Numerous studies have focused on the classification of N6-methyladenosine (m6A) modification sites in RNA sequences, treating it as a multi-feature extraction task. In these studies, the incorporation of physicochemical properties of nucleotides has been applied to enhance recognition efficacy. However, the introduction of excessive supplementary information may introduce noise to the RNA sequence features, and the utilization of sequence similarity information remains underexplored. In this research, we present a novel method for RNA m6A modification site recognition called M6ATMR. Our approach relies solely on sequence information, leveraging Transformer to guide the reconstruction of the sequence similarity matrix, thereby enhancing feature representation. Initially, M6ATMR encodes RNA sequences using 3-mers to generate the sequence similarity matrix. Meanwhile, Transformer is applied to extract sequence structure graphs for each RNA sequence. Subsequently, to capture low-dimensional representations of similarity matrices and structure graphs, we introduce a graph self-correlation convolution block. These representations are then fused and reconstructed through the local-global fusion block. Notably, we adopt iteratively updated sequence structure graphs to continuously optimize the similarity matrix, thereby constraining the end-to-end feature extraction process. Finally, we employ the random forest (RF) algorithm for identifying m6A modification sites based on the reconstructed features. Experimental results demonstrate that M6ATMR achieves promising performance by solely utilizing RNA sequences for m6A modification site identification. Our proposed method can be considered an effective complement to existing RNA m6A modification site recognition approaches. To date, approximately 160 chemical modifications have been discerned in RNA, substantially enriching the diversity of RNA function and genetic information . Among tHigh-throughput sequencing techniques have been extensively utilized for the identification of m6A modification sites, including m6A sequencing , crossliThe computational identification of RNA m6A modification sites can be broadly categorized into two groups: machine learning-based methods and deep learning-based methods. Both approaches share a common core, which is the feature extraction task, aimed at capturing better representations of RNA sequences for improved recognition performance. Machine learning-based methods typically involve two main stages: feature engineering and downstream classification. In feature engineering, various coding approaches have been applied to represent RNA sequences, including k-mer, one-hot coding, accumulated nucleotide frequency , composiThese studies have made significant progress in identifying RNA m6A modification sites. However, they also have limitations. For instance, incorporating additional information, like the physicochemical properties of nucleotides, alongside RNA sequences may introduce potential information interference. Moreover, these methods have mainly focused on learning features from sequential nucleotide distributions, potentially overlooking associations of nucleotides through self-correlations in RNA sequences. To address these issues, we propose a novel approach in this article, named m6ATMR, for RNA m6A modification site recognition. m6ATMR utilizes Transformer  to guideThe main contributions of this article are as follows: First, we propose a sequence-based approach for identifying RNA m6A modification sites without introducing potentially misleading additional information. Second, the similarity matrices of RNA sequences are computed to provide more effective information that can be learned for sequences, and on this basis, the Transformer is used to reconstruct the similarity matrices and further optimize the sequence representations. Third, we propose a graph self-correlation convolution to learn a low-dimensional representation of the sequence without introducing prior information about the nodes. A series of experiments demonstrate the effectiveness of the representation strategies of M6ATMR. For M6ATMR, it is worth noting that relying solely on RNA sequences for m6A modification site identification can reduce the need for additional prior information, while still ensuring high identification accuracy. In addition, the sequence representations learned by our model perform consistently well across different classifiers, indicating that our method is not dependent on the choice of a specific classifier. In conclusion, our experimental results demonstrate that M6ATMR achieves excellent performance in identifying m6A modification sites using only RNA sequences. This highlights its effectiveness as a complementary method for RNA m6A modification site recognition.Arabidopsis thaliana (A101 dataset)   for studM6ATMR implements the identification task of m6A modification sites based on several procedures. First, the RNA sequences were processed into readable coding representation based on k-mer algorithm, and the similarity between RNA sequences was measured by Manhattan distance to further construct the similarity matrix. After that, M6ATMR learned the structural information of RNA sequences through the Transformer encoder, and converts this structural information into the structural graph by Manhattan distance. It then inputted the similarity matrix and structure graph into the self-correlation graph neural network to iteratively update the similarity matrix and obtain the low-dimensional representations of each RNA sequence. These low-dimensional representations were passed through a local-global fusion block to generate the final fusion representation, which was fed into the RF for identification. For the convenience of description, section 2 describes M6ATMR in detail from four parts: similarity matrix calculation, structure graph learning, similarity matrix optimization, and local-global representation fusion. The framework of M6ATMR is shown in For similarity matrix calculation, the key step is to transform RNA sequences into the readable representations using k-mer frequency statistics. Statistical k-mer frequency information can reveal the distribution law of seed sequences and is an important tool to study sequence similarity. In sequence After representing all RNA sequences using statistical k-mer frequency, we attempt to calculate the similarity between each sequence and construct an initial similarity matrix of RNA sequences. We construct matrices between k-mer representations for the following reasons: Firstly, earlier studies on the prediction of biomedical entity associations  have shoWhen obtaining the initial similarity matrices of the RNA sequences, we use a Transformer encoder to capture the structural information and learn the structural graph of RNA sequences. To this end, we apply the encoder part of the Transformer to further process the k-mer sequence. For a given Transformer encoder, there are two basic components: the position encoding block, and the self-attention mechanism. In addition, in order to further explore the structural relationship between RNA sequences, we also calculate the Manhattan distance between vector representations of the output of the Transformer encoding block, and strictly constrain the value of the structure matrix within the set of Position encoding of Transformer is a functional encoder, that is, position vectors are calculated for each element in the sequence:In this regard, we believe that it is a valid way to describe the structural association of RNA sequences through internal relationships captured by the self-attention mechanism. To this end, we also measure this structural associations by the same method in section 2.3 and construct the structure graph For each element To optimize the sequence similarity matrix and update the sequence structure graph, we design a self-correlation graph neural network that does not depend on prior node representations. In traditional graph neural networks, the embedded learning process relies on the existing representations of the nodes or edges in the graph. These prior representations serve as the starting point for the learning algorithm to update and refine the embeddings based on the graph structure. For instance, in some studies related to drug-drug association prediction, the SMILES of drugs are often applied as the prior information to serve as the initial input to the graph neural network. However, for similar matrix and structure graphs of sequences, the initial information of nodes is difficult to be obtained. In addition, both the similarity matrix and the structure graph describe the self-correlation property of the sequence, which makes the introduction of additional prior information may lead to misleading information. Therefore, inspired by the self-attention mechanism, we generate the learnable initial node representations based on the input matrix information. The initial representations are constantly updated and optimized during the process of graph convolution and participate in the optimization of the final embeddings. Taking the processing of similarity matrix as an example, we describe in detail the learning process of representation of similarity graph.For a given similarity matrix In addition, another task of the networks is to get better RNA representations by optimizing the similarity matrix. Hence, we apply the reconstructed similarity matrix and sequence structure graph to calculate the loss of the networks:In order to obtain comprehensive knowledge of RNA sequences, we propose a local-global strategy for fusing learned embedded representations. We process two kinds of representations of the same sequence respectively from the local and global perspectives of embedding representations, and further determine the weight relationship between embedding representations from similarity matrices and embedding representations from sequence structure graphs. Specifically, we design a local information extraction block and a global information extraction block respectively to calculate the weights of the two types of embedded representations.For a given similarity matrix embedding representation We then enter We utilize this weight difference to further integrate the two types of embedded representations to obtain the similar-structural representation In addition, we consider the indispensable role of the k-mer representations of the sequences for the recognition