The world is going through the critical phase of COVID-19 pandemic, caused by human coronavirus, SARS-CoV-2. Worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. However, there have not been many studies focusing on the variations in the 5’ and 3’ untranslated regions and their consequences. Considering the possible importance of these regions in host mediated regulation of viral RNA genome, we wanted to explore the phenomenon.To have an idea of the global changes in 5’ and 3’-UTR sequences, we downloaded 8595 complete and high-coverage SARS-CoV-2 genome sequence information from human host in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID) from 15 different geographical regions. Next, we aligned them using Clustal Omega software and investigated the UTR variants. We also looked at the putative host RNA binding protein (RBP) and microRNA binding sites in these regions by ‘RBPmap’ and ‘RNA22 v2’ respectively. Expression status of selected RBPs and microRNAs were checked in lungs tissue.We identified 28 unique variants in SARS-CoV-2 UTR region based on a minimum variant percentage cut-off of 0.5. Along with 241C>T change the important 5’-UTR change identified was 187A>G, while 29734G>C, 29742G>A/T and 29774C>T were the most familiar variants of 3’UTR among most of the continents. Furthermore, we found that despite the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs.Our results, shows for the first time in SARS-Cov-2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. The knowledge might be helpful in developing anti-viral compounds in future. Outbreak of novel coronavirus SARS-CoV-2 has been proclaimed as a pandemic by the World Health organization (WHO). From the first report of infection in Wuhan, China on December 31, 2019, the virus has infected 7.21M people worldwide till 9th June, 2020. SARS-CoV-2 is an enveloped, positive-sense, single stranded RNA virus of genus Beta-coronavirus (β-CoVs) with the entire genome size of approximately 30kb. This viral genome is composed of about 14 open reading frames (ORF) which encodes both structural and non-structural proteins having a role in their transmission, survival and pathogenesis  and a 3’-noncoding co-termini [3’UTR], identical to the viral genome . Extent HNRNPA1, HNRNPQ is bound to the 3’UTR of beta-coronavirus MHV. Similarly, a host protein, MADP1 (zinc finger CCHC-type and RNA binding motif 1) interacts with the 5′-UTR of SARS-CoV, influencing the RNA synthesis machinery [Molecular evolution in the untranslated region i.e. the variation in the UTR region leads the virus to evolve to a great extent. There are many studies which have considered the mutation in the coding region with respect to geographical location. Here, we have enumerated the variants in the 5’- and 3’-UTR regions of SARS-CoV-2 genome. We have studied a total of 8595 viral sequence samples worldwide for this purpose and found that there are some rare variants of UTR that are distributed over a global spectrum, while some variants are specifically present in a population at a comparatively higher frequency. This drove us to make a systematic catalogue of the UTR variants across six continents of the world that could have a role in emergence of different strains of SARS-CoV-2. We have also looked at the possible regulation of viral genomic RNA through binding of host RNA binding proteins (RBPs) and miRNAs in specific sequences of the viral UTRs. There are experimentally validated evidences of human RBPs binding to the regulated signals within the untranslated region of SARS-CoV RNA in order to control the viral RNA synthesis and turnover. Polypyrimidine tract-binding protein (PTB) is found to bind to the leader sequence and achinery . As the achinery  and inveTable 1). We have downloaded the sequences on 28th May, 2020.We have downloaded complete and high-coverage SARS-CoV-2 genome sequence data in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID), which is a public repository of all influenza virus sequence. Our data involved only the human-host specific viral sequence covering six continents of the world: Asia, Europe, Oceania, North America, South America and Africa. From Asia, we have taken the viral sequence data from China (n = 197), India (n = 205), Japan (n = 123), Thailand (n = 121) and Taiwan (n = 102). From Europe, the data is taken from Italy (n = 109), Spain (n = 671), England (n = 2345), Russia (n = 130) and Germany (n = 202). Viral sequence data is taken only from Australia (n = 522), on behalf of Oceania. Representative countries from North America include USA (n = 3286) and Canada (n = 147). There are no further divisions for South America (n = 281) and Africa (n = 150). Initially the plan was to retrieve viral sequences collected in a span of one month, just after an interval of two month from the date of first report in the corresponding place, in order to catch the diversity in the viral sequence in that region. But, unfortunately, due to less number of sequence deposition in some countries, we had to deviate from the plan and retrieve all the available sequences till May28, 2020 for them (https://figshare.com/articles/Supplementary_Data_Alignment_Files/12649109.The reference sequence from Wuhan was taken as our reference from NCBI (NC_045512.2). All the country specific sequences were aligned with the reference sequence separately using Multiple Sequence Alignment Tool Clustal Omega (EMBL-EBI). As we were interested only in the 5’- and 3’-untranslated region (UTR) and these positions are located at the two extreme ends of the sequences, there is a chance of lower coverage and higher error-rate. To resolve this ambiguity, we have put a high filtering cut-off of at least 70% coverage in a particular location of that area. All the alignment files have been shared in ‘figshare’ and can be accessed using the link: For prediction of human RBPs bound to the UTR of a viral genome, we have used the web-tool ‘RBPmap’ that enabled prediction of RBP binding on genome sequences from a huge list of experimentally validated motifs of RBPmap database. Weighted-Rank algorithm was used for mapping the motifs . We havePutative human miRNA binding sites on viral UTR was assessed using the ‘RNA22 v2 microRNA target detection’ tool . As a taExpression of specific genes was obtained from ‘The Protein Atlas’ respective to normal lung tissue. RNA-seq data from lung tissue generated by the Genotype-Tissue Expression (GTEx) project was reported as mean pTPM (protein-coding transcripts per million), corresponding to mean values of the different individual samples from each tissue. HPA RNA-seq data from lung tissue was reported as mean pTPM (protein-coding Transcripts per Million), corresponding to mean values of the different individual samples from each tissue. Tissue data for RNA expression also obtained through Cap Analysis of Gene Expression (CAGE) generated by the FANTOM5 project, reported as Scaled Transcripts per Million. GEPIA  web-toolS1 Table based on a minimum variant percentage of 0.5. But for the variants that are present in at least four populations, no such variant percentage cut off was taken. The most common high frequency variant is the 241C>T, which lies in the leader sequence of the SARS-CoV-2 viral genome. China, being the ancestral domicile of the virus has a very low percentage of this variant . Europe has the maximum frequency of this variant . Other than China, other countries in Asia also have a lower percentage of this variant . Thus, we can a see a clear effect of regional variation on the frequency of this variant. Another study reported that this variant has co-evolved with three coding region mutations  of nsp3, RNA primase and spike glycoprotein. They have also related this mutation with the increased transmissibility of the virus [Fig 2). Most of the countries in this study seem to have a moderate to significant variation in the 3’UTR of the viral genome.Sequence alignment of over 8500 virus samples collected from over 15 geographical locations with the SARS-CoV-2 reference genome (NC_045512.2) has yielded a total of 74 variants in the 5’ UTR and 83 variants in the 3’UTR. Among them, 28 unique variants have been summarized in the he virus . AnotherFurthermore, we have explored the relationship of these variants with the secondary structure of SARS-Cov-2 viral RNA. 5’UTR variant 187A>G fall within the SL5A stem-loop and 241C>T variant was within SL5B stem-loop. The above mentioned three 3’UTR variants were also within the bulge portion of the stem-loop of viral RNA secondary structure. Wild type nucleotides of all these variants had high SHAPE-reactivity value which suggested that these nucleotides were less likely to form base-pair and hence had an important role in affecting the secondary structure , 18.Figs 2). Even countries in a single continent differ in their UTR variation. Some variations are merely specific to a particular location with a quite considerable percentage. The overall sequence of 5’UTR of SARS-CoV-2 in Italy is exceptionally variable, whereas the 3’UTR is substantially stable with a single variant in the position 29701 (G>T). But England, also being a part of Europe, has a notable number of variations in the 3’UTR having a relatively stable 5’-end. From our analysis, it is evident that Russia had no such frequent variation in the UTR, except the 241C>T variant which was present in 100% of the population. India had two striking variations in the 3’UTR, 29827A>T, 29830G>T, which was found nowhere in this global mutational landscape  , implicating that the variation might have some effect on CUG-BP binding. In order to test that, we used the mutated sequence as ‘input’ and found that the single nucleotide change didn’t have any effect on binding of CUG-BP to its target sequence. In other words, no matter whether the virus has ‘A’ or ‘G’ at position 187 of its 5’-UTR, CUG-BP binds and probably performs its function.In the next step, the logical exploration was to find out whether the most prevalent variations identified in the 5’-UTR of the viral RNA genome could alter binding of any of these RBPs. The ‘A’ to ‘G’ change at position 187 coincided with the binding site of TARDBP binding site created upon this change . TARDBP  is a well characterized RBP which binds to specific ‘UG’ (and ‘TG’) rich sequences of RNA (and DNA) and reported to facilitate translation when bound to 5’-UTR [Fig 3C & 3D) which indicated strong binding of the protein to that particular nucleic acid stretch. Hence, our finding showed strong binding of TARDBP to viral 5’-UTR having ‘U’ at position 241. This could be implicated in facilitating translation of viral proteins resulting in its effective propagation within human host.Then, we focused on the most important change in the 5’-UTR, the position at 241. Looking at the distribution of this variation and the reports related to its association with virulence , we had o 5’-UTR . From prTable 2). As with 5’-UTR, here also we focused on the major variations in 3’-UTR to find out if specific nucleotide change could interfere with the binding of specific RBPs. We found that 3’-UTR variations at positions 29742 and 29774 could affect interaction of SRSF5 and HNRNPA1 respectively. However, the regulation at 3’-UTR is not that simple. Another important factor which needs be taken into account is whether there are host miRNA binding sites present within the viral 3’-UTR region and how the combinatorial interaction of host RBPs and miRNAs could dictate the viral RNA stability.Following the same principle and considering the fact that 3’-UTR is the most important site for regulation of RNA stability and turnover, we also wanted to find out what are the RBPs interacting with this region. The 3’-UTR reference sequence of SARS-CoV-2 was fed into RBPmap and we obtained a list of 3’-UTR binding RBPs selected on the basis of p-value and Z-score (Table 3). As the most important factor for the functional miRNA-target RNA interaction is to have the perfect homology between the seed sequence of miRNA (2nd to 8th nucleotide from 5’ end) and the corresponding target RNA sequence, we also explored the fact in the identified pairs. Furthermore, whether the changes caused by the common variations interfere with the seed sequence binding was also investigated. The interesting part in this scenario is that the reference sequence at a particular position causes a mismatch in the