Here, we identified the synthesis of abortive leaderRNAs (as well as trailer RNAs) that are specifically initiated opposite to (anti)genome nt 2; leaderRNAs are predominantly terminated in the region of nt ~ 60–80. LeaderRNA synthesis requires hexamer phasing in the 3’-leader promoter. We determined a steady-state NP mRNA:leaderRNA ratio of ~10 to 30-fold at 48 h after Ebola virus (EBOV) infection, and this ratio was higher (70 to 190-fold) for minigenome-transfected cells. LeaderRNA initiation at nt 2 and the range of termination sites were not affected by structure and length variation between promoter elements 1 and 2, nor the presence or absence of VP30. Synthesis of leaderRNA is suppressed in the presence of VP30 and termination of leaderRNA is not mediated by cryptic gene end (GE) signals in the 3’-leader promoter. We further found different genomic 3’-end nucleotide requirements for transcription versus replication, suggesting that promoter recognition is different in the replication and transcription mode of the EBOV polymerase. We further provide evidence arguing against a potential role of EBOV leaderRNAs as effector molecules in innate immunity. Taken together, our findings are consistent with a model according to which leaderRNAs are abortive replicative RNAs whose synthesis is not linked to transcription initiation. Rather, replication and transcription complexes are proposed to independently initiate RNA synthesis at separate sites in the 3’-leader promoter, i.e., at the second nucleotide of the genome 3’-end and at the more internally positioned transcription start site preceding the first gene, respectively, as reported for Vesicular stomatitis virus.Transcription of non-segmented negative sense (NNS) RNA viruses follows a stop-start mechanism and is thought to be initiated at the genome’s very 3’-end. The synthesis of short abortive leader transcripts ( leaderRNAs) and predominantly terminated in the region of nt ~ 60–80 near the TSS. The functional role of abortive leaderRNA synthesis is still enigmatic; a role in interferon induction could be excluded. Our findings indirectly link leaderRNA termination to nucleoprotein (NP) availability for encapsidation of nascent replicative RNA or to NP removal from the template RNA. Our findings further argue against the model that leaderRNA synthesis is a prerequisite for each transcription initiation event at the TSS. Rather, our findings are in line with the existence of distinct replicase and transcriptase complexes of RdRp that interact differently with the 3’-leader promoter and intiate RNA synthesis independently at different sites (position 2 or 56 of the genome), mechanistically similar to another NNS virus, Vesicular stomatitis virus.The RNA polymerase (RdRp) of Ebola virus (EBOV) initiates RNA synthesis at the 3’-leader promoter of its encapsidated, non-segmented negative sense (NNS) RNA genome, either at the penultimate 3’-end position of the genome in the replicative mode or more internally (position 56) at the transcription start site (TSS) in its transcription mode. Here we identified the synthesis of abortive replicative RNAs that are specifically initiated opposite to genome nt 2 (termed  Filoviridae family in the order Mononegavirales, causes a severe febrile illness with high fatality rates . The RNA pellet was washed with 70% EtOH, air-dried and finally redissolved in RNase-free water.LeaderRNA detection by Northern Blotting was performed essentially as described [escribed , using aThe strategies for the detection and quantification of the different viral RNAs are schematically illustrated in p values), see figure legends. A statistical significance level of 0.05 was chosen for analysis by the unpaired parametric Welch’s t test or the non-parametric Mann Whitney test.Statistical analysis was performed using GraphPad Prism (version 8.1.1). For details  RNA to detect NP mRNAs. In the small RNA libraries, considerable amounts of short transcripts antisense to the genome 3’-leader sequence were identified. The majority (~70%) was 60–80 nt in length (leaderRNAs by the EBOV polymerase complex. The majority of termination sites coincide with the region of the NP hairpin (HP) structure. The NP HP is predicted to form on the genomic and/or antigenomic RNA level  nucleotide during library construction or sequencing.We performed a transcriptome (RNA-Seq) analysis of EBOV-infected HuH7 cells either enriching for small RNAs <200 nt to detect potential n length , thus ren length . A smallleaderRNAs in EBOV-infected cells by RNA-Seq  RNAs identical to antigenome nt 2–78 and 56–158 as well as genomic nt 154–1  , indicating that NP mRNAs are more abundant than leaderRNAs. As the leaderRNA primer set simultaneously detected leaderRNA and cRNA, we indirectly calculated the ratio of NP mRNA:leaderRNA  used for generating the standard curve , may anneal inefficiently or not at all to leaderRNAs <60 nt. Based on leaderRNAs <60 nt contributing ≤30% to all leaderRNAs  to estimate the molar ratio of NA cRNA; . Notably amounts . To consrd curve . The leaaderRNAs , the mRNleaderRNAs as well  used for generating the standard curve (leaderRNA reads <60 nt (see above).We also performed RNA-Seq using small RNA (<200 nt) preparations derived from cells transfected with monocistronic EBOV MGs. This revealed the presence of abortive  as well , the maj as well . This defication  also amptup Figs  vs. 4B. tup Figs  Figs. A rd curve . Again, 2 [leaderRNA opposite to C2 as well or at another site as shown for RSV  or the first two  nucleotides, as well as a variant carrying an additional 3’-nucleotide  signal, thus also employing a mechanism similar to the stop-start mechanism ubiquitously used by NNS viruses for mRNA transcription at internal genes. To address the possible presence of an alternative GE signal that could explain the production of abortive leaderRNAs in EBOV, we constructed a bicistronic MG mimicking the first two EBOV genes, which had the following features . Note that we expected synthesis of mRNA 2 and thus Firefly luciferase (FF) reporter gene activity only if an alternative GE signal compensated the inactivated NP GE signal at the NP-VP35 gene border. As positive control we included an additional derivative construct in which we replaced the NP hairpin at the 2nd cistron with the VP40 5’-UTR hairpin  that carried the mutant NP GE signal, while our positive control resulted in FF reporter activity comparable and even higher than that of the bici NP-NP MG construct , in line with enhanced leaderRNA and cRNA synthesis in the absence of VP30. As mentioned before, the measured leaderRNA levels are lower estimates due to inefficient RT-PCR amplification of leaderRNAs <60 nt.The NP hairpin structure regulates VP30-dependent transcription initiation at the TSS ,44. To e of VP30 . This in of VP30  compared of VP30 , emphasielations : in the leaderRNA amounts in the absence of VP30 for the wt (NP) MG and the two mutant MGs  MG and the two aforementioned mutant constructs (leaderRNA length profiles for wt (NP) MG and the Δ5’ spacer MG devoid of any secondary structure are very similar, we can conclude that termination of leader transcripts is not influenced by the potential to form RNA structures in the spacer between PE1 and PE2.Overall, 3’-ends of nstructs . Of notenstructs . As the leaderRNA initiation and termination sites in RNA samples derived from cells transfected with the wt (NP) MG versus the NheI NP and Δ5 ‘spacer MGs, either in the presence or absence of VP30. For all three MG variants and independent of VP30, leaderRNA 5’-ends mapped to position 2 . TrailerRNAs were less (~20-fold) abundant than leaderRNAs in RNA-Seq libraries  or Newcastle disease virus (NDV) were reported to induce the antiviral type I interferon (IFN) system by activating the virus sensor RIG-I ,47,48. Lomic RNA . The 65-sition 2 , we furtaderRNAs . A validaderRNAs , as wellA mimics . Success qRT-PCR . Also, i qRT-PCR . The absleaderRNAs (and trailerRNAs). LeaderRNAs, as cRNAs [Rhabdoviridae) and SeV (Paramyxoviridae) that, however, initiate leaderRNAs and cRNAs at the genome’s very 3’-end [In the present study, we identified and characterized, for the first time, abortive EBOV as cRNAs  are inity 3’-end ,51, not leaderRNAs antisense to the genome 3’-leader sequence before transcription initiation at the TSS representing the first GS signal [leaderRNA synthesis, initiates transcription at the first gene and becomes programmed to recognize the GS/GE signals and to cap and polyadenylate the individual mRNA transcripts [leader and NP mRNA, respectively. However, a VSV mutant strain (polR1) produced about twofold more NP mRNA than leaderRNA, suggesting the possibility that the viral polymerase complex might have the capacity to also initiate RNA synthesis directly at the TSS of the NP gene [leader promoter, i.e., at the genome 3’-end and at the more internally positioned TSS preceding the first gene, respectively [In vitro, the isolated replicase or the RNP isolated from the virus, but not the purified transcriptase, synthesized leaderRNA [leaderRNA synthesis followed by N mRNA transcription, thus contrasting the intracellularly observed direct initiation of RdRp at the TSS of the N gene. Thus, pure in vitro studies are at risk of not representing the intracellular situation. The aforementioned findings indicate that leaderRNA synthesis is not necessarily a prerequisite for each transcription initiation event. Rather, these findings support the idea that different functional forms of RdRp complexes interact differently with the 3’-leader promoter, initiating RNA synthesis at different locations. Two distinct RdRp complexes are also conceivable for EBOV, considering that its unique viral transcription factor VP30 supports transcription but is completely dispensable for replicative RNA synthesis.The widely accepted “single-entry and stop-start” transcription