The plant epitranscriptomic field is rapidly growing, emphasizing the role of modifications in low-abundant RNAs. We summarize current knowledge on modified ribonucleotides in RNA polymerase II transcripts and show new perspectives. N6-methyladenosine (m6A) and pseudouridine (Ψ), were initially identified in highly abundant structural RNAs, such as rRNAs, tRNAs, and small nuclear RNAs (snRNAs). Current methods provide the opportunity to identify new types of modifications and to precisely localize them not only in highly expressed RNAs but also in mRNA and small RNA molecules. The presence of modified nucleotides in protein-coding transcripts can affect their stability, localization, and further steps of pre-mRNA maturation. Finally, it may affect the quality and quantity of protein synthesis. In plants, the epitranscriptomic field is still narrow, but the number of reports is growing rapidly. This review presents highlights and perspectives of plant epitranscriptomic modifications, focusing on various aspects of modifications of RNA polymerase II transcripts and their influence on RNA fate.Regulation of gene expression is a complicated process based on the coordination of many different pathways, including epigenetic control of chromatin state, transcription, RNA processing, export of mature transcripts to the cytoplasm, and their translation into proteins. In recent years, with the development of high-throughput sequencing techniques, the importance of RNA modifications in gene expression has added another layer to this regulatory landscape. To date, >150 different types of RNA modifications have been found. Most RNA modifications, such as  Chemical modifications of RNA have been known for over 50 years, with pseudouridine as the first described, also called the ‘fifth nucleotide’ . SeveralN6-methyladenosine (m6A) ( N1-methyladenosine (m1A) (5C)  (5C) (O-methylated nucleosides (Nms)  and may play more important roles in the RNA life cycle. In most cases, the biological meaning of the modifications described still needs to be uncovered.It is clear, however, that some of the modifications mentioned above are much more abundant than others   , ending with an influence on RNA transport and decay. Epitranscriptomic changes affect the structure of non-coding RNAs such as miRNA precursors and potentially may determine the function of deriving small RNAs , were shown to affect R-loop formation in rice (6A (an RNA modification), suggesting a possible interplay between marks on different nucleic acids  and DNA-All RNAPII transcripts are characterized by a specific structure formed at their 5ʹ ends, called the 5ʹ-cap. The eukaryotic mRNA 5ʹ-end cap was first described in the 1970s . It is s7G (N7-methylguanosine) eukaryotic caps are synthesized by three enzymes  that subsequently modify the 5ʹ end of RNA molecules synthesized by RNAPII. All those steps result in the creation of a structure known as cap 0, which can be further modified. Methylation of the 2ʹ-O-ribose of the nucleotide adjacent to cap 0 results in the creation of cap 1, and the same reaction carried out on both the first and second nucleotides next to the cap creates cap 2 , as well as in some small nucleolar RNAs (snoRNAs) and mRNAs , the FAD cap, and the UDP-Glc/UDP-GlcNAc cap (glycosylation cofactor UDP-glucose/UDP-N-acetyloglucosamine caps)  were elevated in these mutants in comparison with wild-type plants. This activation of the immune response to dxo1 loss-of-function Arabidopsis mutants suggests an autoimmunity phenotype , as a crosstalk between decapping activator (DCP5) and UTP:RNA URIDYLTRANSFERASE 1  has been described in Arabidopsis  have been reported in various studies on animals (6A writer complexes) showed that splicing and alternative splicing are not significantly affected by the reduced RNA methylation level in plants  selection  with compromised m6A deposition mechanisms exhibit the disrupted m6A pattern. This observation was associated with altered patterns of mRNA cleavage and polyadenylation  signal sequence AAUAAA, and that m6A inhibits the polyadenylation complex by selecting downstream proximal cleavage and polyadenylation sites in Arabidopsis  and CPSF30-S (28 kDa shorter version)  motifs  that have m6A modification is connected with poly(A) tail structure. Uridylation has been found to repair deadenylated mRNAs in plants, thus preventing degradation (It seems that not only m5C) in animal mRNAs (5C methyltransferase enzyme NSUN2 (NOP/SUN protein 2) and the ALYFEF protein  protein, which functions as an mRNA transport adaptor during export, can specifically recognize and bind 5ʹ-methylcytosine modified nucleotides  and HSC70.1 (heat shock cognate protein 70.1), which were confirmed to be successfully transported in wild-type plants  in either direct or indirect ways  transcripts resulted in their increased stability(6A reader) was shown to affect trichome morphology in Arabidopsis by affecting mRNA stability in the cytoplasm  is another RNA modification correlated with translation. The modification was shown to be located near the translation start site of transcripts and their first splice site, as well as in highly structured 5ʹUTRs  regions, closely after the start codon, suggesting its potential role in translation, although this needs further investigation  tail  are simultaneously 3ʹ truncated and 3ʹ uridylated (O-methylation protects plant miRNAs from 3ʹ–5ʹ truncation and degradation (hen1 mutant (in which miRNA cannot be methylated), most of the miRNAs were uridylated by the HESO1 (HEN1 SUPPRESSOR 1) protein and URT1 nucleotidyl transferase , non-coding RNAs transcribed by RNAPII and encoded by 6A modification is also present in pri-miRNAs, and it affects