of m6A modification sites, and further integrate these representations with the similar-structural representations to obtain the final embedding representations For a binary classification problem, its prediction states can be divided into the four categories: true positive (TP), false positive (FP), true negative (TN), false negative (FN). Thus, we select some predictive indicators to evaluate the prediction effect, and the calculation processes of these indicators are as below. Moreover, we obtain the area under the precision-recall curve (AUPR) and area under the receiver-operating characteristic curve (AUC) for evaluating our model.In our study, we set the learning rate to 0.0001 and set the number of layers as three in the self-correlation graph neural network. Considering the time and complexity of training, we reduced the number of heads from eight to six in the Transformer encoder. We set the embedding size of the encoder to 32, the hidden dimension of the feed-forward layer to 128, and the number of encoder blocks to six.We conduct a rigorous 10-fold cross validation to evaluate the performance of our proposed model on the A101 dataset. The dataset is systematically partitioned into ten subsets of equal size, ensuring non-overlapping test sets in each fold. For each fold, we utilize nine subsets for training and one for testing. During evaluation, we consider six essential indicators: AUPR, AUC, Acc, F1 score, Prec, and Sen. The results of the 10-fold cross validation are presented through PR curves and ROC curves in In some studies, the downstream classifiers have shown to significantly influence the classification performance of RNA sequence representations generated by models, potentially leading to model instability. To demonstrate the stability and efficacy of our proposed model, we conduct a comparison experiments using five additional classifiers: logistic regression, eXtreme Gradient Boosting (XGBoost), Light Gradient Boosting Machine (lightgbm), CatBoost, and support vector machines (SVM). Logistic regression employs maximum likelihood estimation to predict model parameters, yielding binary results by minimizing cross-entropy loss during data training. XGBoost, an integrated classification algorithm, employs multiple simple base learners to iteratively train input data, continually reducing the discrepancy between model and input values. In contrast, lightgbm stands out with its advantage of low memory usage and faster training speed. CatBoost is designed to extract the most information from given data and is particularly effective for small machine-learning datasets. SVM, as a binary classification model, aims to find an optimal hyperplane for sample segmentation. For evaluation, we utilize 10-fold cross validation on the A101 dataset, as mentioned in “Performance on A101 datasets”. The same set of indicators, AUPR, AUC, Acc, F1 score, Prec, and Sen are employed for assessment. The experimental results, presented in In this study, we integrate three types of features for comprehensive analysis, including feature representations from similarity matrices, feature representations from sequence structure diagrams, and k-mer sequence representations. K-mer representations are pre-coded representations based on the frequency count of sequence k-mer substrings, while the other two features are dynamically learned through neural networks. To achieve a holistic understanding of the sequences, we perform local-global integration of these features. To illustrate the importance of combining these three features, we construct two additional features, drop-raw and drop-trans, to validate our model’s performance. Drop-raw represents features without similarity matrix information, and drop-trans represents features without sequence structure graph information. The comparison results of the three types of features are presented in In this study, we conduct a rigorous 10-fold cross validation to compare our model with five other existing methods on the A101 dataset. The compared methods include M6AMRFS, BERMP, RFAthM6A, BERT-m7G , and theIn this article, we commence by reviewing classical methods for identifying RNA m6A modification sites and presenting our own perspectives. Subsequently, we analyze the limitations of these methods, leading us to propose a novel sequence-dependent-only RNA m6A modification site recognition method, named M6ATMR. M6ATMR utilizes the Transformer to guide the reconstruction of similarity matrices for each RNA sequence, thereby optimizing the feature representation of RNA sequences. Comparative analysis with other recognition methods reveals that M6ATMR demonstrates superior predictive performance, as evidenced by improved metrics. Comprehensive experiments further attest to the accuracy and robustness of our model. Additionally, we delve into several critical aspects. First, computing the similarity matrix and optimizing feature generation proves effective in enhancing the recognition performance of RNA m6A modification sites. Second, cooperative updating of similarity matrices and sequence structure graphs in the sequence representation of the same RNA sequence facilitates the retention of richer nucleotide distribution information. Third, the deep fusion of multiple features from both local and global perspectives results in a comprehensive understanding of RNA sequences.However, there remain certain limitations in our study that warrant attention. First, the restriction of RNA sequence length necessitates the selection of the A101 dataset for model verification, rendering our approach less adept at handling short RNA sequences. Second, our current model primarily focuses on nucleotide distribution information, with limited exploration of other sequence properties. Future work will address these issues and explore the application of our model to the identification of other modification types, such as M1A, and modifications on DNA sequences.10.7717/peerj.15899/supp-1Supplemental Information 1Click here for additional data file.


RNA-binding proteins (RBPs) represent a large family of proteins with an extensive array of roles that contribute to coordinating and directing multiple functions in RNA metabolism and transcription. Currently, there are upwards of 1500 RBPs, and unravelling their range of complex functions in cell biology is an ongoing research effort, where there is still much to discover. Although RBPs primarily interact with RNA in both the nucleus and cytoplasm, these interactions often have a wider influence on cell biology, including effects on genomic stability and critical cell protein levels. Recent studies have shed light on specific RBPs and their roles in cancer, but the detailed molecular mechanisms responsible for the reported roles of RBPs in tumorigenesis have hitherto been largely unexplored. This Special Issue, entitled “RNA-Binding Proteins and Their Emerging Roles in Cancer” in the International Journal of Molecular Sciences (IJMS), includes seven contributions, three original articles, and four reviews covering a wide range of highly relevant topics in RBP biology and cancer. These include cancer therapies, post-transcriptional regulation and genome integrity, non-coding RNA regulation, RNA and DNA damage responses, and R-loop biology. A summary of the contributions that make up this recently published IJMS Special Issue are provided below.Nasiri-Aghdam et al.  providedCDKN1A) expression, which is mediated by the effects of MBNL2 on the modulation of p21 mRNA stability. They presented evidence that these effects are independent of the p53 protein [Another small family of RBPs that regulate alternative splicing, such as the CELF RBPs, are the muscleblind-like (MBNL) RBPs, MBNL1, MBNL2, and MBNL3. The multi-functional roles of MBNL RBPs were evidenced in a research article by Cai et al. [The review provided by Sidali et al.  was concLöblein et al.  producedDolicka et al.  presenteUruci et al.  providedNascakova et al.  providedA common theme that is evident in the publications in this Special Issue is that RBPs are present in both the cytoplasm and the nucleus, often shuttling between these two cell compartments and other cytosolic compartments such as stress granules and P bodies. These observations are consistent with multiple potential regulatory functions for individual RBPs in RNA transcription and metabolism. Increasing evidence is emerging that links mutations in RBPs to tumorigenesis. There is evidence that some RBPs can regulate the stability of the RNAs involved in cell cycle control and apoptosis. Additionally, certain RBPs appear to be involved in maintaining genomic stability through direct or indirect mechanisms. At least some RBPs have roles in the regulation of R-loops, which are increasingly considered to be major sources of genomic instability. A picture is emerging of a complicated interplay between the RBPs regulating multiple functions in RNA transcription and metabolism that are also crucial for maintaining genomic stability and avoiding unwanted effects on the genome, such as the generation of R-loops when RNA transcription conflicts with DNA replication. Interestingly, mounting data identify RNA/DNA hybrids as immunogenic species that abnormally accumulate in the cytoplasm following R-loop processing. Future research into the functions of RBPs in controlling these cytoplasmic RNA/DNA hybrids, the resulting innate immune activation, and its associations with many disorders, including cancer, would be intriguing.To date, most RBPs have generally been considered as non-tractable for therapeutic purposes. Novel approaches, such as proteolysis-targeting chimera (PROTAC) strategies, may enable selected RBPs to soon become tractable. It is likely in the future that new therapeutic strategies coupled with more detailed analyses of tumour molecular signatures and the growth of personalised medicine will result in selected RBPs becoming useful therapeutic targets in cancer therapy.As Guest Editors for this IJMS Special Issue, we are pleased to present the collection of riveting articles focused on the topic of RBPs in cancer. This collection of articles develops the idea that RBPs have significant effects on genomic stability and cellular homeostasis, which have an impact on cancer at many levels and through various pathways. We thank the authors for their excellent contributions that add fresh perspectives to this fascinating and vitally important field of genome biology.