model proposes that NNS viruses synthesize S signal –34,52. Fnscripts ,33,35. Tnscripts ,53, wher NP gene . Indeed,ectively ,55. For eaderRNA  providedleaderRNA levels in EBOV-infected and MG-transfected systems  and ~20 to 30-fold (based on qRT-PCR standard curves) NP mRNA excess over leaderRNA during virus infection and a ~70- to 190-fold excess, respectively, of Rluc mRNA over leaderRNA in MG-transfected cells (after 48 h in both cases). Considering that the MG setup provides constant excess amounts of NP molecules, the higher mRNA to leaderRNA excess seems consistent with the abovementioned model predicting that termination of leaderRNA synthesis is suppressed at high NP levels. Yet, the abovementioned model would also imply that replication is boosted in the MG system as most leaderRNAs are elongated to cRNAs. However, the MG system yields ~100-fold higher amounts of mRNA than cRNA [leaderRNAs, far off equimolarity also in the infection model, is consistent with leaderRNAs representing aborted cRNAs, but argues against their synthesis as obligatory pre-products required to initiate mRNA transcription. The only argument to rescue the hypothesis of linked and equimolar synthesis of leaderRNA and NP mRNA for the EBOV system is to assume that leaderRNAs are degraded more rapidly than NP mRNA. This possibility cannot be entirely excluded at present, although coherent evidence in favor of this possibility is lacking, complicated by the fact that, for other NNS viruses, lower leaderRNA levels in cells versus in vitro-transcription assays and different levels in different cell types were observed [leaderRNA in EBOV-infected HuH7 versus MG-transfected HEK293 cells also included cell type-specific contributions. In HEK293 cells infected with Borna disease virus (BoDV), which however replicates in the nucleus compared with cytoplasmic EBOV replication, leaderRNAs were detected in equimolar amounts to the first mRNA [leaderRNAs degradation in this cell line that we also used for our MG system. However, not excludable is the possibility that BoDV leaderRNAs were produced in excess over the first mRNA, but were degraded more rapidly than mRNA, such that the steady-state levels of both RNA species were (fortuitously) equimolar. Altogether, the most straightforward interpretation of our findings is, analogous to VSV, the presence of distinct replicase and transcriptase complexes of EBOV RdRp that interact differently with the 3’-leader promoter and initiate RNA synthesis at distinct locations.We analyzed ems Figs  and 4. Qhan cRNA , suggestobserved ,53. Agairst mRNA . This mileaderRNA levels in the MG system are similar to, though somewhat higher than cRNA levels. Both levels increase in the absence of VP30 . The genome variant Δ1 lacking the Gt genome  in the ct genome  and retaG system . Hence, G system . This vaG system . This ca2), however, is essentially indispensable for transcription and cRNA synthesis [leaderRNA synthesis. We recently proposed a model according to which productive binding of the EBOV polymerase to the 3’-terminal region of the genome, as a prerequisite for any polymerization activity, requires that NP molecules are assembled in the correct register in the region of nt 51 to 80 and further upstream [leaderRNAs in the region of nt ~ 60–80 may then be caused by inefficient removal of NP from the template RNA in this region or inefficient NP encapsidation of nascent leaderRNAs. For viral transcription, NP molecules have to be removed from the TSS (nt 56 to 67), and this may be achieved with the help of VP30 and/or VP35 [leaderRNA termination is in line with the lack of any clear RNA sequence or structure signature for termination of leaderRNAs  relative to leaderRNAs, and why this shift is more pronounced in MG-transfected than EBOV-infected cells . These non-canonical, partly non-templated leaderRNA termination products were generally confined to the nt 70 region, where its proportion was increased in the MG system relative to EBOV-infected cells  at homooligomeric sequence stretches in the EBOV genome, such as the genomic U stretch at the GP mRNA editing site [For pneumoviruses, such as RSV, the genomic leader includes U-rich sequence motifs preceding (3’ of) the TSS. Such sequence elements were previously shown to be important for transcription in some of the NNS viruses –71. As Gynthesis ,53,72–74junction . The majsequence . Beyond  stretch . We obseed cells . Beyond ing site .leaderRNAs might be sheer by-products of replication. Yet, leader transcripts in other NNS viruses were suggested to be recognized by RIG-I, thus inducing an innate immune response [leaderRNAs were suggested to revoke RIG-I recognition by binding to the cellular La autoantigen [leaderRNAs are initiated opposite to genome position 2, it is conceivable that they anneal to non-encapsidated genome 3’-ends during RNA synthesis, generating a duplex with 1-nt 3’-overhang that is unfavorable for RIG-I activation [in vitro and purified by denaturing PAGE, was reported to elicit some RIG-I activation [leaderRNA mimics, including a 73-mer with identical 5’-ppp end and representing a major EBOV leaderRNA length variant, but did not observe any stimulatory or inhibitory effects on innate immunity , mapped their borders, identified hexamer phasing in the leader promoter as a prerequisite for their synthesis, could largely exclude RNA sequence and structure constraints and cryptic gene end signals as determinants of their termination, and were able to largely discard the possibility of a leaderRNA role in IFN induction. We showed that leaderRNA amounts are substantially lower than those of the first mRNA and provided evidence that leaderRNAs, and also trailerRNAs, are synthesized by EBOV RdRp in its replicative mode. Additionally, we observed differential genome 3’-end constraints in viral transcription versus replication, providing first evidence for differences in 3’-leader promoter recognition by the filoviral RdRp in the transcriptional versus replicative mode. Our results favor the model of distinct replication and transcription complexes that directly initiate RNA synthesis at separate sites in the 3’-leader promoter, i.e., at the genome 3’-end and at the more internally positioned TSS preceding the first gene, respectively, as demonstrated for VSV [In summary, we demonstrated, for the first time, the existence of EBOV  for VSV ,55. VP30S1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableFor methodological details, see (XLSX)Click here for additional data file.S4 TableFor more information, see (XLSX)Click here for additional data file.S1 Fig(A) Inside-out primer deletion mutagenesis. (B) Overhang/inside-out primer insertion mutagenesis. (C) Complementary primer mutagenesis for introduction of insertions or substitutions. In approaches A and B the entire plasmid is amplified with 5‘-phosphorylated primers that introduce the desired insertions/deletions, followed by circularization of PCR products and template removal by Dpn I treatment before bacterial transformation. In approach C phosphorylation of 5’-ends and ligation are carried out by bacterial enzymes after DNA transformation.(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 FigleaderRNA amounts in EBOV-infected cells based on the standard curve for the 73-meric leaderRNA mimic, and Rluc mRNA:leaderRNA amounts in MG-transfected cells based on the standard curve for the 65-meric leaderRNA mimic.(A) qRT-PCR standard curves for the individual standard RNAs. The molar amount of the respective RNA fragment introduced into the qRT-PCR reaction is given on the x-axis in logarithmic scale. The equation of the linearized calibration curve is shown as inset. (B) RT-PCR products using total RNA from EBOV-infected or MG-transfected cells and the different primer pairs according to strategy 4 were analyzed on a 2% agarose gel; the PCR products obtained after 40 PCR cycles were stained with GelRed; M, 100 bp ladder  used as size marker. (C) Example calculations of NP mRNA:(PDF)Click here for additional data file.S5 FigRNA-Seq reads representing Renilla mRNA 5’-ends in poly(A) RNA fractions derived from cells transfected with the Δ5’ spacer MG, the NheI HP MG and the wt MG, either in the presence (A to C) or absence (D to F) of VP30. Panels A and D, identical to (DOCX)Click here for additional data file.S6 FigleaderRNA lengths in RNA libraries derived from (A) EBOV-infected cells and (B-G) cells transfected with MGsComparative RNA-Seq analysis of  Click here for additional data file.S7 Figspike-in: a synthetic 5’-triphosphorylated 65-meric leaderRNA  from EBOV-infected cells and from cells transfected with the different MG variants in the presence versus absence of VP30. Regarding the MG libraries, the difference between read numbers in the +VP30 versus –VP30 samples is neither significant if the three +VP30 and the three–VP30 MG libraries are each pooled  nor if the individual MGs constructs are considered . For number and details on biological replicates, see  For the MGs , read numbers in the presence (+) as well as absence (-) of VP30 are given; (DOCX)Click here for additional data file.S8 FigtrailerRNA lengths in RNA libraries derived from (A) EBOV-infected cells and (B-G) cells transfected with MGsComparative RNA-Seq analysis of  Click here for additional data file.S9 FigleaderRNA 65- and 73-mer Click here for additional data file.S10 FigleaderRNA 65-mer (pink columns). The slight reduction in IFN-β and CXCL10 mRNA levels at 500 and 750 ng leaderRNA can be attributed to slightly reduced cellular uptake of VSV RNA at excess amounts of the leaderRNA competitor in the transfection mix . For experimental details, see qRT-PCR reactions were performed with primer pairs specific for IFN-β mRNA , CXCL10 mRNA (dark blue columns), VSV RNA (light green bars) and the EBOV (DOCX)Click here for additional data file.S11 Fig(DOCX)Click here for additional data file.


