Cardiovascular disease still remains the leading cause of morbidity and mortality worldwide. Current pharmacological or interventional treatments help to tackle symptoms and even reduce mortality, but cardiovascular disease cases continue to rise. The emergence of novel therapeutic strategies that precisely and efficiently combat cardiovascular disease is therefore deemed more essential than ever. RNA editing, the cell-intrinsic deamination of adenosine or cytidine RNA residues, changes the molecular identity of edited nucleotides, severely altering the fate of RNA molecules involved in key biological processes. The most common type of RNA editing is the deamination of adenosine residue to inosine (A-to-I), which is catalysed by adenosine deaminases acting on RNA (ADARs). Recent efforts have convincingly liaised RNA editing-based mechanisms to the pathophysiology of the cardiovascular system. In this review, we will briefly introduce the basic concepts of the RNA editing field of research. We will particularly focus our discussion on the therapeutic exploitation of RNA editing as a novel therapeutic tool as well as the future perspectives for its use in cardiovascular disease treatment. Progress in the past two decades in molecular genetics, such as genome-wide association studies, has led to the identification of genes and variants responsible for human disease, including cardiovascular disease (CVD). Results from such studies have been used not only to determine the certain risk variants pose for disease development but also to predict drug efficiency. Despite this progress, many risk factors remain undetected. According to the World Health Organisation, cardiovascular disease complicated by heart attack and stroke represents 32% of all global death and remains the leading cause of death in 2022 . It is eAlu elements within introns or the 3’UTR due to their ability to form double-stranded RNA . In . In 3]. nded RNA , 11. Inv editing , 11, res editing . Anothertly Fig. .Fig. 1MeAdar1 in murine model embryos results in embryonic lethality owing to defective haematopoiesis and liver disintegration at E11.5–12.5. Specifically, further histological analysis of Adar1 ablated embryos shows widespread cell death and ineffective haematopoiesis [The implication of editing by ADARs is that information encoded within DNA may be changed at the RNA level. We also know that all RNA types, not only mRNA, may be subjected to editing. As such, RNA base deaminases seem to have a complex role in the expression, function and stability of RNAs. ADAR1 is indispensable for life . Ablatioopoiesis . ADAR1 hopoiesis , 15.GluR2 mRNA. Under normal conditions, nearly 100% of the GluR-B mRNAs are edited at Q/R (glutamine/arginine) site-607 [GluR-B exhibits high Ca2+ permeability due to the negative charge of glutamine, which also leads to cell toxicity and death [Adar2 is postnatally lethal in mice as these mice suffer from seizures indicating severe neurological issues. However, Adar2 deficient mice are rescued through A > G point mutations introduced in the genomic regions of Gria2 that are normally Adar2 editing sites to simulate the pre-edited condition, supporting the pivotal role of A-to-I RNA editing and ADAR2 for neuron homeostasis and overall mammalian physiology [A well-known substrate of ADAR2 is the glutamate receptor site-607 , 16. Unend death , 17. Delysiology .Site-directed RNA editing (SDRE) is an important set of tools that aims to harness the editing ability of A-to-I and C-to-U RNA base editors and guide them to specific RNA targets in order to correct pathogenic point mutations previously occurred in DNA at the RNA level. SDRE is highly programmable and aims to edit single nucleotides with precision. The SDRE methods discussed in this review can either be designed to recruit endogenously expressed ADARs to a specific mRNA target or engineered to contain a catalytic domain that performs deamination. Due to the transient nature of RNA, such therapeutic methods pose a much smaller clinical risk and ethical concern by not introducing permanent changes. In this review, we discuss the main changes that RNA editing affects in cardiovascular gene expression and biology, as well as we introduce the concept of SDRE tools, the challenges of their application and their perspectives about boasting a therapeutic option against cardiovascular disease.RNA editing is one of the most evolutionary conserved properties of RNAs, and it occurs in many living organisms, including humans –21. We hA(Leu) to CU(I)G(Leu)). RNA editing might also introduce readthrough mutations. For example, the STOP codon UAA may be edited to Trp UGG(II) by ADAR1 on the EGFP mRNA [RNA editing is predominantly carried out by adenosine to inosine (A-to-I) editors, which are members of the ADAR protein families and cytidine to uridine (C-to-U) editors, which are members of the AID/APOBEC family . There aGFP mRNA . HoweverGFP mRNA . JantschGFP mRNA .The editGFP mRNA .Additionally, certain point mutations might introduce novel sites for RNA deaminases. Recently, A:C mismatches in RNA:DNA hybrids induced by point mutations at telomeric R-loops have been proven preferred targets of ADAR1p110 . In thisAlu regions [Alu elements, which are repetitive elements, within the 3’UTR of CTSS mRNA allowed RNA-binding protein (RBP) to the edited mRNA and thereby increased mRNA stability as well as CTSS expression [Alu region of CTSS, was strongly correlated with CTSS expression and cardiovascular disease clinical outcomes in the biomaterial of patients with different stages of ASCVD.Admittedly, most RNA editing occurs within non-coding regions and especially within  regions , 40, 41. regions , 42. Edipression . As suchADAR1 resulted in embryonic lethality, whilst tamoxifen-inducible knockout of ADAR1 expression in cardiomyocytes resulted in acute heart failure and lethality. RNA-seq data revealed an upregulation of apoptotic genes linked to the observed reduced trabecular compaction, reduced proliferation and increased in the numbers of apoptotic cardiomyocytes [. showed that editing within sequences that are not binding sites for Drosha and Dicer does not affect microRNA maturation [Adar2 KO mouse hearts [MicroRNAs are short non-coding RNA molecules that regulate the expression of protein-coding genes by complementarily base-pairing via their seed region to specific sequences within the 3’ UTRs of mRNAs . Currentmyocytes , attribumyocytes . Restorimyocytes . A-to-I myocytes , 52. In myocytes . Of noteturation . Interese hearts . The pree hearts , was alse hearts . In anote hearts .Neat1 lncRNA, well established for promoting pro-inflammatory gene expression and has also been linked with atherosclerotic cardiovascular disease [Neat1 lncRNA protects the lncRNA expression by facilitating the recruitment of AUF1, an RNA-binding protein (RBP) with stabilising properties [Apart from miRNAs, long non-coding RNAs (lncRNAs) shape a great deal of the non-coding genome. LncRNAs play important and multispectral roles in cardiovascular disease . Recentl disease . RNA edioperties .. identified approximately one hundred high-confidence splicing events that are altered by ADAR1 and/or ADAR2 [. reported approximately 500 native editing sites of ADAR1 within the 3’ splice site acceptor sequences of highly conserved protein-coding genes with very important cellular functions [Splicing may also be affected by RNA editing enzymes. Tang et alor ADAR2 . Hsiao eunctions . Such chunctions , 61. Cirunctions . Notablyunctions . ADAR1-munctions . TherefoConclusively, the discussed examples underscore the importance of RNA editing for cardiovascular gene expression and, in turn, cardiovascular biology. Although further research is required to determine the relevance of the millions of edited sites, it is not too early to start considering RNA editing from a different prism: its therapeutic exploitation which can be based on the tenets of genome editing recently introduced in the field of cardiovascular disease as well . An incrUntil recently, precision medicine has been associated mostly with DNA therapeutics –68. The The primary concern of such gene therapies remains the long-lasting effects of the permanent alteration of DNA material, which is also subsequently inherited through cell division . This reSeveral RNA-based therapeutics, some of which we describe here, have already been proven effective in treating various disorders, including cardiovascular disease. Their success fuelled the subsequent and currently ongoing development of site-directed RNA editing treatment approaches, which are at the epicentre of this review and have not yet been studied for cardiovascular treatment.Current RNA therapeutic approaches use both coding and non-coding RNA molecules. Coding RNA therapeutics involve the delivery of mRNAs containing instructions for specific proteins to be expressed. The most notable mentions are the two mRNA vaccines developed for the prevention of COVID-19 , 76. TheNon-coding RNA therapeutics are antisense oligonucleotides (ASOs) , small iMost currently approved RNA therapies are ASOs and have already proven successful in treating human diseases, including cardiovascular disease . The firFurthermore, inclisiran is a long-acting RNAi therapeutic agent which targets and inhibits PCSK9 synthesis, thereby lowering blood LDL-particle concentrations , 90. IncExisting RNA therapeutics are efficient in inhibiting or inducing mRNA expression. However, many genetic disorders, including CVDs, are caused by changes in single nucleotides . Site-diSDRE methods aim to recruit endogenous molecules or deliver engineered base editing constructs to mRNA targets Fig. , B. MostThe key notion considered in SDRE using ADARs is that ADARs target dsRNAs Fig. . TherefoDue to their simplicity and quite high efficiency, the most promising SDRE therapeutic methods are currently focused on harnessing the editing abilities of endogenously expressed ADARs, to perform A-to-I editing at mRNA sites that are not their natural targets Fig. . These mThe stability of the gRNA is a concern, and various modifications can ensure the gRNA is not degraded. For example, a series of chemical modifications have been shown to increase the gRNA’s resistance to enzymatic degradation and editing efficiency  . CirculaAlthough reasonably efficient, SDREs using only gRNAs rely on expression levels of RNA deaminases. Of course, as we have seen in the case of GluR2-ADAR, expression of the gRNA can be accompanied by overexpression of ADARs, for increased editing efficiency , 103. Ho). These additional elements can be individually engineered and modified in order to achieve more specific outcomes. Therefore, these methods are more programmable [Most engineered SDREs also use gRNAs. However, they include other components for target recognition, as well as either ADAR/APOBEC deaminase domains Fig. . These arammable . For exarammable . Such merammable , 111 andrammable , 94, 112These Cas13 RNA editing systems have proven quite effective. However, they raise two main concerns: cell toxicity due to the bacterial origin of Cas13 and difficulty in vivo delivery due to the large size of the SDRE constructs. However, there has been conflicting evidence regarding the former, suggesting different cell types, organisms and methods of administration may play a part in the level of toxicity of Cas13 , 114. ToResearch also suggests that endogenous RNAs are competing with engineered gRNAs for binding to the effector proteins used as part of the SDRE constructs, including Cas13 . TherefoCurrently, there are no SDRE clinical trials to our knowledge. However, during a recent investigation, a system that achieves 50% A-to-I editing in vivo, in non-human primates, using a gRNA similar to the GluR2-ADAR method, described above, with no bystander editing, has been developed . InjectiThe most significant limitation for developing SDRE therapies is probably determining target mutations that could most significantly benefit from being corrected. Some SDRE methods do show multiplexing capabilities and can therefore be engineered to edit multiple residues by co-transfection of multiple gRNAs , 108. HoPCSK9 A > G mutations have been achieved in non-human primates, through the delivery of DNA base editors in vivo, via a CRISPR editing system [. The results presented by Musunuru et al. show around 60% PCSK9 suppression in the liver, which is significant in reducing LDL cholesterol and improving the familial hypercholesterolaemia phenotype caused by gain-of-function PCSK9 variants [. was engineered [PCSK9 mRNA and perform SDRE. Since the GluR2-ADAR method has already been tested in non-human primates as well, where it showed 50% editing efficiency, and since this method would only require the engineering of a gRNA, it is certainly worth being tested as a potential therapeutic approach.Recently, single nucleotide loss of function g system . The resvariants . The DNAgineered . Intrigugineered , includigineered   and C-to-U editing using adenosine and/or cytidine RNA deaminases as well as present comparisons between SDRE and other precision medicine approaches, such as DNA editing and RNA inhibition.Manipulation of adenosine and cytidine deaminases is at the epicentre for the development of site-directed RNA editing. Selective targeting is achieved through Watson–Crick base-pairing between the editing system  and the RNA target. The wide variety of SDRE methods allows for applications in various cellular and tissue models, and they can either direct endogenous editors to desired targets or deliver engineered constructs to target mRNAs. Currently, clinical interest is mostly invested in the former type of SDRE, due to its relative simplicity regarding delivery and synthesis, compared to the rather complex engineered constructs of methods using CRISPR, for example, which contain multiple parts and are quite large.Further non-site-specific therapies are also currently underway in development or clinical trials. For example, pharmaceutical inhibitors against RNA modifying enzymes, including ADAR1, are being developed and scheduled to enter the phase I clinical trial already this year . AlthougRNA therapeutics are no news to the era of precision medicine. Antisense oligonucleotides have been approved for treating disease as early as 1998, and there are a number of drugs that are currently being used for cardiovascular disease as well. SDRE strategies are anticipated to make subtle but decisive changes in mRNA sequences adequate to reverse the disease course. So far, such therapeutic methods have not been established for the treatment of cardiovascular disease. Nevertheless, our knowledge in terms of synthesis and delivery of oligonucleotides for the purpose of targeting RNA can be applied to site-directed RNA editing methods described in this review. Therefore, site-directed RNA editing approaches are deemed to feature as novel applications replacing existing “not very precise” therapeutic strategies in the near future.