Here, we analysed the importance of the methyltransferase METTL3 for the life cycle of this virus. We found that METTL3 interacts with the EBOV nucleoprotein and the transcriptional activator VP30 to support viral RNA synthesis, and that METTL3 is recruited into EBOV inclusions bodies, where viral RNA synthesis occurs. Analysis of the m6A methylation pattern of EBOV mRNAs showed that they are methylated by METTL3. Further studies revealed that METTL3 interaction with the viral nucleoprotein, as well as its importance for RNA synthesis and protein expression, is also observed for other haemorrhagic fever viruses such as Junín virus (JUNV) and Crimean-Congo haemorrhagic fever virus (CCHFV). The negative effects on viral RNA synthesis due to loss of m6A methylation are independent of innate immune sensing, as METTL3 knockout did not affect type I interferon induction in response to viral RNA synthesis or infection. Our results suggest a novel function for m6A that is conserved among diverse haemorrhagic fever-causing viruses , making METTL3 a promising target for broadly-acting antivirals.N Methylation occurs co-transcriptionally and is mediated by the m6A writer complex, consisting of the methyltransferase dimer of METTL3 (the active methyltransferase) and METTL14  . S. SMETTL3After having demonstrated the importance of METTL3 for viral RNA synthesis using life cycle modelling systems, we next confirmed these data in the context of an infection with authentic EBOV. Parental, negCtrl and METTL3 KO 293 T cell lines were infected with recombinant EBOV expressing firefly luciferase from an additional transcriptional unit , and rep6A methylation pattern of both cellular and EBOV mRNA by extracting mRNA from rgEBOV-infected negCtrl and METTL3 KO 293 T cells and subjecting it to miCLIP analysis [6A-specific antibody with methylated RNA allows specific purification of m6A-methylated RNA, but impairs reverse transcription, resulting in either the introduction of mutations within the DRACH motif, or in truncation of the transcript near the methylation site. As expected, we observed a strong reduction in the number of reads mapping to cellular mRNAs in the METTL3 KO cells compared to those in the negCtrl cells, confirming that we had functionally impaired the m6A methylation pathway in the KO cells . Further, we readily observed reads that aligned with EBOV sequences in infected negCtrl cells, but hardly any such reads were found in METTL3 KO cells  V, which is known to be a strong inhibitor of both IFN-β induction and signalling  Figure D and CCH6A influences their viral life cycle, albeit with very different outcomes. Antiviral roles have been shown for some viruses, such as flaviviruses or Kaposi sarcoma virus [It has been described for many viruses that mma virus ,41, but ma virus ,16,36. H6A in cellular mRNAs in our KO cell lines. Furthermore, we only used low passages of our KO cell clones, as extensive passaging increases the chances for a functional complementation. Also, the METTL3 KO clones showed reduced growth in comparison to parental and negCtrl cell lines, suggesting that METTL3 is absent in these low passages. Finally, inhibition of METTL3 with STM2457 led to the same results as METTL3 KO, confirming the results obtained in the KO cell lines.Recently, generation of METTL3 KO cell lines has been shown to result in expression of alternative METTL3 versions from alternative spliceoforms . While w6A methylation of RNAs has been described to inhibit the cellular immune response, and specifically IFN-β induction, either by masking viral RNAs so that they resemble host RNAs or by preventing the formation of dsRNAs that would trigger activation of RIG-I and MDA5 [6A methylation of viral RNAs has a different function in the life cycles of EBOV, JUNV and CCHFV than has been described for other NSVs. Rather, other known functions of m6A methylation are more likely responsible for the negative effect of METTL3 KO on the life cycles of the analysed viruses, such as its impact on RNA stability, translational efficiency and/or mRNA export [The phenotypes observed in our study show parallels to those observed upon methyltransferase depletion or deletion in other NSV infections ,16,36. Hand MDA5 ,16. Our A export ,9,43–45.6A methylation of cellular mRNAs can promote their nuclear export via the nuclear RNA export factor 1 (NXF1) pathway through interaction of the m6A reader YTHDC1 with the export adapter SRSF3 [6A methylated viral RNAs from the nucleus [In particular, mer SRSF3 . Also, h nucleus . We coul nucleus ,47, and  nucleus . Indeed, nucleus .6A readers like YTHDF to m6A mRNA has been shown to cause liquid–liquid phase separation of YTHDF-mRNA complexes [6A mRNA might support the phase separation process or help to separate mRNAs from other RNA-binding proteins like NP, which might also explain the localization of METTL3 in punctae within the inclusion bodies.Furthermore, binding of momplexes . As inclomplexes ,50,51, t6A [6A has been suggested to enhance translation through different methods, all involving recruitment of eukaryotic initiation factor 3 (eIF3), which has also been shown to interact with EBOV VP35, although the functional consequences of this interaction are not well understood [Reduction of mRNA stability was one of the first described functions of m6A , but accderstood ,53.6A, has been shown to protect mice from an otherwise lethal EBOV infection [6A. In order to identify the exact function of m6A in the life cycles of these viruses, further studies assessing the importance of other components of the m6A writer complex like RBM15 and WTAP, which we identified as important host factors for EBOV RNA synthesis in a genome-wide siRNA screen [6A reader proteins will be required. Nevertheless, the broad range of viruses for which m6A methylation seems to play an important role in facilitating their life cycle suggests that the m6A machinery is a promising target for broadly-acting antiviral drugs.Taken together, we have shown that METTL3 methylates EBOV mRNAs, is recruited to EBOV inclusion bodies as the sites of viral RNA synthesis, and plays an important role in viral RNA synthesis of not only EBOV but also other haemorrhagic fever-causing NSVs (i.e. JUNV and CCHFV). Further, we show that a METTL3 inhibitor, which has been shown to be effective against acute myeloid leukaemia and in an improved version is currently undergoing clinical trials for solid tumours (identifier NCT05584111) , leads tnfection . HoweverA screen , but alsClick here for additional data file.Click here for additional data file.


Reconstruction of postglacial sea-level rise using reef cores recovered from Tahiti during IODP Expedition 310 showed that the first major acceleration, known as Meltwater Pulse 1a (MWP-1a), was a 12–22 m rise in 340 years starting at 14.65 ka BP. Although it was reported that the pulse did not drown Tahitian reefs, the subsequent discovery of a fringing reef at the base of several cores implies that its timing, magnitude and impact require revision. Here, we report facies and paleodepth data from this reef, revise sea level, and revisit reef response. We find its reef crest is dominated by surf-adapted corals to a depth of 2.5 m and show that it retreated upslope over an approximately 1000-year interval from 16 ka. Reef development then apparently ceased at 15 ka at −106 m and remained absent for approximately 600 years, before resuming at 14.4 ka further upslope at −93 m. This absence is consistent with reef drowning and requires that MWP-1a had a smaller magnitude of 13.8 ± 1.3 m, and may have started 300 years earlier than previously reported. It confirms MWP-1a was a global event, drowning reefs on Tahiti as well as those in other oceans. One oAcropora palmata, which extend to depths of 5 m or less, and are mixed with other corals below this [The drowning of shallow reefs is dependent upon the rate of SL rise exceeding the rate of reef-accretion for an extended period of time, until the crest is submerged below its habitat zone and enters the adjacent zone further downslope . As a colow this –19. If tlow this .et al. [The requirement for a distinct and depth-restricted zonation in reef-crest assemblages has posed difficulties in identifying and constraining meltwater pulses in other postglacial reef sequences. At Tahiti, cores from the barrier reef at Papeete recovered a thick reef-crest sequence of robust acroporid and pocilloporid corals intergrown with crustose coralline algae (CCA) and vermetids ,20. Baseet al.  used theet al. ,25.in-situ corals and their reefal units, and refine the minimum SL reconstruction. We then reassess the impact of MWP-1a on fringing-reef development.This uncertainty in identifying the onset of MWP-1a at Tahiti has been compounded by the discovery of a fringing-reef unit at a higher elevation in cores from marginal sites , which w. 2. 2.1et al. [et al. [et al. [et al. [The IODP Expedition 310 to Tahiti recovered reef cores mostly from two offshore sites: Tiarei on the north coast with 20 cores and Maraa on the south coast with 15 . These het al.  reported [et al.  and Desc [et al.  reported [et al.  combined. 2.2et al. [in-situ corals, from detrital units composed predominantly of grain-supported clasts  given that this is minimal over the approximately 2 ka timescale considered here . We usee common , but note common . By combe common ,28, we de common . Elevati [et al.  in coresollowing ,31).. 2.3Although reconstruction of coral paleodepth in postglacial reef sequences has relied on the depth habitat of their modern counterparts ,33, indi. 3. 3.1We characterized detrital and framework units at the base of 23 cores recovered from Tiarei (16 holes) and Maara  bioeroded colonies of in-situ Pocillopora spp.  with closely spaced, short, compact branches and large basal attachments, commonly intergrown with thin encrusting sheets of Montipora sp.  wavy sheets of encrusting montiporids with millimetre-thin crusts of CCA intergrown with small vermetids, including the larger Dendropoma maxima.At Marginal Sites 10, 11 and 12, and the Inner-Ridge Site 23, the basal unit is a 0.7–1.0 m-thick coralgal framestone with a dense biofabric and a cemented interstitial matrix of pebbly skeletal-volcanic sand . Two biopora sp. . Seconda 3.2. 3.2.1.Pocillopora biofacies is only found in Hole M0007A where it forms a thin layer (25 cm) with a dense biofabric of small, highly bioeroded colonies encrusted by millimetre-thin intergrowths of CCA and vermetids in a cemented skeletal sand matrix  layer of coralgal framestone composed of either the compact-branch pocilloporid or encrusting montiporid biofacies . The Pocd matrix . In Holecolonies .Figure . 3.2.2Overlying the coralgal framestones at Sites 5 and 7, and forming the basal layer at Sites 15, 17 and 18 is a consolidated and thinner version (0.3–0.8 m) of the gravelly coralgal bindstone found at Tiarei. At Sites 15 and 17, this facies also contains small- to medium-sized head corals .. 3.3Pocillopora (40%) and Acropora (13%), with subordinate encrusting Montipora (19%), but that corals only have a live cover of approximately 20%, with the rest of the substrate covered by CCA [Pocillopora found at Sites 7, 12 and 23 are particularly useful as environmental indicators because inter-branch spacing is related to environmental gradients in wave energy [P. damicornis, for example, changes to a compact-branching morphospecies as it shallows into the breaker zone [Pocillopora found at Sites 7, 12 and 23 can be reliably interpreted as having developed under surf-zone conditions. The extent of this habitat can be calculated independently from the wave climate around Tahiti, where fair-weather waves of approximately 1.5 m [The dense coralgal framestone facies, with its biofabric of encrusting and compact-branch corals, is analogous to assemblages that today colonise high-energy shore-attached fringing reefs around Moorea, Tahiti's sister island . Early ed by CCA . Althouge energy . The comker zone –39. Furtillopora % and Acrly 1.5 m  produce in-situ corals in these units with the highest coeval corals in all cores (Montipora framestone biofacies ranges in depth from 1–5 m (n = 5). The association of encrusting montiporids sheets with the surf-adapted pocilloporids shows that they inhabit the surf zone, but their presence as a capping layer over surf-zone corals at Sites 12 and 23 implies that this biofacies extends below surf-base to a depth of approximately 5 m. It is more difficult to interpret why compact-branch pocilloporids are absent in the encrusting Montipora framestone biofacies in Holes 10A and 11A at Tiarei, and 5D and 7B at Maraa. One possibility is a higher sediment flux in the surf zones at these sites, which also explains the absence of acroporids and limited development of CCA in these facies compared to the modern analogue.For the basal facies that lack surf-adapted corals, we reconstruct paleowater depth by comparing age-elevation data of ll cores . This rein-situ corals in the gravelly coralgal bindstone facies, which forms the basal unit at all other sites, grew at depths of 0–15 m (n = 7). The presence of encrusting sheets of Montipora in this predominantly detrital facies confirms their tolerance to high sediment flux and suggests an inter-reefal habitat at or below fair-weather wave base, where the sea floor was dominated by intermittent sediment deposition  in Hole 9A  and terminating shortly before 14.4 ka (14.31 ka BP), giving a minimum rise rate of 23 mm yrt Tahiti . The amo (Le Roy ), amount. 