their structure and processing into mature miRNAs (A. thaliana mutants with lower levels of active MTA. This observation leads to the suggestion that MTA is involved in the regulation of biogenesis of some miRNAs at the initial stages of this multi-step process (Recently, it was shown that m6A in plant miRNA biogenesis, it was found that in soybean, 21 nt long miR1510 is only partially 2ʹ-O-methylated at the 3ʹ terminus (Apart from the role of mterminus .6A has been shown to affect the cleavage of pre-miRNA by Dicer enzyme, which leads to the promotion of miRNA maturation (There are several differences between plant and animal miRNA biogenesis pathways including: gene organization, processing steps, proteins involved in the pathway, and the way in which mature miRNA functions. The effects of RNA modifications on animal miRNA biogenesis and function have been widely described. For example, mO-methylation is important for miRNA stability (6A, present in some miRNA precursors, is required for their proper structure and processing (In recent years, the number of reports pointing to the key role of RNA modifications has rapidly grown. This trend is particularly noticeable for mRNAs. Modified nucleotides provide another stage that could impact the final product of genetic information—the level of functional protein. By inducing or repressing transcription and affecting the stability or maturation of the transcript, RNA modifications can shape the proteome and, as a consequence, change the fate of the cell . This isocessing . It is a


The reactivity of RNA 2′-OH groups with acylating agents has recently been investigated for high-yield conjugation of RNA strands. To date, only achiral molecules have been studied for this reaction, despite the complex chiral structure of RNA. Here we prepare a set of chiral acylimidazoles and study their stereoselectivity in RNA reactions. Reactions performed with unfolded and folded RNAs reveal that positional selectivity and reactivity vary widely with local RNA macro-chirality. We further document remarkable effects of chirality on reagent reactivity, identifying an asymmetric reagent with 1000-fold greater reactivity than prior achiral reagents. In addition, we identify a chiral compound with higher RNA structural selectivity than any previously reported RNA-mapping species. Further, azide-containing homologs of a chiral dimethylalanine reagent were synthesized and applied to local RNA labeling, displaying 92% yield and 16 : 1 diastereoselectivity. The results establish that reagent stereochemistry and chiral RNA structure are critical elements of small molecule-RNA reactions, and demonstrate new chemical strategies for selective RNA modification and probing. The 2′-OH groups of RNA are shown to react differentially with opposite enantiomers of chiral acylating agents, with applications in conjugation and mapping of the biomolecule. Selective control of reactions at specific sites along the RNA enables functionalization of both small and large RNAs for future biotechnological and biomedical applications.4,5 For high-yield preparative reactions, acyl small-molecule reagents -ribose sugars that comprise the phosphodiester backbone. Chirality in nucleic acids is widely discussed and has been leveraged in numerous studies for selective recognition of small molecules with DNA;17–21 for example, chiral metal intercalator complexes have long been studied for their selectivity in associating with right-handed B-DNA helices versus left-handed structures.17 However, for RNA much less attention has been paid to chiral recognition by small molecules.22 A chief example has been the testing of RNA on solid support as a stationary phase for chiral chromatography.23,24 Chiral fluorinated diamines have also been tested as NMR probes of RNA.25 RNA aptamers selected for binding a naturally occurring small molecule have in a few cases also been shown to be selective for that enantiomer over the opposite one.26,27 While the number of small molecule ligands known to bind RNA is growing rapidly, the R-BIND database28 lists no enantiomer pairs among 131 ligands. However, in a recent example of chiral recognition by synthetic compounds, ligands for a bacterial RNA riboswitch have been shown to be stereoselective in binding.29RNA can fold into complex three-dimensional shapes, and is inherently chiral due to the  with an alanine derivative.34,35 As solid-phase RNA/DNA synthesis was not yet available, no defined RNA sequences or folded contexts were tested. Thus it remained unknown whether preparatively useful yields could be achieved with chiral compounds, whether higher levels of selectivity are possible, and whether folded RNA structure might affect stereoselectivity. Given this lack of information, multiple questions arise: to what degree can reagent stereochemistry influence reactions at 2′-OH? Second, does the addition of steric bulk that confers asymmetry near the reactive carbonyl hinder RNA reaction? Finally, and importantly, how does RNA folded structure locally influence diastereoselectivity?Although large oligonucleotide conjugates have been investigated more recently,To begin to address these questions, we report here the study of enantiopairs of aqueous-soluble acylimidazole reagents having varied asymmetric substitution near their reactive carbonyl groups. Our findings show that with appropriate substitution, reagents can demonstrate high diastereoselectivity and preparatively useful yields in RNA reaction. The results establish new strategies for selective RNA recognition, with applications in conjugation, modification, and mapping of the biopolymer.To initiate a survey of stereoselectivity, we selected a range of small