The ability of nucleic acids to form double-stranded structures is essential for all living systems on Earth. Current knowledge on functional RNA structures is focused on locally-occurring base pairs. However, crosslinking and proximity ligation experiments demonstrated that long-range RNA structures are highly abundant. Here, we present the most complete to-date catalog of conserved complementary regions (PCCRs) in human protein-coding genes. PCCRs tend to occur within introns, suppress intervening exons, and obstruct cryptic and inactive splice sites. Double-stranded structure of PCCRs is supported by decreased icSHAPE nucleotide accessibility, high abundance of RNA editing sites, and frequent occurrence of forked eCLIP peaks. Introns with PCCRs show a distinct splicing pattern in response to RNAPII slowdown suggesting that splicing is widely affected by co-transcriptional RNA folding. The enrichment of 3’-ends within PCCRs raises the intriguing hypothesis that coupling between RNA folding and splicing could mediate co-transcriptional suppression of premature pre-mRNA cleavage and polyadenylation. Functional RNA secondary structure is important for the pre-mRNA processing including splicing, cleavage and polyadenylation, and RNA editing. Here the authors present a catalog of conserved long-range RNA structures in the human transcriptome by defining pairs of conserved complementary regions (PCCR) in pre-aligned evolutionarily conserved regions. Many RNAs adopt functional secondary structures, and messenger RNAs (mRNAs) are no exception, although their main role is to encode proteins6. In eukaryotes, RNA structure affects gene expression through modulating all steps of pre-mRNA processing including splicing7, cleavage and polyadenylation8, and RNA editing9. The loss of functional RNA structure has been increasingly reported as implicated in human disease13.A double-stranded structure is a key feature of nucleic acids that enables replicating the genomic information and underlies fundamental cellular processes14. However, recent progress in high-throughput sequencing techniques enabled novel experimental strategies to determine RNA structure in vivo19. In particular, photo-inducible RNA cross-linking and proximity ligation assays revealed that long-range base pairings are highly abundant in the human transcriptome24. Currently, the applicability of these assays for large-scale profiling of RNA structure is still limited, and computational identification of long-range RNA structure remains a great challenge in RNA biology.To date, a few dozens of functional RNA structures have been characterized in the human genome Table . Many of26. Previous reports presented complex methodologies that implement simultaneous alignment and folding to detect RNA elements with divergent sequences that are nevertheless conserved at the secondary structure level30. However, a substantial fraction (3.4%) of intronic nucleotides in the human genome are highly conserved at the sequence level, which raises a compelling question of whether their function may be related to RNA structure. This motivated us to revisit this problem with the “first-align-then-fold” approach14, one which finds pairs of conserved complementary regions (PCCRs) in pre-aligned evolutionarily conserved regions. In this study, we developed a method named PrePH (PREdiction of PanHandles) to efficiently find long, nearly perfect complementary matches in a pair of input sequences, and applied it to all pairwise combinations of CIRs located at a certain distance limit from each other. Subsequently, we analyzed MSAs that had a sufficient amount of variation to detect compensatory substitutions within PCCRs.Comparative genomics provides a powerful alternative to de novo RNA folding by detecting signatures of evolutionary conservation31. We additionally provide a transcriptome-wide characterization of RNA bridges32 and exon loop-outs33, two particular mechanisms of alternative splicing regulation by long-range RNA structures.Multiple lines of evidence indicate that a large proportion of PCCRs indeed have a double-stranded structure, for example, significant decrease of icSHAPE nucleotide accessibility, high abundance of adenosine-to-inosine (A-to-I) RNA-editing sites, significant overlap with long-range RNA contacts identified by proximity ligation assays, and frequent co-occurrence of the so-called forked enhanced cross-linking and immunoprecipitation (eCLIP) peaks (see below). They also have other characteristic features such as occurrence within introns proximally to splice sites, avoidance of branch points, and obstructive effect on cryptic and inactive splice sites. At the same time, the method has a substantial false-positive rate, which, as we show, cannot be reduced without losing sensitivity to bona fide validated RNA structures Table . The cat34, which defines genomic intervals that are highly conserved between 100 vertebrates . This resulted in a set of 1,931,116 short fragments with the median length 17 nts, which will be referred to as conserved intronic regions (CIRs).To identify conserved complementary regions (CCRs), we considered nucleotide sequences of human protein-coding genes excluding all constitutive and alternative exons, repeats, noncoding genes residing in introns, and other regions with selective constraints that may not be related to base pairings Fig. A. The reL nucleotides apart from each other and belong to the same gene using PrePH, a k-mer-based method  and the median length of CCRs were −17.2 kcal/mol and 13 nts, respectively, with the frequency distribution decaying towards longer and more stable structures , indicating that less stable structures are not less functional or less interesting than the others. For the purpose of presentation here, we chose the distance limit L = 10,000, which by the order of magnitude corresponds to long-range RNA structures listed in Table ATE1 gene demonstrated that functional RNA structures may spread over much longer distances36. We, therefore, additionally explored how the number of PCCRs changes with increasing the distance limit and found that it grows approximately fourfold with increasing L up to 100,000 nts  and thus tend to occur in more constrained regions. Gene ontology (GO) terms associated with genes that have PCCRs were enriched with terms related to morphogenesis and development of the central nervous system as compared to genes of the same length, but without PCCRs  that were cut out from the MSA of 100 vertebrates34 by PCCRs (see “Methods”). The deviation from the null hypothesis that pairwise covariations in a PCCR are not due to conservation of RNA structure was estimated as a product of E values reported by R-scape for all base pairs within the PCCR. These products were adjusted using Benjamini–Hochberg correction.Compensatory mutations, that is, pairs of nucleotide substitutions that individually disrupt base pairings, but restore them when introduced in combination, play a central role in the evolution of RNA structureE value, and only 3204 of them had E value below 5%  spanning 700 nts in the first intron of PIGL gene ; s2 is the average phastCons score within CCR and 300 nts around it ; s3 is the length of a CCR relative to the length of its parent CIR . However, neither of the three metrics reached extreme values for the base pairings listed in Table A remarkable feature of standalone RNA regulatory elements is their high level of conservation, as opposed to that of their flanking sequences41. We compared the average icSHAPE reactivity within CCR with that in a control set of intervals located nearby , and the difference increased by the absolute value with the increase in the structure free energy 22 , ligation of interacting RNA followed by high-throughput sequencing (LIGR-seq)23 , and RNA in situ conformation sequencing (RIC-seq)24 . Towards this end, we considered the interacting pairs in the experimental data that were located intramolecularly within CIR not >10,000 nts apart from each other, and restricted the set of PCCRs to the underlying CIRs that overlap the experimental dataset (see “Methods”). The precision (P) and recall (R) metrics were defined as the proportion of PCCRs supported by the experimental method and the proportion of experimental interactions supported by PCCRs, respectively. In addition, we computed π, the conditional probability of predicting the interacting CCR partner correctly given that another CCRs in a pair has been predicted correctly . However, the breadth of these findings is limited by small sizes of the true-positive sets , because structural assays sparsely cover the transcriptome at cell line-specific conditions and focus on intermolecular interactions.Chemical RNA structure probing can reveal which bases are single- or double-stranded, but it cannot determine which nucleotides form base pairs29. Unlike PrePH, IRBIS follows the “first-fold-then-align” strategy to simultaneously detect conserved complementarity and sequence homology, but at stricter conditions. Overall, the predictions of the two programs had a large intersection relative to IRBIS predictions indicating that the current method generally outputs a superset of IRBIS predictions both in terms of the number of nucleotides and the number of base pairs  reflecting the fact that, unlike IRBIS, PrePH allows for short internal loops and bulges. Nevertheless, a small fraction of IRBIS predictions is missing from the list of PCCRs presented here, which relies on the conserved regions from 34. Without this limitation, however, the current approach would be impractical from the computational standpoint, and it was our intention to limit the search to conserved regions at the expense of losing some structures that are misaligned by structure-agnostic phylogenetic analysis.Finally, we explored how the set of predicted PCCRs relates to a similar list that was reported previously by IRBIS29. Here, we used the same strategy by running the pipeline on chimeric sets of sequences that were sampled randomly from different genes while controlling for nucleotide composition and length, which confound this comparison . The resulting FDR figure of 47% indicates that FDR cannot be improved without loss of sensitivity with respect to bona fide structures. On the one hand, it implies that more than half of the group I predictions could be false positives. On the other hand, this is a pessimistic estimate since the re-wiring method is known to greatly overestimate the FDR29. We, therefore, interpret 47% as an excessively conservative FDR estimate and proceed to the statistical characterization of the full PCCR set with the mindset that at least half of the predictions are true positives.Many PCCRs listed in Table 29. To elaborate on this, we used the classification shown in Fig. Previous reports indicate that long-range base pairings are positioned nonrandomly with respect to splicing signals28, we repeated the same analysis using random gene control, and the enrichment and depletion remained significant . This indicates a large regulatory potential for splicing since a large fraction of CCR that overlaps PPT (43.2%) also blocks the acceptor splice site. In addition, there were on average 20% fewer overlaps of CCR with intronic branch points42 than would be expected from random shift control, that is, CCRs tend not to overlap intronic branch points.The frequency of intronic PCCRs decays with increasing the distance to intron ends in all four energy groups partially reflecting the decrease of sequence conservation Fig. E. Howeve43. CCRs overlapping highly expressed (active) splice sites were depleted, while CCRs overlapping splice sites with low read support (inactive) were enriched , in agreement with the previous findings that cryptic splice sites tend to be suppressed by local RNA secondary