4Previous analysis of the basal unit reported that a fringing-reef unit was present in all cores from Tiarei , but ourPrevious assessment of SL from the Tahitian cores reported that the onset of MWP-1a occurred at 14.7 ka 14.65 ka BP), based on basal corals and their encrusters at Maraa Site 15, and mid-sequence corals at Tiarei Site 24 5 ka BP, ,43,44, w−1 [We argue that the onset of MWP-1a occurred between 15.0 and 14.7 ka (14.94 and 14.65 ka BP). This interval represents the age difference between the youngest surf-adapted coral in the 12A fringing reef, and the ages of corals in Holes 15A and 24A used previously to constrain its onset . The unc−1 .et al. [Acropora sp. prior to approximately 14 ka, this supports a drop-out sequence in reef-crest assemblages, with acroporids and crustose corallines being the least tolerant to sedimentation, pocilloporids moderately tolerant, and montiporids most tolerant of all. This might also explain the restriction of fringing-reef development to the Tiarei Marginal Sites, where the steeper gradient of the Pleistocene substrate likely led to a reduced sediment flux compared to level substrates around the Inner- and Outer-Ridge Sites.The response of fringing-reef development to this meltwater pulse can be further assessed by considering the offset between its accretion rate and that of SL rise. For example, Blanchon et al.  conjectuet al. ,46. This−1, but over this interval the accretion rate of the fringing-reef unit is 4 mm yr−1 or less, signifying that it was forced to retreat upslope to keep pace with the rise. This retreat was facilitated by the 2.5 m-depth habitat in which surf-adapted corals survive, which provided a buffer to SL rise and gave the reef sufficient time to leave a deposit as it retreated upslope. This is because the residence time of the reef surface in the habitat range is controlled by the rate of SL rise, and so there is an inverse relation between the thickness of the fringing-reef deposit and the rate of SL rise [−1 during MWP-1a, which was apparently sufficient to prevent the fringing reef from leaving a deposit. But visiting Tahiti in the midst of the pulse would still have given a normal picture of reef development, with surf-zone corals fringing the coast. The only difference would be that drilling into those zones would show a thin intermittent layer of corals encrusting the substrate, rather than a substantive, metre-thick reef framework.The suppressed accretion rate of the fringing reef also explains its upslope retreat in response to SL rise. As shown by our reconstruction in  SL rise . When th−1, would have accreted 4 m in 250 years, reducing water depth over the crest to between 4 and 7 m, which is insufficient to completely submerge it below its 6 m habitat range and into a deeper assemblage. Instead, the only evidence of the pulse would be an increased accretion rate over that interval.The threshold at which the rate of SL rise could induce the drowning of a reef therefore depends on both the depth habitat of the crest assemblage and its accretion rate. The best combination for constraining the timing and magnitude of a MWP-like event would be a shallow-depth habitat and/or low accretion rate, which would promote the more rapid onset of drowning. Both of these characteristics are present in the Tiarei fringing-reef unit, making it particularly well-suited for constraining MWP-1a. Conversely, for a rapidly accreting reef-crest assemblage with a larger depth habitat, like that of Tahiti's barrier-reef sequence at Papeete, a similar meltwater pulse would take longer to cause functional drowning because of the longer residence time in the deeper reef-crest habitat. Therefore, there will be an accretion-depth threshold above which the MWP will not be detected in the sequence because it was neither rapid nor large enough to submerge the reef surface into the adjacent habitat zone. This may explain the absence of MWP-1b in the Papeete barrier-reef sequence , especia. 5Previous assessment of the onset timing, rate and magnitude of MWP-1a from Tahiti's offshore reef sequence was not based on the identification of a distinct, depth-restricted reef-crest assemblage, but instead used deeper corals that colonised the sea floor after the meltwater pulse had started. The discovery of a fringing-reef unit with surf-adapted corals in Marginal Sites at Tiarei, and its detailed dating and sedimentary analysis presented here, now shows that MWP-1a had a smaller 13.8 ± 1.3 m magnitude, started as much as 300 years earlier (15 ka), and led to the drowning and back-stepping of this fringing reef. Even though the meltwater pulse may have lasted longer than previously thought, it still produced the same drowning response in shallow reef-crest habitats on Tahiti as it did on reefs elsewhere, like Hawaii and Barbados.It follows therefore that distinctive, depth-restricted reef-crest units, like those reported here, are key to identifying and accurately constraining the rate and magnitude of SL rise during postglacial meltwater pulses. This stems from the restricted amount of accretion that can occur as SL rise submerges the reef-crest zone into the adjacent zone further downslope. In the optimal case, a unit of surf-adapted corals growing within 2–3 m of SL has a low accretion potential during a meltwater pulse and fails to produce a recognizable deposit, thereby undergoing functional drowning. In the less-optimal case, a more diverse reef-crest assemblage occupying a deeper habitat with a higher accretion potential cannot be fully submerged into the downslope zone by the same pulse. Sub-metre dating of these units could potentially identify pulses from increased accretion rates, but the accuracy of this approach is limited by the larger depth range of these reef-crest habitats. Therefore, it should be expected that different reef-crest assemblages with different accretion rates will produce different responses to the same meltwater pulse, with some able to maintain reef crests within their depth habitats, and others failing to do so and subsequently drowning. In the case of MWP-1a, the rate and magnitude of the pulse was clearly sufficient to cause reef drowning on a global scale.


Arabidopsis, corn, and rice) and their interacting pathogens, the key transporters responsible for sugar competition have been identified. However, the regulatory mechanisms of sugar transporters, especially in the microbes require further investigation. Here, the key transporters that are responsible for the sugar competition in the host and pathogen have been identified and the regulatory mechanisms of the sugar transport have been briefly analyzed. These data are of great significance to the increase of the sugar distribution in plants for improvement in the yield.As the primary energy source for a plant host and microbe to sustain life, sugar is generally exported by Sugars Will Eventually be Exported Transporters (SWEETs) to the host extracellular spaces or the apoplast. There, the host and microbes compete for hexose, sucrose, and other important nutrients. The host and microbial monosaccharide transporters (MSTs) and sucrose transporters (SUTs) play a key role in the “evolutionary arms race”. The result of this competition hinges on the proportion of sugar distribution between the host and microbes. In some plants (such as  The contradiction between the growing population’s demand for food and the safety of food production is becoming increasingly severe. Furthermore, plant diseases continue to seriously threaten the safety of food production worldwide . The glovia photosynthesis and assimilated in the mesophyll , and osmotic pressure . Althougesophyll . During esophyll  (Naseem esophyll .SWEETs function as susceptible (S) genes and are induced during pathogen infection, resulting in increased pathogen virulence  were up-regulated following Rhizoctonia solani AG1-IA strain inoculation (SWEETs (OsSWEET11 and 14), of the susceptible Pujiang6 rice line were up-regulated  after nine days following the inoculation with Ustilaginoidea virens P4 strain  of the resistant Hui1586 and susceptible Nipponbare (NIP) lines responded to Magnaporthe oryzae Guy11 strain induction  were up-regulated by Xanthomonas oryzae pv. oryzae P3 and P6 strains following inoculation. However, in the resistant NSIC RC154 line, seven SWEET genes  were up-regulated following inoculation while two were not (OsSWEET4 and 7d)  and Os11N3/OsSWEET14 (targeted by TALC) are induced by various transcription activator-like (TAL) effectors secreted via type III secretion system of Xoo, which cause bacterial leaf blight. Xa25/OsSWEET13 has been shown to be induced by Xoo PXO339 strain as a susceptible allele  and increasing the virulence of fungi through nutritional competition  fungi, MtSWEET1b is also induced to promote a glucose efflux carrier  are one of the three known sugar transporter families, including monosaccharide transporters (MSTs) and sucrose transporters (SUTs). They arirulence . The traculation    encoding a CWIN is a putative domestication gene, which is required for carbon distribution at the early stage of grain filling in rice  are divided into the following categories according to subcellular localization and cleavage of sucrose into fructose and glucose: cell wall invertase (CWIN), cytoplasmic invertase (CIN), and vacuolar invertase (VIN). CWIN plays a key role in the production of crops, particularly in the development of seeds and fruits . GIF1  ) also play an important role in sugar transport, specifically in the process of phloem loading and unloading, pathogen defense, and yield formation  from apoplasts ormation  The MST ormation .+ symporter and is characterized into five groups: SUT1 to SUT5. SUT1 is present in a specific branch of dicotyledonous plants and members of SUT2 and SUT4 branches are found in both monocotyledonous and dicotyledonous plants (via the SWEET family sugar transporters in the sink cells and then absorbed or loaded into the phloem by SUT or SUC sugar transporters. Sucrose transported via the companion cells-sieve elements (CC-SE) complexes will eventually be exported or unloaded into the phloem apoplasmically by SWEETs and then absorbed into the sink cells by the action of sucrose/H+ cotransporter. Alternatively, this sucrose can be hydrolyzed by the CWIN to produce hexose and then enter the surrounding source cells via the hexose transport protein , the pattern recognition receptors (PRRs) expressed on the cell surface, and BRASSINOSTEROID INSENSITIVE 1–associated receptor kinase1 (BAK1) interacts with STP13 by phosphorylating it at the threonine 485 site, resulting in improved hexose absorption capacity by the plant. The pathogenicity of bacteria is therefore weakened by limiting the nutrient absorption from the host  monosaccharide transporter GpMST1 is a proton cotransporter and contains a very low GC content and has the highest affinity for monosaccharides, including glucose, mannose, galactose, and fructose  combines with the orphan governor XccR, the homologous protein of cognate receptors LuxR, to activate the proline aminopeptidase-encoded gene PIP and enhance virulence  with low sucrose affinity and energy dependence to absorb sucrose from the apoplast of the host directly and avoids the plant immune response triggered by monosaccharide leakage    and thatsorption . Therefosistance . AtSWEETsistance . Therefoelopment . Conseqund maize . There iCWIN genes in response to signals of pathogen invasion, accompanied by local hydrolysis of sugars and promoting immune responses (The processes by which hosts and pathogens recognize each other are as important as the processes involved in competition. Previous studies have demonstrated changes in the expression of plant esponses . Glucoseesponses . TherefoOryza sativa)  , can be YX, SY and GX designed this work, JC, YX, MS, RS and CZ wrote the manuscript. All authors contributed to the article and approved the submitted version.