chiral carboxylic acid precursors for which both enantiomers are readily available . Structu+/Mg2+/MOPS buffer (pH 7.5) with 20% DMSO at 0 °C for 2 h, using a target single-stranded 20 nucleotide (nt) RNA of mixed sequence. Yields of adducts were assessed by MALDI-TOF mass spectrometry  for alanine derivative 2, and 2 : 1 for structurally related proline derivative 8. Interestingly, the stereochemical preference varied, with α-amino species showing preference for the R isomer , while others  reacted selectively as the S enantiomer.Among these chiral scaffolds, we observed that several yielded significant levels of diastereoselectivity in reaction with RNA . Note thR,S)-N,N-dimethylalanine imidazolide (2), in more detail by investigating the effects of time and temperature on yields (R) enantiomer, but its mirror image achieves only <20% conversion even at extended times. Diastereoselectivity (dr) was 10 : 1 at 50% conversion and 5.3 : 1 at 90% conversion. The (R) enantiomer of 2 reacts at remarkably low concentrations -2 also unexpectedly increases its reactivity with RNA.We next examined the most selective scaffold in the initial survey, -2 react equally with an achiral alcohol  favored) as compared with the dimethylamino compounds ((R) favored).For an initial test of whether RNA folded structure can influence diastereoselectivity of 2′-OH acylation, we compared reactions of acylimidazole scaffolds 1 and 2 with three RNAs: single-stranded RNA, a hairpin containing a 3 nt loop, and a hairpin containing a small 1-nt bulge loop -1 and -2, and reacted the four compounds individually under identical conditions  with a folded library of 43 RNAs containing systematically varied loop sizes and loop types.12 Loops were constructed of polyU and polyA sequence, to test extremes of base stacking and helicity. We employed deep sequencing to quantify and pinpoint sites of reaction by reverse transcriptase (RT) stops (ca. 0.1–1.0% of each reactive 2′-OH), so the data provide a measure of kinetic selectivity of acylation at early timepoints of reaction.The above experiments measured conversion yields in aggregate over multiple 2′-OH positions, and thus the selectivity at specific sites remained unknown. This prompted the question of how selectivity and reactivity at each local 2′-OH can vary depending on diverse folded RNA structural contexts. To evaluate this, we selected two enantiopairs of reagents that exhibited relatively high selectivity in the initial screening, compounds (T) stops . CompariT) stops  and S10†3Plots of the data for positional reactivity of the two pairs of chiral molecules are shown in e.g., B4, B5) and internal loops  are the most reactive contexts for the chiral reagents in general, and indeed are more reactive than nucleotides in single-stranded RNAs (e.g. ss-polyA and ss-mixed sequence RNA (“ss-combo”)  that were highly reactive for achiral reagents proved to be relatively unreactive for the current chiral reagents. Also noteworthy for the chiral compounds is that small uridine loops are generally more reactive than those composed of adenosine nucleotides  ). Unlikes Fig. S9, which iR,S)-1, -2) for acylation of unpaired loop residues over paired ones (stems) in the folded RNA library. This ratio has been used as a marker to evaluate acylating reagents for structure mapping; a high loop/stem (L/S) reactivity ratio indicates elevated signal over background. The data -1 showing higher L/S values than -2. The data for -1 are particularly remarkable, with a higher value for (S)-1 than its mirror image; indeed, the L/S value of 8.5 for (S)-1 is considerably higher than that of all structure mapping acyl reagents currently in use.12,40Finally, we measured overall (averaged) selectivity of each compound ((The data  show thad)-RNA. Notably, positional diastereoselectivity varies widely with RNA structure (S) enantiomer is generally favored for scaffold 1, while the (R) enantiomer of 2 is favored at most positions  exhibit relatively low diastereoselectivity in reactions with scaffold 1 , and a ted polyU  and S10†12 The results suggest that compounds with greater asymmetry can better sense differences in local chiral environment around each 2′-OH.To further explore the role of reagent stereochemistry in local nucleotide reactivity across this RNA library, we examined nucleotide-by-nucleotide positional correlations of reactivity of enantiomers of 1 and 2 Fig. S12. InteresAveraging diastereoselectivity of reagent scaffolds 1 and 2 over all loops  revealedS) enantiomer of 1 (L/S = 8.5) as compared with the (R) variant  enantiomer exhibits over twice the signal over background of the (R) enantiomer in mapping this intracellular RNA  and 5% (S) for the two enantiomers, yielding diastereoselectivity of >16 : 1 , recent studies have investigated the properties and applications of enantiomeric (“mirror image” or “spiegelmer”) RNAs, with l-ribose as the source of chirality.42–47 Given the favorable properties of enantio-RNAs in applications such as aptameric ligands in biological settings, we investigated a further test of chiral selectivity in conjugation and labeling of enantiomeric RNA. We carried out reactions of d- and l-enantiomers of a short RNA having a reactive bulge loop structure (sequence B4A from the above library) using each enantiomer of the dimethylalanine reagent 2. MS data (R) with d-RNA; (S) with l-RNA), and considerably lower 5–8% conversions for the “mismatched” pairs. Diastereoselectivity was over 100 : 1 at early time points and averaged 24 : 1 at ∼70% conversion. Thus, highly enantioselective reaction was seen for both native and mirror-image RNAs using reagents of appropriately matched handedness. Extending this to a