Efficient virus multiplication depended on 3′-CITE activity. In CABYV isolates belonging to the three phylogenetic groups identified so far, the 3′-CITEs differ, and recombination prediction analyses suggest that these 3′-CITEs have been acquired through recombination with an unknown donor. Since these isolates have evolved in different geographical regions, this may suggest that their respective 3′-CITEs are possibly better adapted to each region. We propose that translation of other polerovirus genomes may also be 3′-CITE-dependent.Most plant viruses lack the 5′-cap and 3′-poly(A) structures, which are common in their host mRNAs, and are crucial for translation initiation. Thus, alternative translation initiation mechanisms were identified for viral mRNAs, one of these being controlled by an RNA element in their 3′-ends that is able to enhance mRNA cap-independent translation (3′-CITE). The 3′-CITEs are modular and transferable RNA elements. In the case of poleroviruses, the mechanism of translation initiation of their RNAs in the host cell is still unclear; thus, it was studied for one of its members, cucurbit aphid-borne yellows virus (CABYV). We determined that efficient CABYV RNA translation requires the presence of a 3′-CITE in its 3′-UTR. We showed that this 3′-CITE requires the presence of the 5′-UTR  Lactuca sativa) and fodder beet (Beta vulgaris), and several weeds [Aphis gossypii and Myzus persicae [Luteoviridae family that was recently abolished. While the luteovirus genus was reassigned to the Tombusviridae family, the polero- and enamovirus genera were reassigned to the Solemoviridae family [Cucurbit aphid-borne yellows virus (CABYV) was first described in the early 1990s in France ,5,6,7,8.persicae . Plants e family . The CABe family ,17,18. Re family ,21. Phyle family ,22,23. Le family ,22,24,25Tombusviridae family lack both the cap and poly(A) tail, and several of its species have been shown to harbor modular functional RNA elements in their 3′-end able to control their cap-independent translation, called cap-independent translational enhancer elements or 3′-CITEs [Tombusviridae) isolate able to overcome the eIF4E-mediated resistance in melon, MNSV-N [Although most eukaryotic mRNAs depend on the 5′-cap structure and the 3′-poly(A) tail for their translation, only about 20% of known positive-strand RNA viruses have genomic and subgenomic RNAs including these features . Thus, v3′-CITEs ,30. The 3′-CITEs ,32. To d, MNSV-N . MNSV pr, MNSV-N ,35. The , MNSV-N . This imin cis but do not need eIF4E for their activity, and that they are required for efficient virus multiplication.Since the RNA sequence from CABYV acquired by MNSV-N was shown to be a functional RNA element able to enhance the cap-independent translation of this viral genome, we hypothesized that it would also act as a 3′-CITE in its donor. Thus, in this manuscript we show that the CABYV 3′-UTR is able to enhance the cap-independent translation of a reporter gene, and identify different 3′-CITEs, depending on the group to which the CABYV isolate belongs. We show that these 3′-CITEs need the presence of the 5′-UTR Following phylogenetic analyses of the complete genome of CABYV isolates, these were classified into two groups, Asian and Mediterranean . We perfThese previous results, together with the alignments shown here, suggested that the variable first nucleotides of the CABYV 3′-UTRs (60–90 nts) of Asian and Mediterranean isolates could harbor different 3′-CITEs. To study the possible role of the CABYV 3′-UTRs in cap-independent translation, the firefly luciferase (luc) gene was flanked by the 5′- and/or 3′-UTRs of CABYV. The 3′-UTRs of the genome of Spanish and French CABYV isolates are identical, representing the Mediterranean isolates (CABYV-Sp) here, while the 3′-UTRs of Asian CABYV isolates are highly conserved , with CAvice versa, last two columns in Deletion of the first 60 nt of the 3′-UTRs in these 5′-luc-3′-UTR constructs reduced the translation efficiency to a similar level as that obtained in the absence of the complete 3′-UTR (sixth and ninth columns). On the other hand, RNAs with only the first 60 nt of the 3′-UTRs added to the 3′-end of the 5′-UTR-luc constructs showed similar translation efficiencies as the RNAs with both complete 3′-UTRs (seventh and tenth columns). These results allowed us to conclude that these 60 nt were sufficient for cap-independent translation control. Thus, these results confirm that the 3′-CITE identified as active RNA element in MNSV-N  is also working as a 3′-CITE in Asian CABYV isolates, while Mediterranean isolates also have a 3′-CITE that differs in sequence, now named CMTE (M for Mediterranean). Additionally, Nicotiana benthamiana protoplasts, the translation efficiency of the 5′-luc-3′-UTR construct including the CXTE, was higher than that including CMTE , was detected by RDP4, strongly suggesting that this first part of the 3′-UTR of Mediterranean CABYV, including the 3′-CITE, was acquired through recombination. A nucleotide BLAST search with the recombined new sequence did not result in any matches apart from Mediterranean CABYV. Several authors have hypothesized, and support has been provided by the first direct proof provided with our studies on MNSV-N, that 3′-CITEs are modular transferrable RNA elements in nature ,39. ThusThe CXTE in MNSV-N was shown to be active in the absence of eIF4E , giving http://www.major.iric.ca/MC-Fold), we obtained the mFold secondary structure predictions shown in http://www.unafold.org/mfold/applications/rna-folding-form.php). The nucleotide variations found in the CXTEs of the other Asian isolates support the structure, as they are either in unpaired regions, or if they involve nucleotides proposed to be paired, they do not affect complementarity. The structure obtained for the CXTE of CABYV-Xin was similar to the one of the CXTE identified in MNSV-N [The alignment in n MNSV-N , foldingNicotiana benthamiana leaves. We analyzed the infiltrated leaves by Northern blot to be able to study virus multiplication through the detection of the subgenomic RNA (sgRNA). N. benthamiana, as well as in melon, the mutant virus without a 3′-CITE multiplied inefficiently, while the chimeric one containing the CXTE replicated with an efficiency higher than the wild-type virus (with its own CMTE). Similar results were obtained with both CABYV clones expressed from the two different binary plasmids. These results were in line with the ones from the in vivo translation experiments obtained with the luciferase constructs shown above , turnip yellows virus (TuYV), beet western yellows virus (BWYV), pepper vein yellows virus (PVYV), faba bean polerovirus (FBPV), beet chlorosis virus (BChV), and beet mild yellowing virus (BMYV) . Apart fin cis but do not need eIF4E for activity. Congruently, as these RNA elements enhance virus protein synthesis, they are required for efficient virus multiplication. We have not been able to identify a 5′–3′-interaction that is functionally important for 3′-CITE activity yet.In this report, we study CABYV protein synthesis in host cells. We started this study after the identification of a sequence inserted in the 3′-UTR of a new MNSV isolate (MNSV-N) able to break eIF4E-mediated resistance in melon that was highly similar to the first 60 nucleotides of the 3′-UTR of CABYV . This seLuteovirus, until recently belonging to the same family, 3′-CITEs were also identified, all similar to that of barley yellow dwarf virus (BYDV) translational enhancer (BTE) [How polerovirus proteins are translated in the host cell is still unclear . We proper (BTE) ,51,52. Aer (BTE) ,53.N. benthamiana leaves, an additional mechanism for cap-independent translation of its proteins must exist.Here, we showed that uncapped CABYV-RNA is able to translate and multiply in melon protoplasts with a similar efficiency as capped CABYV-RNA, suggesting that the 5′-cap is not needed for initiation of infection from this input RNA. Polerovirus genomes are supposed to be uncapped . OtherwiVPgs from different viruses have been shown to interact with several eukaryotic translation initiation factors, suggesting a possible role in translation . On the Luteoviridae family, are recombination prone, and they have been proposed as having emerged from intergeneric recombination events. Thus, several recombinants between poleroviruses and luteoviruses have been described, and CABYV itself has been proposed to derive from recombinations between polero- and enamo-like viruses [Here, we identified different 3′-CITEs in each of the phylogenetically determined CABYV groups, Asian, Mediterranean, and Brazilian. Our recombination prediction analyses suggest that these 3′-CITEs have been acquired through recombination. The modularity and transferability of 3′-CITEs have been proposed before, as different types of 3′-CITEs have been found in a single genus, and the same type of 3′-CITE appeared in different virus genera . Additio viruses ,58,59. M viruses ,10,24,463′-CITEs that differ in sequence and structure coincide in the general mechanistic steps involving recruitment of translation initiation factors at the 3′-CITE, and delivery of these near the translation start site through communication with the 5′-UTR . Thus, wWe can conclude that an RNA element located in the first set of nucleotides in the 3′-UTR of the CABYV polerovirus genome enhances its translation in host cells in the absence of a 5′-cap or VPg. Isolates from the three different phylogenetic CABYV groups, which evolved in different geographical regions, have different 3′-CITEs. Translation of other polerovirus genomes may also be 3′-CITE-dependent.The 20 nt long 5′-UTR was cloned after hybridization of two complementary primers including the T7 promoter followed by the 5′-UTR sequence and flanked by KpnI and NcoI restriction sites, after its digestion with these enzymes. The resulting fragment was directionally cloned into the KpnI/NcoI sites of the T7-luc plasmid  and transcribed in vitro . Sequences of transcripts shown in Melon protoplasts were isolated from cotyledons as described in . Brief dThe protocol for the determination of the secondary structure of RNA fragments in solution by selective 2´-Hydroxyl acylation, analyzed by primer extension (SHAPE) was described in detail . BrieflyThe mutant viruses were constructed based on a full-length clone from an unpublished Spanish CABYV isolate infecting melon, cloned in pTOPO. The deletion of the first 60 nt of the 3′-UTR and the interchange of the CMTE in the 3′-UTR of CABYV-Sp for the CXTE from CABYV-Xin was performed by in vitro mutagenesis, using complementary primers including the sequence flanking both sides of the first 60 nt of the 3′-UTR, without this 60 nt, or with the CXTE 60 nt in between. Subsequently, DpnI digestion was used to digest the input plasmid and select for the mutant plasmids . The obtN. benthamiana leaves using the agro-infectious clones. The agro-infiltration and Northern blot protocols described in [N. benthamiana) or 10 μg (melon) of total RNA were loaded on a denaturing gel. Viral genomic and subgenomic RNAs were detected by digoxigenin-labelled RNA probe complementary to a highly conserved CP-gene fragment [CABYV wild-type and mutants were infiltrated into the abaxial side of expanded melon cotyledons or ribed in  were folfragment . For CABYV multiplication detection in melon protoplasts (see above), 5 μg of capped or uncapped RNA (transcribed in vitro,