Phytophthora infestans, we varied the grinding time of the Tissue Lyser and used TRIzol and beta-mercaptoethanol to inhibit the RNase. In addition, we tested the mortar and pestle grinding of mycelium in liquid nitrogen, with this method showing the most consistent results. During the sample grinding with the Tissue Lyser device, adding an RNase inhibitor proved to be a prerequisite, and the best results were achieved using TRIzol. We considered ten different combinations of grinding conditions and isolation methods. The classical combination of a mortar and pestle, followed by TRIzol, has proved to be the most efficient.RNA isolation from fungi and fungus-like organisms is not an easy task. Active endogenous RNases quickly hydrolyze RNA after the sample collection, and the thick cell wall prevents inhibitors from penetrating the cells. Therefore, the initial collection and grinding steps may be crucial for the total RNA isolation from the mycelium. When isolating RNA from  The isolation of RNA from fungi and fungus-like organisms appears to be a non-trivial task. Despite their external similarity, these organisms differ in physiology and often require individual approaches to supposedly routine procedures. Complications with RNA isolation are primarily due to the thick cell wall and active endogenous RNases, both issues having to be addressed at the earliest stages of the procedure, i.e., during cell grinding and lysis . The sucPhytophthora infestans is a parasitic oomycete affecting significant economic crops, such as potatoes and tomatoes. Though not related to true fungi, it exhibits a certain convergent similarity, forming a non-septic mycelium with a cell wall of cellulose and beta-glucans [P. infestans, but we could not find any specific publications on RNA isolation from this species. Those studies where the RNA isolated from oomycetes was used often reported TRIzol [P. infestans.-glucans . The dif-glucans  are alsod TRIzol  or the Rd TRIzol  as the cTo achieve this goal, we ground the mycelium using two methods: automatically on the Tissue Lyser LT with stainless steel beads and manually in a mortar with liquid nitrogen. We tested how effectively TRIzol and beta-mercaptoethanol inactivate the RNase and prevent RNA degradation during grinding. TRIzol , the RNEasy Plant Mini Kit , and their combination were attempted for RNA isolation after grinding. To assess the quality of the isolated RNA, we used the RNA integrity number (RIN)  and measP. infestans strain VZR18 was obtained from the All-Russian Institute of Plant Protection and grown on Petri dishes with Rye B medium [Mycelium of B medium  for 6 weP. infestans for each technique. For the NB_T-15 and B_T-15 methods, the number of samples was reduced because of the insufficient amount of mycelium material in the relevant Petri dishes, with one sample of B_TQ-15 excluded because of suspected contamination.For each of the 10 grinding and extraction techniques listed in After receiving the interim data, the RNA extraction was repeated with 3 more mycelium samples for the MP_T and B_TQ-15 methods. With a mortar and pestle in liquid nitrogen.The frozen material was subjected to grinding using Tissue Lyser LT : oscillation frequency of 50 Hz, processing times of 15 s, 30 s, and 1 min, bead material–stainless steel. The rotor was cooled down to −70 °C. Immediately after grinding, 1 mL TRIzol  or 450 μL RLT buffer  with beta-mercaptoethanol for RNase inhibition was added to the samples.Similar to point 2, but with TRIzol and RLT buffer  added before grinding.Different techniques and parameters of mycelium grinding were attempted during the experiment:All parameter combinations used with the number of samples are shown in After grinding and adding the lysis agent, the samples were thoroughly mixed and incubated on ice for 10 min.A standard TRIzol protocol according to the manufacturer’s recommendations .RNEasy Plant Mini Kit  according to the manufacturer’s protocol.A combination of TRIzol and RNEasy spin column  methods.Three different methods were attempted for RNA isolation:For each method, RNA elution was performed with 50 uL of RNase-purified water. The aliquots for the RIN determination and concentration measurements were selected immediately after isolation. The RNA was stored at −80 °C. A complete protocol for the combined TRIzol and spin column RNA isolation is given in The concentration of isolated RNA was measured using a Qubit 4 Fluorimeter  with an HS RNA Assay kit . A 2100 Bioanalyzer  and RNA 6000 pico chip  were used to assess the integrity and the length distribution of isolated RNA. The analysis was carried out in the Genomics Center at the Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences . The 2100 Expert Agilent software  uses an algorithm to evaluate RNA integrity based on capillary electrophoresis data with a specific dye. As a result, the sample is assigned an RNA integrity number (RIN) ranging from 1 to 10, with 1 representing completely degraded RNA .car [ggpubr R packages. We used one-way ANOVA to measure the effect of experimental conditions on the RIN and the concentration of extracted RNA. Pairwise comparisons between groups were conducted using a t-test .For each group of experimental conditions described above , we perfcar  and ggpuThe RIN measurements for all protocols are presented in We have not succeeded in finding statistically significant differences in the RIN between the three best techniques , but theAn example of the 2100 Bioanalyzer data obtained during the experiment can be found in The concentration measurement results are averaged in p = 0.116), as shown in Among the methods mentioned above, the highest amount of RNA was isolated with a mortar and pestle. Despite the apparently greater efficiency of TRIzol compared with the RNEasy Plant Mini Kit when processed on the Tissue Lyser, the one-way ANOVA suggests these differences are not statistically significant  = 3.02, P. infestans comparable to TRIzol: when using a mortar and pestle, this method proves equally efficient (Thus, the RNEasy Plant Mini Kit can provide a concentration of RNA isolated from fficient .Phytophthora. At the same time, in contrast to the data [P. infestans RNases.Given the results obtained, a mortar and pestle method can be considered to be the best choice. This method provides cooling in liquid nitrogen throughout the grinding process and has been used by default in works for the isolation of very high-quality RNA from fungi ,3. Neverthe data , beta-meIn this study , TRIzol However, the RNA with an RIN ranging from 6 to 7 can also be used for library formation . In partOne of the reasons for the reduced integrity of isolated RNA could be the long process of collecting mycelium from the nutrient medium, which is associated with considerable damage. Therefore, using alternative cultivation techniques, such as those in ,6,16, maP. infestans. Thus, grinding mycelium with a mortar and pestle in liquid nitrogen is preferable both in terms of the RIN and the concentration of isolated RNA. If it is necessary to scale up the experiment, it is acceptable to use a bead-beating system, with the mandatory addition of lysis buffer before grinding, as well as minimization of the exposure time.This work allowed us to determine the most promising approaches to mycelial grinding and RNA isolation from


Saccharomyces cerevisiae.As a master regulator in cells, RNA-binding protein (RBP) plays critical roles in organismal development, metabolism and various diseases. It regulates gene expression at various levels mostly by specific recognition of target RNA. The traditional CLIP-seq method to detect transcriptome-wide RNA targets of RBP is less efficient in yeast due to the low UV transmissivity of their cell walls. Here, we established an efficient HyperTRIBE (Targets of RNA-binding proteins Identified By Editing) in yeast, by fusing an RBP to the hyper-active catalytic domain of human RNA editing enzyme ADAR2 and expressing the fusion protein in yeast cells. The target transcripts of RBP were marked with new RNA editing events and identified by high-throughput sequencing. We successfully applied HyperTRIBE to identifying the RNA targets of two yeast RBPs, KHD1 and BFR1. The antibody-free HyperTRIBE has competitive advantages including a low background, high sensitivity and reproducibility, as well as a simple library preparation procedure, providing a reliable strategy for RBP target identification in  RNA-binding protein (RBP) specifically interacts with RNA in cells and is widely involved in the regulation of gene expression . RBP–RNAThe importance of RNA–protein interactions has facilitated rapid development of profiling methods for RBP targets at the transcriptome level. These methods include RNA-immunoprecipitation sequencing (RIP-seq), high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) and its variants, Targets of RNA-binding proteins Identified By Editing (TRIBE), HyperTRIBE, RNA tagging, and Surveying Targets by APOBEC-Mediated Profiling (STAMP) ,10,11,12C. elegans, adds a poly(U) tail to the 3′ end of target RNA and the U-tailed mRNA is then identified by paired-end sequencing. As poly(U) polymerase modifies the 3′ end of the mRNA poly(A) tail, RNA tagging is supposed to work better for RBPs that bind to the 3′ UTR since the region is close to 3′ terminus. STAMP [Rattus norvegicus with an RBP and then determine cytosine to uridine transitions in target mRNA by sequencing. Current research has indicated that STAMP works well in human cells but it is hard to applied in Drosophila cells [In recent years, three antibody-independent methods have been developed, all by fusing an RNA modification enzyme to an RBP to mark RBP targets, thereby providing alternative strategies for the study of RBPs. TRIBE and RNA tagging were developed in late 2015 to early 2016 and STAMP was established in 2021 ,8. In TRs. STAMP  in mammas. STAMP  from Ratla cells . FurtherS. cerevisiae homolog of mammalian heterogeneous nuclear ribonucleoprotein K (hnRNP K) [Yeasts are remarkable eukaryotic model organisms for basic studies and bioengineering . To datehnRNP K) , and inchnRNP K) ,28, mosthnRNP K) . In addihnRNP K)  while ithnRNP K) . During hnRNP K) ,33,34, KhnRNP K) .Another multi-functional RBP, BFR1, is involved in the uneven distribution of mRNA during yeast budding and responsible for target mRNA localization in the bud tip . BFR1 isS. cerevisiae and investigated the target transcripts of two RBPs, KHD1 and BFR1. Our results showed that HyperTRIBE performed well with a low