promega) followed by cleaning using an RNAeasy kit  prior to transfection. The GFP RNA was detected with primers and probe specific to the GFP reporter gene  . The houeviously .® v.9  from at least three independent experiments. A statistical value p < 0.05 was considered significant.Unless specified, the effects of rCP on the reporter gene expression, amount of RNA, or antibiotic resistant cells were presented by the quantitative comparison using the data from experiments with supplemented rCP to the experiments with supplemented rE2 (rCP/rE2). All data were graphed using Microsoft Excel or GraphPad Prism v.9. Kolmogorov–Smirnov test and Student’s T-test were performed using GraphPad Prism