RNA recombination is a major driver of genetic shifts tightly linked to the evolution of RNA viruses. Genomic recombination contributes substantially to the emergence of new viral lineages, expansion in host tropism, adaptations to new environments, and virulence and pathogenesis. Here, we review some of the recent progress that has advanced our understanding of recombination in positive-strand RNA viruses, including recombination triggers and the mechanisms behind them. The study of RNA recombination aids in predicting the probability and outcome of viral recombination events, and in the design of viruses with reduced recombination frequency as candidates for the development of live attenuated vaccines. Surveillance of viral recombination should remain a priority in the detection of emergent viral strains, a goal that can only be accomplished by expanding our understanding of how these events are triggered and regulated. RNA viruses cause significant morbidity and mortality to both humans and livestock on a global scale. This extensive group of viruses includes severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) , Ebola vRecombination is critical in the biology and evolution of RNA viruses. Accumulated evidence has shown that RNA virus recombination is one of the main approaches to virus rapid adaptation and evolution. However, there is a lack of in-depth understanding of the effects and the methods of studying virus recombination. This review provides a detailed summary of RNA virus recombination.Recombination in RNA viruses can be defined as an exchange of genetic material between at least two separate viral genomes. Recombination occurs when two or more viruses co-infect a single cell and exchange segments, often giving rise to viable hybrid progeny. To date, recombinant RNA viruses have been found in both positive-strand RNA viruses and negative-strand viruses . RecombiVarious animal and plant viruses exhibit different forms of recombination, some of which are unique to RNA viruses and different from DNA viruses. Based on the structure of viral RNA molecules and the sites where recombination occurs, RNA virus recombination can be classified into three types: homologous recombination, aberrant homologous recombination, and non-homologous recombination . HomologFlaviviridae family. HCV recombinants could occur at all levels, including between genotypes, between subtypes of the same genotype, and even between strains of the same subtype  is a positive-sense, single-stranded RNA genome of the  subtype , and a s subtype . The rec subtype . NotablyViral RNA recombination facilitates the ontogeny of viral variants with increased virulence and pathogenesis. The emergence of new variants could be the result of selective pressure or a positive selection. The new variant may respond to host immune pressure and develops advantageous mutations or recombination to make it more transmissible in susceptible populations or better able to escape our natural immune responses. Meanwhile, specific variants may be favored by having higher fitness in that context. The variant with higher replicative capacity can be selected and could be less virulent to the host, and the result does not necessarily have to be detrimental to the host. The impact of the new variant on the host may be double-sided. Understanding the propensity for RNA recombination between similar viruses infecting different host species, for example, establishes an important framework in the control of disease spread between endemic regions and could be used to model and predict the risks of future RNA virus infections.Accumulating evidence supports the hypothesis that recombination could result in an expansion of cellular tropism and even host tropism. For example, studies have suggested that Western equine encephalitis virus (WEEV), a zoonotic virus that causes encephalitis in humans and severe diseases in horses, seems to be the result of the recombination between an Eastern equine encephalitis virus (EEEV)-like virus and a Sindbis-like virus , exhibitMore recently, the SARS-CoV-2 pandemic has resulted in high morbidity and mortality in the human population. Despite extensive research, the origin of SARS-CoV-2 remains unclear. The receptor-binding domain (RBD) in its spike protein is almost identical in sequence to that of Pangolin-CoV , suggestRecombination in beta coronaviruses, including natural populations of MERS-CoV , SARS, aRecombinant viruses can preserve or increase the pathogenicity of the parental strains . For insVirulent variants of animal viruses can be generated by recombination. Recombination occurs between wild-type PRRSV strains and between the modified live vaccine (MLV) strain and wild-type PRRSV . In 2009Recombination can create considerable changes in the viral genome, allowing for antigenic shifts, host jumps, and fitness modifications . For virRecombination can generally generate virus variants to be able to escape host immune responses. For example, poliovirus recombinants have been isolated from vaccine-associated poliomyelitis patients. Certain recombinants, particularly between type 3 and type 2 poliovirus vaccine strains, are frequently isolated . RecombiAccumulating evidence has shown that RNA recombination occurs at specific RNA secondary structures or homologous sequences between the two parental viruses . TherefoIn addition to the host interferon system combating viruses early upon infection, vertebrate organisms employ RNA interference (RNAi) as an antiviral defense mechanism. In this regard, positive-stranded RNA viruses can undergo strand-switching to rapidly excise genomic material through recombination as an intrinsic defense against antiviral RNAi , renderiVarious molecular mechanisms have been proposed to describe the recombination events in RNA viruses. To date, both the replicative copy-choice model and the non-replicative breakage-rejoining model have gained widespread acceptance. Most RNA viruses recombine through a copy-choice mechanism, which involves the formation of recombinant RNA molecules when the RdRp switches from one template to another during genome replication . HoweverThe viral RdRp plays a central role in mediating template-switching recombination. The fidelity of viral RdRp has shown to be a fundamental determinant of recombination frequency in both cell-based and biochemical assays . Viral RNucleotide homology, nucleotide composition, and secondary structures of viral genomic sequences could all affect the frequency of recombination . RecombiRNA secondary structures were confirmed to convey critical functional information during the recombination process . It has Virus recombination can be affected by hosts. Several plant viruses, including TCV, tomato bushy stunt virus, cucumber necrosis virus, and potato virus X, have been shown to undergo non-homologous RNA recombination between a defective viral RNA and a helper wild-type virus RNA . As suchNidoviruses, including SARS-CoV-2, exhibit discontinuous transcription and produce a set of sub-genomic RNAs that contribute to high recombination rates . These vIn addition, bacterial infection may also affect viral recombination. The ability of enteric RNA viruses to undergo homologous recombination is variable and depends, at least in part, on bacterial infections that facilitate co-infection of different viral genotypes within the same cell .in vitro  . This meThe above approach is a more classic recombination detection method, and while the results are more accurate, the process is time-consuming with a huge workload.in vitro transcribed RNAs that alone cannot produce viable viruses. In this system, the “donor” RNA is a replication-competent, sub-genomic RNA encoding a luciferase reporter gene in place of the viral capsid sequences. In turn, the “acceptor” RNA is a replication-incompetent, genomic RNA that has a defective cis-acting replication element, and only after successful recombination between these two parental genomes, viable progeny recombinant viruses can be produced and quantified.A cell-based recombination assay (termed CRE-REP) established by the Evans laboratory has drastically advanced studies of enterovirus recombination . This apPicornaviridae have used this kind of assay to identify the recombination rate. Whether this method can be applied to other virus families has not been investigated.Since its emergence, this technique is currently extensively utilized for virus recombination studies in the poliovirus . ResearcA recent study of poliovirus recombination utilizes two different biochemical assays to distinguish copy-choice recombination from forced copy-choice recombination . In bothRecent advances in genome sequencing by third-generation sequencing technologies (TGS), including PacBio single-molecule real-time sequencing and nanopore sequencing technology, can generate reads of >10 kb in length . The lonWith the advancement and maturation of sequencing technology, an increasing number of experiments are opting for third-generation sequencing for the research. Although third-generation sequencing is certainly speedy and convenient for recombinant virus analysis, its high cost and greater error rate have led to some laboratories using Sanger sequencing instead.Bioinformatics analysis of genomic sequence data contributes to an in-depth understanding of the evolutionary process, including the impact of genomic structure , phenotyMost RNA viruses can recombine either by homologous or non-homologous recombination, both of which play an important role in the biology and evolution of RNA viruses. Recombination is a major driving force for viral diversity and has significant biological consequences on viral biology. Recombination generates novel viral strains, which expands virus genetic diversity, increases virulence and pathogenesis, and promotes the emergence of new circulating viruses. In addition, recombination can facilitate the evolution of antiviral resistance, host immunity escape, and associate with alterations or expansions of viral host tropism. Thus, it is important to emphasize that viral recombination detection and surveillance need to be prioritized for viral disease control and prevention. Understanding the mechanisms of virus recombination and the factors that affect recombination can ultimately help us design live attenuated vaccines unable to recombine with circulating strains.Simultaneously, recombination can be used as a beneficial tool for research. Reporter viruses are useful tools for screening susceptibility and resistance to antiviral compounds. However, the stability of reporter viruses has been challenged by viral RNA recombination, leading to the deletion of the reporter gene during viral replication. Lethal mutations upon recombination have been engineered to create a stable Zika virus harboring a NanoLuc-reporter gene . The devTA and XHC contributed to conception of the study. HW and XYC wrote the draft. TA revised the draft. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