structure45.To test whether PCCRs interfere with splicing signals, we identified intronic motifs with high similarity to donor and acceptor site consensus sequences (cryptic splice sites) and analyzed the expression of splice sites in a large compendium of RNA-sequencing (RNA-seq) samples from the Genotype Tissue Expression Project (GTEx)46. A comparison with the set of circRNAs from the tissue-specific circRNA database (TCSD)47 by random shift and random gene controls revealed a pattern opposite to that of linear introns, in which PCCRs were enriched outside circRNAs and depleted inside circRNAs 49. Since ADAR editing occurs in double-stranded RNA substrates51, we asked whether A-to-I editing sites are enriched among CCRs  for PCCR with higher evidence of compensatory mutations, but it did not depend significantly on the PCCR spread. These results were concordant with each other for the datasets of A-to-I editing sites from RADAR and REDIportal databases54 and can be regarded as an additional support for double-stranded structure of PCCRs.A widespread form of post-transcriptional RNA modification is the enzymatic conversion of adenosine nucleosides to inosine, a process called A-to-I editing, which is mediated by the adenosine deaminase acting on RNA (ADAR) family of enzymes8, and that competing RNA base pairings could be involved in alternative splicing and polyadenylation in the 3′-variable region55. In this section, we characterize the relationship between PCCRs, 5′-end, and 3′-end mRNA processing using the data from transcript annotation databases56 and clusters of poly(A)-seq and cap analysis of gene expression (CAGE) tags58.Recent reports indicate that RNA structure is important for the 3′-end processing of human mRNAsFirst, we estimated the number of transcripts that start or end within CCRs, including all incomplete and aberrant isoforms that are annotated in GENCODE Fig. D. Both 5Thus, we asked whether the annotated transcript ends, CAGE tags, and poly(A)-seq clusters occur more frequently in introns that contain PCCRs. However, introns with PCCRs tend to be also longer than introns without PCCRs, which may affect the above frequencies. Hence, we subdivided all annotated introns into two groups, introns with PCCR (IWP) and introns without PCCR (IWO), and selected two samples from IWP and IWO, 38,119 introns each, with matching intron lengths Fig. . Of thes59. In order to estimate the impact of human population polymorphisms on PCCRs, we analyzed single-nucleotide polymorphisms (SNPs) from the 1000 Genomes project60 and compared SNP density in CCR with that in the remaining parts of CIR. Germline SNPs were significantly underrepresented in CCR . Next, for each SNP that occurs in a PCCR, we calculated the free energy change caused by the mutation and compared it to the free energy change that would have been observed if the same mutation occurred at a different position of the same CCR , suggesting that SNPs generally tend to minimize their destabilizing impact on RNA structure.Mutations generally lower the stability of RNA secondary structure, and some of them are under evolutionary selection owing to their effects on the thermodynamic stability of pre-mRNACCR Fig. . It turn−5% for the randomized set. From this we conclude that, despite compensatory mutations within PCCR are quite rare in the human population, they are significantly enriched .Compensatory mutations may also occur in the human population, but at a substantially lower frequency. To estimate the frequency of compensatory population polymorphisms, we identified PCCRs that contain SNPs with an allele frequency of 1% and higher. Out of 64,074 bp in PCCRs that were affected by SNPs, only 12 showed sufficient support for compensatory mutations. After randomization, in which the pairs of complementary nucleotides were randomly switched, the respective average values were 64,132 and 0.06 , which is crucial for co- and post-transcriptional RNA processing, depends on RNA structural contextCRs Fig. 68,69.Figp value <10−12) indeed have a substantially higher likelihood of binding close to a CCR given that they bind the other CCR in a pair -binding protein itself, but interacts with FUS, which is capable of binding dsRNA70. Similarly, ILF3, which showed a depleted RNA binding within CCRs, nevertheless is positively associated with forked eCLIP peaks, suggesting its binding at single-stranded regions adjacent to PCCRs. This indicates, on the one hand, that RBP binding is inseparable from the surrounding RNA structure and, on the other hand, that eCLIP peaks may not correctly reflect the actual binding positions of RBPs since they are affected by intramolecular base pairings and interactions with other players.One of the features of the eCLIP protocol is that an RBP can crosslink with either RNA strand that is adjacent to the double-stranded region. Hence, we expect a higher chance of observing an eCLIP peak near the CCR given that the other CCR in the pair contains a nearby peak, a situation that will be referred to as forked eCLIP peak. To estimate the magnitude of this association, we computed the respective OR and found that the vast majority of RBPs  of the same factor using the data on RBP KDs produced by the ENCODE Consortium (see “Methods”)65. This procedure yielded a set of 296 candidate RNA bridges  upon RBFOX2 KD. However, we also find a nested PCCR with the hybridization energy −20.4 kcal/mol, which suggests that the RNA bridge extends much further than it was reported originally. As a novel example, we describe a candidate RNA bridge in the 3′ end of RALGAPA1 gene, which encodes the major subunit of the RAL-GTPase-activating protein75. In this gene, a group of nested PCCRs approximates binding sites of RBFOX2 and QKI to the penultimate exon  and via http://arkuda.skoltech.ru/~dp/shared/PrePH/.The tables listing PCCRs for GRCh37 and GRCh38 human genome assemblies are available in BED format as Supplementary Data Files 76. However, since RNA structure affects, and is itself strongly affected by RBP binding, a reliable prediction of long-range RNA structure in full-length eukaryotic transcripts does not seem feasible at the current state of the art. Instead of the detailed structure, here we consider as a proxy for RNA structure the core of highly stable and evolutionarily conserved double-stranded regions, different combinations of which may represent one or several physiologically relevant folds.It has been increasingly acknowledged that RNA structure plays a critical role in the regulation of eukaryotic gene expression at all steps from transcription to translation, but little attention has been paid to long-range RNA structure. From the thermodynamic standpoint, long-range base pairings contribute to the enthalpy of RNA folding as much as local base pairings do, and the corresponding energy figures exceed by an order of magnitude the typical folding energies of globular protein domains7, including our earlier reports29. The trends that were proposed for smaller sets also hold for the extended catalog of PCCRs presented here, namely, the preference of PCCRs to be positioned within introns proximally to splice sites27, lower inclusion rate of looped out exons77, circumscription of circRNAs78, avoidance of intronic branch points80, and generally obstructive effect on splice sites that are implicated in double-stranded structure83. We additionally observed a remarkable overlap of PCCRs with A-to-I RNA-editing sites and multiple associations with forked eCLIP peaks, which reveal traces of multimolecular complexes with patterns that are specific to double-stranded regions. These findings reconfirm the well-known mechanism of ADAR-mediated pathway84 and show the importance of RNA structure for the assembly of RNA–protein complexes, with a preference for some RBPs and avoidance for the others66. At the same time, they may also be regarded as independent support for double-stranded structure of PCCRs in addition to the evidence from experimental RNA structure profiling and compensatory substitutions.The existence of associations between long-range RNA structure and splicing has been noted in the previous studies85. Slow RNA Pol II elongation generally opens a window of opportunity for weak splice sites to be recognized, leading to higher inclusion of alternative exons, although in some cases the effect can be quite opposite87. Slow RNA Pol II elongation may also influence poly(A) site choice by enhancing the recognition of suboptimal polyadenylation signals (PASs)88. Consistent with this, we observe an increased inclusion of exons that are preceded by shorter introns. However, we also observe an additional component to this general trend, one in which structured and unstructured RNAs respond differently to RNA Pol II slowdown. This observation indicates that long-range RNA structure could coordinate the interaction between spatial and temporal components of splicing regulation. The example of long-range RNA structure in the Ate1 gene demonstrates that mechanisms similar to bacterial attenuation may also take place in eukaryotic cells90.The components of RNA-processing machinery operate in a strict coordination not only in space but also in time. The kinetic profile of RNA Pol II elongation has a significant impact on alternative splicing91. Functional RNA structures in 5′-untranslated regions are also implicated in the regulation of translation11. While these reports mostly concern local RNA structure, an intriguing finding of this work is the link between long-range RNA structure and pre-mRNA 3′-end processing, which is manifested by the enrichment of poly(A)-seq clusters in the inner part of PCCRs. It indicates that mechanisms other than sequestration or spatial convergence of PAS and cleavage sites may be involved93. In fact, human genes contain thousands of dormant intronic PASs that are suppressed, at least in part, by U1 small nuclear ribonucleoproteins in a process called telescripting94. While the exact mechanism of this suppression is not known, many intronic PASs were found to be associated with CstF64, a ubiquitous pre-mRNA 3′-processing factor95, suggesting that cleavage and polyadenylation machinery may actually operate in all introns constitutively. Could it be that RNA structure helps to suppress premature intronic polyadenylation?RNA structure is implicated in the recognition of PASs by cleavage and polyadenylation specificity factor (CPSF) and facilitates the 3′-end processing by juxtaposing PAS and cleavage sites that are otherwise too far apartNmnat gene in D. melanogaster27.Figure 24. However, these data reflect gene expression patterns that are specific to cell lines, in which they were generated, and have strong undercoverage bias in intronic regions, which are spliced out and degraded. The reduction of the intronic signal is also a common problem for RNA cross-linking and immunoprecipitation experiments96. In addition, it was meaningful to compare PCCRs only to intramolecular RNA contacts that belong to CIRs and do not exceed the distance limit of 10,000 nts. Consequently, the validation with respect to these assays was possible only for a small number of PCCRs, on which the comparison, however, showed a concordant result  contain tracks for both GRCh37 and GRCh38 assemblies.February 2009  and December 2013  assemblies of the human genome were downloaded from Genome Reference ConsortiumAte1 gene36. Next, these regions were intersected with the set of conserved RNA elements (99) using bedtools100. We additionally excluded genomic regions that overlap intervals from the UCSC Genome Browser tRNA track105, sno/miRNA track112, TFBS Conserved track, which consists of Conserved Transcription Factor Binding Sites generated using the Transfac Matrix and Factor databases113, RepeatMasker track114  and LINE (long interspersed