functionalizable reagent, we reacted the enantiomers of azidoethyl-2 with the d- and l-RNAs, resulting in preparative conversions of 85% and diastereoselectivities averaging 8 : 1 . Our results add support to early reports of amino acid reactions with dinucleotides,33–35 confirming that amino acid derivatives can indeed react at 2′-OH. The current work identifies new reagents and RNA folded structures that exhibit considerably higher yields and greater diastereoselectivity than previously seen, establishes new reagents that enable high-yielding and highly selective RNA modification, and demonstrates new applications of chirality.Our experiments establish that chiral acylimidazole reagents with appropriate asymmetric substitution can exhibit substantial levels of diastereoselectivity in RNA reaction. This is, to our knowledge, the first report of chiral selectivity for acylation of non-homopolymer RNAs. Multiple surprising findings arise from the study regarding unusual reactivity and selectivity -1, that exhibits exceptionally high loop/stem selectivity relative to its enantiomer, and indeed considerably higher than that of any reported agent to date. No previous chiral compound has been employed in mapping of folded RNA structure. In our preliminary tests, this resulted in markedly improved signal over background in intracellular RNA mapping. Further exploration of this finding seems warranted, with the aim of developing future high-performing generations of transcriptome-probing reagents.Our new results add the dimension of asymmetry as an important influencing factor in reagent design, going beyond the effects of alkyl/aryl substitution and steric bulk.5,45,48 however, it is significantly challenging, due to the large number of chemically similar groups in the biopolymer. In this light, our observation of the highly selective stoichiometric labeling reaction of (R)-azidoethyl-2 seems promising  exhibit the opposite diastereoselectivity for RNA, for the most part, as the structurally analogous α-alkoxy compounds . This suggests that the two classes of compounds adopt distinct conformations that favor approach of 2′-OH to the opposite faces of the reactive carbonyls. As a possible explanation -2, and the (R) enantiomer in particular, in acylation of RNA. Indeed, (R)-2 is the most highly RNA-reactive compound yet described in the literature to our knowledge. An experiment benchmarking the reactivity of enantiomers of 2 in comparison to the most commonly applied previous acylation scaffolds employed in RNA research (1M7 and NAIN3) underscores this marked difference  than structurally related tetrahydrofuran scaffold 10. Comparisons of reactivity of (S)-2 and dimethylglycine (DMG) acylimidazoles (which differ by only a methyl group) reveal similar RNA reactivity and half-lives for hydrolysis  has little effect on carbonyl reactivity with water in this isomer. However, the (R) enantiomer of 2 shows higher reactivity for RNA than the (S) isomer (by a factor of roughly ten), and acylates the biopolymer with >90% conversions even at the remarkably low concentration of 100 μM, one thousand-fold lower than common concentrations of RNA acylating agents. This suggests that the methyl group substitution in the (R) configuration confers specially elevated reactivity with RNA.Also remarkable and unexpected is our finding of exceptionally high reactivity by compounds -2, we considered whether the compound might bind RNA noncovalently. We followed up by performing a titration of concentration vs. conversion, and by testing the effects of competition by a chiral nonreactive analogue of (R)-2 -2 in multiple loop types  in RNAs, enabling improved site selectivity in localized conjugation and labeling of larger RNAs. Future work will be required to explore these possibilities.The datasets supporting this article have been uploaded as part of the ESI.R. S. synthesized all compounds, performed screening and optimization of acylating agent reactions with RNA, and determined the stereoselectivity of all compounds. L. X. performed experiments to probe chiral selectivity on folded RNA, performed in-cell RNA structure mapping, and performed site-selective RNA modification experiments. S. C. synthesized, purified, and characterized L-RNA oligonucleotides. E. T. K. designed the project and supervised experiments. R. S., L. X., and E. T. K. wrote the paper with input from all authors.There are no conflicts of interest to declare.SC-014-D3SC03067A-s001


Escherichia coli. Such genes are typically encoded only by bacteriophages supporting the model that PBVs infect bacterial hosts. Recognition of PBVs as RNA phages in the human gut would completely shift models of how PBVs could impact human health. In addition, expanding the RNA phage world beyond the two recognized clades to three clades has implications for our understanding of the evolution of RNA viruses.Picobirnaviruses (PBVs) are double-stranded RNA viruses frequently detected in human and animal enteric viromes. Associations of PBVs with enteric graft-versus-host disease and type I diabetes during pregnancy have been established. Since their discovery in 1988, PBVs have been generally assumed to be animal-infecting viruses despite the lack of culture system, animal model, or detection in animal cells or tissues. Recent studies have proposed that bacteria or fungi could be the hosts of PBVs based on genomic analysis. Here, we functionally demonstrate that multiple PBVs of different genome organizations encode bacterial lysins that lyse  Picobirnaviridae family (Picobirnaviruses (PBVs) are small, double-stranded RNA viruses belonging to the e family . Human de family  and typee family . Since te family . It has Recent