La-related proteins (LARPs) comprise a family of RNA-binding proteins involved in a wide range of posttranscriptional regulatory activities. LARPs share a unique tandem of two RNA-binding domains, La motif (LaM) and RNA recognition motif (RRM), together referred to as a La-module, but vary in member-specific regions. Prior structural studies of La-modules reveal they are pliable platforms for RNA recognition in diverse contexts. Here, we characterize the La-module of LARP1, which plays an important role in regulating synthesis of ribosomal proteins in response to mTOR signaling and mRNA stabilization. LARP1 has been well characterized functionally but no structural information exists for its La-module. We show that unlike other LARPs, the La-module in LARP1 does not contain an RRM domain. The LaM alone is sufficient for binding poly(A) RNA with submicromolar affinity and specificity. Multiple high-resolution crystal structures of the LARP1 LaM domain in complex with poly(A) show that it is highly specific for the RNA 3′-end, and identify LaM residues Q333, Y336 and F348 as the most critical for binding. Use of a quantitative mRNA stabilization assay and poly(A) tail-sequencing demonstrate functional relevance of LARP1 RNA binding in cells and provide novel insight into its poly(A) 3′ protection activity. The family of La-related proteins (LARPs) are RNA-binding proteins with multiple functions in the regulation of gene expression ,2. Each 7Gppp cap  from deadenylation and associated mRNA stabilization. Both interact with poly(A)-binding protein (PABPC1) via a PABP-interacting motif 2 (PAM2)  using the purified LARP1 La-module  RNA, but not with poly(U), poly(C), or poly(G) . High afa-module .In the present study, we use a variety of biophysical techniques to investigate the nature of the LARP1 La-module. Unexpectedly, we find that the module does not contain a RRM domain. NMR and ITC studies demonstrated the stand-alone LaM domain binds RNA with submicromolar affinity and preference for A-rich sequences. High-resolution crystal structures of LARP1 LaM in complex with poly(A) sequences of different length reveal the molecular basis for specificity for the RNA 3′-end and residues required for RNA binding. Using cellular assays of mRNA levels and poly(A) tail length, we demonstrate that high affinity binding by the LaM domain is critical for mRNA stabilization by LARP1.Escherichia coli BL21(DE3) in rich (LB) medium as a fusion with N-terminal His-tag using induction with 1 mM IPTG for 4 h at 30 ºC. For NMR experiments, the recombinant protein was isotopically labeled by growth of E. coli BL21 in M9 minimal medium with 15N-ammonium sulfate as the sole source of nitrogen. Doubly labeled protein was prepared from cells grown in 15N-ammonium sulfate and 13C-glucose. Cells were harvested and broken in lysis buffer  containing 1 mM PMSF, 0.1 mg/ml lysozyme, 0.01 mg/ml DNase, 5 mM imidazole. The His-tagged proteins were purified by affinity chromatography on Ni2+-charged chelating Sepharose resin. The proteins were eluted with lysis buffer containing 500 mM imidazole. The resulting proteins contained an N-terminal MGSSHHHHHHS extension and were additionally purified and exchanged into the final buffer using size-exclusion chromatography using a HiLoad 16/600 Superdex 75 PG column (Cytiva) with HPLC buffer .Plasmids for bacterial expression of the LARP1 fragments 323–410, 323–417, 323–439, 323–509, 417–509 and 399–540 were obtained by mutagenesis of 310–540 fragment cloned into pET28a vector introducing deletions and stop codons at the appropriate positions using QuikChange Lightning Site-Directed Mutagenesis Kit (Thermo Fisher Scientific). LARP1 point mutants were obtained by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit (Thermo Fisher Scientific). DNA sequencing was used to verify all sequence modifications. Proteins were expressed in 25, A11, A6 and C2UCU4C2GUG2CGC2UC were obtained from Sigma-Aldrich. A2UA3, A2CA3, ACA4, A5A(p), A5A(2′-OMe) were from Integrated DNA Technologies. Commercially obtained RNA samples were used without additional purification. A3, A2dA, A4, A5U, A4UA, A3UA2, A5C, A4CA, A3CA2 and additional samples of A6 and A25 were synthesized on 2 × 2 μmol scale using an ABI 3400 synthesizer with standard β-cyanoethylphosphoramidite chemistry on long chain alkylamine controlled pore glass  with standard synthesis protocols. Oligonucleotides were purified either by preparatory denaturing PAGE or ion-exchange HPLC and desalted using C-18 SEP PAK cartridges as previously published (Aublished .15N/13C) labeled protein were used to assign the backbone resonances. The assignments were deposited in Biological Magnetic Resonance Data Bank (BMRB) under accession number 51255. For NMR titrations, A2 and A6 RNAs were added to 15N-labeled LARP1 fragments to the final molar ratios of 1:2 or 1:1, respectively. Chemical shift perturbations were calculated as the weighted sum of proton and nitrogen shifts using the equation (ΔδH2 + (ΔδN/5)2)1/2. All NMR experiments were performed at 25 ºC using Bruker 600 MHz spectrometer. NMR spectra were processed using NMRPipe  experiments on doubly . The syringe was typically loaded with 300 μM protein, while the sample cell contained 30 μM RNA . All exp3,  for A4,  or  for A6,  for A11,  for A3UA2. Crystals grew in 3–14 days at 20ºC. For data collection, crystals were cryo-protected by soaking in the reservoir solution supplemented with 30% (v/v) ethylene glycol in conditions using PEG3350 or with 25% glycerol otherwise.Initial crystallization conditions were identified utilizing sitting drop vapor diffusion with the Classics II and Nucleix screens (QIAGEN). The best LARP1 LaM domain crystals were obtained by equilibrating a 0.6 μl drop of the protein (residues 323–410) at 20 mg/ml in HPLC buffer , mixed with 0.6 μl of reservoir solution containing 0.2 M ammonium sulfate, 0.1 M Bis–Tris pH 6.5, 25% (w/v) PEG 3350. Crystals grew in 30–40 days at 20ºC. The best LARP1 LaM domain/RNA complex crystals were obtained by equilibrating a 0.6 μl drop of the LaM domain (residues 323–410) with oligonucleotide in a 1:1.1 molar ratio (10 mg/ml of protein) in buffer , mixed with 0.6 μl of reservoir solution containing  or  for ADiffraction data from single crystals of LARP1 LaM domain and its RNA complexes were collected at the Canadian Light Source (CLS), Cornell High-Energy Synchrotron Source (CHESS) and Advanced Photon Source (APS) . Data pret al. (The wild-type LARP1 cDNA corresponding to isoform-1 (1019 aa) version as in Mattijssen et al.  was subc5 cells per six-well plate were seeded one day prior to transfection with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Because the LARP1 mutant accumulates to lower levels than wild-type LARP1 (not shown), more plasmid was added, and the difference was made up to 1 μg with empty pFLAG-CMV2 plasmid. For wild-type LARP1, 125 ng was transfected plus 875 ng empty plasmid; for LARP1-Q333A, 312.5 ng plus 687.5 ng empty plasmid. In addition, 100 ng of pcDNA3.1-β-globin−TNFα−ARE, 100 ng of pcDNA-TPGFP and 25 ng of pVA1 were cotransfected. 24 h post transfection, cells were split 1:4 into multiple wells, allowed to grow for another 24 h and then harvested for protein and RNA.HEK293T LARP1 KO cells were a gift of Carson Thoreen  and previously characterized for TOP mRNA and related translation factors . Transfe32P-labeled oligo probes were added, and hybridization was overnight at Ti. Oligo probes used and their Ti can be found in Mattijssen et al  following manufacturer's instructions, except that the RNA pellet was washed 3 times with 1 ml 75% EtOH instead of once. Total RNA was then separated on a 1.8% agarose-formaldehyde gel and transferred to a GeneScreen-Plus membrane (PerkinElmer) overnight. After crosslinking with UV and vacuum-baking for 2 h at 80ºC, the membrane was prehybridized in hybridization solution  for one hour at hybridization incubation temperature (Ti). en et al . VA1 RNACells were washed twice with PBS and cell lysis was performed directly in RIPA buffer (Thermo Scientific) containing protease inhibitors (Roche). Proteins were size separated using SDS-PAGE and transferred to a nitrocellulose membrane. Primary antibodies used were anti-FLAG , anti-actin  and anti-GFP . Secondary antibodies from LI-COR Biosciences, which were conjugated to either IRDye 800CW or 680RD and western blot were scanned using the Odyssey CLx imaging system (LI-COR Biosciences).2O per cartridge. Protein was analyzed by western blot to confirm equal levels of wild-type and mutant FLAG-tagged LARP1.Single-molecule polyadenylated tail sequencing (SM-PAT-seq) was performed along with data handling and analysis as previously described ,42. For Arabidopsis thaliana, Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, Escherichia coli, Homo sapiens, Mus musculus, Rattus norvegicus and Saccharomyces cerevisiae, Danio rerio, Drosophila melanogaster and Mus musculus, Schizosaccharomyces pombe elegans and other metazoa were noted to lack an RRM ((Multiple members of the LARP super family from a variety of species were previously demonstrated to have tandem LaM and RRM domains with both engaged in RNA-binding activities . Althoug an RRM ( and refeSecondary structure prediction of human LARP1 with JPred4  shows th15N-labeled proteins purified for NMR spectroscopy. The 1H–15N correlation spectrum of residues 323–509 encompassing the LaM-RRM (numbered according to the 1019-residue long isoform) showed a mixture of weaker, dispersed signals and a central band of intense signals  yielded a well-dispersed spectrum that closely matched the weaker signals in the (323–509) NMR spectrum including the Trp406 indole proton  representing the putative RRM alone, produced a spectrum with poorly dispersed signals that matched the unfolded set of signals in the 323–509 fragment  with little difference compared to LaM 323–439 or 323–410  to measure the affinity of the LARP1 La-module for RNA. ITC thermograms with LARP1 (323–509) and AM Figure  with a sM Figure . SimilarM Figure , B, and M Figure . These d0 Figure . This co0 Figure ,22.11, A6 and A4 bound with roughly the same affinity as A25. The small variations in affinity and binding stoichiometry are due to uncertainties in the ligand concentration and a slight tendency of the LARP1 (323–410) fragment to precipitate during ITC experiments.We carried out additional ITC experiments to determine ligand binding specificity of the LaM. Comparison of shorter oligonucleotides showed that three or four nucleotides were sufficient to capture most of the binding affinity Figure . Notably25 suggest an absence of LaM binding to the middle nucleotides of the poly(A) RNA. Specificity for the 3′-end was determined by experiments with RNAs modified at the 3′-ribose. Addition of a 3′ phosphate on A6 led to a 200-fold loss of affinity  had no effect on Kd  assigned using standard triple resonance techniques  shows most of affected residues are in the N-terminal half of the domain.We used triple resonance and ZZ-exchange experiments to assign the signals for the LaM-Ag Figure . PlottinWe employed X-ray crystallography to investigate the molecular mechanisms responsible for the binding specificity of LARP1 Figure . CrystalUnsurprisingly, the structure is highly similar to previous LaM module structures but without the RRM domain Figure . One con3  nucleotide ribose O2′ makes a hydrogen bond with the ordered water while the adenine base is flipped out and away from the 3′-nucleotide. The A(–2) base stacks against the side chain of Tyr336 and