nuclear elements)), SimpleTandemRepeats located by Tandem Repeats Finder115, and Human Nuclear mitochondrial sequences118. Genomic regions marked as “snRNA,” “miRNA,” “miscRNA,” “snoRNA,” “rRNA,” and “tRNA” in GENCODE genome annotation were also excluded. The resulting set of CIRs was used as an input for the next step, which is described below.The intronic regions were defined as the longest continuous segments within genes that do not overlap any annotated exons (including exons of other genes). The intronic regions were extended by 10 nts into the flanking exons to enable the identification of CCRs that overlap splice sites such as regions R1–R5 in the human 119.The first reference to long-range RNA structure appeared in the literature under the term “panhandle structure,” which was coined in by virologists in the 1980s to refer to a complementary base pairing between the 5′ end and 3′ end of the RNA genome of several segmented negative-stranded virusesk-mer-based technique, which is similar to previously published IRBIS method29, to identify all pairs of nearly perfect complementary regions in a given pair of sequences. At the preparatory step, PrePH pre-computes a 4k × 4k table containing helix hybridization energies for all pairs of k-mers (default k = 5) that are either Watson–Crick complementary or contain a few GT base pairs (by default at most two) using energy tables from Vienna RNA package120. The dynamic programming matrix is computed by local Smith–Waterman algorithm using a limited set of structural rules: initiating a k-nt-long helix, extending a helix by a stacking base pair, and adding to a helix a short internal loop or bulge with up to m nucleotides in each strand (default m = 2), followed by another helix. To speed up back-tracking, PrePH uses auxiliary matrices to store intermediate structures, and reports nonintersecting pairs of complementary regions passing the energy threshold . Here, two pairs of complementary regions, x1 complementary to y1 and x2 complementary to y2, are referred to as intersecting if x1 has common nucleotides with x2 and also y1 has common nucleotides with y2. That is, two pairs of complementary regions may intersect by only one, not both interacting strands. The reduced scoring scheme, optimized back-tracking, and indexing of the initial sequences by k-mers result in a great improvement of computation speed. The detailed description of PrePH is exempt to https://github.com/kalmSveta/PrePH) (codes are available at: 10.5281/zenodo.4601044).The PrePH utility uses a 121, RIsearch2122, RNAplex123, DuplexFold124, and bifold124. PrePH was run with the following parameters: k-mer length is 5 nts, maximal distance between complementary regions is 10,000 nts, the minimal length of the aligned regions is 10 nts, the energy threshold is −15 kcal/mol, and the maximal number of Wobble pairs in a k-mer is 2. The parameters for the other programs are listed below.We compared the accuracy and runtime of PrePH to those of other programs such as IntaRNA2.0outOverlap parameter was set to B, which allowed overlap for interacting subsequences for both target and query; n parameter was set to 100 to limit the maximal number of suboptimal structures; qAcc and tAcc were set to N to omit the computation of accessibility. fast-folding parameter was set to f2 to allow the structure to be computed based on the approximated model. To benchmark the time efficiency, we use a set of 1000 pairs of randomly chosen conserved intronic sequences from the human genome. The sequences were 50 to 500 nts long and contained nearly perfect sequence complementarity. All the programs were run with the energy threshold set to −15 kcal/mol. s) Table . At thats) Table .S1 is the set of base pairs predicted by PrePH, S2 is the set of base pairs predicted by the other program, and S1 ∩ S2 is the common set of base pairs . The number of base pairs that were common between PrePH and each of the other programs as a fraction of the number of base pairs predicted by PrePH alone was used as a measure of specificity .x, y] in the containing gene , where x, y, a, and b are genomic coordinates on the plus strand, was calculated as p was used instead.The relative position of a genomic interval [126 between these two gene sets was calculated with clusterProfiler R package127.GO enrichment analysis was performed by controlling for the gene length since genes with PCCRs tend to be longer than genes without PCCRs. We randomly matched each gene with PCCRs to a gene without PCCR of approximately the same length. The enrichment of GO terms128 were mapped from GRCh38 to GRCh37 human genome assembly by LiftOver129. The reactivity score of a CCR was calculated as the average reactivity of its base-paired nucleotides, for which the icSHAPE reactivity score was available. The background reactivity was calculated as the average reactivity of the same number of nucleotides chosen at random outside the CCR, but in the same CIR. Wilcoxon’s signed-rank test was applied to matched samples of reactivity score differences to test for departures from zero.The icSHAPE reactivity scores22  were mapped from GRCh38 to GRCh37 by Liftover129. The coordinates of base-paired regions from LIGR-seq data  were used in the GRCh37 human genome assembly23. The coordinates of intramolecular base-paired regions from RIC-seq data24  were kindly provided by Prof. Xue by request . For α-amanitin (Sigma) treatments, 1 and 2 μg/mL of α-amanitin was added to cells at 50–70% confluency. After 24 h of treatment, cells were harvested, total RNA was isolated using PureLink RNA Mini Kit (Thermo Fisher Scientific). Poly(A)+ mRNA was purified using Dynabeads Oligo(dT) 25 (Thermo Fisher Scientific) following the manufacturer’s instructions.A549 cell line  was maintained in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 medium containing 10% fetal bovine serum, 50 U/ml penicillin, and 0.05 mg/ml streptomycin  at 37 °C in 5% COIllumina cDNA libraries were constructed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs) following the manufacturer’s protocol with the only modification: the change of fragmentation time from 15 to 10 min. Complementary DNA libraries were sequenced using the NextSeq500  instrument; 33–41 million raw reads were obtained for each sample with a 75 bp read length. The results of RNA-seq have been deposited at Gene Expression Omnibus under the accession number GSE153303.+ RNA for the HepG2 cell line (accession numbers ENCFF670LIE and ENCFF074BOV) were downloaded in BAM format from the ENCODE Consortium website130. Short-hairpin shRNA-KD of 250 RBPs followed by RNA-seq data65 were downloaded in BAM format from ENCODE data repository131 (Table + RNA from wild-type Amr and Rpb1 C4/R749H mutant HEK293 cells treated with α-amanitin for 42 h were downloaded from the Gene Expression Omnibus (GSE63375)71. RNA-seq data of poly(A)+ RNA data from the A549 cell line treated with α-amanitin were obtained as explained below and mapped to the GRCh37 human genome assembly using STAR aligner v2.3.1z with the default settings132. The coordinates of circRNAs expressed in liver tissue and their associated SRPTM metrics (the number of circular reads per number of mapped reads per read length) were obtained from the TCSD database47. The genomic coordinates of adenine branch point nucleotides were selected from the validated set of branch points expressed in K562 cells42.RNA-seq data of poly(A)31 Table . Poly(A)133. Split read counts were filtered by the entropy content of the offset distribution, annotation status, and canonical GT/AG dinucleotides at splice sites with the default settings, and pooled between bioreplicates. The exon inclusion rate  was calculated according to the equationexc is the number of reads supporting exon exclusion. Ψ values with the denominator <10 were considered unreliable and discarded. Differential exon inclusion between a pair of conditions (shRNA-KD vs. non-specific control and α-amanitin vs. untreated control) was assessed as described previously134.RNA-seq experiments were processed by IPSA pipeline to obtain split read counts supporting splice junctions43. Splice sites with the canonical GT/AG dinucleotides were called from split read alignments and ranked by the total number of supporting split reads pooled across all 8551 samples. The top 2%  of splice sites among those supported by at least three split reads were referred to as active . In order to identify cryptic splice sites, we applied the same strategy as in ref. 135 by scanning the intron sequences for any sites that have a MaxEntScan score >800 for donor sites and >950 for acceptor sites136 and excluding splice sites that were detected in GTEx or present in the genome annotation. MaxEntScan score thresholds were chosen to have a comparable number of splice sites as in active and inactive sets above.Cryptic and actively expressed splices sites in the human transcriptome were identified using genomic alignments of RNA-seq samples from the GTEx Consortium57. Similarly, clusters of human CAGE tags expressed in the HepG2 cell line that were supported by RPKM of at least 10 and intersected with the annotated transcript starts in GENCODE database were used137.The genomic coordinates of human poly(A) sites profiled by high-throughput sequencing of 3′ ends of polyadenylated transcripts (poly(A)-seq) that were supported by 20 or more reads and intersected with the annotated transcript ends in GENCODE database were used53 and REDIportal54 databases. To compare the density of RNA editing sites in CCR and that in the adjacent conserved regions, we computed the number of adenosine residues that are RNA-editing sites within CCR and compared it to the respective figures for CIRs of the same length outside of CCR. OR was calculated for the contingency table of the number of adenosine residues that are/are not RNA-editing sites within/outside CCR.A-to-I RNA-editing sites were obtained from RADAR131  present in >0.1% of individuals) in stacked nucleotide pairs of CCR and compared it to the density of SNPs in conserved regions outside CCRs .To evaluate the enrichment of population polymorphisms in CCR, we used genotyping data from phase 3 of the 1000 Genomes Project for 2504 individuals120 and compared it to the respective energy of the structure with SNPs of the same substitution type as observed originally, but introduced at a different position. We selected PCCRs with the free energy change >2 kcal/mol by absolute value and compared the two energy sets using Wilcoxon’s signed-rank test.To assess the impact of SNPs on RNA structure stability, we calculated PCCR energy for the mutated sequence using energy parameters1T1 and A2T2 were replaced by A1T2 and A2T1 and applied the same procedure again, that is, estimated the number of base pairs, in which compensatory mutations defined by SNVs occurred in >1% of donors  located on the same chromosome with the same number of SNP donors. Then, we randomly interchanged (“re-wired”) base pairs within each group, for example, Aors Fig. . This ra99 of all its nucleotides and the average PhastCons conservation score of the same number of nucleotides in its flanking regions within the same To assess the degree of evolutionary conservation of a CCR, we computed the difference between the average PhastCons conservation score139. For each PCCR, we extracted two parts of the MSA corresponding to two CCRs using Bio.AlignIO.MafIO module from biopython library140. The organisms that had indels compared to the reference organism (hg19) in any of the two CCRs were removed. The number of orthologous sequences for each PCCR ranged from 15 to 99. The two alignment blocks were merged through an additional spacer containing ten adenine nucleotides, resulting in an MSA STOCKHOLM format with a secondary RNA structure generated by PrePH. Next, we restrict the phylogenetic