seed resulting in inefficient binding of a miRNA, but the variant base at that position creates a perfect binding site. However, considering the complexity of the region, all these probabilities could not be assessed only in terms of miRNAs and hence we set out to probe into the interaction between the host RBP-miRNAs and viral sequence variants in 3’-UTR region in a comprehensive manner.In order to identify whether there are any host miRNA binding site present in viral 3’-UTR and if so, what they are, we used the viral 3’-UTR sequence as ‘input’ in the miRNA prediction software ‘RNA22 v2’. The algorithm predicts several of such miRNAs and we selected them based on folding energy for heteroduplex formation and p-value . This clearly indicated a prevalent competition between RBMS3 and miR-34b-5p-RISC complex for their respective binding site, with one of them simply presenting a steric hindrance to the other one by blocking the access. This is quite common scenario in regulation of mammalian mRNA stability where the final verdict whether the mRNA is degraded or stabilized largely depends on the spatio-temporal expression or the availability of both the molecules (RBP and miRNA).We investigated the interaction in a stepwise manner. The first situation explored was the case where host miRNA miR-34b-5p targets viral RNA and no mutation has been identified in the ‘seed’-corresponding region. This means that all the viral sub-types will be vulnerable to degradation by this miRNA, provided it is expressed in host lungs tissue. Careful scanning of the binding site and also looking at the RBP binding information we identified a Fig 4B). This is clearly an advantage to the virus as the variation would help it to get rid of the miRNA mediated degradation. As this overlapping region also had a binding site for RBP HNRNPA1, we wanted to find out what happens to HNRNPA1 binding when there is a change from ‘C’ to ‘A’. Interestingly enough, we discovered that HNRNPA1 binding is also not affected, implicating an ensured protection of viral RNA by RBP HNRNPA1, no matter whether miR-9-5p is functional or not. Again, this situation is also quite common in mammalian mRNA regulation where we see some amount of degeneracy among the RBP target sequences, when change of a single nucleotide is often tolerated. The virus uses this phenomenon to its benefit where in spite of UTR sequence variation, protection from same RBP is ensured.Second scenario was the variation in position 29774. In this case, we identified that change from ‘C’ to ‘A’ disrupted the binding of miR-9-5p to its corresponding target sequence at the ‘seed’ region , which means that the virus having the variant nucleotide will be prone to degradation by this miRNA. We had already commented about the possible binding of SRSF5 in this region and found that the variation under investigation had no impact on SRSF5 binding. Again, it indicated that despite creation of miR-3664-5p binding site, the viral RNA is safe until SRSF5 is available to bind and compete with miR-3664-5p-RISC complex. However, as evident from our 3’-UTR analysis, a significant proportion of viral sequence had ‘G’ to ‘T’ change at position 29742. While SRSF5 binding site was still retained, the miR-3664-5p site creation did not take place due to this change.In case of the third incidence, we looked at position 29742, where the ‘G’ to ‘A’ change creates a binding site for miR-3664-5p . This means that there is probably no host factor to protect viral RNA from miR-4701-3p mediated degradation when the virus carries a ‘C’ at position 29734. However, this entirely depends on the expression status of miR-4701-3p in host tissue and there are evidences that suppression of miRNA expression falls under viral strategy to safeguard its genome.The fourth scenario was found to be very different from what have been mentioned so far. We identified that change of ‘G’ to ‘C’ at position 29734 creates a binding site for miR-4701-3p, but no RBP with a overlapping target sequence could be identified  and in the second approach we used the TCGA dataset for lung adenocarcinoma (LUAD) from GEPIA and compared the expression of the selected RBPs in adjacent normal lung tissue . To our excitement, we found a decent to huge expression of all the RBPs in normal lung tissues, clearly indicating their availability to protect viral RNA from host decay machinery. As the viral infection will follow a huge inflammatory condition in the lungs and by the time we know that in severe cases this prolonged inflammation leads to pulmonary fibrosis, we also wanted to check whether there are supporting evidences relating the expression of these RBPs and miRNAs to inflammation or more specific to pulmonary inflammation. As shown in Table 4, almost all the RBPs under investigation were either reported to have induced by inflammation or responsible for fibrosis in lungs or other organ. Expression of some of them was also reported to be modulated by viral infection. This finding supports our hypothesis and indicates that from the stages of early viral infection to severe advanced conditions SARS-CoV-2 could utilize the host stabilization factors for its own benefit.The phenomenon of viral RNA utilizing host stabilizing factors and escaping RNA decay machinery is well-established. But, nothing is known so far for SARS-CoV-2. As mentioned before, the actual incident what is taking place after viral infection largely depends on the expression status of the interacting RBPs and miRNAs in particular host tissue. Since, lungs is the primary site of infection for SARS-CoV-2, we wanted to find out what is the nature of expression of these molecules in normal lungs tissue. In the first approach we tested their expression in ‘The Protein Atlas’ portal . On the other hand, there exists multiple possibilities in case of 3’-UTR, where we first see direct competition over access to target sites between RBMS3 and miR-34b-5p (arm:B). Lung inflammation causes induction of miR-34b-5p [The most significant aspect of this study was to identify miRNA binding site in the viral 3’-UTR and deciphering the cross-talk between RBPs and miRNAs along with the nucleotide variations at specific sites. Our results elaborated five distinct types of such interactions, as summarized in R-34b-5p  which coHNRNPA1 and SRSF5 is high in lungs, further supporting their possible protective effect on viral genome. The incidence described in arm: E is different as it creates a miRNA binding site without an RBP protection. We have seen that expression of miR-4701-3p is less in lungs, provoking the thought that despite having its site created, there might not be enough miRNA to act on the viral RNA at this site.The effect of variation in viral sequence could either disrupt or create miRNA binding site, keeping the RBP binding site intact. This result in non-functional miRNA site in one case (arm:C) and competition between RBP and miRNA in the other (arm: D). Expression of both CUG-BP, TARDBP, RBMS3, HNRNPA1 and SRSF5 with host miRNAs miR-34b-5p, miR-9-5p, miR-3664-5p and miR-4701-3p and showed how the interactions changed along with sequence variations at specific positions of untranslated regions of viral genome. Our findings elaborate complex relationship between host RNA stabilization/ decay factors and SARS-CoV-2 RNA genome highlighting how the virus could manipulate host machinery. The knowledge could also be used to develop antiviral compounds following further experimental studies.Thereby, we have characterized SARS-CoV-2 5’ and 3’-UTR sequences for existing variations in these regions prevailing at major countries of infection spread over all the continents. We further identified interactions between host RBPs like S1 Table(XLSX)Click here for additional data file.


With the global threat of SARS-CoV-2, much effort has been focused on treatment and disease control. However, how coronaviruses react to the treatments and whether the surviving viruses have altered their characteristics are also unanswered questions with medical importance. To this end, bovine coronavirus (BCoV), which is in the same genus as SARS-CoV-2, was used as a test model and the findings were as follows. With the treatment of antiviral remdesivir, the selected BCoV variant with an altered genome structure developed resistance, but its pathogenicity was not increased in comparison to that of wild type (wt) BCoV. Under the selection pressure of innate immunity, the genome structure was also altered; however, neither resistance developed nor pathogenicity increased for the selected BCoV variant. Furthermore, both selected BCoV variants showed a better efficiency in adapting to alternative host cells than wt BCoV. In addition, the previously unidentified feature that the spike protein was a common target for mutations under different antiviral treatments might pose a problem for vaccine development because spike protein is a common target for antibody and vaccine designs. The findings derived from this fundamental research may contribute to the disease control and treatments against coronaviruses, including SARS-CoV-2. Coronavirinae, family Coronaviridae, order Nidovirales [Coronavirinae is divided into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Among the seven known human coronaviruses (HCoVs), HCoV-OC43, SARS-CoV-1, MERS-CoV, and SARS-CoV-2 belong to Betacoronavirus, whereas HCoV-229E and HCoV-NL63 belong to Alphacoronavirus [Coronaviruses (CoVs) are single-stranded, positive-sense RNA viruses with a genome size of 26–32 kilobases (kb) and belong to the subfamily ovirales ,2,3,4. Bonavirus . In addiSince the outbreak of severe acute respiratory syndrome (SARS) , Middle Viruses are constantly under the pressure of immune surveillance. Of the antiviral immune responses, innate immunity, which involves nonspecific immune defense mechanisms, is the very first line of cell defense against virus infection . To estaBecause of the lack of proofreading capability, the RNA-dependent RNA polymerase (RdRp) encoded by RNA virus has high mutation rates during RNA synthesis, leading to a diverse population of viruses or quasispecies ,38. AccoBetacoronavirus) than HCoVs HCoV-229E and HCoV-NL63 (Alphacoronavirus) based on the phylogenetic tree analysis [Betacoronavirus [With the global threat of SARS-CoV-2, much effort has been focused on treatments and prevention for the disease; however, how coronaviruses react to the treatments and whether the surviving viruses have altered their characteristics such as pathogenicity after weathering different pressures are also important questions that remain unanswered. Because bovine coronavirus (BCoV) (i) has been intensively used in replication study of coronavirus ,46,47, (2. Bovine coronavirus (BCoV) strain of Mebus (GenBank accession no. U00735), which was also obtained from David A. Brian, was plaque-purified [Human rectum tumor (HRT)-18 cells  and Mouspurified ,50 and gFor selection pressure with antiviral remdesivir (GS-5734), HRT-18 cells were infected with wild type (wt) BCoV at a multiplicity of infection (MOI) of 1 and after 1 h of infection, HRT-18 cells were treated with GS-5734 with final concentration of 10 μM based on the study showing that the antiviral activity of GS-5734 at this concentration is able to inhibit replication of SARS-CoV-1 and MERS-CoV . This st2 for 72 h. Viral plaques were visualized by haemadsorption with mouse red blood cells. Blood was collected in Alsiever solution and centrifuged at low speed. The supernatant was discarded followed by washing with phosphate-buffered saline. The virus titer was determined by the number of haemadsorption foci.Plaque assay for determining the titer of BCoV was described previously . In brieThe terminal sequence of BCoV genomic RNA was identified using a head-to-tail ligation method as described previously . The hea32P and bound to BCoV 3′ UTR. The probed blot was exposed to Kodak XAR-5 film .Ten microgram of TRIzol-extracted total cellular RNA collected from the aforementioned experiments was electrophoresed through a formaldehyde-agarose gel for 3 h. By vacuum blotting, RNA was transferred from the gel to a Nytran membrane followed by UV cross-linking. BCoV RNA was detected with the oligonucleotide, which was 5′-end labeled with ® green amplification mix  and oligonucleotides were used according to the manufacturer’s protocol. In these experiments, dilutions of plasmids