background, good sensitivity and reproducibility. Additionally, combined with the identification of differentially expressed genes (DEGs) after depletion of KHD1 or BFR1, we identified key targets of KHD1 and BFR1 and their functions. Our work established an effective method to identify RBP targets in yeast with a simple procedure and will be beneficial for the field.Because the rigid cell wall leads to less efficiency of UV cross-linking for CLIP in organisms like plants, fungi and bacteria, TRIBE could be a good choice there. In addition, multiple available tools for RBP target examination in one species will be conducive to the confident identification of RBP targets as well as to studying several cooperative RBPs in a large complex simultaneously. Thus, we pioneered HyperTRIBE in S. cerevisiae was validated by Western blotting | > 0.2). Among them, 17 genes overlapped with the 835 KHD1-HyperTRIBE targets  and non-targets  term analysis showed that the target genes of KHD1 are related to filamentous growth, cell growth and adhesion C. Interear plate , indicatS. cerevisiae cells, the proteins of hADAR2cd (Hyper-only) and BFR1-hADAR2cd (BFR1-Hyper) were expressed well after induction  domain and lacks any classical RNA-binding domains , it is wnduction . Then, tnduction A. The ednduction . A signinduction . The idenduction . In addinduction , and ~61nduction , indicatThe frequency histogram of edited sites per BFR1 target gene showed that most target genes have only one edited site A. When tbfr1-heterozygous mutant (+/−bfr1) strain and wild type (WT) strain from NCBI, and obtained 1451 differentially expressed genes (FPKMavg ≥ 2 and FPKM ratio (+/−bfr1/WT) multiples greater than 1.5) [+/−bfr1 and WT from RNA-Seq data . To date, no evidence has indicated that BFR1 can directly bind to RNA; thus, we speculate that indirect association of BFR1 on RNA makes motif identification difficult and/or its binding on RNA does not require a strict consensus motif. GO term analysis of BFR1 targets revealed that most of its targets were involved in metabolic processes, cytoplasmic stress granules and the membranous organelle-related pathways   ,37 so weembrane) C, which embrane) B and a pkhd1-knockout yeast, such as increased filamentous growth, improved cell adhesion both between cells and to the plate [Our KHD1 and BFR1 HyperTRIBE data are consistent with these RBPs’ functions published in previous research. The roles of the KHD1 HyperTRIBE targets are enriched in the processes of filamentous growth, cell growth and adhesion C; thus, he plate . AdditioAs master regulators in cells, RBPs take part in gene regulation at multiple levels mostly by specifically recognizing RNA. To reveal the underlying mechanisms of RBP–RNA interactions, the profiling methods of this interaction network have been rapidly advancing. These methods each hold their own advantages. HITS-CLIP and its variants can detect the RNA targets from endogenous RBPs with relatively accurate binding site information. Instead, TRIBE, RNA tagging and STAMP are antibody-free methods, avoiding the problems associated with antibody specificity. These three methods also avoid the low UV-crosslinking efficiency problem, making them applicable in cells with cell wall structures, such as plants, fungi and bacteria. Of the three, TRIBE and STAMP require a lower number of starting cells and undergo standard RNA-seq library preparation protocol, making them easier for lab execution and accessible for single-cell level research. In addition, the research on a complex comprising multiple RBPs will benefit from combining different techniques in the same organism. Specifically, different RBPs, fused with different RNA modification enzymes, can be expressed in the same cells to mark the common and unique RBP targets. Thus, the development of various strategies for profiling RBP–RNA interactions will greatly contribute to research in the field of RNA biology.Drosophila used the fusion protein of Drosophila ADARcd (dADARcd) with an RBP [The fusion protein-based system is a cornerstone of RBP target identification methodologies. The key challenge for establishing this kind of method in an untested species is obtaining an RNA modification enzyme with high activity in that particular species. The development of TRIBE in h an RBP , while th an RBP ,14,15. Ih an RBP . To adaph an RBP  to constRBPs of interest were cloned into pcDNA3-3HA-hADAR2cd-E488Q  to build600 < 0.2. When the OD600 was close to 1, uninduced control cells were collected for Western blotting (WB). Glucose was eliminated by resuspending yeasts three times in SC/-His media (without dextrose) as it could inhibit GAL1-initiated transcription [600 = 1 for several hours. The expression of TRIBE constructs was examined by WB and the verified samples were used for RNA preparation.For the plasmids with a PGK1 promoter, the transfected cells were grown in SD/-His media  for 12~16 h. For the plasmids with a GAL1 promoter, the selected cells were maintained in SD/-His first, then saturated cultures were seeded into SD/-His media at ODcription . The inc® Ultima Dual-mode mRNA Library Prep Kit for Illumina®, Yeasen, Shanghai, China). Hieff NGS® Complete Adapter Kit for Illumina®  was used for sample pooling. The quality of the NGS library was monitored using the Qubit dsDNA 915 kit  for Fragment AnalyzerTM  [The HyperTRIBE RNA-sequencing library was prepared followed the manufacturer’s instructions from the Illumina Stranded mRNA Prep kit in which dUTP was included during second strand cDNA synthesis   and qPCRhttps://cutadapt.readthedocs.io/en/stable/, accessed on 18 April 2023) was used for adaptor dislodgment. Trimmomatic (0.38)  was changed to the paired-end sequencing model for inferior read filtering. The clean data were aligned to the Saccharomyces cerevisiae genome (version R64-1-1) from SGD by STAR2.5.3a  (--outFilterMismatchNoverLmax 0.10 --outFilterMatchNmin 16 --outFilterMultimapNmax 1), while alignment index was generated from Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa and Saccharomyces_cerevisiae.R64-1-1.103.gtf. After removing PCR duplicates, editing events were identified with the indicated threshold of reads and editing percentages and single-nucleotide polymorphisms were avoided using the wtRNA–RNA approach.The raw data were processed according to a published protocol  with modhttps://www.yeastgenome.org/reference/S000127887, accessed on 19 September 2008). KHD1 targets detected in CLIP-seq were obtained from the database of the POSTAR3 CLIPdb Module. The differentially expressed genes (DEG) of KHD1 were calculated from microarray data from the wild type strain S288C and khd1-knockout strain (hek2Δ) from NCBI GEO Sample GSM843507 . The RNA-tagging data for BFR1 were from SGD , and the RIP-Chip data of BFR1 were from SGD . The differentially expressed genes (DEG) of BFR1 were calculated by comparing RNA-Seq data from the wild type strain BY4743 with the BFR1 heterozygote mutant BFR1/bfr1Δ::GFP (+/−bfr1) obtained from NCBI GEO Sample GSM4240918 .RNA IP-Chip data for KHD1 were from the Saccharomyces Genome Database (Saccharomyces_cerevisiae. R64-1-1), the 100 bp upstream and 100 bp downstream regions of the edited sites were selected to carry out motif analysis. The same length (201 bp) and same number of background regions were randomly selected from non-target genes. The analysis was carried out for two biological replicates and two similar high-confidence motifs for KHD1 were observed each from the MEME algorithm [https://meme-suite.org/meme/tools/meme, accessed on 18 April 2023, parameters: -discriminative mode -zoops mode -0-order model of sequence -minw 10 -maxw 15) and XSTREME algorithm [https://meme-suite.org/meme/tools/xstreme, parameters: -Ray2013 S. cerevisiae database -minw 10 -maxw 15 -zoops mode).In the reference genome . The ID type of GENENAME was converted to ENTREZID with function bitr loaded with org.Sc.sgd.db  before proceeding to use the enrichGO [GO enrichment was analyzed using the R Programming Language (clusterProfiler , http://enrichGO  function


No study appraised the knowledge gaps and factors impacting men’s sexual and reproductive health (SRH) in MENA (Middle East and North Africa). The current scoping review undertook this task.We searched PubMed and Web of Science (WoS) electronic databases for original articles on men’s SRH published from MENA. Data was extracted from the selected articles and mapped out employing the WHO framework for operationalising SRH. Analyses and data synthesis identified the factors impacting on men’s experiences of and access to SRH.A total of 98 articles met the inclusion criteria and were included in the analysis. The majority of studies focused on HIV and other sexually transmissible infections (67%); followed by comprehensive education and information (10%); contraception counselling/provision (9%); sexual function and psychosexual counselling (5%); fertility care (8%); and gender-based violence prevention, support/care (1%). There were no studies on antenatal/intrapartum/postnatal care and on safe abortion care (0% for both). Conceptually, there was lack of knowledge of the different domains of men’s SRH, with negative attitudes, and many misconceptions; as well as a deficiency of health system policies, strategies and interventions for SRH.Men’s SRH is not sufficiently prioritized. We observed five ‘paradoxes’: strong focus on HIV/AIDS, when MENA has low prevalence of HIV; weak focus on both fertility and sexual dysfunctions, despite their high prevalence in MENA; no publications on men’s involvement in sexual gender-based violence, despite its frequency across MENA; no studies of men’s involvement in antenatal/intrapartum/postnatal care, despite the international literature valuing such involvement; and, many studies identifying lack of SRH knowledge, but no publications on policies and strategies addressing such shortcoming. These ‘mismatches’ suggest the necessity for efforts to enhance the education of the general population and healthcare workers, as well as improvements across MENA health systems, with future research examining their effects on men’s SRH.The online version contains supplementary material available at 10.1186/s12889-022-14716-2. Historically, sexual and reproductive health (SRH) and rights were viewed as a woman’s issue . One of The Reproductive Health Working Group established in 1988 in Cairo to advance research in the Arab countries and Turkey began with a focus on women . With tiTherefore, the current scoping review aimed to outline the