tree for the original MSA139 to have only the organisms available for the given PCCR and pass the tree and MSA to R-scape v1.2.340 with the following parameters: -E 1 -s –samplewc –nofigures. The output .out files of R-scape were parsed by custom scripts to extract E values of individual base pairs. The E value of the PCCR was defined to be equal to the product of E values of the base pairs that were marked as having significant covariations by R-scape. As a result of this procedure, E values were obtained for 909,146 PCCRs; 539,264 E values for PCCRs that were <1 were adjusted using Benjamini–Hochberg correction. MSA and the phylogenetic trees were downloaded from the UCSC Genome Browser website (http://hgdownload.cse.ucsc.edu/goldenpath/hg19/multiz100way/). Structural alignments were visualized using tableGrob function from gridExtra R package.To assess the number of complementary substitutions in PCCRs and their statistical significance, we used global MSAs of 99 vertebrate genomes with human genomep values are reported throughout the manuscript unless the contrary is specified. The levels of significance 5%, 1%, and 0.1% in all figures are denoted by *, **, and ***, respectively.The data were analyzed and visualized using R statistics software version 3.4.1 and ggplot2 package. Nonparametric tests were performed by built-in R functions using normal approximation with continuity correction. MW denotes Mann–Whitney sum of ranks test. Boxplots in all figures are represented by the median, upper and lower quartiles, and upper and lower fences; outliers are not shown. Error bars in all figures and the numbers after the ± sign represent 95% confidence intervals. Two-sided Further information on research design is available in the Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Description of Additional Supplementary FilesReporting Summary


However, there is limited knowledge regarding the relationship between nanoparticles and m6A RNA modification, but evidence is beginning to emerge. Therefore, a summary of these aspects from current research on nanoparticle-induced m6A RNA modification is timely and significant. In this review, we highlight the roles of m6A RNA modification in the bioimpacts of nanoparticles and thus elaborate on the mechanisms of nanoparticle-induced m6A RNA modification. We also summarize the dynamic regulation and biofunctions of m6A RNA modification. Moreover, we emphasize recent advances in the application perspective of nanoparticle-induced m6A RNA modification in medication and toxicity of nanoparticles to provide a potential method to facilitate the design of nanoparticles by deliberately tuning m6A RNA modification.With the increasing application of nanoparticles (NPs) in medical and consumer applications, it is necessary to ensure their safety. As m For e2-NPs)  and tita [2-NPs)  have the [2-NPs) ,7. Quant [2-NPs) . Nanopar [2-NPs) . Additio6A RNA modification, which is one of the most abundant mRNA modifications.Because of the widespread use of NPs, their biological effects on organisms and the mechanisms involved have been extensively studied over the past few decades using many models, both in vitro and in vivo ,13,14,156A RNA modification as well as its relationship to NP-induced bioimpacts, and we discuss future research directions with the goal of providing a theoretical foundation based on currently available literature for further investigating the relationship between m6A RNA modification and nanoparticle bioimpacts.In this review, we provide a comprehensive overview of recent research progress on the dynamic regulation and bioimpacts of m6A RNA modification [6A RNA modification and diseases indicates its potential application in toxicity- and medication-involved NPs.There have been more than 150 various RNA modifications reported in different organisms; since the first modification was found in 1957, the most abundant modification is the mfication . As the 6A and the short half-life of most types of RNA, m6A RNA modifications have been considered to be stable and unchanged for a long time. The discovery of the first authentic m6A demethylase, namely, fat mass and obesity associated protein (FTO) [6A RNA modification is a dynamic process that is tightly regulated. As more studies have been performed, it is known that m6A RNA modification is involved in “writers”, “erasers” and “readers”, which is a dynamic process to add or remove a methyl group to adenosine and usually occurs in the RRACH sequence of adenines. To visualize the location and distribution of these proteins involved in m6A, we outlined the types of proteins involved in m6A RNA modification (Due to the shortage of proteins related to min (FTO) , resolvefication .6A RNA modification is modified by “writers”, including adenosine methyltransferases, methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms tumour 1-associated protein (WTAP), KIAA1429 and RNA-binding motif protein 15/15B (RBM15/RBM15B). Well-known methyltransferases can add a methyl group to adenosine. The METTL3-METTL14-WTAP methyltransferase complex can add m6A, and METTL3 has been previously discovered to be an S-adenosyl-L-methionine (SAM)-binding site in this multiprotein complex, which is widely expressed and highly conserved. Through knockdown of Mettl3 in blastocysts achieved by mating Mettl3+/− mice, it has been shown that METTL3 is a regulator for terminating murine naive pluripotency [6A RNA modification. METTL14 and METTL3 are close homologues, and they always work together as METTL3-14 complexes. This complex subsequently interacts with WTAP to regulate the activity and precise localization of m6A methyltransferase [6A RNA modification in mammals. During mammalian female development, loss of function of RBM15/RBM15B results in defective XIST-mediated gene silencing on the X chromosome [mnsferase . Additionsferase , requireromosome .6A mRNA demethylase to be discovered, confirming that m6A RNA modification is not unalterable but can be removed. Later investigations revealed that the dysregulation of FTO may be associated with obesity, brain malformations and growth retardation [6A RNA modification may have essential regulatory functions in these diseases. Moreover, in human cell lines, knockdown of ALKB homologue 5 (ALKBH5), which belongs to the same ALKB family of FTO, causes higher m6A levels [6A-methylated adenosine instead of indirect oxidative demethylation [FTO was the first mardation ,34,35,36A levels . Howeverhylation .6A RNA modification works in one of two ways [6A mRNA translation [6A RNA to accelerate RNA translation and decay [6A mRNA [6A “readers” have been reported, such as insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs), heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1), heterogeneous nuclear ribonucleoprotein C (HNRNPC) and heterogeneous nuclear ribonucleoprotein G (HNRNPG). As more m6A “readers” are discovered, the knowledge of m6A RNA modification will be more comprehensive.mtwo ways , as follnslation , and YTHnslation . In addinslation . YTH domnd decay  as well [6A mRNA  independ6A over the past few decades, the vital roles of m6A RNA modification have been verified in a number of essential cellular events, including the fate of mRNA, the maintenance of stemness and even tumorigenesis [6A RNA modification have also been linked to certain diseases [6A RNA modification as well as the discovery of additional m6A-related proteins are expected.Due to breakthroughs in transcriptome sequencing of migenesis . The “wrdiseases ,46,47, s6A regulates the processing and alternative splicing of precursor mRNA (pre-mRNA) in the nucleus as well as the translation, degradation and decay of mRNA in the cytosol  binding with RNA, thereby increasing the number of target exons included. These data provide strong evidence of the essential role of m6A RNA modification during mRNA splicing.For mRNA, m cytosol . Processand mRNA , thus re6A is one of the factors because the presence of m6A in exons and surrounding stop codon regions may have an impact on protein production. Mettl3 ablation significantly enhances translation efficiency in mouse embryonic stem cells (mESCs) and embryoid bodies (EBs) [6A may play a translational regulatory role. Additionally, another m6A “reader”, YTHDF1, has been reported to increase translational output by interacting with initiation factors and ribosomes [6A in RNA translation. Identifying more m6A “readers” will aid in better understanding the regulatory role of m6A in the translation process [As proteins are executors of biological functions, their synthesis and translation processes are vital to life and are regulated by various factors , and m6Aes (EBs) , indicatibosomes , providi process .Mettl3/14 modestly increases the stability of target mRNAs in mESCs [6A RNA modification. RNA degradation has been verified to be enhanced by YTHDF2 through the transportation of mRNA for degradation [6A RNA modification in mRNA degradation. The same conclusions have been reached in Hnrnpc [Alkbh5 [The flexible and rapid degradation of mRNA is critical for the response of living organisms to changeable environments. Knockdown of in mESCs , demonstradation , revealin Hnrnpc  and Alkb [Alkbh5  knockdow6A RNA is important in both physiological and pathological circumstances, including cellular stress, differentiation, development, immune response and tumorigenesis  production through the Keap1–Nrf2 antioxidant pathway in mouse renal tubular epithelial cells (mRTECs) [6A demethylation at the 3′UTR of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), an important modulator of mitochondrial metabolism, which increases the stability and enhances translation of PGC1α mRNA, thereby causing increased production of ROS [Keap1–Nrf2 pathway, as its knockdown induces the overexpression of NRF2 and increases the production of antioxidants in human bronchial epithelium (Beas-2B) cells [Ythdf2 causes heat shock stress by increasing HSP90, HSP60 and HSPB1 mRNA expression [6A “writer”, METTL16 has been confirmed to be recruited to DNA damage sites after 20–30 min of UV exposure and to methylate RNAs transported to DNA damage sites, which may be related to the nucleotide excision repair (NER) mechanism [HSP70 and other genes related to ER stress through the NF-κB pathway in osteoblasts [6A level of Atg5 and Atg7, which was recognized by YTHDF2 [Cellular stress includes oxidative stress, hypoxia, metabolic stress, heat shock, DNA damage ER stress and autophagy . Overexp(mRTECs) . A recenn of ROS . YTHDF1 B) cells . In hepaB) cells . Depletipression ,59. As aechanism . Additioeoblasts . Moreovey YTHDF2 . FTO alsy YTHDF2 .6A RNA modification plays an important role in haematopoietic and progenitor stem cell (HSPC) differentiation as well as in haematopoietic development in vertebrates. Depletion of Mettl3 prevents double-stranded RNAs (dsRNAs) from being synthesized and activates MDA5 and RIG-I [6A levels, METTL3 stimulates c-MYC, PTEN and BCL2 mRNA translation, which inhibits the proliferation of HSPCs [Ccnd1, c-Myc and Axin2, YTHDF2 suppresses this signalling pathway, thereby inhibiting the proliferation and differentiation of HSCs [Notch1a [6A RNA modification governs haematopoietic stem cell-directed differentiation.mnd RIG-I , ultimatof HSPCs . By degr of HSCs . In HSPC[Notch1a , providi6A RNA modification regulates the development of the central nervous and reproductive systems. First, through the JAK/STAT and PI3K/AKT signalling pathways, YTHDF2 [m, YTHDF2  and FTO , YTHDF2  increase, YTHDF2 . YTHDF1 , YTHDF2 .6A levels. YTHDC1 [CYR61, thus resulting in spontaneous abortion [In addition, METTL3  and ALKB. YTHDC1  and YTHD. YTHDC1  promote . YTHDC1 . Knockdoabortion .6A protects RNA from being recognized by TLR3 [6A modification plays an indispensable role in the immune response by influencing