containing the same gene as the detected genome and subgenome were always run in parallel with the quantitated cDNA for use in standard curves (dilutions ranged from 108 to 10 copies of each plasmid). The amount of synthesized RNA was normalized to the levels of internal control 18S rRNA.To determine the synthesis efficiency of BCoV RNA, 2 μg of TRIzol-extracted total cellular RNA was used for the RT reaction. To quantitate the synthesis of BCoV genome, oligonucleotides binding to the leader sequence and sequence in the nsp1 gene were used. For measurement of subgenome N synthesis, oligonucleotides binding to the leader sequence and sequence downstream of the start codon for N gene were used. For qPCR, SYBRThe harvested cell lysates were separated using 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After electrophoresis, samples were electrotransferred onto nitrocellulose membranes . An antibody against BCoV nsp1, nucleocapsid (N) protein, or β-actin was used as the primary antibody followed by goat anti-mouse IgG conjugated to horseradish peroxidase (HRPO) as the secondary antibody . Detected protein(s) was visualized using Western Lightning™ Chemiluminescence Reagent  and Kodak XAR-5 film .t-test was used for the analysis of the data using Prism 6.0 software . The values in the study are presented as the means ± standard deviations (SD) (n = 3); * p < 0.05, ** p < 0.01 and *** p < 0.001.Student’s Antiviral therapy with drug such as nucleos(t)ide analogues targeting the viral polymerase can inhibit viral replication and subsequent disease progression. Conversely, viruses may also undergo mutations for survival under the treatment of antivirals, leading to the emergence of drug resistance and treatment failure. To test whether the genome structure was altered under antiviral selection pressure, a series of wt BCoV passages in the presence of antiviral remdesivir GS-5734 were performed. The virus collected at virus passage (VP) 10 in the absence of GS-5734 was designated Wt10 while the virus collected in the presence of GS-5734 was designated WtGS10. In comparison with the genome sequence of wt BCoV (Wt), few nt mutations were identified but no nonsynonymous mutations were found in Wt10. However, as illustrated in 7.54 pfu/mL) of wt BCoV in the presence of GS-5734  in the absence of GS-5734  was lower by ~13-fold in comparison to that of Wt10  (108.02 pfu/mL). The amounts of viral RNA synthesis collected from WtGS10-infected HRT-18 cells were also decreased by ~30% in comparison to those from Wt10-infected HRT-18 cells in the absence of GS-5734 (7.44 pfu/mL) of WtGS10 + GS (in the presence of GS-5734) was ~3-fold higher than that of WtGS10 , but still ~four-fold lower than that of Wt10. The amounts of viral RNA synthesis of WtGS10 + GS were also increased by ~20% when compared with those of WtGS10 (To test whether GS-5734 had an inhibitory effect on the replication efficiency of wt BCoV (Wt) in HRT-18 cells, a plaque assay and Northern blot analysis were employed. The titer  environment, but may not be advantageous for increasing the pathogenicity of the virus in the absence of GS-5734 in HRT-18 cells. In addition, the WtGS10 selected by antiviral GS-5734 adapted to ML cells with better efficiency than wt BCoV (Wt10) and thus may have implication in interspecies transmission and disease control.In addition to antiviral drug, whether the constant pressure of innate immunity could lead to the alterations of the genome structure and subsequent viral characteristics remained to be determined in coronaviruses. Thus, HRT-18 cells were respectively transfected or mock-transfected with poly IC, a stimulant of innate immunity with a double-stranded RNA structure, followed by wt BCoV infection. At VP10, the virus (designated WtIC10) and RNA were collected for subsequent analyses. After sequencing analysis, in comparison with the genome sequence of Wt10, multiple nt mutations in WtIC10 were identified and mostly occurred in genes of replication-associated protein and S protein A. In add7.58 pfu/mL, Wt + IC, 8.09 pfu/mL, Wt, 7.84 pfu/mL) was lower by ~two-fold in comparison to that of Wt10  (108.05 pfu/mL). The amounts of viral RNA synthesis collected from WtIC10-infected HRT-18 cells were also decreased by ~30% in comparison to those collected from Wt10-infected HRT-18 cells in the absence of poly IC  than WtIC10 . The amounts of viral RNA statistically showed no significant difference between WtIC10 and WtIC10 + IC  in the presence of poly IC  was collected to infect fresh ML cells (VP1). At VP10, both viral RNAs collected from Wt10- and WtIC10-infected ML cells were detected C in ML cThese results together suggest that BCoV does not develop resistance and increased pathogenicity after the repeated treatment of poly IC-induced innate immunity in HRT-18 cells. However, as demonstrated with WtGS10 in Betacoronavirus) as SARS-CoV-1, SARS-CoV-2, and MERS-CoV, as a test model to explore how coronavirus reacts to the treatments of antiviral remdesivir GS-5734 and poly IC-induced innate immunity in light of alterations of the genome structure and subsequent virus fitness. We anticipate that the results may have medical importance on pathogenicity, treatments, transmission, and antiviral design for coronavirus, and are discussed below.In this study, we employed BCoV, which is in the same genus (After a series of passages of mouse hepatitis virus A59 (MHV-A59) in the presence of GS-5734 in DBT cells, 4 aa mutations in the replication-associated proteins and 2 aa mutations in S protein are found . HoweverIn terms of function-orientated selection, the selected mutations for WtIC10 were expected to be associated with resistance to poly IC treatment. However, the mutations in WtIC10 showed almost no effect on replication efficiency  BCoV. In contrast, under the selection by poly IC-induced innate immunity, the genome structure is also altered; however, neither resistance develops nor pathogenicity increases for the selected BCoV variant. Furthermore, both selected BCoV variants show a better efficiency in adapting to alternative host cells than wt BCoV, highlighting the diverse features of coronavirus genome. Thus, the results may provide information contributing to disease control and treatments against coronavirus infection.


Influenza is an important research subject around the world because of its threat to humanity. Influenza A virus (IAV) causes seasonal epidemics and sporadic, but dangerous pandemics. A rapid antigen changes and recombination of the viral RNA genome contribute to the reduced effectiveness of vaccination and anti-influenza drugs. Hence, there is a necessity to develop new antiviral drugs and strategies to limit the influenza spread. IAV is a single-stranded negative sense RNA virus with a genome  consisting of eight segments. Segments within influenza virion are assembled into viral ribonucleoprotein (vRNP) complexes that are independent transcription-replication units. Each step in the influenza life cycle is regulated by the RNA and is dependent on its interplay and dynamics. Therefore, viral RNA can be a proper target to design novel therapeutics. Here, we briefly described examples of anti-influenza strategies based on the antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA) and catalytic nucleic acids. In particular we focused on the vRNA structure-function relationship as well as presented the advantages of using secondary structure information in predicting therapeutic targets and the potential future of this field.  Influenza is a significant research subject for scientists around the world due to its serious threat to public health. Generally, influenza A virus (IAV) is a human and animal pathogen causing seasonal epidemics and sporadic pandemics resulting in high morbidity and mortality. It is estimated that due to the Spanish flu in 1918/1919 up to 100 million people died .The IAV belongs to the Orthomyxoviridae family of enveloped viruses. Its genome consists of eight negative-sense viral RNA (vRNA) segments that encode for 11 proteins including glycoprotein hemagglutinin (HA) and neuraminidase (NA), a nucleoprotein (NP), two matrix proteins (M1 and M2), three polymerase complex proteins: polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase acidic protein (PA) as well as four non-structural proteins  . HA and The vRNA is replicated into positive-sense complementary RNA (cRNA) and transcribed into viral messenger RNA (mRNA) inside infected host cells. The vRNA and the cRNA are shielded with viral NP. In viral particles, the vRNA is associated with the polymerase, consisting of three subunits: PB1, PB2 and PA . The vRNThe viral RNA is involved in many processes through activities which relate to its secondary and tertiary structures. The correct packaging of all eight vRNA segments into one viral particle is suggested to be conferred at least partially via structured RNA motifs of vRNA protruding from vRNP. Within the viral particles, the vRNPs present a highly flexible double helical structure, with the polymerase at one end and a closing loop at the other . It is kThe segmented nature of IAV RNA serves as a good target for potential new antiviral strategies. Due to the high genetic variability of the influenza virus and the ease of its transmission, protective vaccination and anti-influenza drugs currently used are not fully effective. Therefore, there is a necessity to search for new approaches to limit the influenza spread. These days, the only anti-influenza drugs approved by the FDA are small ligands that bind to the surface of glycoprotein neuraminidase. Previously used ion channel M2 inhibitors are no longer recommended in therapy due to the emergence of the resistant strains . Antigenic shift and drift as well as adapting mutations allow the influenza virus to effectively evade host immune response. Since the variable nature of the influenza virus makes development of an effective vaccine challenging, its formula must be changed seasonally. Moreover, it cannot provide protection against future pandemic strains . This suThe design of currently used therapeutics is based on the prediction of a three-dimensional structure of viral proteins and interactions between potential inhibitor and protein(s). Intensive crystallography, cryo-EM and computational studies have provided new insights into secondary, tertiary and quaternary structure of the influenza proteins and the potential inhibitory small molecule ligands ,17,18. HAs an alternative, advances in primary and secondary RNA structure determination have led to the development of novel and effective anti-influenza agents. RNA-based approaches can potentially restrain spreading of the infection by interacting with vRNA, cRNA or mRNA ,10,19. TSequence and structural motifs of influenza RNAs are partially conserved between strains due to the combination of evolutionary constrains. Therefore, various oligonucleotide tools are designed to alter RNA expression and function, thus allowing to influence processes beyond protein or receptor involvement ,23. TheyAntisense oligonucleotides (ASOs) are synthetic, single stranded, short (12–30 nucleotides long) DNA or RNA fragments. They selectively bind to complementary regions and block target genes by several different mechanisms. Based on their mode of action, ASOs can be divided into three main groups: steric blockers of translation, modulators of splicing and recruiters of protein factors  that lead to the degradation of target RNA. The functions of target RNA as well as the selected interaction site determine the modulatory effect of ASOs . Thus, t2 nanoparticles, which allowed a complete replication inhibition of different influenza subtypes in MDCK cells [Liposomal encapsulation was used to deliver ASOs binding to functionally important sequences of packaging signals at the 5′-ends of PA and PB1 segments . AlthougCK cells ,32. In cCK cells ,32 in coCK cells .Currently, most antisense strategies involve particular ASO binding only to one target in the viral genome. Innovative approach was investigated by Duan et al., who designed the universal phosphorothioate antisense oligonucleotide, specific for the 5′-terminal conserved sequence found in all eight vRNA segments of the influenza virus . In MDCKAnother specific delivery strategy in influenza treatment was developed by Zhang et al., who conjugated single chain variable fragment (scFv) directed against influenza HA antigen with truncated protamine (tP) ,33. ASOsThus far, most studies on the ASO strategy towards the inhibition of the influenza proliferation have been reported as successful in both cell culture and animal models. Radavirsen (AVI-7100) was the first and currently only ASO in influenza treatment that entered clinical trials. Radavirsen is a 20-mer PMOplus®-based ASO, designed to bind highly conserved regions responsible for splicing M1 and M2 transcripts of M (+)RNA segment. The initial experiments in preclinical ferret, mouse, rat and monkey models revealed the antiviral activity of ASO and allowed to assess its toxicity. Next, a single and multiple ascending dose phase I clinical study was conducted in healthy volunteers. Since Radavirsen was well tolerated with no safety concerns identified, the results obtained by Beigel et al. are promising for the use of oligonucleotide drugs in influenza therapy ,36.Recent advances in ASO chemistry and delivery methods have led to the discovery of various promising candidates for influenza treatment. Remarkably, preventing viral proliferation with the ASO strategy has been achieved by targeting other elements than influenza RNA segments, including host-encoded hemagglutinin-activating protease TMPRSS2  and lympSmall interfering RNA (siRNA) is an oligonucleotide approximately 21–25 nt long formed through intracellular Dicer enzyme cleavage of the excises of exogenous double-stranded RNA. The siRNA induces sequence-specific degradation of targeted mRNA and can specifically inhibit the replication of influenza virus . In geneIt has been shown that synthetic siRNAs can specifically silence the influenza virus mRNA and inhibit the virus replication at different levels . Ge et aAn interesting observation was made by McMillen et al., who described that the NS1 gene-specific siRNAs caused decreasing mRNA level, while in case of siRNAs targeting NA and M genes, mRNA and protein expression levels were upregulated in culture lysates . This caGiardia intestinalis Dicer and evaluated the efficacy of these molecules in preventing viral infection in human primary monocyte-derived macrophages and dendritic cells. These siRNAs contained more than 100 different antiviral RNAs targeting the most conserved regions of the IAV genome. Results revealed that the replication of different IAV strains was significantly inhibited by IAV-specific siRNAs. The authors observed an increase in inhibition of viral RNA expression by up to seven orders of magnitude and a reduction of IAV protein synthesis as well as virus production. This research showed that the wide antiviral spectrum makes the siRNA a potentially efficient tool against multiple strains of influenza virus, acting by targeting their conserved gene regions [Recently, it has been demonstrated that siRNA molecules can efficiently inhibit the replication of human seasonal and avian influenza viruses in various types of human cell systems . Jiang e regions . Interesting research has been conducted by the Vasin group, who developed a highly effective hybrid microcarrier system for the delivery of antiviral siRNA mixtures . They usTo summarize, on the one hand, the siRNAs have high specificity, strong selectivity and silencing efficiency. Therefore, these molecules have been developed and used in the studies as antiviral tools against influenza, including highly pathogenic and avian influenza virus strains. On the other hand, siRNAs can be responsible for several problems, resulting in off-target effects. Hence, the current studies of the anti-influenza approach based on the siRNA-mediated RNAi process are limited to cells, chick embryos and animals and have not yet entered clinical trials.MicroRNAs (miRNAs) are small noncoding RNAs that control the translation and transcription of its target genes. These molecules can also regulate various biological processes, including viral infection. Emerging studies indicate that the host miRNAs play an important role in the intricate host-pathogen interaction network ,47. CellThe Kumar group presented that the miRNA-485 (miR-485) can regulate antiviral immunity and restrict viral replication via targeting host and influenza virus transcripts . They deIn 2017, Peng et al. reported that the endogenous miRNAs mediate antiviral defense against IAV . They scPrevious studies revealed a novel antiviral miRNA targeting the NS1 of the pandemic human influenza virus strains . BavagnoApart from the miRNA-based tools aimed at the target sequence of IAV genome, this approach can also be used to enhance biosafety of gain-of-function IAV research. For instance, tenOever’s group described a species-specific attenuation strategy, which allowed to restrict transmission in one species while maintaining transmission in the other hosts . The autCollectively, the examples of the studies described above indicate that host cells can lower their viral loads by controlling miRNA pathways, which may provide new opportunities for anti-influenza approaches. Investigation of miRNA molecules that directly target the conservative viral sequences can lead to the discovery of new therapeutics against influenza virus infection.Ribozymes and deoxyribozymes are two types of nucleic acids that have catalytic activity. Ribozymes have been shown to play an important role in biological processes such as splicing, RNA processing and replication of RNA genomes ,55. SomeDNA enzymes, also known as DNAzymes, were identified using in vitro selection methodology . Two DNAInteresting study by Kumar et al. presented a new approach by constructing siRNA-ribozyme chimera as a potential inhibitor for influenza virus . The desMotard et al. engineered ribozyme construct based on the hepatitis delta virus (HDV) and a specific on/off adapter (SOFA), which increases the specificity of the cleavage while reducing the non-specific effect . They prIn another study by Toyoda et al. reported the usage of 10-23 DNAzymes targeting the AUG initiation codon of PB2 mRNA of IAV . They prIn another study, Kumar et al. prepared modified 10-23 DNAzymes, targeting various regions of the M2 gene and testing their ability to specifically cleave the target RNA . The preInvestigations described above indicated that catalytic nucleic acids could be a useful tool to control viral infection. The ribozymes and appropriately modified DNAzymes possess potent anti-influenza activity and can provide an opportunity to enhance the ASO strategy. In fast-evolving viruses, such as influenza, the development of effective therapeutics to limit disease spread is challenging. ASOs, siRNAs, miRNAs or catalytic nucleic acids tools afford the potential tractable strategies to eradicate the influenza virus. It is because they are easy to design, can be chemically modified to improve their stability and are directed against multiple virus strains by targeting the most conserved RNA regions. However, these tools act in a sequence-specific manner, which makes these approaches not highly effective in inhibiting the proliferation of influenza virus. Hence, the designing and development of new inhibitory oligonucleotides should be based on additional criteria using knowledge on the viral RNA secondary structure. As previously mentioned, influenza virus genome is organized into eight segments of single-stranded viral RNA. Each segment is composed of conserved, partially complementary 5′- and 3′-ends, segment-specific untranslated regions (UTRs) and open-reading frames (ORFs) . During The secondary structure of vRNA is an important regulator of virus biology, because it plays a role in assembling the different vRNPs into infective virions and in directing reassortments between IAV subtypes. The terminal fragments at the 5′- and 3′-ends of the vRNA segments  nucleotides.In order to inhibit viral proliferation, Lenartowicz et al. used information about secondary structure of protein-free (in vitro) influenza A genomic segment 8 (vRNA) to design ASOs . PredictA similar strategy for selecting ASO-susceptible RNA regions for another influenza genome segment was employed by Michalak et al. . A summaThe significant role of secondary structure determination preceding the design of sites available for ASO applies not only to the genomic influenza segments, but also to (+)RNA. In the paper published by Soszynska-Jozwiak et al., influenza virus segment 5 (+)RNA secondary structure was established and validated by bioinformatics and experimental studies . Nine ofOverall, the results revealed above illustrate strong correlations between the predicted ASO binding regions and the ability to inhibit influenza virus replication. However, in each study some ASOs targeting single stranded regions exhibited moderate efficiency. This can be due to the dynamic nature of the structure of influenza RNA segments. Additional reasons, including local structure stability as well as interactions with other RNAs or proteins, also contribute to the antisense potency of ASO in the cells (in vivo). Nevertheless, besides playing an important role in designing efficient hybridization sites for ASOs, RNA secondary structure determination significantly contributes to understanding the influenza virus life cycle and replication mechanisms. The RNA structure-based target site selecting method provides novel prospects for other antiviral therapeutic agents such as small molecules.According to several publications, the RNAi efficiency is influenced by the target RNA structure ,90,91. TInteresting research concerning the inhibition of influenza viral replication by targeting structural motifs of vRNA has also been conducted by Kesy et al. . It is kAnother structure-based approach uses the ability of small molecules to target specific RNA structures. In general, small molecules have the potential to recognize and bind to RNA targets based on their secondary or tertiary structures. Bottini et al. reported specific recognition of the vRNA promoter region (panhandle structure) by small molecules as a potential novel drug target and tool against IAV infection . The autIn summary, we presented examples of studies concerning ASOs, siRNAs, miRNAs and catalytic nucleic acids approaches as potential antiviral tools against IAV. We described investigations that focused on the viral RNA structure-function relationship and genomic conserved RNA regions as targets for oligonucleotides strategies. Moreover, we also discussed a new criterion for choosing and designing target sites in IAV segmented RNA that is based on information about its secondary structure.Nowadays, given the large number of new imaging, sequencing and biochemical techniques, the influenza virus biology  is relatively well understood. Recent advances in next-generation sequencing (NGS) technology provide new insights into the IAV genome architecture . NGS tecHowever, an efficient oligonucleotide tools delivery to the target cell still remains a challenge. To promote the intracellular uptake, many effective approaches are being developed, including oligonucleotide chemical modifications, bioconjugation of various delivery-promoting moieties or usage of nanocarriers . In addiIn the last few years, the CRISPR-Cas system has become one of the most powerful gene editing tools. Interestingly, several studies using a CRISPR-Cas13-based strategy have been performed on potential inhibition of RNA viruses, including IAV  and the In light of the continuous evolvement of IAV and its increasing resistance to existing drugs and vaccines, finding target sites in IAV RNA that are sequentially or structurally conserved and less prone to drug resistance is a very important issue. That conclusion could be extended to other RNA viruses, including SARS-CoV-2. A novel therapy concerning the regulation of pathogenic RNA by using RNA-targeting strategies is still being developed. The flexible and dynamic nature of viral RNA structures is a challenge associated with targeting RNAs with potential therapeutics. Therefore, information regarding the molecular and structural basis behind influenza virus biology, including knowledge about RNA secondary structure, could be used to design and develop structure-based approaches that will inhibit influenza replication. The ability to identify oligonucleotides or molecules that specifically bind to RNA targets on the basis of its structure provides a viable path to create new drugs against influenza.