knowledge gaps and considerations that impact on men regarding SRH in MENA. Specifically, we appraised the range of factors that bear on men’s SRH in terms of clients/users, healthcare providers (HCPs), healthcare system and sociopolitical environment.The purpose of a scoping review is not to localize and account for every published information on the topic . Rather,The review team systematically synthesized the findings and summarized and presented the charting results as they relate to the review questions and objectives. This included extracting the aims, populations, findings and conclusions of each included study development of an excel sheet. Then groupings were created based on the eight domains of SRH as outlined by WHO  frameworThe theoretical framework for analysis we employed involved two interrelated schemes. The first is WHO’s (2017) framework for operationalising sexual health and its linkages to reproductive health, comprising 8 domains: antenatal, intrapartum and postnatal care; comprehensive education and information; contraception counselling and provision; gender-based violence prevention, support and care; fertility care; prevention and control of HIV and other sexually transmissible infections (STIs); safe abortion care; sexual function and psychosexual counselling .Then, for each of these domains, we further employed Kilbourne et al’s (2006) framework to outline the health service perspectives on the appreciation of health and healthcare disparities, highlighting the factors influencing men’s experiences, including: individual (HCPs and users); interpersonal ; organisational ; and the larger influence of the community and public policies . Our searches of men’s SRH yielded very sparse articles on healthcare encounter and contact characteristics, therefore our domains are organized under 3 categories, namely clients/ HCPs; healthcare system factors; and sociopolitical factors .Figure Men’s SRH topics were grouped using the WHO framework, illustrating the intertwined features between sexual health and reproductive health . The greThe identified studies included children/adolescents, youth, school/university students, HCPs, particular patient groups, general public or special populations e.g., industrial/tourist workers, refugees, truck drivers, refugees, seafarers, alcohol/drug abusers, men having sex with men and female/male sex workers, and people living with HIV/AIDS (PLWHA) or healthy children/ adolescents of HIV-positive parents . Generally, these studies identified a knowledge lack regarding issues relating to men’s SRH in MENA.A total of 10% of the studies we identified were dedicated Most studies of the review assessed the knowledge of a range of population groups pertaining to prevention and control of STIs and HIV/AIDS. More focus was directed towards knowledge level among clients/ users than of HCPs. Overall, there was low knowledge level across most of the groups that were assessed. Knowledge level was influenced by factors including age, sex, level of education and experience with age. Knowledge deficits often translated into negative attitudes towards HIV/AIDS or PLWHA. In addition, little is known about the perception of the magnitude of HIV/AIDS in MENA Table .In terms of the healthcare system, for HIV, much had been achieved in linking to and retention in care, antiretroviral therapy coverage and viral suppression, despite obstacles in prevention programs e.g., deficient funding and infection control due to lack in supplies and procedures as well as insufficient data/surveillance , 59. TheSeveral HIV awareness programs have been implemented. At hospital level, introduction of multi-disciplinary team (MDT) approach in managing HIV patients resulted in statistically significant control of the disease . On the As for the social factors, high stigma and discrimination toward PLWHA were present , 39, rooSome end-users displayed limited/inadequate knowledge about the concept, availability and benefits of SRH, voiced by the need for more information and quality services . HealthcHealthcare system-related factors suggested that improvements in quality of infertility management required evidence-based training, supplies, laboratory/radiology support, improved communications with specialists, and availability of guidelines . Youth aThe knowledge and acceptance of family planning varied across MENA. Generally, good awareness of contraceptive approaches was mainly for women’s methods but not for male contraception. However, such awareness was not translated into increased application/use of family planning in many MENA countries due to religious and sociocultural norms surrounding this topic. The lack of knowledge about male contraception was also including HCP e.g., pharmacists Table .Little information exists on gender-based violence in MENA, and our search yielded only 1 article. In Egypt, the majority of street children experienced more than one risk including harassment or abuse by police and other street children, drug abuse, and, among sexually active 15–17-year-olds, most reported multiple partners and never using condoms, and most girls had experienced sexual abuse . Such beAddressing SRH of men alongside that of women’s is essential. However, it has not received the attention it deserves worldwide. We outlined the current knowledge, knowledge gaps and considerations that impact on men’s SRH in MENA, and appraised the HCPs’, users’, healthcare systems’ and social factors affecting such services. To our knowledge, this is the first comprehensive scoping review of men’s SRH in MENA.Our main findings unearthed a strong HIV/AIDS focus of the published outputs, but a much weaker focus on issues related to fertility care, sexual dysfunctions/ counselling, and gender-based violence. The general population, different clientele groups, and a range of HCPs exhibited many SRH knowledge gaps, that subsequently lead to a high prevalence of unfavorable attitudes towards men’s SRH conditions, stigmatization, and the emergence of many misconceptions. Generally, across the range of countries under examination, the quality of SRH services could be improved. Surprisingly, we could not find published data on legislation, government policies or national SRH strategies. More importantly, we observed several paradoxes in terms of the lack of congruence between many of the domains that the published outputs addressed on the one hand; and the actual ‘on the ground’ situation across MENA on the other.The first paradox pertained to the strong HIV/AIDS focus across the published literature, despite the low prevalence/ burden in MENA region (0.1%) , 98. WhiThe second paradox was the weak focus of the published articles on the two topics of fertility care and sexual dysfunctions/counselling, despite the high prevalence of fertility and sexual problems in MENA (22.6%) . Such miThe third paradox we observed was related to gender-based violence. The current review found that publications of gender-based violence prevention, support and care represented only 2% of the retrieved studies. This is despite that women’s exposure to male domination has long been normalized in the Arab world , 106. ThThe fourth paradox was that the present review found no studies pertaining to men’s SRH that addressed the domain of antenatal, intrapartum and postnatal care (0%), despite that men’s involvement in maternal health programs is a key to increase utilization of maternal health services . This coThe fifth paradox was that a great proportion of studies discussed knowledge levels and gaps pertaining to men’s SRH in MENA, identifying a range of factors that influence knowledge. Surprisingly, there was no parallel body of literature debating effective interventions and their implementation in order to remedy/overcome such gaps. Similarly, we found no articles dedicated to policies and strategies to address such shortcomings at state, health care system and public health policy making level, when certainly improving men’s access to SRH requires state, health care system and health care providers interventions and policies , 101.Likewise, the present review found no studies of men’s SRH that addressed safe abortion care (0%), not surprising given that abortion is illegal across most of the region, and there are various legal and societal barriers to the practice in the countries where it is allowed . Very feA unique characteristic across MENA is the prevalent political instabilities and refugee situations . MENA haThe current scoping review has limitations. Surveys and reports about MENA health issues are mainly published in local languages and hardly accessible through electronic databases . The stuIt is important for men to access SRH care. The available literature from the MENA region suggests that men’s SRH is not sufficiently prioritized. A detailed landscape of men’s experiences in SRH care across these countries remains to be explored. A number of pertinent ‘mismatches’ were evident in the literature. There was strong focus on HIV/AIDS, when MENA has the lowest prevalence of HIV in the world; a much weaker focus on fertility and sexual dysfunctions, despite that these conditions were much more prevalent in MENA countries; no publications on men’s involvement in sexual gender-based violence, despite women’s exposure to its various forms across the MENA region; and no studies pertaining to men’s SRH in terms of their involvement in antenatal, intrapartum and postnatal care, despite the prevailing international literature to the value of such involvement. Such incongruencies might serve to provide future direction for the formulation and implementation of comprehensive strategies to help tackle men’s SRH challenges in MENA region. These could include strengthening the current policies, strategies and interventions to enhance and improve the attitudes, behaviors and education of the general public, the youth, men in general as well as HCPs. Furthermore, strengthening the health systems over time in terms of political commitment, structures, organization, funding and interventions to address core issues and to more formally respond to men’s SRH challenges in MENA would be welcomed. Future research should examine the influence of policies and the healthcare service delivery and organization on men’s access and experiences in SRH care.Additional file 1: Supplementary Box 1. Search terms used in the current scoping review. Supplementary Table 1. Included articles on men's experiences of sexual and reproductive healthcare in MENA countries [ountries –154.