genomic studies have shown that a high proportion of PBV genomes harbor bacterial ribosomal binding site upstream of their open reading frames (ORFs), which suggested that PBVs could be bacteriophages –7. A fewA key step toward defining the host would be to identify a PBV protein functionality unique to one of the hosts. One critical step in phage lifecycles is the lysis of bacterial hosts which is necessary for release of progeny. Of the two families of known RNA phages, both encode bacterial lysins . TherefoThe current reference PBVs are composed of two RNA segments. PBV segment 1 encodes the viral capsid and 1 or 2 other ORFs  5′ to the capsid that have unknown function and no homology with known proteins. PBV segment 2 encodes only the RNA-dependent RNA polymerase (RdRp). In addition to the conventional genome organization, there are two other genome organizations identified more recently. One has an unconventional segmented genome whose segment 2 encodes not only RdRp but also 1 or 2 other ORFs  that have unknown function; the other organization is a nonsegmented genome that encodes the ORFs of unknown function, capsid, and RDRP on the same segment. If PBVs were to encode lysins, logical candidate genes would be the ORF1 or ORF2.Escherichia coli, and monitored cell growth for 600 min. Of the three tested ORFs, ORF1 from ND_152547 PBV dramatically reduced bacterial growth comparable to that of the RNA phage MS2 lysin  from 10 representative reference PBVs with the conventional genome organization to determine whether any of these proteins would alter bacteria growth. Three of 16 ORFs, from chicken, rat, and gray teal PBV, reduced bacteria growth with one showing higher activity than the other two C. We alsi growth D. The 13i growth E. Of notNext, we used microscopy and propidium iodide (PI) staining to examine the bacterial morphologic phenotypes and membrane integrity resulting from expression of the 13 functional PBV ORFs. PI binds to DNA but is excluded from live, intact bacteria. All the ORFs showed morphological changes by microscopy and loss of membrane integrity by PI staining. There were diverse morphological phenotypes observed, and the representative ones are shown in E. coli. It is worth noting that phages and their lysins usually have high host specificity  and the other with trisegmented double-stranded RNA genomes (cystoviruses). The identification of at least a subset of PBVs as RNA phages significantly expands the world of RNA phages. This suggests that RNA phages are far more commonly found in human and animal enteric and respiratory tracts than previously recognized. Moreover, the existence of additional RNA phage taxa is important in the models of the evolutionary history of RNA viruses. For example, one analysis proposed a possible origin for cystoviruses from eukaryotic dsRNA viruses ; howeverSI Appendix.XL1-Blue competent cells were transformed with pBAD24 plasmids encoding corresponding ORFs. Growth curves and microscopy were recorded before and after induction of the ORF expression. Detailed descriptions are provided in Appendix 01 (PDF)Click here for additional data file.


Recent evidence from whole-exome and whole-genome sequencing approaches revealed a complex genomic landscape for many cancers. In addition to somatic mutations and alternative splicing changes, genetic information can also be altered by RNA editing, which enables alterations of genome information in a very dynamic and flexible way. Influenced by both external factors and microenvironmental signals, RNA editing deeply contributes to cancer morphological plasticity.Coding and noncoding RNA molecules play their roles in ensuring cell function and tissue homeostasis in an ordered and systematic fashion. RNA chemical modifications can occur both at bases and ribose sugar, and, similarly to DNA and histone modifications, can be written, erased, and recognized by the corresponding enzymes, thus modulating RNA activities and fine-tuning gene expression programs. RNA editing is one of the most prevalent and abundant forms of post-transcriptional RNA modification in normal physiological processes. By altering the sequences of mRNAs, it makes them different from the corresponding genomic template. Hence, edited mRNAs can produce protein isoforms that are functionally different from the corresponding genome-encoded variants. Abnormalities in regulatory enzymes and changes in RNA-modification patterns are closely associated with the occurrence and development of various human diseases, including cancer. To date, the roles played by RNA modifications in cancer are gathering increasing interest. In this review, we focus on the role of RNA editing in cancer transformation and provide a new perspective on its impact on tumorigenesis, by regulating cell proliferation, differentiation, invasion, migration, stemness, metabolism, and drug resistance. The genome-wide complexity is greatly expanded by RNA processing features, including RNA editing. The output information deriving from a single gene depends on how the pre-mRNA is processed, which is strictly connected to the specific cell type, location, and fate, indicating that the cellular environment determines in time and space which splice sites are recognized and which codons are edited . In thiscoxII) [RNA editing comprises different types of irreversible post-transcriptional processing events that modify the RNA sequences and introduce mismatches, thus modifying the coding capacity of a given transcript. The term ‘RNA editing’ was first referred to as the insertion and deletion of uridine nucleotides in the trypanosome mitochondrial mRNA encoding the cytochrome oxidase subunit II (coxII) . Later ocoxII) ,5, thus