Quercus robur L.). The newly identified pathogen is associated with leaf symptoms such as mottle, chlorotic spots and ringspots on diseased trees. High-throughput sequencing  was used to explore the virome of a ringspot-diseased oak that had chlorotic ringspots of suspected viral origin on leaves for several years. Bioinformatic analysis of the HTS dataset followed by RT-PCR enabled us to determine complete sequences of four RNA genome segments of a novel virus. These sequences showed high similarity to members of the genus Emaravirus, which includes segmented negative-stranded RNA viruses of economic importance. To verify the ends of each RNA, we conducted rapid amplification of cDNA ends (RACE). We identified an additional genome segment (RNA 5) by RT-PCR using a genus-specific primer (PDAP213) to the conserved 3´ and 5´termini in order to amplify full-length genome segments. RNA 5 encodes a 21-kDa protein that is homologous to the silencing suppressor P8 of High Plains wheat mosaic virus. The five viral RNAs were consistently detected by RT-PCR in ringspot-diseased oaks in Germany, Sweden, and Norway. We conclude that the virus represents a new member of the genus Emaravirus affecting oaks in Germany and in Scandinavia, and we propose the name “common oak ringspot-associated emaravirus” (CORaV).We report the complete nucleotide sequence of the genome of a novel virus in ringspot-diseased common oak (The online version contains supplementary material available at 10.1007/s00705-021-04955-w. Quercus robur L.) [Quercus [In Germany, chlorotic ringspots, mottling and chlorotic spots have been observed for decades on leaves of common oak (obur L.) . Mechaniobur L.) . A viralobur L.) . Attempt[Quercus .Emaravirus is part of the family Fimoviridae  and includes plant viruses with a segmented, negative-stranded RNA genome. At least four genome segments are present in all members described to date [Here, we report the complete genome sequence of a novel emaravirus based on its initial identification in a ringspot-diseased common oak tree in Kreuztal, Germany, using high-throughput sequencing , first employing a random sequencing approach, followed by an emaravirus-enrichment strategy for library preparation . In the  to date . We obta to date , 8. PDAP to date , jujube  to date , and blu to date . We deteThe putative functions of proteins encoded by RNA 1 to RNA 4 of this virus were deduced based on the presence of conserved domains related to those of other emaraviruses. The RNA-dependent RNA polymerase (RdRP) is encoded by RNA 1 , while RNA 2 encodes the glycoprotein precursor . The viral nucleocapsid protein  is encoded by RNA 3, and RNA 4 encodes the movement protein   of CORaV contains one major open reading frame, coding for a protein of 179 aa P5, 21.1 kDa). We identified sequence similarity to the putative silencing suppressor P8 of HPWMoV  and to p1.1 kDa. Q. robur samples, we confirmed the presence of viral RNAs by RT-PCR applying species-specific primers (Table S1) targeting all five genome segments. We observed chlorotic ringspots and spots on diseased trees as the most abundant symptoms , and mottle was found only in a few samples. However, in seven trees displaying ringspot symptoms, we did not detect any of the tested CORaV genome segments. In leaf material collected from trees without any symptoms (n = 28), no viral genome segment could be detected. We obtained the same results for leaf samples from 25 trees that had never shown chlorotic ringspots but had either a regular chlorotic pattern or partial chlorosis of leaves . The latter symptoms were therefore considered atypical of CORaV and not associated with the ringspot disease. We consider it likely that CORaV contains five genome segments and is associated with the ringspot disease in common oak. Since virus infections represent a significant predisposition for other diseases and stress, further studies are needed to fully understand the biology and epidemiology of CORaV as well as its impact on the health status of trees, a prerequisite for effective disease management.We correlated the detection of all five viral RNAs to the occurrence of the ringspot disease of oaks. Leaf material from healthy and ringspot-diseased oaks at over 25 locations in Germany, Sweden, and Norway was sampled from landscape, nursery-grown, and urban trees. In 191 out of 198 Supplemental Fig. S1 Symptoms observed on diseased Q. robur trees, with CORaV-associated chlorotic ringspots  and atypical regular chlorotic patterns (right). Supplemental Table S1 Primer pairs used for RT-PCR-based amplification of missing sequence information of the novel emaravirus identified in common oak including RACE and detection of viral RNA 1 - RNA5 (DOC 547 KB)Below is the link to the electronic supplementary material.


Innovations in high-throughout sequencing approaches are being marshaled to both reveal the composition of the abundant and heterogeneous noncoding RNAs that populate cell nuclei and lend insight to the mechanisms by which noncoding RNAs influence chromosome biology and gene expression. This review focuses on some of the recent technological developments that have enabled the isolation of nascent transcripts and chromatin-associated and DNA-interacting RNAs. Coupled with emerging genome assembly and analytical approaches, the field is poised to achieve a comprehensive catalog of nuclear noncoding RNAs, including those derived from repetitive regions within eukaryotic genomes. Herein, particular attention is paid to the challenges and advances in the sequence analyses of repeat and transposable element–derived noncoding RNAs and in ascribing specific function(s) to such RNAs. Xist  approaches (a.k.a. the growing menagerie of “…-seq”s) to the study of RNA. Coupled with revolutionary advances in long-read sequencing, genome assembly, and annotation methods, comprehensive cataloging of nuclear ncRNA is underway with a view towards understanding the cellular functions of these heterogeneous and fascinating biomolecules. This review focuses on some of the recent technological developments that have enabled isolation of both chromatin-associated RNAs and DNA-associated RNAs. Moreover, computational approaches and initiatives to achieve chromosome-level genome assemblies are discussed in light of the challenges in studying such RNAs.noncoding; in other words, what exactly is a ncRNA? The phrase “noncoding RNA” at face value could refer to any RNA molecule that does not lead to a translated protein. However, this would include spliced introns, degradation products, and RNA debris, as well as RNAs that are predictably transcribed and have a structured transcription unit, such as rRNAs and tRNAs. Current nomenclature distinguishes ncRNAs rather arbitrarily as either small RNAs of 200 nt and less, or RNAs 200 nt and longer, referred to as long or large ncRNAs (lncRNAs) and long intergenic noncoding RNAs (lincRNA). Small RNAs are further classified into groups based on function, biogenesis, and/or other biochemical features    vs evolutionary (why does the ncRNA exist) contexts. Recently, a new lexicon to clarify “function” has been proposed, referred to as the “Pittsburg model of function”. In this model, ncRNAs are further classified into five categories based on the depth and context of genetic information available to support functional classification , ignoring repeat-derived and intergenic ncRNAs, thus rendering only a partial view of transcriptome dynamics. HTS approaches support transcriptome-scale sequencing (RNA-seq) that include ncRNAs by capturing potentially all RNAs present in a given sample, representing newly transcribed RNAs, stable RNAs, and RNAs heading for imminent decay. While RNA-seq was the first global transcriptomic approach enabled by HTS, new techniques have been developed to score the density of RNA polymerase II binding across the genome or to measure nascent, active transcription, and delineate transcription start sites (TSSs), eliminating the need to account for the variable half-life of different RNAs. Sequencing data outputs are subsequently mapped to a reference genome and intersected with gene annotations to tease apart mRNAs from cell-specific ncRNAs.Immunoprecipitation of RNA polymerase II  and regional  scales, one must consider Direct RNA-DNA interactions occur through complementary base pairing of DNA with RNA, resulting in the formation of a three-stranded structure consisting of a DNA:RNA hybrid and the displaced complementary DNA strand  to RNA:DNA duplex structures specifically, independent of nucleic acid sequence  , indicating that a single RNA species may affect many loci across the genome that share a similar sequence composition, such as repeated elements and satellite arrays. The single stranded DNA binding protein RPA (replication protein A) was recently identified at human centromeres. While RPA is known to participate in ATR (ataxia telangiectasia mutated and Rad3-related) kinase activation targeting DNA damage and stalled replication forks , is mediated by a triplex containing the Fendrr lncRNA , and LINEs within opossum. The lineage-specific convergence in redundant motif sequences shared between lncRNAs involved in X chromosome inactivation and X-linked LINEs may indicate that lncRNA-LINE triplexes are essential for inactivation of the X in females . Three techniques employing the ONE vs MANY approach, ChIRP (chromatin isolation by RNA purification) , an approach to isolate chromatin-proximal RNAs, uses nuclear fractionation followed by RNA deep sequencing (Werner and Ruthenburg MmeI whose recognition sequence is within the bridge but cuts 18–20 bp away) allows size selection prior to sequencing to enrich for fragments containing RNA-DNA ligations , is enabled by a bivalent linker containing a biotinylated bridge sequence, ligating the RNA portion to nascent RNA and the double-stranded DNA portion to proximal DNA. The MANY vs MANY techniques that rely on this type of proximity ligation, RNA-DNA heteroduplex capture include Fig. : MARGI , GRID-seons Fig. , left  and applications such as Hi-C, accompanied by increasingly accessible high-coverage short-read sequencing are supporting efforts to complete telomere-to-telomere (T2T) assemblies for a reference human genome. Successes in this approach have been realized for the X chromosome (Miga et al. Such advances will enable a full appreciation of the dynamic and diverse RNA-chromatin relationships that exist in eukaryotic genomes. However, a major challenge will be to “carryover” exiting datasets developed to study RNA-chromatin interactions to new assemblies and repeat annotation pipelines as they emerge. Furthermore, the diversity of genomes across individuals within a population should be incorporated into studies exploring the role of ncRNAs in instability and disease. The lack of T2T-scale genomes that support comprehensive comparative approaches must be overcome (Doolittle