related factors, such as methylating a specific mRNA. A recent study has found an m6A score is useful to measure immune features of tumorigenesis in clinicopathology, such as inflammatory stages [Socs mRNA and thus regulate the TLR4/NF-κB pathway, which is related to inflammation [Mettl14 in dendritic cells (DCs) severely impairs B cell development, which affects the processes of IL-7-induced pro-B cell proliferation and causes abnormalities in gene expression critical for B cell development [In the immune response, research has shown that m by TLR3  and TLR7 by TLR3  as antigy stages . METTL3 ammation . By inacelopment , confirmelopment , which celopment .6A RNA modification and cancer [6A regulates the proliferation, invasion and migration of tumours as well as the maintenance of the stemness of cancer stem cells by targeting several oncogenes [6A levels of ADAM19 and increasing its expression in glioblastoma stem cells (GSCs), downregulation of METTL3/14 stimulates the proliferation and self-renewal of GSCs, ultimately resulting in GBM [6A RNA modification of oncogenes and tumour suppressor genes plays an important role in many stages of tumorigenesis, such as proliferation and differentiation of cancer cells as well as self-renewal and cellular death of cancer stem cells [6A levels and induce translation, and SP1 subsequently activates c-MYC, a notorious oncogene, resulting in the formation of AML. Moreover, another study has found that a decreased copy number of Alkbh5 is common in AML and has been linked to TP53 mutations, which predict a poor prognosis for AML patients [Recent studies have shown a strong correlation of md cancer , includincogenes . By decrg in GBM . During em cells ,89,90. Fem cells . CEBPZ rpatients .6A modification in diabetes has been confirmed. In 2019, β-cell-specific Mettl14 knockout mice were used to mimic the features of human type 2 diabetes (T2D). Some transcripts involved in the regulation of cell cycle progression and the secretion of insulin are hypomethylated and were detected by m6A sequencing. This study highlights the importance of RNA methylation in regulating metabolism of glucose and provides a potential therapeutic targeting of m6A modulators in type 2 diabetes [Lately, the significant role of mdiabetes . In HepGdiabetes .6A bases is difficult because the base pairing properties remain unchanged and cannot be distinguished from regular bases by reverse transcription. Methylated RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq), also known as m6A RNA immunoprecipitation sequencing (m6A-seq), has been widely used to detect m6A at a resolution of 110–200 nt since it was developed in 2012 [The direct detection of m in 2012 .6A sequencing (PA-m6A-seq) [6A individual nucleotide resolution cross-linking and immunoprecipitation sequencing (miCLIP-seq) [6A bases by m6A-sensitive RNA endoribonuclease-facilitated sequencing (m6A-REF-seq) [In the past decade, several approaches with the resolution of a single base, including photo crosslinking-assisted mm6A-seq) , m6A indLIP-seq) , site-spLIP-seq) , have beREF-seq)  with hig6A in RNA [6A dot blot analysis and high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Furthermore, directly detecting the effect of changing any site and in any organism is promising with the advancement of genome engineering.Moreover, several methods have been developed to detect the total amount of mA in RNA , includiNPs have been widely used in production and life due to their distinct physicochemical features, which is accompanied with concerns over their bioimpacts and mechanisms involved. Therefore, there is a need to study and comprehend the impacts in terms of the cellular and molecular mechanisms of the bioimpacts of NPs. The mechanisms involved are partly clarified, including oxidative stress, cytotoxicity, neurotoxicity and genotoxicity , which d6A RNA modification is less well understood, but evidence is beginning to emerge in the past two years. Therefore, we will introduce these studies and summarize the role of m6A RNA modification in the bioimpacts induced by NPs.However, there is more evidence suggesting the possibility of NP-induced genetic toxicity. DNA methylation, histone modifications and RNA modifications as well as many other epigenetic mechanisms have been proposed and widely studied in recent years. As more research has been performed and more knowledge is available, the roles of epigenetics in the biological function of NPs are gaining increasing attention. Most notably, epigenetic dysregulation has been linked to clinical disorders, including cancer and neurological diseases. Thus, it is possible that NP-mediated epigenetic modulations can also contribute to pathogenesis to a certain level. One of the characteristics of cancer is global DNA hypomethylation, which can be passed down from generation to generation . For ins6A RNA modification. Surprisingly, the global m6A level was increased after BPQD exposure in lung cells, and after the protein expression of m6A-related proteins was examined, we further clarified the significant role of ALKBH5 in this event; the expression of “writers” and other “erasers” did not change, but ALKBH5 decreased with BPQD exposure [6A levels and the aberrant expression of genes related to ferroptosis involving ER stress, iron homeostasis and iron homeostasis, which has also been verified by evidence of iron overload, lipid peroxidation, glutathione peroxidase 4 (GPX4) downregulation and glutathione (GSH) depletion in vitro. Moreover, the mechanism of the downregulation of antiferroptosis-related genes is that YTHDF2, a m6A “reader”, recognizes and promotes the decay of mitochondrial homeostasis-containing and antilipid peroxidation genes modulated by m6A, which has been verified by m6A RNA immunoprecipitation (RIP)-qPCR in lung cells. Our study reported the roles of m6A RNA modification in BPQD-induced ferroptosis and for the first time revealed the mechanism involved, thereby providing a promising biomarker and a drug target for BPQD-induced ferroptosis  . Interesexposure . Transcrroptosis .6A RNA modification [6A level of pri-miRNA-126 and its binding with DGCR8. Additionally, another study has reported that CBNP exposure during pregnancy influences maternal behaviours and partially causes abnormal neurobehaviors and the development of the reproductive system in offspring, all of which are linked to m6A RNA modification [6A level. Further studies need to be performed to provide more information about this intriguing phenomenon.Carbon black nanoparticles (CBNPs) have been reported to have a close correlation with many diseases, some of which are involved in reproductive systems and abnormal behaviours. A recent study has shown the relationship between CBNP-induced pulmonary fibrosis and mfication . The profication . This stArabidopsis thaliana [6A RNA modification and the bioimpacts of MECNTs  in macrophages infected by vesicular stomatitis virus (VSVs) polarize macrophages and stimulate immunological responses related to METTL14 [Moreover, a previous study on the bioimpacts of high dots of multiwalled carbon nanotubes (MWCNTs) on thaliana  has suggf MECNTs . In deta METTL14 . Further2-NPs and m6A RNA modification was reported [2-NPs by inhalation during pregnancy in female mice and to reveal an epitranscriptomic mechanism that contributes to this phenomenon. The results showed that in offspring, the enzymatic function of mPHGPx is considerably decreased, and the m6A levels are increased. These findings attribute the abnormally high m6A level of mPHGPx to reduced antioxidant capacity and the resulting mitochondrial and cardiac impairments that last into adulthood after gestational nTiO2 exposure through inhalation.In 2021, research related to TiOreported . In this6A RNA modification, there is some research about electrochemical immunosensors for m6A detection based on nanotechnology. Silver nanoparticles and SiO2 nanospheres are used in the electrochemical immunosensor, providing a promising detection platform for m6A [6-methyladenosine (m6A) mediated by METTL3 upregulated long noncoding RNA LINC00958, which subsequently promoted HCC progression through the miR-3619-5p/HDGF axis [6A modification functions as a robust tool to modify RNA used in cancer therapy, as the veil of RNA m6A modification in the bioimpacts of NPs is lifted completely.In addition to those studies related to bioimpacts of NPs induced by m for m6A . MoreoveDGF axis . Therefo6A modification. Epigenetic modifications can be utilized to study nanoparticle toxicity and, more crucially, to predict their toxicity in preclinical research, according to recent evidence from both experimental and epidemiological studies. A link between nanotoxicity and epigenetic changes following exposure to specific nanoparticles has been demonstrated in many experimental studies in vivo and in vitro [6A modifications may function as biomarkers not only to detect and predict the toxicity of NPs but also to evaluate the process of diseases triggered by NPs as the role of RNA m6A modification in the bioimpacts of NPs is further understood.Despite a large number of findings connected to the functional roles of epigenetics in the bioimpacts of NPs , many viin vitro ,114,115.in vitro . Therefo6A modification is lacking. Moreover, most studies focus on the bioeffects of one type of NP on RNA m6A modification, whereas clarifying the process involved in RNA m6A modification and NPs in greater depth could provide broader insights into the understanding of nanomaterials. The precise impacts of the physicochemical features of NPs, including their size and surface charge, in modulating RNA m6A modification are also unknown to some extent.The roles of DNA methylation and histone acetylation in the biological functions induced by NPs have been reported in great detail ,25,117. 6A modification are another gap that must be filled. Studies involving longer research periods are needed to illustrate the consequences of RNA m6A modification correlated with NPs and to determine whether these biological effects can be passed from generation to generation [Danio rerio, can serve as efficient in vivo models for determining the potential epigenetic mechanisms in the toxicity triggered by NPs and the genetic effects of the bioimpacts of NPs in this field [The genetic effects of NP-induced bioimpacts related to RNA mneration . Severalis field ,120. Zeb6A modification has been linked to some pathological diseases, such as obesity, as well as several mental disorders, such as neurodegenerative diseases, and even cancer [6A modification can similarly lead to some diseases to a certain extent. Therefore, addressing the roles of RNA m6A modification in the bioimpacts of NPs and the involved mechanisms as well as using various practical in vivo and in vitro models are important challenges for the future.Finally, dysregulation of RNA mn cancer ,42. Recen cancer . Thus, i6A-methylation-based epitranscriptomics using the nanoparticle as an “epigenetic drug” for cancer therapy.Last but not least, with the remarkable development of RNA nanotechnology, RNA nanoparticles can be used as carriers in drug delivery systems . In orde