coxII) . This prcoxII) ; but, lacoxII) .The A-to-I transition, which is the most common editing feature across Metazoa, was the last editing process identified . This coAlu repeats represent privileged sites for editing, since they tend to pair and form double-strand structures [There are two main forms of A-to-I editing: site selective and hyperediting. The site-selective editing targets determined adenosines within a short region of the transcript, without other neighboring deaminated adenosines nearby. This type of editing is less common and occurs in conserved coding regions displaying adjacent stem-loop structures shaped by bulges and mismatches ,22. Convructures .In addition to uridine insertion/deletion, a cytosine to uridine transition (C to U) was identified in the mRNA encoding the intestinal Apolipoprotein B-48 (APOB), leading to a premature termination codon ,30. APOBcis- and trans-acting RNA elements, as well as RNA-binding proteins (RBPs), can modulate RNA-editing efficiency in vivo. In addition, duplexed inverted Alu repeats act as ADAR recruitment elements, enhancing editing efficiency at adjacent sites [RNA editing plays a key role in generating molecular complexity in eukaryote transcriptomes. In a cell, not all adenosines at a given site are edited in every transcript, thus leading to a mixture of cellular transcripts. Rather than a static control of gene expression, A-to-I editing allows for dynamically rewiring the genetic code in a cell-type-specific manner. During normal development, and in both neurological diseases and cancers, the extent of RNA editing does not directly correlate with levels of the substrate mRNA or the editing enzymes, but cellular factors are required for the spatiotemporal regulation of editing efficiency. Recent studies have suggested that nt sites ,39. Alu elements can shape. Unlike genetic mutations, the genetic variation introduced through editing occurs at a low evolutionary cost since production of the wild-type protein is retained and both isoforms (edited and nonedited) exist and may play a role in shaping the transcriptomic landscape.Remarkably, the low basic level of editing detected throughout the transcriptome is a source for adaptive evolution, where cellular factors and hairpin structures made by Deregulation of the RNA-editing process plays a pivotal role in cancer pathogenesis . An alteThe expression of the adenosine-to-inosine (A-to-I) RNA-editing regulator ADAR was found altered in different types of cancer ,42,43,44SLC22A3 gene operated by ADAR2 was found associated with reduced RNA transcription in familial esophageal squamous cell carcinoma (ESCC) [ATM, GINS4, and POLH. The 3′UTRs regulatory regions play crucial roles in mRNA stability and translation efficiency and are responsible for the recruitment of different protein complexes onto the mRNA [ATM and POLH transcripts was able to modulate their expression rate, whereas editing of GINS4 was associated with a decrease in mRNA stability [CDC14B editing with a concomitant increase in its expression [A-to-I editing in the a (ESCC) . Since Sa (ESCC) . ADAR1 oa (ESCC) . In partthe mRNA . A-to-I tability . Whetherpression . The phopression .2+ permeability and affect the activity of the receptor, as observed in tumor-derived tissues compared to a control [GluR-6 transcripts was also analyzed in pediatric astrocytoma [GluR-B and GluR-6, compared to control tissues. Loss of editing in the GluR-B transcript was sufficient to increase astrocytoma invasiveness in vivo [In A-to-I editing, the edited inosine is recognized as guanosine during the translation, resulting in amino acid substitution and conferring to the RNA-editing process the ability to produce protein isoform variability. In glioblastoma multiforme (GBM), which is the most aggressive form of astrocytoma; the glutamate receptor subunit B (GluR-B) transcript was found underedited at the Q/R site . The act control . The levrocytoma , where d in vivo . Althoug in vivo , astrocyBLCAP was associated with cervical cancer [BLCAP altered the motif involved in the binding to the SH2 domain of the signal transducer and activator of transcription 3 (STAT3), with the subsequent loss of inhibition of STAT3 phosphorylation. Hence, editing of BLCAP could represent a driver event in the progression of cervical carcinogenesis [Remarkably, in astrocytoma, the human bladder cancer-associated protein (BLCAP) transcript showed decreased editing . These el cancer . In thisogenesis .AZIN1 transcript resulted in a serine-to-glycine substitution at residue 367 [FLNB  and a lower editing level of COPA (coatomer protein complex subunit α) were found to be associated with pathogenesis [PODXL  gene at codon 241, causing His-to-Arg substitution, indicated that PODXL is one of the ADAR2-editing targets responsible for its tumor-suppressive function [AZIN1 [AZIN1 RNA editing in fibroblasts mediated by ADAR1 expression [In human hepatocellular carcinoma (HCC), ADAR1-editing activity on idue 367 . This caidue 367 . The binogenesis . A similogenesis  and to rogenesis  and gastogenesis . Moreovefunction . In colon [AZIN1 , accordipression .RHOQ) is higher in cancer tissue compared with normal tissue. As a result, the substitution of asparagine with serine at residue 136 (RhoQ N136S) increases RHOQ activity responsible for actin cytoskeleton reorganization and a higher invasion potential [GLI1) factor, a key terminal effector of the hedgehog signaling pathway. The editing on the adenosine 2179 of GLI1 mRNA leads to an amino acid change (from arginine to glycine at position 701) of the GLI1 protein that impacts its activity [In addition, the RNA editing of the Ras homologue family member Q transcript  mRNA by