RNA modifications have recently emerged as a widespread and complex facet of gene expression regulation. Counting more than 170 distinct chemical modifications with far-reaching implications for RNA fate, they are collectively referred to as the epitranscriptome. These modifications can occur in all RNA species, including messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). In mRNAs the deposition, removal, and recognition of chemical marks by writers, erasers and readers influence their structure, localization, stability, and translation. In turn, this modulates key molecular and cellular processes such as RNA metabolism, cell cycle, apoptosis, and others. Unsurprisingly, given their relevance for cellular and organismal functions, alterations of epitranscriptomic marks have been observed in a broad range of human diseases, including cancer, neurological and metabolic disorders. Here, we will review the major types of mRNA modifications and editing processes in conjunction with the enzymes involved in their metabolism and describe their impact on human diseases. We present the current knowledge in an updated catalog. We will also discuss the emerging evidence on the crosstalk of epitranscriptomic marks and what this interplay could imply for the dynamics of mRNA modifications. Understanding how this complex regulatory layer can affect the course of human pathologies will ultimately lead to its exploitation toward novel epitranscriptomic therapeutic strategies. RNA molecules can undergo more than 170 different chemical modifications . These mA broad set of RNA-binding proteins (RBPs) determines the mRNA epitranscriptome: Modifications are induced by writers, and several can be reverted by erasers. Eventually, some modifications need readers to be decoded. . ThroughN6-methyladenosine (m6A), and pseudouridine (Ψ), for which we provide flashcards . Furthermore, we will provide an overview of detection methods and discuss emerging evidence on the interplay of different modifications, proposing potential avenues to improve our understanding of these pervasive RNA regulators.In this review, we will describe mRNA modifications and their increasingly appreciated role as drivers of human pathologies. We will give particular focus on the most abundant ones, namely RNA editing (A-to-I and C-to-U), ashcards –4 summarADAR and APOBEC gene families likely originate from the adenosine deaminase acting on tRNA (ADAT) genes  enzymes . AlongsiT) genes , whose eT) genes .In humans, A-to-I editing  is mediaGRIA2) and the serotonin 5-hydroxytryptamine (2C) (2c5-HT) receptors , a rare autosomal genetic disorder of the skin, but the pathogenetic mechanisms are not yet clear  mRNA was identified in the small intestine  and 14 (METTL14) (6A at specific transcripts. Fat mass and obesity-associated protein (FTO) and Alkb homolog 5 (ALKBH5) are the demethylases or “erasers” of m6A  , effectiMETTL14) . Other wMETTL14) , are imp” of m6A , suggestNA fates .6A plays a central role in several biological and pathological processes  which in turn, facilitated METTL3 expression by inhibiting miRNA let-7g which targets METTL3 for subsequent degradation  (6A levels and promoting breast tumorigenesis.The mderstood . For insradation . In addihenotype . Howeverhenotype . Therefoor genes . In addior genes . Hypoxicor genes . The expls (CSC) . In addils (CSC) . The mouls (CSC) , and hen6A levels might influence global expression programs that lead to malignant phenotypes, and the crosstalk among m6A readers, erasers and writers critically regulates the expression of key transcripts to maintain cellular homeostasis  complex comprised of four conserved proteins, namely NHP2, GAR1, NOP10, and dyskerin . In contTo date, only a few biological functions of Ψ on mRNAs have been identified . PreviouPUS7 that segregate with intellectual disability and microcephaly lead to the abolishment of pseudouridylation not only in tRNAs but also in mRNAs  cap, the second nucleotide in many mRNAs can be methylated at the 2′-hydroxyl group; if the transcription start nucleoside is 2′-O-methyladenosine (Am), its N6 position can be further methylated to form N6, 2′-O-dimethyladenosine (m6Am) (Adjacent to the e (m6Am) .6A, the biological function of m6Am and its role in cellular homeostasis are still poorly understood.This modification stabilizes the mRNA by preventing DCP2-mediated decapping and microRNA-mediated mRNA degradation . Unlike 6Am is a reversible modification catalyzed by the writer PCIF1/CAPAM  confers a positive charge that can influence the local structure of the RNA or its interaction with RBPs. It can be found in several RNA species, including tRNA, rRNA and mRNA  was initially discovered in rRNAs and tRNAs. More recently, high-throughput techniques have revealed its presence in mRNAs, although its prevalence is limited  family of proteins (NSUN1–7) and DNMT2, being all of them specific for distinct RNA species. Although NSUN2 was originally described as a tRNA methyltransferase  consists in the transfer of a methyl group at the 2′-hydroxyl of the ribose of all RNA species, predominantly of rRNA and tRNA (reviewed elsewhere in Its deposition on mRNA was thought to occur only at the first 2 ribonucleotides (N1 and N2) within the 5′ cap structure  being deIn addition, the mechanism of 2′-O-Me deposition on coding transcripts is still to be revealed, but it might be guided by box C/D snoRNAs (SNORDs) as in the case of rRNA or directly mediated by single methyltransferases known to methylate other RNA species, such as FTSJ3 .1 prevents transcripts’ degradation by blocking decapping and exoribonuclease activities of DXO which degrades defectively capped pre-mRNAs  and m6A individual-nucleotide-resolution crosslinking and immunoprecipitation (mi-CLIP or m6A-CLIP)  signatures can process both inosines (reading them as guanines) and uracils, so a discrepancy between RNA and DNA sequences can be detected by RT-PCR or high-throughput sequencing . Moreovechniques B such as6A-CLIP) , have reNA 5′UTR . To over6A-SEAL, C, or enz6A-SEAL,  have beereatment .p-toluenesulfonate (CMC), a covalent adduct that blocks the RT activity  carbodiimide methyl-activity . Among tactivity . Non-CMCactivity .1A-induced RT mismatch signature  and METTL3/14 (m6A) methylate many coding and ncRNA species  is required for the recognition of all three modifications, albeit the affinities for m1A and m5C are lower than those for m6A. This lower affinity, coupled to the scarcity of both marks in mRNAs, leaves the actual occurrence and phenotypic impact of this “integration” as a question still to be answered. If confirmed, also other YTH domain-family proteins could behave similarly (Supplemental Table S1, we collect alterations of RNA modifications and editing in several diseases, obtained from the literature on this topic. As shown there, several tumor types are associated with the altered deposition of multiple modifications. This catalog could allow the identification of disorders associated with multiple RNA modifications and thus possibly affected by their interplay.On the other end, RBPs controlling the life cycle of RNA modifications appear to interact with marks other than their canonical one. For instance, the YTHDF2 m and m5C . A conseimilarly , as mighimilarly . One mayimilarly . Few stuimilarly , and howOverall, it appears that RNA marks may “talk” through direct cooperation or competition, and through the integration of multiple modifications by their reader proteins. Nevertheless, further work will be necessary to demonstrate the actual occurrence and elucidate the amplitude of this interplay, the complexity of the induced regulatory networks and its importance in shaping cell physiology. Is this behavior a form of “epitranscriptomic signaling,” allowing to coordinate the outcome of different pathways? And do changes in the RNA secondary structure interact to alter the transcript life cycle or are reader enzymes required for this crosstalk to modulate cell phenotypes? The answers could bring a new layer of complexity to epitranscriptomics, leading the field into uncharted avenues of even greater possibilities.drivers or mere passengers? It is clear that in some cases alterations in the mediators of these RNA marks have direct effects on cellular tumor-promoting features (Supplemental Table S1 we have highlighted the presence of opposite roles that can be played by RNA marks-regulating factors at an intra- and intertumor level. Can the affected pathways explain these disease-specific behaviors? Furthermore, tumors with potential alterations in multiple RNA marks have been identified. Can alterations to multiple enzymes controlling the life cycle of the different modifications be regarded as “double hits” leading to oncogenesis?Since RNA modifications represent such a basic layer in the biology of the cell, loss of these modifications is often fatal  and mighfeatures . As suchAnswering these questions will greatly expand our knowledge on the role of epitranscriptomics in disease onset and progression, ultimately enabling us to design novel, highly specific therapeutic strategies against still incurable diseases.Supplemental material is available for this article.


Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity.Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from  S-adenosylmethionine ) ) .pfl motif, as it is frequently associated with a gene coding for pyruvate-formate-lyase  and P3 (Zsw15) was investigated to monitor pseudoknot formation in trans . Primers with high annealing temperatures exceeding 60 °C were chosen for Zsw101 as they led to significantly better PCR results. Primers for Zsw81–91 contained 2′-methoxy modifications to ensure 3′-end homogeneous transcription products  using home-made Phusion polymerase. As DNA template, we used either 0.1 μl of 1 μM plasmid solution containing the full-length riboswitch sequence or 0.1 μl of PCR-amplified template. Sample integrity was confirmed by native PAGE or agarose gel electrophoresis.Transcription templates were generated by PCR from pUC57 vector that contains the full-length native sequence of the ZMP-sensing riboswitch from products . PCR was15 were prepared by in vitro transcription with T7 RNA polymerase (RNAP). PCR-reactions were directly used for transcription without purification. Transcription reactions contained transcription buffer (100 mM Tris/glutamate pH 8.1), 2 mM spermidine, 20 mM dithiothreitol (DTT), 20% (v/v) DMSO, 5 mM of each NTP, 12.5 mM Mg(OAc)2, 0.2 u/ml yeast inorganic pyrophosphatase (New England Biolabs®) and 144 nM home-made T7 RNAP. Unlabeled NTPs were purchased from Carl Roth GmbH + Co. KG (Germany). 13C,15N labeled NTPs were purchased from Silantes GmbH (Germany).All RNAs except Zsw81–91 were purified according to the protocol developed by Helmling et al.  and washed repeatedly with water until the elute did not show a significant signal at 180–220 nm in the UV-VIS spectrum . The solution was concentrated to 1 ml, mixed with 30 % glycerol and loaded onto a 15% polyacrylamide (PAA) gel (7 M urea). The RNA band was excised from the gel and eluted in 0.6 M NaOAc, pH 5.5. The RNA was precipitated once with cold EtOH and twice with 2 % (w/v) LiClO4 in acetone. After resuspending in water, buffer exchange to NMR buffer (as described above) was performed on a centrifugal concentrator.The RNA constructs Zsw15 RNA was bought from Dharmacon GmbH. The RNA was deprotected according to the supplier protocol. Following HPLC purification, the HPLC buffer was removed on a 1000 MWCO centrifugal concentrator. After one EtOH and two LiClO4 precipitations (see above), the RNA was resuspended in water and the buffer exchanged to NMR buffer. The NMR buffer contained 25 mM potassium phosphate buffer adjusted to a pH of 6.2 and 50 mM KCl. The RNA was thermally refolded before sample preparation.Zsw2O and 7.5 nmol DSS as internal reference to RNA stock solutions in NMR buffer (see above). All spectra were recorded of 280 μl samples in Shigemi NMR tubes (Shigemi Inc.).NMR samples were prepared by adding 10 % D15N-editing in 1H,1H-NOESY was achieved by implementing X-filter schemes before and after t1 chemical shift evolution  and sparky 3.14 . Water svolution . Analysi® MicroCal iTC200 instrument with RNA sample concentrations of 60 μM and ligand concentrations of 600 μM (for Zsw77 and Zsw91) or 300 μM (for constructs Zsw81–83). Raw data were exported and analyzed using NITPIC  of 0.06 ppm. Strikingly, however, Zsw48+Zsw15 showed clear ligand binding , respectively, before adding two equivalents ([RNA]:[ZMP] = 1:2) of ligand. In both cases, CSPs to the downfield region were observed for all proton signals after addition of Mg2+. However, these shifts were accompanied by signal broadening at 10 mM Mg2+. After adding two equivalents of ligand, signal line widths decreased and the signals shifted non-uniformly for the 10 mM Mg2+ sample. In contrast, the 3 mM Mg2+ sample showed no spectral changes upon ZMP addition  and 13% (Zsw81), while in NMR measurements, a decrease from 70% to 43% was observed , leads to partial ZMP binding incompetence, which we also observe via ITC and NMR spectroscopy. However, for this construct, absolute values differed largely between ITC and much slower NMR measurements. We therefore suspect very slow folding kinetics on a timescale of minutes between a binding incompetent pre-terminator state back to a binding competent state if binding competent RNA is removed from the equilibrium by ligand interaction. This would render the pre-terminator state kinetically trapped in a biological context. Consequently, it is expected that RNA displaying formation of a single terminator base-pair could already be predominantly binding incompetent in vivo, where fast progress of transcription limits the refolding time available. In comparison, Zsw82 displays strongly reduced binding competence and an almost full population of the terminator state, leaving Zsw81 as the only construct with considerable population of both states. Therefore, in a biological context, the transition to the binding incompetent state is expected to be comparably sharp in contrast to other examples like the deoxyguanosine-sensing riboswitch, which displays a smoother transition , which could be an interesting premise for further kinetic experiments. With the terminator fold being thermodynamically more stable than the ligand-bound antiterminator fold, the stabilization of the pseudoknot established by ZMP addition can be considered temporary and therefore kinetically driven. This type of regulation is a common motif observed in riboswitches such as fluoride- (We propose a model Figure  in whichHAPE-seq . The timluoride- , FMN- (4luoride- , deoxyguluoride-  or guanoluoride-  riboswitpfl riboswitch from T. carboxydivorans and their structural characteristics at atomic resolution.To resolve these kinetics, real-time NMR measurements can be performed by rapid addition of interactors or by introduction and subsequent elimination of photolabile protecting groups. However, these studies require sufficient exploration of the underlying system in equilibrium state. We herein provided the NMR spectroscopic basis for such studies, showing important key players of the conformational landscape of the gkaa427_Supplemental_FileClick here for additional data file.


HSPG2 is implicated in the human diseases Schwartz-Jampel Syndrome type 1 and Dyssegmental Dysplasia, Silverman-Handmaker type . The mutated unc-52 gene expresses the phenotype for uncoordinated movement (“Unc”), which involves progressive paralysis and retarded sarcomere construction . The UNC-52 protein is localized in the striated muscle dense bodies and the basement membrane, where it plays an important role in developmental processes such as cell adhesion, cell migration, and signal transduction . Within the unc-52 gene, an RGD (Arg- Gly- Asp) sequence is located at amino acid locations 746, 747, and 748 in exon 7. Exon 7, containing RGD748, is included in all three major isoforms, short (S), medium (M), and long (L) . The RGD sequence is part of the Laminin IV type A domain and primarily functions as a cell attachment and adhesion site for integrins . In this study, two separate mutations were performed on this unc-52 RGD sequence using CRISPR-Cas9 technology. The unc-52(kq748) mutation replaced the aspartic acid (D) located at the 748 amino acid position with a glutamic acid (E) . The unc-52(kq745) mutation removed the RGD sequence by deleting amino acids 746, 747, and 748. Previous studies have shown that unc-52 gene mutations cause the disorganized distribution of pat-3 β integrin, which is a receptor for extracellular matrix proteins . In order to study the cellular phenotypes of the unc-52(kq748) mutation, a double mutant was created: pat-3::GFP; unc-52 (kq748). Staining showed that localization of the pat-3::GFP reporter in the double RGD mutant appeared normal, with dense bodies and M-lines alternating along the muscle filaments . In order to visualize the actin filaments in the body wall muscles of N2 wild type and unc-52(kq748) mutants, staining was performed using 0.4 U/mL rhodamine-conjugated phalloidin, Thermo Fisher Scientific, Waltham, MA . This unc-52(kq748) staining showed no obvious abnormalities outside of interruptions due to fixation and slicing procedures (unc-52(kq748) mutants compared to the N2 worms, and a 33% decrease in unc-52(kq745) mutants  and unc-52(kq745) groups differed significantly from the N2 wild-type group, as shown by p-values of 7.1×10-6 and 4.63×10-8 respectively. These results show that there was a statistically significant decrease in motility for both unc-52(kq748) and unc-52(kq745) mutants.The ocedures . Additio mutants . A one-wunc-52(kq748) and unc-52(kq745) mutations. For the unc-52(kq748) mutation, the DNA repair template (UNC52RGE748), crRNA (UNC52RGE748), tracrRNA (cat. #1072532), Cas9 nuclease (cat. #1081058), and a dpy-10 crRNA co-CRISPR marker were mixed and micro-injected into wild-type N2 worms, which served as the parent (P0) generation. F1 worms that carried the mutation were then identified and isolated using the dpy-10 marker  and underwent PCR genotyping using mutant specific primers (UNC52RGE748SEQF and UNC52RGE748SEQR) to screen for the RGE mutation. The F2 generation then also underwent PCR screening using both a mutant specific primer (UNC52RGE748SEQF) and a wild-type specific primer (UNC52RGD748WTF) to isolate non-dumpy homozygous mutants. These homozygous mutant PCR products were sequenced to validate the success of the mutation . This process was repeated for the unc-52(kq745) mutation using the DNA repair template (UNC52RGD748D) and the primers (UNC52RGD748DF and UNC52RGDWTF). The DNA repair template and all other oligos were designed at IDT Inc., Coralville, IA. A double mutant was then created by crossing unc-52(kq748) II males with NK358 pat-3::GFP III hermaphrodites. Homozygous double mutants from the F2 generation were identified as green-fluorescent progeny and confirmed through PCR genotyping. Staining was performed by collecting mutants and fixing them with ice-cold methanol for 5 minutes. Images were captured using a Nikon Eclipse Ni-U epifluorescence microscope and processed with NIS Elements (version 5.02). Once the homozygous mutants were identified and isolated, phenotypic assays were conducted on 50 young adult worms from both homozygous mutants and N2 wild-type worms in order to characterize the phenotypes for both the unc-52(kq748) and unc-52(kq745) mutations. A one-way ANOVA test was then run to confirm statistical significance of the results. Since the p-value for the ANOVA test was less than 0.05, post hoc tests were run to determine which groups differed .CRISPR-Cas9 technology was used to create both the crRNA sequences (5’ to 3’)UNC52RGD748 TTTCTATTGATTATGCTCGT(dpy-10) ZQDP10A GCUACCAUAGGCACCACGAGRepair templates (5’ to 3’)PrimersUNC52RGE748SEQF TTCCTTGCTTCTGCTCAGGTUNC52RGE748SEQR TGATCGGAGTTGCCATTTCCAUNC52RGD748WTF TTCTATTGATTATGCTCGTGGGGATUNC52RGE748F TCAATCGACTACGCAAGAGGCUNC52RGD748DF GTCAATCGACTACGCAAGAGATCAAunc-52(kq748) II, BU745 unc-52(kq745) II, and BU111 unc-52(kq748); pat-3::GFP are available upon request. NK358 pat-3::GFP III was purchased from Caenorhabditis Genetics Center, Minneapolis, MN.BU748