the adenine N3 forms a hydrogen bond with the side chain of Gln333, which may contribute to the base selectivity. The A(–3) nucleotide is flipped back where it stacks against the A(–1) base. The phosphate between the –2 and –3 nucleotides is stabilized by Arg345 . This st4 bound  nucleotide is partially disordered and does not make any contacts with the LaM. Instead, the nucleotide A(–4) stacks on A(–1). This stacking arrangement (–4/–1) was observed in four of the seven ligand structures  and A(–4) nucleotides  RNA, which results from tight binding to the LaM domain Figure –J. The gleotides . The str1H–15N correlation spectra. All the mutants yielded NMR spectra similar to the wild-type protein consistent with small perturbations restricted to the environs of the mutations  and tested their affinity for RNA by ITC Figure . We veriutations . Single 4  cells to monitor the ability of LARP1 to stabilize a widely used type reporter β-globin mRNA containing a 38 nucleotide AU-rich element (ARE) of instability in its 3′ UTR that was taken from the TNFα mRNA (t1/2) from several hours to 75–90 min  RNA. ITC experiments showed that the LaM specifically recognizes the 3′-end of RNA with a clear preference for A-rich sequences Figure . Poly(C)4 binds with only 3-fold better affinity than A3. We further used the crystal structures to design point mutants that specifically block RNA binding. Loss of invariant Gln333, whose side chain hydrogen bonds with the adenine at position –2, decreased RNA binding affinity 20-fold with no effect on the global fold  sequences interacting with LARP3 . In LARPfactor B ,29. The factor B ,6,23. LAlization ,40,41,53The RNA binding behavior of the LARP1 LaM in the context of the full-length protein is less well-understood. While there is agreement that poly(C) RNA does not bind, a study of LARP1 (310–540) using EMSA binding assays showed a TOP20 pyrimidine-rich sequence bound with 40 nM affinity . Pull-doLARP1 contains a second RNA-binding DM15 domain at its C-terminus. This domain, which is unique to LARP1 and LARP1B, binds the mRNA 5′-cap and TOP sequences to negatively regulate TOP mRNA translation in response to mTORC1 inhibition ,10–12,15t1/2, ≤1.5 h), and GFP, a long-lived, stable mRNA   . The seq, ≥6 h . Cumulative data in this report lead us to propose that LARP1 protection of β-glo-ARE and GFP mRNA reflects the ability of LARP1 to bind poly(A) 3′ ends and protect them from deadenylases. The PAT-seq data for these mRNAs fits with the biphasic kinetics model of deadenylation and is consistent with this interpretation. Specifically, the difference between LARP1-WT and LARP1-Q333A in PAT length protection of the GFP mRNA substrate Figure  is greatIn conclusion, our studies add new information and insight into the structural basis of mRNA stabilization by LARP1. Although full-length LARP1 has a mass of > 100 kDa, a single mutation in the LaM domain significantly impacts its ability to stabilize mRNA. Our previous observation that a 27 kDa fragment (residues 310-540), containing the LaM domain and PAM2 motif, was sufficient for PAT protection and mRNA stabilization highlights the central role of the LaM domain . The ideThe coordinates were deposited into the RCSB Protein Data Bank with the accession codes: 7SOQ, 7SOR, 7SOR, 7SOS, 7SOU, 7SOT, 7SOP, 7SOV. NMR signal assignments for LARP1 LaM domain have been deposited as BMRB entry 51255.gkac696_Supplemental_FilesClick here for additional data file.


Pseudoophonus rufipes [De Geer]) were offered either fresh or 1‐day‐old dead Drosophila melanogaster fruit flies (carrion). The detectability of prey RNA and DNA in the beetles' regurgitates was assessed with diagnostic Drosophila‐specific RT‐PCR and PCR assays at 0, 6, 12, 24 and 48 h post‐feeding. After fresh fly consumption, prey RNA and DNA were detectable equally well at all times. When carrion prey was consumed, the detection strength of prey RNA immediately after feeding was significantly lower than that of prey DNA and reached zero in most samples within 6 h of digestion. Our findings provide evidence that prey RNA allows distinguishing between the consumption of fresh and scavenged prey, thereby overcoming a long‐known weakness of molecular diet analysis. The assessment of prey RNA offers a generally applicable approach for examining the importance of scavenging in food webs to unravel its functional consequences for populations, communities, and ecosystems.Facultative scavenging by predatory carnivores is a prevalent but frequently underestimated feeding strategy. DNA‐based methods for diet analysis, however, do not allow to distinguish between scavenging and predation, thus, the significance of scavenging on population dynamics and resource partitioning is widely unknown. Here, we present a methodological innovation to differentiate between scavenging and fresh prey consumption using prey RNA as a target molecule. We hypothesized that the rapid post‐mortem breakdown of RNA in prey tissue should lead to a significantly lower detection probability of prey RNA than DNA when carrion rather than fresh prey is consumed. To test this hypothesis, ground beetles ( Cups were stored in a climate cabinet with an artificial day‐night rhythm of L:D 14:10 h and an alternating temperature regime of 22 and 12°C, respectively. They were ventilated daily and moistened tissue was renewed every second day. The beetles were maintained on a diet of mealworms  until being starved for 4 days before the feeding experiments. At the end of the study, all carabids were released unharmed to a suitable habitat.Adult beetles of the species 2.2D. melanogaster   served as animal prey. Fly colonies were obtained from the laboratory rearing of the Molecular Biology Division, Medical University Innsbruck, Austria, and propagated on a cornmeal‐yeast diet in the same climate cabinet described above until enough flies had emerged.Common fruit flies, Shortly before the feeding experiments, adult flies were freeze‐killed at −16°C. To simulate two prey types in the experiment, frozen flies were offered to carabids either immediately after defrosting (subsequently referred to as fresh prey) or after decaying in the climate cabinet for 24 h (subsequently referred to as carrion prey).2.3For the feeding experiments, carabids were placed individually in clean screw‐lid cups, containing one fly, representing either fresh or carrion prey, and a drop of water. Carabids were allowed to feed for 1 h in darkness in the climate cabinet. In case carabids had not consumed the fly, they were granted another hour of feeding, up to three times. If carabids had consumed the entire fly, they were included in the experiment and stimulated to regurgitate at different points in time after feeding. To do so, beetles were placed individually headfirst in 1.5 ml microtubes. Regurgitation was then elicited through heat stress by repeatedly dipping the tip of the tube into hot water for less than a second  with minor alterations: the VXL‐buffer mastermix was prepared without carrier RNA and total nucleic acids were eluted in molecular grade water instead of AVE buffer to facilitate subsequent DNA digestion.Regurgitates were mixed with 200 μl DNA/RNA Shield (Zymo Research) and total nucleic acids were extracted using the IndiSpin Pathogen Kit  on a BioSprint96 automatic extraction platform (Qiagen) according to the manufacturer's instructions ‐PCR. Thus, to allow for a comparison of the presence of prey DNA and RNA in the samples, the extracts of total nucleic acids were split, and one portion was treated with DNase to get pure RNA extracts as described below. Total DNA/RNA extracts will further be referred to as “DNA extracts” and digested split samples as “RNA extracts”. DNA/RNA were extracted in two independent rounds. The first batch included 92 samples; the second batch had 23 samples. In each batch, two extraction negatives (molecular grade water) were included to check for possible cross‐contamination.2.5The Monarch RNA Cleanup Kit (New England BioLabs Inc.) was used as follows: a mix of 22.2 μl DNA/RNA extract, 2.5 μl DNase I reaction buffer and 0.3 μl DNase I enzyme was prepared on ice before incubation at 37°C for 20 min. Then, 0.5 μl of 0.25 M EDTA was added to each sample, followed by heat inactivation of DNase at 75°C for 15 min. Incubation and heat inactivation were both done in an Eppendorf Mastercycler nexus (Eppendorf AG).Drosophila DNA target molecules with the PCR protocol described for prey DNA detection below, but with a stricter detection threshold: in case any Drosophila DNA was still amplified in an RNA sample, the DNA digestion was repeated with more enzyme and a longer incubation. To do so, 22.0 μl fresh DNA/RNA extract, 2.5 μl DNase I reaction buffer, 0.5 μl DNase I enzyme were incubated at 37°C for 30 min. Inactivation and screening for residual DNA were the same as above. After this extended DNA digestion, no Drosophila DNA was detectable in any of the RNA samples.The digested samples were thereafter screened for residuals of 2.6Drosophila spp., that can detect initial prey DNA amounts of as little as 0.02 pg  and Droso‐A381 (5′‐GTAATACGCTTACATACATAAAGGTATA‐3′), targeting a 240 bp fragment of the nuclear 18S rDNA of Drosophila DNA were screened with RT‐PCRs, also employing the primer pair Droso‐S391/Droso‐A381. For RT‐PCR, 10 μl reactions contained 2 μl RNA extract, 1× OneStep RT‐PCR Buffer (Qiagen), 400 μM of each dNTP, 0.6 μM of each primer, 0.4 μl Qiagen OneStep RT‐PCR Enzyme Mix and molecular grade water. Thermocycling included a reverse transcription step of 30 min at 50°C followed by denaturation at 95°C for 15 min, 35 cycles of 94°C for 30 s, 62°C for 60 s and 72°C for 60 s, and final elongation at 72°C for 10 min.The RNA extracts without residual All samples were screened without technical replicates. Within each PCR/RT‐PCR, one negative control (molecular grade water) and one positive control (fly DNA/RNA) were included to check for carry‐over contamination and amplification success, respectively.n = 14) of beetles fed with carrion prey, one of the extraction negatives tested positive for Drosophila DNA and RNA. To allow for all samples to be used in the analysis despite this contamination, we subtracted the RFU value measured in the extraction‐negative from the values of the samples in batch 2  before any further data processing. All PCR negative controls, however, were clean.Undiluted PCR/RT‐PCR products were visualized by capillary electrophoresis on the QIAxcel Advanced System (Qiagen), with the method AM320 and an injection time of 30 s. Peaks above a threshold of 0.1 relative fluorescent units (RFU) were considered positive, and the RFU value of each PCR product was recorded as a relative estimate of DNA or RNA content in the sample. In the extraction batch 2, which was comprised of regurgitates taken after 24 h (8 of 13 samples), and all taken after 48 h  with a LOGIT link function. DNA and RNA detections were therefore translated into a binary response variable (cutoff ≥0.1 RFU) and digestion time was used as a predictor for detection probability of each target molecule for each prey type, separately. To test for the effect of prey type, the overall detection probabilities of prey DNA and prey RNA were calculated in combined models using the variables digestion time and prey type as predictors. Using the