APOBEC-1 creates an in-frame translation stop codon with a consequent reduction of its tumor-suppressor function [The C-to-U editing in the function . Interesfunction .WT1 transcript in nonprogenitor umbilical cord blood mononuclear cell samples (CBMCs), compared to acute myeloid leukemia (AML) [In ER-positive breast cancer, the expression of the cytosine deaminase APOBEC3B  inversely correlates with the response to tamoxifen treatment , suggestia (AML) .Editing events discussed in the RNA editing extensively impacts alternative splicing choices. Editing can alter regulatory motifs, RNA–protein interactions, and RNA secondary structures, thus playing pivotal roles in modulating RNA processing . SeveralHNRNPLL pre-mRNA by ADAR1 p110 and ADAR2 creates a binding site for SRSF1, leading to the inclusion of exon 12A and the consequent production of an hnRNPLL isoform displaying oncogenic activity in kidney and bladder tumors [cis-acting elements, which, in turn, impacts splicing factors binding affinity. A-to-I editing of the r tumors . The crer tumors , revealiCCDC15, located between introns 8 and 9, thus improving its strength and repressing exon 9 inclusion by enhancing the binding of the splicing repressor SRSF7 [RELL2, therefore impeding the recognition of the 3′splice sites by U2AF65. As a consequence, exon 3 is skipped and causes a frameshift and premature stop codon, making the transcript susceptible to nonsense-mediated RNA decay (NMD) [ADAR proteins can influence splicing decisions by distinct mechanisms. Particularly, it was shown that ADAR1 binds to and edits an intronic silencer of or SRSF7 . Conversay (NMD) . In thisay (NMD) . These ePTPN6 pre-mRNA was shown to give rise to a novel PTPN6 transcript retaining intron 3, thus causing a nonsense translation of PTPN6 mRNA and contributing to the production of a nonfunctional protein [PTPN6 processing in leukemogenesis [RNA-specific adenosine deamination of a branch site in  protein . Notablyogenesis . Alu elements, both in sense and antisense pairs of Alus [Alu elements are unique primate-specific retrotransposons that occur in over one million copies of the human genome [Alu repeats, flanked by introns also encompassing Alu, are often targets of the editing machinery in their acceptor splice site, which is converted from AG to IG and interpreted as GG. The strength of the acceptor splice site is then dramatically reduced, causing the skipping of the exons [Alu elements within introns can result in the skipping of the adjacent exons. Insertion of an Alu element in the NF1 caused skipping of the exon immediately downstream of the intron of insertion and, consequently, shifts in the reading frame, leading to neurofibromatosis [Human transcripts contain excess editing over mouse, rat, chicken, and fly transcripts , and mor of Alus . Alu elen genome . Exons che exons . Consistomatosis . Alu sequences can turn them into functional splice sites, recognized by the splicing machinery, thus contributing to the “exonization” process, even in a tissue-dependent fashion [NARF Alu-exon 8, “exonized” via tissue-dependent RNA editing [Alu elements in the NARF gene (encoding the nuclear prelamin A recognition factor), leads to the insertion of 46 in-frame additional amino acids in the encoded protein [Alu sequences in introns can also create a canonical 5′ splice donor site GU  and/or a canonical 3′ splice acceptor site AG  which can influence the splicing outcome. This phenomenon has been observed in several transcripts, such as alternative splicing of exon 15a in the GPR107 gene due to editing of the AluSx in intron 15 [Alu sequence between exons 1 and 2 in the SARS gene (encoding seryl-tRNA synthetase) was also described to prevent aberrant “exonization” [Random mutations in  fashion , as desc editing . In part protein . Similarntron 15 . Editingization” . HoweverAlu elements can increase the coding capacity of human genes while maintaining the original protein repertoire. However, to what extent this process affects transformation has not been deeply elucidated yet. Recently, it was shown that editing of the PODXL pre-mRNA promotes the inclusion of an alternative exon [PODXL long isoform is more prone to protease digestion and reducing cell migration and cisplatin chemoresistance [PODXL transcript and the inclusion level of the PODXL alternative exon were strongly associated with overall patient survival in kidney renal clear cell carcinoma (KIRC) [Thus, these processing events deeply contribute to evolutionary transcriptomic processes, including the creation and deletion of exons . In thisive exon . The ressistance . Remarkaa (KIRC) . Overall, the creation or elimination of splice sites and branch points by RNA editing can explain several editing-dependent splicing events ,72,76,80Editing events affecting splicing decisions were listed in Editing of miRNA precursors displays significant implications on miRNA fate and function, affecting both the expression of the miRNA and its ability to recognize target sequences . A-to-I ADARB1 gene is associated with reduced processing of miRNA-15/-16, suggesting a novel oncogenic mechanism in CLL [Recently, the aggressiveness of thyroid cancer has been associated with the overedited miRNA200b by ADAR1 . The 3′ m in CLL . In addim in CLL . Furtherm in CLL . Importam in CLL .ARHGAP26) [ARHGAP26 transcript are destroyed by the ADAR1 editing activity leading to increased ARHGAP26 expression, cell spreading, and migration [A-to-I editing can induce changes in the specific miRNA target sequences, as observed for the transcript encoding the Rho GTPase activating protein 26 (RHGAP26) . In cancigration . OverallAs discussed above, the most studied mechanism of RNA editing