Polycyathus chaishanensis is a symbiotic caryophyllid coral described from a single population in a tidal pool off Chaishan, Kaohsiung, Taiwan. Due to its rarity, P. chaishanensis was declared a critically-endangered species under the Taiwan Wildlife Protection Act. In May 2017, a P. chaishanensis colony was discovered in the intertidal area of the Datan Algal Reef, Taoyuan, Taiwan. To determine whether this is a stable population in the algal reef, a demographic census—including data on occurrence, distribution, and colony size—was carried out in the algal reef in southern Taoyuan. Intertidal censuses and sediment collections were conducted at five different sections—Baiyu, Datan G1, Datan G2, Yongxing, and Yongan algal reefs—during the monthly spring low tide from July 2018 to January 2019. In total, 84 colonies—23 in Datan G1 and 61 in Datan G2—were recorded from a tidal range of − 160 to − 250 cm, according to the Taiwan Vertical Datum 2001 compiled by the Central Weather Bureau. No P. chaishanensis was found in Baiyu, Yongxing, or Yongan. The P. chaishanensis colony sizes ranged from 2.55 to 81.5 cm in diameter, with the larger P. chaishanensis present in the lower intertidal zone. Sediment was extremely high, with monthly site averages ranging from 3,818.26 to 29,166.88 mg cm−2 day−1, and there was a significant difference between sites and months, both of which affected the distribution of P. chaishanensis in the algal reef. Our study confirms the existence of a second population of P. chaishanensis in Taiwan, highlighting the importance of the Datan Algal Reef for the survival and protection of this critically-endangered caryophyllid coral and why it is so urgent that the reef should be conserved. Polycyathus chaishanensis is a caryophylliid coral described from the coast of Chaishan, Kaohsiung, Taiwan1. The other known Polycyathus species are found in the Pacific and Atlantic Oceans from the sublittoral zone (> 12 m) to depths greater than 400 m5; P. chaishanensis, however, was discovered from a single population composed of fewer than 50 small encrusting colonies in a tidal pool (< 3 m) during high tide, which represents the shallowest Polycyathus recorded so far. This makes Polycyathus, including P. chaishanensis, a model genus for understanding the evolution of scleractinian corals, such as whether Polycyathus evolved from shallow to deep waters or vice versa. P. chaishanensis is the only one of the 22 known Polycyathus species6 associated with the Symbiodiniaceae algae Cladocopium C11. P. chaishanensis was declared a critically-endangered coral species in May 2017, and is now protected by the Taiwanese Wildlife Act (TWA)7.P. chaishanensis. Reconnaissance surveys of the holotype locality in Chaishan showed that its environment is different from those of other tropical reef-building corals, which usually include clean water and relatively low sedimentation rates8. For example, the normal sedimentation rate of a healthy coral reef is around 10 mg cm−2 day−1 maximum9. Values above such a rate would cause some degree of reef degradation, and above 50 mg cm−2 day−1 would have catastrophic consequences10. The Chaishan coast is a sandy beach mixed with various sizes of carbonaceous rocks, which originated from nearby coastal hills made by an uplifted Pleistocene reef developed about 0.4–0.6 Mya11. The rock pool is highly turbid and the water column contains a high concentration of sand and particles formed by erosion, waves, and tide, which may have deterred most other scleractinians, even when a hard substratum is available1. P. chaishanensis was found on well-lifted reefs dominated by green and crustose coralline algae (CCA), suggesting that P. chaishanensis may prefer marginal habitats like this sandy rock pool.Apart from the taxonomic description, little is known about the baseline ecology or environment of P. chaishanensis was discovered in the intertidal area of the Datan Algal Reef, Taoyuan, Taiwan12  (mean ± SE) was not significantly different from that of Datan G2   = 9.684, p < 0.05, R2 = 0.106, Fig. −2 day−1  as opposed to month  or site  alone. The sedimentation rate was significantly different across sites  and months . Sedimentation rate was significantly correlated with wind speed  in individual traps ranged from 1,416.56 to 36,592.10 mg cmdf = 31) as opposed to month  or site  alone. The mud ratios were highly significantly different across the months , but not among sites  or the interaction between site and month   and Yongan in November 34.09%), respectively  and colony size indicate that P. chaishanensis forms a stable population in the Datan Algal Reef, reiterating the necessity for conservation action.Baseline ecology and environmental data are urgently needed to accurately illustrate the conservation status of P. chaishanensis mostly inhabits shallow waters1. All Polycyathus species, except P. chaishanensis and P. fulvus18, are azooxanthellate, and P. fulvus (30–50 m), as well as all Polycyathus species except P. chaishanensis, are found in deep waters; e.g., P. mullerae contributes to the formation of the mesophotic reef at 55 m deep in the Mediterranean19. P. mayae and P. senegalensis are found at 127–309 and 12–143 m deep, respectively, in the Gulf of Mexico5. In Colombia, both species are recorded from 75–217 and 73–152 m deep, respectively4. At the holotype locality in Chaishan, Kaohsiung, P. chaishanensis is distributed in an intertidal pool estimated to be less than 300 cm deep during high tide and − 50 cm during low tide, according to the Taiwan Vertical Datum 2001 (TWVD 2001). P. chaishanensis was found at a similar tidal level in the Datan Algal Reef (below − 160 cm), confirming that P. chaishanensis can adapt to a shallow-water environment with high turbidity and a water column with a high concentration of sand and particles due to erosion, waves, and tide1.Our study confirmed the previous finding that Polycyathus species, P. chaishanensis has adapted to shallow tidal pools or the intertidal area and is symbiotic with the Symbiodiniaceae Cladocopium (C1). Corals in disturbed environments tend to associate with stress-tolerant symbionts in the genus Durusdinium and some Cladocopium species (C3 and C15). In contrast, corals from highly sedimented environments in Hong Kong and Singapore were also found to associate with Cladocopium C121. The reason it associates with Cladocopium C1—even in environments with high sediment—is that, although stress tolerant, Durusdinium sp. is considered a selfish genus that does not provide efficient nutrient translocation to its coral host. However, Baker et al.22 showed that Cladocopium sp. might be involved in efficient nitrogen assimilation and carbon translocation to the hosts. On the other hand, high turbidity might provide protection from light stress (elevated levels of irradiance), thereby reducing the impact of thermal bleaching events23. Highly turbid environments might also help corals obtain nutrients in the form of suspended particulate matter, thereby making the coral less dependent on the symbiont (irrespective of the type of symbiont) for its energy requirements and allowing it to exist in shallow and turbid environments.We hypothesized that, unlike other Polycyathus chaishanensis in the Datan Algal Reef was distributed in the lower intertidal zone, and larger colonies were found towards the sublittoral zone. It was suggested that two abiotic gradients—disturbances from wave damage and light availability—determine the distribution and growth of reef-building corals in tropical waters25 and species diversity in the rocky intertidal zone27. Similar abiotic factors plus substratum availability can explain the distribution pattern of P. chaishanensis in the Datan Algal Reef. The algal reef stretches along the intertidal sand flat towards the sublittoral zone along the Taoyuan coast, and the Datan Algal Reef its best-developed section14  might be more tolerant to sand scouring or burial in a high sediment environment31. A study using the CCA collected at rocky sites along the northwestern coast of the USA showed that CCA can survive lengthy anoxic burial for three months with only slightly discoloration32. A similar experimental approach can be conducted to examine how long CCA can survive in the Datan Algal Reef under sand scouring or burial conditions. Furthermore, it is vital to understand how burial conditions affect the growth rate of CCA, the fundamental reef builder in the algal reef.The sedimentation rate was significantly correlated with wind speed on the intertidal algal reef in Taoyuan. Coral communities in the fringing reefs of Magnetic Island, northeastern Australia are also influenced by wind regimes, and the height of locally produced, short-period wind waves control the magnitude of near-bed suspended sediment concentrationsP. chaishanensis in Taiwan in the Datan Algal Reef, which has proven to be an unprecedented ecosystem not only for Taiwan, but around the world33. The P. chaishanensis population in Datan is the only existing healthy and stable population in Taiwan, since the holotype locality of P. chaishanensis, Chaishan, was destroyed by coastal development in Kaohsiung34. In addition, the recent discovery of an unexpected large colony of P. chaishanensis (110 cm long and 80 cm wide) at the − 250 cm tidal level in Datan G2 confirms the urgent need for conservation action35, including measures that allow the public to participate in decisions involving energy-related construction issues and better advising around the government’s policy decisions36. Unfortunately, the Datan Algal Reef is currently facing destruction from the development and construction of liquefied natural gas (LNG) receiving and storage terminals and ports17, which also severely threaten the survival of the merely extant local population of P. chaishanensis. For example, there are at least seven colonies of P. chaishanensis located within 200 m of the scheduled trestle bridge in the LNG terminal in Datan G1, with the closest one being only 52 m away , for protecting the algal reef is higher than for the gas receiving station in Taiwan36; nonetheless, both the Datan Algal Reef and P. chaishanensis face regional extinction if serious conservation action is not taken against coastal development.In conclusion, our study confirms the existence of a second population of 14. When exposed to the air during the low tide, the reef can be classified as a continuous and porous structure  compiled by the Central Weather Bureau. This − 160 cm tidal level is also the most common monthly low tide level, except during the extreme spring tides at the end of the year. We therefore conducted the surveys at the − 160 to − 250 cm tidal range. P. chaishanensis was identified in situ, and the colony locality was recorded by a GPSMAP 64st and mapped onto Google Earth to measure the horizontal distance to the scheduled trestle bridge of the LNG terminal  modified from the design described in English (1997)37. Each sediment trap was attached with a star picket for deploying the trap and maintaining it around 30–40 cm above the bottom. Three sediment trap replicates were deployed at the − 160 cm tide level at low tide in each reef section for 48 h to cover two full tidal cycles during the monthly spring tide period from July to December, 2018. Sediment from each trap was rinsed with distilled water to remove salts, filtered through a 100 μm filter, and air dried in an oven at 60 °C for 48 h before weight measurement. Sediment larger than 100 μm in diameter was defined as “sand,” while the rest  was defined as “mud.” The mud ratio—weight of mud divided by weight of total sediment (mud plus sand)—was calculated to examine the contribution of sand and mud to water quality across the five algal reef sections.Two indices—the distribution pattern, in terms of the distance of each colony to the shore, and the colony size frequency distribution—were utilized to compare the characteristics of P. chaishanensis colony sizes between the two populations, colony size was first converted into size class with a 10-cm interval, then Pearson’s chi-square test was applied. Yate’s continuity correlation38 was also applied due to the presence of size classes with fewer than five individuals. A Student’s t-test was used to test for significant difference in mean colony size between the two populations. Normality and homogeneity of variance were tested using the Shapiro–Wilk test39 and Levene’s test40, and log (x + 1) data transformation was applied when necessary.The distribution pattern, in terms of the distance of each colony to the shore, between Datan G1 and Datan G2 populations was compared using the Mann–Whitney test. To test for significant difference in lme4 (v. 1.1.19) and lmerTest (v. 3.1.0) packages in R-3.4.4 were applied41.A generalized linear mixed model (GLMM) was used to explore the spatial and temporal variation in the dry weight of sediment and mud ratio in sediment traps, separately. The full model included fixed effects of the site  and month (six levels: from June to December). We then compared three alternative models—(i) mud ratio ~ Site, (ii) mud ratio ~ Month, (iii) mud ratio ~ Site × Month—using Akaike information criterion (AIC). In all cases, we included sediment traps as a random effect (~ 1|Trap) to account for intra-site differences of mud ration in the sediment trap level. Kruskal–Wallis test was used to determine the spatial variation of the mud ratio for each month and temporal variation within each site. The Supplementary file1 (DOCX 345 kb)