The SARS-CoV-2 coronavirus is the focus of attention as it has caused more than three million human deaths globally. This and other coronaviruses, such as MERS-CoV, have been suggested to be related to coronaviruses that are hosted in bats. This work shows, through a bibliographic review, the frequency of detection of coronavirus in bats species of the Americas. The presence of coronavirus in bats has been examined in 25 investigations in 11 countries of the Americas between 2007 and 2020. Coronaviruses have been explored in 9371 individuals from 160 species of bats, and 187 coronavirus sequences have been deposited in GenBank distributed in 43 species of bats. While 91% of the coronaviruses sequences identified infect a single species of bat, the remainder show a change of host, dominating the intragenera change. So far, only Mex-CoV-6 is related to MERS-CoV, a coronavirus pathogenic for humans, so further coronavirus research effort in yet unexplored bat species is warranted. Bats have been involved in at least three instances of coronavirus spillover to humans, most recently SARS-CoV-2, the cause of the coronavirus disease COVID-19 and the current pandemic. This virus emerged in late December 2019 in the province of Wuhan, China, and has infected more than 178 million people worldwide, resulting in more than three million deaths ,4. Ten yPaguma larvata), raccoon dogs (Nyctereutes procyonoides), and camels (Camelus dromedarius), to infect humans [The probable source of the coronavirus emergence has been proposed to be related to other genetically similar viruses detected in bats. However, scientific evidence indicates that the SARS-CoV, MERS-CoV, and SARS-COV-2 coronaviruses could probably jump to humans through other intermediate hosts, such as palm civets ; Betacoronavirus (β-CoV), containing coronaviruses related to MERS-CoV, SARS-CoV, and SARS-CoV-2; Gammacoronavirus (γ-CoV); and Deltacoronavirus (δ-CoV). The first two are found in mammals, including bats, while the latter two have been recorded mainly in wild and domestic birds [Coronaviruses are single-stranded positive-sense RNA viruses with genomes of 16 to 31 kb, belonging to the order of Nidovirales and to the Coronaviridae family, which is subdivided into the families ,13,14. Tic birds ,13,14.Bats are the group of mammals that harbor the largest number of coronavirus species . As of 2In this work, we review the available studies on natural infection  and experimental infection  of coronavirus in bats in the Americas up to 2020. We compiled a list of coronavirus host species and the number of positive individuals and showed the coronavirus-host patterns that predominate in the Americas. The results of this study contribute to forming a baseline to identify the information gaps in matters of zoonotic disease management and pandemic control.http://www.mgc.Ac.cn/DBatVir/; accessed on 19 September 2020) and mammals compiled by [http://doi.org/10.5281/zenodo.596810; accessed on 19 September 2020). The search included the following words in Spanish and English: Alphacoronavirus, America, Betacoronavirus, Coronaviridae, coronavirus, CoV, host, MERS, bats, pandemic, chiroptera, reservoir, SARS, viroma, and zoonosis. Only works that addressed natural and experimental infection were considered for this study. Studies that did not present the taxonomy of the species and the number of individuals examined were not considered for the analysis.A review of the existing literature on bat and coronavirus studies in the Americas was conducted through a systematic search in Google Scholar, Web of Science, PubMed, NCBI , and PMC . We also searched the world databases of viruses associated with bats , and bibliographic reference. All sequence accession numbers were corroborated in the GenBank genetic sequence database, which helped fill in information not available in the articles. A map of the American continent was prepared in the ArcGis Desktop  version A presence/absence matrix of the viral sequences and the coronavirus host bat species was constructed. From this matrix, a bipartite network made up of nodes and edges or links was generated, which allows graphically observing the interaction between two groups , in thisThe bibliographic search yielded 25 articles published between 2007 and 2020 in 11 countries of the Americas (76% in the decade from 2011 to 2020) . CountriPhyllostomidae family, 27.90% to Vespertilionidae, 18.60% to Molossidae, and 6.97% to Mormoopidae. Experimental infection had been carried out in nine species of bats with the Alphacoronavirus hCoV-NL63 and the Betacoronavirus MERS-CoV and hCoV-EMC, coronaviruses that mainly affect humans  and Myotis lucifugus . Of the 160 species that have been examined over time, in 70 (43.8%) only 1 to 5 individuals were examined, while in 21 species (13.1%) more than 100 individuals were evaluated , Molossidae (25.1%), and Mormoopidae (5.4%)  . Bats fre (5.4%) . The PhyMyotis lucifugus (n = 42), Tadarida brasiliensis (n = 39), and Molossus molossus (n = 38), while in 10 bat species only one coronavirus sequence was recorded. The network shows a low connection (0.02) with a moderate asymmetry (0.6), and a low nesting of the matrix (2.3). Further, robustness is moderate (0.5), vulnerability low, and 16.5 species of bats per coronavirus sequence. Specialization is zero (0.0), indicating a high degree of generalization (0.16).To date, the 43 coronavirus-positive bat species harbored an average of 7.5 coronaviruses sequences, with the highest values recorded in Miniopterus pusillus in Hong Kong [Nyctinomops laticaudatus from Mexico, which is phylogenetically related to MERS-CoV [Since the SARS pandemic in China in 2002, and after the first report of coronavirus in bats ong Kong , the numong Kong ,52. Sincong Kong ,20,27). MERS-CoV , increasAdvances in molecular techniques have made it possible not only to identify the coronavirus host species but also to determine the mechanisms by which bats allow or restrict the replication of these viruses . For exaPhyllostomidae family and 27.90% to Vespertilionidae. This may be related to the fact that they are the families with the highest number of species in the Chiroptera order and are best studied in ecological studies. For example, of the 160 species considered so far, more than 70% are species of the Phyllostomidae and Vespertilionidae family and only the Artibeus, Carollia, and Sturnira genera represent more than 43% of all individuals, which, contains abundant species in the Neotropics both in conserved habitats and urban areas, in addition to being frequent in captures with fog nets, a method reported with greater frequency in the studies of this research [The 160 species of bats in which coronaviruses have been tested represent more than 45% of the more than 350 species that are distributed in the Americas and the Caribbean . In generesearch .Regarding the type of samples analyzed, the results show that coronaviruses have a positive tropism toward the intestinal tract, as they are detected in a higher percentage in fecal samples (43.1%) and intestines (35.1%) compared to liver tissue samples, lung, spleen, and kidney (20.3%), saliva (1.2%) or blood (0.3%). Intestinal tropism has been reported in previous studies in other geographic areas ,17,57. TLichonycteris obscura, Lonchorhina aurita, Cynomops planirostris, and Myotis riparius the number of individuals examined was very low , a coronavirus was identified. In contrast, in other species such as Saccopteryx bilineata, Sturnira hondurensis, and Phyllostomus hastatus for which more than 100 individuals were examined, none were positive for coronavirus. These varying rates indicate that some species of bats may be more susceptible to coronavirus infection than others. According to recent estimates, future studies should consider and include fecal samples from 154 to 400 individuals per species to maximize the possibility of detecting all the coronaviruses that a species harbors [Leptonycteris nivalis and Myotis lucifugus (classified as endangered according to the IUCN) [In relation to the specimens sampled, of the 160 evaluated species that this study compiled, in 43.8% of them the number of individuals examined was less than five, which could have limited the probability of detecting at least one coronavirus. However, even though in he IUCN) .The factors that could favor the natural infection of coronavirus in bats have been little addressed, but it has been suggested that juvenile individuals or lactating females captured in the dry season of the year are more likely to be positive for coronavirus ,35,59. IThe results obtained from the bipartite network showed moderate asymmetry (0.6) and low connectivity (0.02). This may be because the number of coronavirus sequences nodes was four times greater than the number of nodes of the host bat species, and therefore nesting was also low (2.3) since specialist coronaviruses those that have been detected in a single species of bat—have little interaction with generalist bats species of bats which have more than one different sequence of coronavirus. The bipartite network of this review showed that bats that have been studied in the Americas can host up to 7.5 coronaviruses sequences on average. Furthermore, considering that 75% of the living genera of bats are found in the Americas , greaterNyctinomops laticaudatus in Mexico, was closely related to MERS-CoV, a virus that is pathogenic for humans. The foregoing places Mexico as a potential focus of emerging diseases, although since only one of the 187 viral sequences described so far presents this relationship, a low risk of spillover from coronavirus pathogens for humans is suggested in the Americas [Until 2020, only one of the viral sequences, Mex-CoV-6, which infects Americas ,30,31. HAmericas ,46,47,48Artibeus jamaicensis, which presented histopathological changes in lung cells [In prior studies, none of the individuals naturally infected by coronavirus presented signs of disease. Most of the individuals infected in laboratory experiments also did not show obvious clinical manifestations of the disease, except for ng cells . Ref. [3ng cells  showed, ng cells . Researcng cells ,61.Coronavirus research in bats around the world will continue to be important to characterize and understand the circulation of coronaviruses to identify potential sources of spread or spillover into the human population . Recent


Caenorhabditiselegans UNC-62 homothorax/Meis/TALE homeodomain protein functions sequentially to regulate general identity of the AWC olfactory neuron pair and the stochastic choice of asymmetric AWC subtypes during embryogenesis. Here we analyze the expression pattern of unc-62 during AWC development using an integrated unc-62::GFP fosmid rescuing transgene. UNC-62::GFP was not detected in AWC neurons in early or late embryos. These results are consistent with previous single-cell RNA sequencing data and also suggest an undetectable level of unc-62 expression and/or low stability of UNC-62 protein in AWC neurons during embryogenesis.The  An integrated unc-62::GFP fosmid transgene, in which all UNC-62 protein isoforms are tagged with GFP at the C- terminus  (unc-62(lf) mutant phenotypes of AWC general identity, determined by odr-1p::DsRed expression, and AWC asymmetry, determined by str-2p::GFP expression . These results suggest that UNC-62::GFP fusion protein expressed from the unc-62::GFP fosmid transgene is functional for AWC development. It has been shown that this integrated unc-62::GFP fosmid transgene is expressed in sensory neurons, touch neurons, interneurons, ventral nerve cord motor neurons, and head motor neurons, but it is not expressed in AWC in late-stage larvae or young-stage adult worms using the multicolor transgene NeuroPAL .The UNC-62 homeodomain protein regulates AWC general identity and subsequently plays a cell autonomous role, determined by mosaic analysis, in AWC asymmetry during embryogenesis  , rescuedet al., 1983; Chuang and Bargmann, 2005). To determine whether unc-62 is expressed in AWC neurons at the embryonic stages of AWC development, the expression pattern of the integrated unc-62::GFP fosmid transgene  . Together, these results suggest that unc-62 may be expressed at an undetectable level and/or UNC-62 protein may have a very short half-life in embryonic AWC neurons.The AWC neurons are born near the end of gastrulation; AWC asymmetry is established around the 1.5-fold and 3-fold embryonic stage  , was ana embryos -D. Consi