Viruses rely on hosts for life and reproduction, cause a variety of symptoms from common cold to AIDS to COVID-19 and provoke public health threats claiming millions of lives around the globe. RNA editing, as a crucial co-/post-transcriptional modification inducing nucleotide alterations on both endogenous and exogenous RNA sequences, exerts significant influences on virus replication, protein synthesis, infectivity and toxicity. Hitherto, a number of host-mediated RNA editing sites have been identified in diverse viruses, yet lacking a full picture of RNA editing-associated mechanisms and effects in different classes of viruses. Here we synthesize the current knowledge of host-mediated RNA editing in a variety of viruses by considering two enzyme families, viz., ADARs and APOBECs, thereby presenting a landscape of diverse editing mechanisms and effects between viruses and hosts. In the ongoing pandemic, our study promises to provide potentially valuable insights for better understanding host-mediated RNA editing on ever-reported and newly-emerging viruses. RNA editing, as a crucial co-/post-transcriptional modification inducing nucleotide alterations on both endogenous and exogenous RNA sequences –3, playsViruses rely on hosts for life and reproduction, cause a variety of symptoms from common cold to AIDS to COVID-19 and provoke public health threats claiming millions of lives around the globe. Over the past decades, a number of studies have experimentally revealed complicated mechanisms between various viruses and their hosts mediated by RNA editing  and double-stranded RNA (dsRNA) viruses according to the Baltimore Virus Classification. In this study, we summarize the research progresses of host-mediated RNA editing with experimentally validated evidence by grouping viruses into positive-sense single-stranded RNA (+ ssRNA), negative-sense single-stranded RNA (-ssRNA), single-stranded RNA reverse transcribing (ssRNA-RT), double-stranded DNA (dsDNA), and double-stranded DNA reverse transcribing (dsDNA-RT), and accordingly delineate a landscape of the involved RNA editing mechanisms (deamination dependent/independent) and molecular effects  between viruses and hosts Fig. .+ssRNA viruses infecting cellular hosts rely upon positive-stranded RNA as their primary genetic material . HithertPicornaviridae family, with genome size of about 7.4 kb nucleotides. EV71 can target the human central nervous system and cause hand-foot-mouth disease and herpangina, commonly in children under 5 years old [EV71, first described in 1974, is a kind of + ssRNA viruses that belongs to the ears old . Multiplears old , 37. Firears old , indicatears old . Thus, RFlaviviridae family with genome size of about 9.6 kb nucleotides [HCV, first isolated in 1989, is a member of the leotides . It is cleotides . Studiesleotides , 43, 45.leotides , which ileotides  and the leotides . While, leotides , indicatrhesus macaque [Flaviviridae family, it can be transmitted through blood, placenta, and sex [ZIKV, with genome size of about 10.8 kb nucleotides, was first isolated in 1947 from  macaque . As a me and sex , and cau and sex . Specifi and sex . This pr and sex . Of note and sex .Coronaviridae family with the largest genome (~ 30 kb) among RNA viruses, caused the global pandemic due to its highly adaptive genomic variants [SARS-CoV-2, as a member of + ssRNA viruses belonging to the variants , 57. Albvariants –62  for assembly and release of HDV particles [AG” stops the translation of HDAg and produces HDAg-S that can promote virus replication, whereas the edited codon “UGG” encodes tryptophan and yields HDAg-L that can inhibit virus replication. Thus, ADAR1 provokes an antiviral effect after HDV infection by producing HDAg-L [HDV, belonging to the articles . Infectiarticles , 90; ADAarticles  and latearticles , 96, 97.g HDAg-L . During g HDAg-L , while Ag HDAg-L , 84. Asig HDAg-L .Paramyxoviridae family with genome size of about 15.9 kb nucleotides, is thought to emerge in the 6th century BCE (Before Common Era) [KO cells [MeV, belonging to the mon Era) . Infectimon Era) . It has mon Era) . FurtherKO cells  and the KO cells .Aside from HDV and MeV, RNA editing has also been reported in several other –ssRNA viruses, such as influenza A virus (IAV) , 100, dessRNA-RT viruses can establish infection through integrating a DNA copy of the viral genome into host cell chromosome . Till noHuman immunodeficiency virus (HIV), first isolated in 1983 from human, is an enveloped RNA retrovirus with genome size of about 9.2 kb nucleotides  that canIn addition to HIV, other ssRNA-RT viruses, albeit with limited experimental evidence, are also reported to be affected by host-mediated RNA editing through APOBEC3s and ADARs. It is indicated that life activity of caprine arthritis encephalitis virus (CAEV), feline immunodeficiency virus (FIV), murine leukemia virus (MLV), mouse mammary tumor virus (MMTV), and porcine endogenous retrovirus (PER) can be antagonized by host-mediated RNA editing (with or without requiring deamination), potentially owing to degradation of APOBEC3s by vif protein , 119. SpUL54 gene through APOBEC1 deamination activity are essential impaired accumulation of HSV DNA copy numbers and mRNA transcripts [In addition to RNA viruses, dsDNA viruses, such as BK polyomavirus (BKPyV), Epstein-Barr virus (EBV), human cytomegalovirus (HHV), human papillomaviruses (HPV), and herpes simplex virus (HSV), have been reported to be targeted and impacted by host-mediated RNA editing, in which APOBECs are the main mediators –131. Fornscripts .E6 gene [E2 gene of HPV [To be specific, studies have documented that host-mediated RNA editing influences viral load of HPV , 131 thae of HPV . In totadsDNA-RT viruses, albeit well studied with respect to innate immunity , have liEvidence has shown that APOBECs can influence HBV replication by complex host-mediated RNA editing mechanisms Fig. D. First,Through synthesizing the current knowledge of host-mediated RNA editing in a variety of viruses, it is clearly shown that RNA editing can induce proviral or antiviral effects by promoting or inhibiting virus replication, protein synthesis and/or infectivity with or without requiring deamination. According to the synthesized knowledge, deamination-dependent RNA editing is mainly found in -ssRNA viruses, whereas deamination-independent regulation is observed in the majority of + ssRNA viruses, indicating their divergence in RNA editing mechanisms. This is presumably due to the difference in viral replication mechanism and life cycle, as the genetic materials of + ssRNA viruses could be translated as mRNA, while -ssRNA viruses need additional steps for transcription/reverse transcription , 103. SpAs also indicated in this report, host endogenous RNAs and exogenous viral RNAs could be distinguished by differential editing patterns mediated by ADAR enzymes . EndogenHost-mediated RNA editing, albeit relatively weak in viruses, may be an important contributor to virus evolution . It has Undoubtedly, existing studies on RNA editing in viruses have limitations. RNA editing sites in viruses can be missed or miscalculated due to low sequencing coverage, sequencing error or/and short read length. Specifically, as multiple RNA editing sites may be co-edited and exert complex effects, it is challenging to identify/verify the coexistence of adjacent RNA editing sites based on short sequencing reads, especially for exogenous RNAs. In the future, long reads generated by the third-generation sequencing technology have great potential to capture a complete picture of RNA editing sites and to detect cooperativity among RNA editing sites during dynamic viral life cycles.In addition, given the ongoing pandemic of COVID-19, it would be desirable to conduct a SARS-CoV-2 cohort study to systematically investigate the host-mediated RNA editing mechanisms and molecular effects on SARS-CoV-2 and further explore the editing heterogeneity among infected people with mild, moderate and severe symptoms. Notably, a model of RNA editing on SARS-CoV-2 transcriptome was proposed ; althougTaken together, our study delineates the landscape of RNA editing-associated mechanisms and effects in different classes of viruses, which would provide potentially valuable insights and also call on worldwide collaborative efforts, particularly during the pandemic caused by SARS-CoV-2 as well as monkeypox virus, for better understanding the host-mediated RNA editing on both ever-reported and newly-emerging viruses.