function “lrtest” of the r‐package “lmtest”  test.We calculated a within‐sample ratio of RFU3A total of 116 regurgitates were screened and used for data analysis . Prey RN3.1Drosophila‐specific prey RNA ranged from 0 to 3.67 RFU. The relative amount of prey RNA, measured as signal strength , was frequently higher in regurgitates with fresh than in those with carrion prey . At a fixed time after feeding, the odds of detecting prey RNA in regurgitates with carrion was 92% lower than in samples with fresh prey .Overall, the detection probability of prey RNA was significantly negatively affected by both, digestion time and prey type . Within p = .005) lower after carrion prey consumption. The odds of DNA detection decreased by 10%  per passing hour of digestion.For DNA, the overall detection probability significantly decreased over time of digestion and was lower after carrion prey consumption. The odds ratio of DNA detection was 83%  and 6 h after feeding   . At 24 h4We demonstrated that the detection probability and signal strength of prey‐specific RNA was significantly lower when carrion prey had been consumed. This supports our core hypotheses that prey RNA detection success is reduced in carrion prey due to a continuous breakdown of RNA within the carcass. Surprisingly, detection of prey RNA and DNA worked equally well when fresh prey had been consumed, conflicting with the common expectation that RNA should deteriorate faster than DNA  intron , which is a feasible and sensitive method for relative target fragment quantification (Thalinger et al., Apart from methodological aspects that need to be considered in future studies with prey RNA, several biological factors might affect the detectability of prey RNA. First, the age of carrion will be an important factor that needs to be addressed. Previous studies have shown that prey DNA detectability decreases with carrion age (Foltan et al., In conclusion, we demonstrated that prey RNA can be detected in gut content samples after extended periods post‐feeding if fresh prey was consumed, but only very briefly and at strongly reduced amounts after carrion prey consumption. The analysis of prey RNA in parallel to prey DNA, therefore, holds great promise to differentiate between trophic links involving feeding on carrion and fresh prey. The prey RNA approach will apply to diagnostic and metabarcoding techniques alike and bears relevance for a wide range of different food web systems. While further research on this topic is anticipated, we suggest this novel approach will be of high value to assess the significance of scavenging for populations, communities, and ecosystems.Michael Traugott came up with the conceptual idea for the present research, Michael Traugott and Veronika Neidel led the funding acquisitions, Daniela Sint and Corinna Wallinger developed the methods, Veronika Neidel and Corinna Wallinger carried out the feeding experiments, Veronika Neidel carried out the molecular and statistical analysis and led the writing of the manuscript.The authors declare no conflict of interest.Benefits from this research derive from sharing the data and results on a database as described above.Table S1‐S5Click here for additional data file.


Real-time polymerase chain reaction analysis showed that overexpression of long noncoding RNA Gm9866 in RAW264.7 cells further promoted the expression of M2 markers MRC1 (macrophage mannose receptor 1) and MRC2 (macrophage mannose receptor 2). Western blotting analysis demonstrated inhibition of nuclear factor-κB (NF-κB) expression. EdU (5-ethynyl-2′-deoxyuridine) and TUNEL (TdT-mediated dUTP nick-end labeling) staining assays revealed that overexpression of long noncoding RNA Gm9866 promoted cell proliferation and inhibited apoptosis. These findings thus indicated that long noncoding RNA Gm9866 promoted macrophage polarization and inhibited the nuclear factor-κB signaling pathway. Thus, long noncoding RNA Gm9866 may serve as a potential diagnostic and therapeutic target for polarization-related diseases such as infectious diseases, inflammatory diseases, liver fibrosis, and tumors.Macrophages are a type of immune cells with high levels of plasticity and heterogeneity. They can polarize into M1 or M2 functional phenotypes. These two phenotypes exhibit a dynamic balance during polarization-related diseases and play opposing roles. Long noncoding RNAs (lncRNAs) play an important role in biological processes such as cell proliferation, death, and differentiation; however, how long noncoding RNAs affect the cellular functionality of macrophages remains to be studied. Long noncoding RNA Gm9866 was found to be closely related to macrophage polarization through bioinformatics analysis. In this study, by conducting real-time polymerase chain reaction analysis, it was observed that long noncoding RNA Gm9866 expression significantly increased after treatment with interleukin-4 but significantly decreased after treatment with lipopolysaccharide. Fluorescence  Macrophages play a dual role in immune response. They have high plasticity and can polarize into different phenotypes and play different functions under the action of different immune immunological conditions , 2. Lipoclerosis , inflammclerosis , and insclerosis  and prome repair . Intervee repair –9. Severe repair . In anote repair . Wang ete repair . Howevere repair  and screκB (NF-κB) pathway. Thus, the present study is aimed at identifying the roles of long noncoding RNA Gm9866 in cell proliferation, polarization, and apoptosis and to further explore its potential mechanisms during M2 polarization of RAW 264.7 cells. These experimental results revealed a novel role of long noncoding RNA Gm9866 in the polarization of macrophages, which may contribute to the development of targeted therapeutic strategies for macrophage polarization-related diseases such as atherosclerosis, inflammatory bowel disease, insulin resistance-related obesity, and tumors.It is hypothesized that long noncoding RNA Gm9866 promotes cell proliferation and M2 polarization and inhibits cell apoptosis in macrophages via the nuclear factor-p value less than 0.05 was considered significant. Next, the common differentially expressed long noncoding RNAs from the two datasets were identified by overlapping them [To screen for the functional long noncoding RNAs involved in macrophages M2 polarization, we selected two microarray datasets (GSE107952 and GSE107979)  from Gening them .2 [RAW 264.7 cells were purchased from the American Type Culture Collection . The cells were cultured in Dulbecco's modified Eagle medium  supplemented with 10% heat-inactivated fetal bovine serum , 1% penicillin/streptomycin, 1% sodium pyruvate, and 3.7 g/L sodium bicarbonate at 37°C under 5%CO2 .μg/mL, 2 μg/mL, 4 μg/mL, and 8 μg/mL; and (3) IL-4  at concentrations of 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, and 5 ng/mL. Each treatment was for an additional 24 h [RAW 264.7 cells were serum-starved for 18 h and then divided into three groups: (1) control; (2) LPS  at concentrations of 100 ng/mL, 1 nal 24 h .in situ hybridization assays . The cells were fixed with 4% paraformaldehyde , prehybridized, and then immersed in hybridization solution containing the long noncoding RNA Gm9866 probe marked with cyanine 3 (Cy3) and incubated overnight at 37°C. Then, the cells were stained with 4′,6-diamidino-2-phenylindole . Images were then acquired by laser scanning confocal microscopy  [Subcellular localization of long noncoding RNA Gm9866 was detected by fluorescence Germany) .2 incubator for 24 h [The long noncoding RNA Gm9866 was overexpressed in RAW 264.7 cells by transfecting the cells with a long noncoding RNAGm9866-overpressing plasmid  with Advanced DNA RNA Transfection Reagent™  in accordance with the manufacturer's protocol. In brief, RAW264.7 cells were placed on the surface of culture plates 1 day in advance and allowed to grow to 60−80% confluency. Then, the plasmid was directly mixed with transfection regent (1 : 1) and mixed by using a pipette (10–15 times). Following incubation at room temperature for 15 min, the mixture was added to the cell culture plates, mixed gently, and incubated in a COfor 24 h .The relative gene expression data were analyzed using a previously reported method . Total Rβ-Actin  was used as a loading control. The primary antibody specific for NF-κB (nuclear factor-κB) was obtained from AbMART . Anti-rabbit and anti-mouse second antibodies were obtained from Signalway Antibody . An Odyssey two-color infrared laser imaging system  was employed to scan the blots. The grey values were quantitatively analyzed using ImageJ software  [Radioimmunoprecipitation assay (RIPA) buffer  was used to lyse cells, and the BCA kit  was used to quantify protein levels. IH, USA) .μM EdU (5-ethynyl-2′-deoxyuridine) solution for 24 h, followed by fixation with 4% paraformaldehyde for 30 min and permeation using 0.5% TritonX-100 for 10 min. The treated cells were stained with an Apollo fluorescent dyeing solution and Hoechst 33,342 for 30 min and observed with an inverted fluorescence microscope . All experiments were performed in triplicate [Cell proliferation was monitored by EdU (5-ethynyl-2′-deoxyuridine) assays . In brief, cultured RAW264.7 cells were incubated with 50 iplicate .TUNEL (TdT-mediated dUTP nick-end labeling) staining kit  was used to determine apoptosis of RAW264.7 cells. The experimental methods were performed strictly in accordance with the manufacturer's instructions. A fluorescence microscope  was used to observe the features of apoptosis. The cells stained with TUNEL (TdT-mediated dUTP nick-end labeling) were considered apoptotic. The total number of apoptotic cells was calculated using DAPI (2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride) staining .t-test and one-way analysis of variance (ANOVA). Probability (p) values < 0.05 were considered statistically significant [Data of the three independent experiments are presented using themean ± standarddeviation. SPSS version 25.0  and GraphPad Prism (GraphPad Software Inc.) were used for all statistical analyses. Data were compared with the Student's nificant .in vitro experiment with RAW 264.7 cells. We also found that interleukin-4 upregulated but lipopolysaccharide (LPS) downregulated the expression of long noncoding RNA Gm9866 in RAW 264.7 cells. Moreover, the phenotypic markers of M2 macrophages increased significantly after the overexpression of long noncoding RNA Gm9866 in RAW 264.7 cells, which promoted M2 polarization and decreased nuclear factor-κB protein expression. The overexpression of long noncoding RNA Gm9866 also promoted the proliferation of RAW 264.7 cells and inhibited apoptosis. These findings suggested that the upregulation of long noncoding RNA Gm9866 may contribute to the progression of macrophage polarization-related diseases. Finally, laser confocal microscopy showed that long noncoding RNA Gm9866 was expressed in the cytoplasm and nucleus of macrophages and mainly in the nucleus.The present study demonstrated that long noncoding RNA Gm9866 expression was upregulated during the polarization of macrophages toward the M2 type as noted in the published transcriptome dataset and verified in the The GSE107979 and GSE107952 datasets  were usep < 0.05 for 1 ng/mL vs. 4 ng/mL, 1 ng/mL vs. 5 ng/mL, 2 ng/mL vs. 4 ng/mL, 2 ng/mL vs. 5 ng/mL, 3 ng/mL vs. 5 ng/mL, and 4 ng/mL vs. 5 ng/mL).To investigate whether long noncoding RNA Gm9866 expression was associated with macrophages polarization, unpolarized macrophages, lipopolysaccharide-induced M1 macrophages, and interleukin-4-induced M2 macrophages were obtained. The expression level of long noncoding RNA Gm9866 was then determined. The expression level of long noncoding RNA Gm9866 was significantly increased after interleukin-4 treatment but was significantly decreased after lipopolysaccharide treatment as compared to that of nonpolarized macrophages Figures ; these fκB family was involved in the process of macrophage polarization. On the basis of the above-mentioned result, it was clear that long noncoding RNA Gm9866 promoted macrophage polarization; hence, we speculated that the nuclear factor-κB pathway was likely to participate in macrophage polarization promoted by long noncoding RNA Gm9866. Therefore, the nuclear factor-κB pathway was selected for further analysis to clarify its specific mechanism. Western blotting assay showed that the expression of nuclear factor-κB protein decreased after overexpression of long noncoding RNA Gm9866, thus suggesting that the nuclear factor-κB pathway plays a role here; in other words, the specific mechanism by which long noncoding RNA Gm9866 promoted macrophage polarization to M2 phenotype could be determined by inhibiting the nuclear factor-κB pathway  and MRC2 (macrophage mannose receptor 2) was significantly increased after the overexpression of long noncoding RNA Gm9866 Figures . Previou pathway .in situ hybridization analysis was conducted to confirm the subcellular localization of long noncoding RNA Gm9866 in RAW24.7 cells. The long noncoding RNA Gm9866 was expressed in the nucleus and cytoplasm, but mainly in the nucleus (Fluorescence  nucleus . This reThe effect of long noncoding RNA Gm9866 on the proliferation of RAW264.7 cells was detected by EdU (5-ethynyl-2′-deoxyuridine) assay. The results showed that long noncoding RNA Gm9866 upregulation led to enhanced proliferation of RAW264.7 cells . Chen etTUNEL (TdT-mediated dUTP nick-end labeling) assay was performed to determine the effect of long noncoding RNA Gm9866 on the apoptosis of RAW264.7 cells. The results showed that long noncoding RNA Gm9866 upregulation suppressed the apoptosis of RAW264.7 cells .in vitro and promoted tumor growth in vivo, while knockout of long noncoding RNA ARSR impaired the exosome-mediated polarization of macrophages [in vitro cell model and an in vivo mouse model of Mycobacterium tuberculosis infection and conducted negative pressure treatment. They found that negative pressure treatment after M. tuberculosis infection promoted macrophage polarization to proinflammatory M1 phenotype by regulating the long noncoding RNA XIST/micro-RNA-125b-5p/A20/nuclear factor-κB axis [in vitro experiment, lipopolysaccharide and interleukin-4 were used to induce cell polarization. The results showed that the expression levels of long noncoding RNA Gm9866 decreased when macrophages were polarized to M1-type and increased when macrophages were polarized to M2-type; this finding was in line with the screening results of the bioinformatics analysis. Cell transfection experiments were also conducted. After transfection of long noncoding RNA Gm9866-overexpressing plasmids, the levels of the M2-specific phenotypic markers MRC1 (macrophage mannose receptor 1) and MRC2 (macrophage mannose receptor 2) were increased, thus suggesting that macrophages are polarized to M2 phenotype, which may play an anti-inflammatory role. Subsequently, a FISH (fluorescence in situ hybridization) analysis was conducted to locate long noncoding RNA Gm9866 in RAW264.7 cells. Long noncoding RNA Gm9866 was found to be expressed in the nucleus and cytoplasm, but mainly in the nucleus. This finding suggested that long noncoding RNA Gm9866 mainly played a functional role in epigenetic regulation and transcriptional regulation. These results laid a foundation for more direct and in-depth research on the molecular mechanism of long noncoding RNA Gm9866. Some previous studies [κB family is involved in macrophage polarization. We therefore speculated that long noncoding RNA Gm9866 may act through the nuclear factor-κB signaling pathway. Western blotting assay was then performed to detect the protein levels of nuclear factor-ΚB. The results showed that the nuclear factor-κB protein level decreased after the overexpression of long noncoding RNA Gm9866, which further confirmed the previous speculation. Taken together, the present study suggested that long noncoding RNA Gm9866 promoted M2 macrophage polarization through the nuclear factor-κB signaling pathway. However, the specific mechanism of this phenomenon and the involved binding sites need to be further studied.Macrophages are immune cells that play the role of “scavengers” and participate in various cellular and molecular immune pathways . Macrophrophages . Luo et -κB axis . Most tr-κB axis . Althoug-κB axis  and foun-κB axis . Present-κB axis . Another-κB axis . These s studies , 43–45 hin vivo experiments and research on specific diseases. The biological function of long noncoding RNA Gm9866 can be further verified by combining pathway enhancers or blockers. Finally, bioinformatics analysis can also be used to predict the direct target genes of long noncoding RNA Gm9866 for more in-depth and direct research on the underlying mechanism. Despite these limitations, the present study is the first to explore the relationship between long noncoding RNA Gm9866 and macrophage polarization. These results provided new insights into the molecular mechanism by which long noncoding RNA Gm9866 was involved in macrophage polarization-related diseases.The present study had some limitations that should be addressed in future research. First, bone marrow-derived macrophages should be isolated to further improve the verification of long noncoding RNA Gm9866 through the loss of function experiment. Second, the findings of the present study can be strengthened by κB pathway. Therefore, targeting long noncoding RNA Gm9866 and nuclear factor-κB can serve as a new therapeutic strategy for treating macrophage polarization-related diseases.In conclusion, the present study revealed the potential mechanism by which long noncoding RNA Gm9866 promoted macrophage proliferation and polarization and inhibited its apoptosis by inhibiting the nuclear factor-


The Xuwen coral reef, as a potential refuge for corals to adapt to high-temperature migration, is an ideal “laboratory” to determine the impact of human activities on coral reef ecology in the northern South China Sea (NSCS). Global warming, coastal eutrophication and the decrease in herbivores has influenced coral reef degradation; compared with human activities, global climate change is the stronger influence on coral reef degradation on a large scale. For the Xuwen coral reef, which is in the coastal zone, anthropogenic stressors are more significant to the rapid degradation of its coral reef ecosystems. Under these circumstances, more evidence is required to understand the response of high-latitude reefs under natural and anthropogenic stresses. In this study, we investigated the ecological features of the scleractinian coral assemblage and their correlation with environmental factors on the Xuwen coral reef in 2020. Based on the data acquired by investigation and the implementation of statistical analysis methods, potential environmental conditions favoring coral communities in Xuwen were summarized. These results of the survey are expected to provide a basis for bridging the gap between economic development and sustainable management.Goniopora and Porites. The coral morphology was massive, and the diversity of the coral community  was 2.87. The distribution of coral was uneven. The competition among some dominant species of coral was intense. (2) The mass coral bleaching event in the NSCS in August 2020 did not cause severe coral death in the short term on the Xuwen coral reef. (3) The growth of the coral community in 2020 might be greatly affected by high suspended solids and nutrient levels, which were related to the current, mariculture, and coastal erosion. (4) Anthropogenic activities such as coastal aquaculture and fishing were the major factors leading to the reduction in coral coverage on Xuwen coral reef in the past 10 years.Taking the coral communities of the Xuwen coral reef in 2020 as the research object, we analyzed the species composition, diversity, and interspecific Spearman correlation of the scleractinian coral communities, investigated the features and spatial distribution of the scleractinian coral community, and discussed the correlation between the community composition and environmental factors to identify the affecting factors and their sources. These results showed that (1) compared with the survey in 2004, the coverage of corals in 2020 had significantly decreased, while the dominant genera were still  The 7200 aBP , making 7200 aBP . This su7200 aBP . Global 7200 aBP .Most large-scale coral reef ecological surveys in Xuwen began in 2000. Wang et al. reported eight families and 25 species of scleractinian corals from the Dengloujiao in 2000 ,10. Lu e−1), a loss of 50.7% of the initial coral cover [Coral assemblages have declined significantly due to anthropogenic stressors and climate change in the last few decades ,18. On tal cover . The coral cover ; off Yonal cover ,22. Inteal cover . Pandolfal cover  argued tal cover ,14,15,16al cover .Under these circumstances, more evidence is required to understand the response or suitability of the high-latitude reefs under natural and anthropogenic stresses ,26. EspeTo answer these, a comprehensive study was carried out on the Xuwen coral reef in 2020. Based on the data acquired by investigation and the implementation of statistical analysis methods, potential environmental influence factors for coral communities on Xuwen coral reef were proposed. The results are expected to provide a basis for bridging the gap between economic development and the sustainable management of the Xuwen coral reef.2 [The Xuwen coral reef  is located in the NSCS , which iUsing video transect  and photThe species identification of coral was based on close-up photographs, using the taxonomic framework developed by Veron  and Zou The suitable habitat of coral was defined as the area from the upper to lower limits of coral growth by scuba diving, which was calculated by the geometry area calculation function of ArcMap software, using China Geodetic Coordinate System 2000, with Gauss Kruger 3 Degree projection, the central meridian was 111° E.4+-N, NO2−-N, NO3−-N), inorganic phosphate (PO43−-P), total nitrogen-(TN), total phosphorus (TP), oils and Chla. There were 405 water samples in our survey. Sample collection, storage, and transportation were conducted according to the Specification for Oceanographic Survey (GB/T 12763-2007) [2−-N and NO3−-N, PO43−-P, TN, and TP were analyzed using the flow analysis method. The NH4+-N values were determined using the hypobromite oxidation method. The oils values were analyzed using the ultraviolet spectrophotometry method. The Chla values were tested using the fluorescence spectrophotometry method.In addition, water sampling was performed once in 16 sites, including surface (0.5 m) and bottom (Water layer 2 m from the bottom) layers. The samplings of water parameters were temperature (T), salinity (S), pH, transparency, suspended solids (SS), dissolved oxygen (DO), chemical oxygen demand (COD), dissolved inorganic nitrogen  . Briefly63-2007) . The seain is the number of colonies of the coral species i in each quadrat, ic is the cover of the coral species i in each quadrat, if is the frequency of the coral species i in each quadrat, iRA is relative abundance, iRC is relative coverage, and iRF is relative frequency, respectively.The importance values (IVs)  of varioH’), evenness  and species richness  were used to analyze the diversity characteristics of coral communities as follows [iP is the proportion of the coverage of species I in the total coverage of the community, S is the total number of species in the transect, and N is the coverage of total number of individuals.The species diversity H′We adopted the method of Spearman rank, correlation coefficient and significance methods to analyze the relationships among the dominant coral species . ClusterDipsastraea was the genus with the most species present.The records of species at the Xuwen coral reef are shown in 2 in 2020, and living coral cover ranged from 5.2% to 26.1%, with an average of 12.1%. The corals of Xuwen coral reef had an uneven spatial distribution, were mainly distributed within 5.0 m depth, and the highest percent of living coral cover was near Shuiwei and Fangpo  was the most dominant coral species, and Bernardpora stutchburyi (8.7% IVs), Montipora turgescens (8.0% IVs), Turbinaria peltata (7.9% IVs), and Porites lutea (7.7% IVs) were codominant species. According to importance values (IVs), the dominant coral species, genera, and families are shown in p < 0.05) was 6, accounts for 7.7%, indicating that the interspecific association of the coral community was relatively loose. On the other hand, some species were highly correlated, such as the significant positive correlation between Favites abdita and Platygyra daedalea, and between Porites lutea and Goniopora sp. , and the sites with H’ > 3 were the XW4, XW13 and XW20 sites. At 4 m, the range of H’ was 2.40~3.69, with an average of 2.85. A total of 8.6% of sites with H’ > 3 were the XW8 and XW10 sites.The Shannon–Wiener index H’,  ranged f2. Juvenile corals were mainly distributed at the XW1 and XW14 sites. Most species of juvenile corals were in the Merulinidae family, which was the same as the overall dominant species. The mean mortality rate of recently dead corals was 2.8%. The site with the highest mortality rate was the XW1 site, which was close to Liusha Bay, where living coral cover was also the lowest (5.2%). A total of 62.5% of the sites were found to have coral bleaching, and the highest was at the XW16 site, and the mean coral bleaching rate was 1.60% (in July 2020)  was 3.01 ind./mly 2020) .Caulerpa racemosa, Caulerpa sertularioides, etc. The height of macroalgae ranged from 18 cm to 20 cm. The mean macroalgae cover was 0.7%, and the highest was near Liusha Bay  and Dengloujiao (in the XW13 site). There were six families and seven species of coral reef fish, mainly of the Pomacentridae family. The dominant species was Neopomacentrus bankieri. The mean density of coral reef fish was 8.38 ind./250 m2, and 96.7% of coral reef fish were less than 10 cm in length. The mean density of invertebrates was 155.06 ind./100 m2. The dominant group was sponges, whose density accounted for 59.73%  and PCA tests  are shown in Correlation and multiple linear regressions were used to analyze the relationship between ecological features and major environmental factors . The RDA4+-N . For dominant genera, the significant environmental impact factors  were nutritional salt  and suspended solids (p = 0.022). For dominant species, the significant environmental impact factor  was nutritional salt . At the genus level, Goniopora and macroalgae (mainly Caulerpa racemosa) had formed a large competitive relationship . Goniopora gracilis and Bernardpora stutchburyi were positively correlated with NH4+-N, which indicated the possibility that these corals could grow well in water with high NH4+-N content. The number of coral species was negatively correlated with chlorophyll a . The coral diversity index (H’) was significantly negatively correlated with Chla . Our results are shown in Goniopora sp. was the dominant species from 2004 to 2020. These dominant species often grew slowly and are distributed widely, which were features of a unique northern coral reef ecosystem [H’) was 2.87, species distribution in the coral community was uneven, and there was still a trend of population differentiation and succession. The reason may be that human activities  have disturbed the mechanism of interspecific niche complementarity in recent years, weakening the positive effect of species diversity on community stability [Past research ,14,15,16cosystem . These mcosystem . The restability , suggesttability  investigtability . The cortability .Porites lutea and Galaxea fascicularis on the Xuwen coral reef, which was 29.5 °C. During this study, the water temperature was 31.75 °C–32.70 °C, which was higher than the suitable temperature of most corals. The habitat fragmentation of the Xuwen coral reef was significant. It should be noted that naturel disturbances may play a more visible role in the dynamics of coral assemblages in small areas than in larger areas . For mos2 emissions continue to rise as usual), two severe bleaching events per decade will be familiar scenarios at 25 of the 29 World Heritage reefs (86%) by 2040 [The World Meteorological Organization “Global Climate Conditions 2020”  had repo by 2040 . In prev by 2040  speculat by 2040  also fouFrom July 2020 to July 2021, a mass coral bleaching event was observed. In July 2020 , 62.5% of sites experienced coral bleaching, with 1.6% mean bleaching. In August 2020, a mass coral bleaching event was recorded in NSCS . Ten sitLyu et al.  also repIt is suggested that the reason why global warming failed to cause severe coral death in the short term on the Xuwen coral reef was more likely the unique features of ocean currents . There iThe major human-induced stressors on coral reefs have been known for decades, including sewage, siltation, industrial discharge, urban development, and destructive fishing methods . Local a4+-N . The number of coral species was negatively correlated with chlorophyll a . The coral diversity index (H’) was significantly negatively correlated with Chla . The high Chla content indicated high phytoplankton biomass, which suggested that there was a high nutrient content in the water. In addition, the fish density was positively related to the abundance and diversity of coral species and the most dominant genera. High fish density  can affect macroalgal growth, which is conducive to coral growth. Based on these statistical results, it is suspected that the growth of the coral community in 2020 might have been mostly affected by suspended solids and nutrients.Most corals at the Xuwen coral reef were distributed in shallow water, where the concentrations of suspended solids were high in our survey. In these areas with high concentrations of inorganic nitrogen and phosphate in the local area, macroalgae flourished. Our results showed that there was a significant negative correlation between live coral cover and NHAs mentioned above, the environment was almost identical between the XW16 and XW8 sites, though XW16 site had a higher concentration of Chla and sand coverage. It might indicate XW16 site was not suitable for coral growth or recovery.The seawater of Xuwen coral reef was turbid. The average concentration of suspended solids on the Xuwen coral reef was 17.0 mg/L, where approximately 68.8% of survey sites had an average concentration of more than 10 mg/L. Elevated turbidity can reduce light, thus affecting the photosynthesis of zooxanthellae . Suspendp< 0.05), which was consistent with the results of Wang et al. [Porites sp. can strongly resist to suspended solids in water [Acropora sp. is sensitive to the content of suspended solids in water [Porites sp. [Favites sp. has a larger coral cup and higher heterotrophic capacity (to capture organic particles as food), and resists high suspended solids content more strongly [Dipsastraea, Goniopora and Favites) were massive corals, all of which can grow well in water with high level of suspended solids.The statistical results in 2020 showed that the suspended solids were significantly positively correlated with coral bleaching 0.567, , p< 0.05g et al. , e.g., Pin water . Acroporin water , and it ites sp. . Favitesstrongly . Our resIt is possible the high suspended solids content is the result of the hydrodynamic conditions, coastal erosion, and traditional salt production in Xuwen. The frequent exchange and mixing of seawater and coastal water outside the Qiongzhou Strait , the tur3−-N) is 0.1–0.5 μmol/L, the ammonium salt (NH4+-N) 0.2–0.5 μmol/L, and the inorganic phosphorus less than 0.3 μmol/L [4+-N , 1.52 μ in 2006 ), 1.79 μ in 2006 ), all of in 2006 . Eutroph in 2006 ,79, and  in 2006 . Thurber in 2006  presente4+-N values were negatively correlated with most coral communities, which was similar to the results of the previous study [4+-N (20 μmol/L), the photosynthetic efficiency (Fv/FM) of corals decreased significantly, the host protein content increased evidently, and the symbiotic pigmentation (Chla content) also had obviously increased. This NH4+-N content may mostly come from the excretion of organisms and the decomposition of organic matter. The DIN was transferred from the seawater to the sediment, participating in microbial N cycling, microalgal uptake and sedimentary adsorption [3−-N was the primary component of DIN during the daytime, which changed to NH4+-N during the nighttime [4+-N and the coral community in this study.Our results showed that NHus study . After lsorption . The NO3ighttime . Increasighttime . Our resighttime , which mGoniopora and Porites. Bernardpora stutchburyi, Turbinaria peltata and Plesiastrea versipora were common dominant species on the Xuwen coral reef, which all showed resistance to interference. The coral morphology was massive, with a diversity of 2.87 of the coral community , which indicates the distribution of corals was loose.Based on the ecological investigation of the Xuwen coral reef in July 2020, the ecological features and impact factors of scleractinian corals on Xuwen coral reef were analyzed. These results showed that compared with 2004, the area and coverage of corals were significantly decreased, and corals were distributed in patches, mainly in the core zone of the reserve. The dominant genera were still The mass coral bleaching event happened in the NSCS in August 2020 did not cause severe coral death in the short term on the Xuwen coral reef. The comprehensive statistical results suggested that the coral community in 2020 might be greatly affected by high suspended solids and elevated nutrient levels, which were related to the current, mariculture and coastal erosion in and around Xuwen coral reef. Anthropogenic activities such as coastal aquaculture and fishing had been the major factors causing the reduction in coral coverage on Xuwen coral reef in the past 10 years.