in cancer is the deamination of adenosine due to the ADAR family members. Several studies have highlighted abnormal RNA-editing levels and the aberrant expression of the editing enzymes in cancer specimens compared to normal tissues, indicating that aberrant editing could play a role in cancer development and progression. However, A-to-I editing levels correlate with an enhanced or reduced tumoral phenotype depending on the specific cancer type ,96. AlthIn mice, ADAR1 deletion has been associated with a loss of leukemic cells and a reduction of the leukemic cell burden in the spleen . In addiCCDC15 and RELL2), thereby leading to the production of pro-oncogenic protein isoforms [Another interesting aspect to consider for the treatment of cancer is the connection between RNA editing and splicing ,117,118.isoforms . Interesisoforms , the stuisoforms . ASOs arisoforms , that maisoforms . Notablyisoforms , and theisoforms . Hence, RNA editing opens a novel layer of complexity to the intricate process underlying cancer development, progression, and resistance to therapy. RNA-editing events occurring in tumors have allowed for the identification of the pathological mechanisms specifically linked to the different stages of cancer progression. Understanding these signatures could contribute to the development of more efficient patient-tailored anticancer therapies. RNA-editing events could even be used diagnostically as early biomarkers for ongoing tumor diversification and relapse. On the other hand, pharmacological modulation of writers, readers, and erasers by specific inhibitors or activators could have huge therapeutic potential. In this study, we have reviewed editing changes occurring in cancer; some of them represent only an accompanying phenomenon, not necessarily a contributing factor to cancer progression, while others may play a direct role in cancer development and affect therapeutics. Therefore, some RNA-editing changes might be considered as cancer biomarkers but not therapeutic targets. To date, more effective strategies to identify, validate, and analyze editing events are still needed to allow for precision medicine based on epi-transcriptome signatures that are tailored to specific tumor types. Hence, more efforts should be made in the coming years to characterize editing in normal and cancer conditions, providing the opportunity to develop more sophisticated diagnostic and prognostic procedures in clinical practice.


Here, we use simulation and experiment to discover an innate and sophisticated mechanism of control of RNA lifetime. By simulation we characterized RNA Goldilocks landscapes and their dependence on cleavage and folding parameters. Experiments with yeast tRNAPhe and the Tetrahymena ribozyme P4–P6 domain show that structured RNAs can inhabit Goldilocks peaks. The Goldilocks peaks are tunable by differences in folded and unfolded cleavage rate constants, Mg2+ binding cooperativity, and Mg2+ affinity. Different folding and cleavage parameters produce Goldilocks landscapes with a variety of features. Goldilocks behavior allows ultrafine control of RNA chemical lifetime, whereas non-folding RNAs do not display Goldilocks peaks of protection. In sum, the effects of Mg2+ on RNA persistence are expected to be pleomorphic, both protecting and degrading RNA. In evolutionary context, Goldilocks behavior may have been a selectable trait of RNA in an early Earth environment containing Mg2+ and other metals.Magnesium, the most abundant divalent cation in cells, catalyzes RNA cleavage but also promotes RNA folding. Because folding can protect RNA from cleavage, we predicted a ‘Goldilocks landscape’, with local maximum in RNA lifetime at Mg  Universal biopolymers  are ephemeral ,2. Biopo2+ directly increases RNA cleavage rates by one mechanism and indirectly decreases cleavage rates by a different mechanism. Mg2+, the most abundant divalent cation in cells , does not display Goldilocks behavior.In our experiments, Goldilocks behavior is observed for well-established model RNAs, including yeast tRNAPhe ,26 and t6 domain ,28. An e2+-mediated shifts into and out of Goldilocks peaks and by remodeling Goldilocks landscapes via sequence and chemical modification. Goldilocks behavior of RNA is consistent with its selection in a primordial world of stringent and conflicting evolutionary demands.Goldilocks behavior of RNA suggests intrinsic sophistication, allowing ultrafine control of chemical lifetime by a variety of inputs ,29. RNA 2+] on folded and unfolded RNA cleavage rate constants which both contribute to an observed cleavage rate constant kobs. Lifetime is the reciprocal of kobs.We mathematically modeled the effect of [Mgff and the fraction unfolded is fu. We used the Hill equation to describe extent of folding  is proportional to the second-order rate constant (k) and [Mg2+] ::2+]    32)::kobs)  of Mg2+ with RNA has a rate constant of k ≈ 1010 s−1, near the rate of diffusion . rU20 was purchased from Integrated DNA Technologies. T7-transcribed, stabilized P4–P6 RNA was produced as in Athavale et al. . Theta values were baseline-corrected for the effects of dilution, evaporation, and cleavage over the course of CD data acquisition. Yeast tRNAPhe showed little cleavage during CD data acquisition. Some P4–P6 RNA cleavage was observed, consistent with the longer CD acquisition time and greater RNA length .Extent of folding was quantified by CD spectroscopy. A solution of 10 μM tRNA, 8 μM P4–P6 RNA, or 10 μM rU2+-dependent folding  to observe Mg folding . Solutio2+ were reverse transcribed in parallel with omission of the ddNTPs for identification of cleavage sites. 1 μl of each reverse transcription was combined with 1 μl GenefloTM 625 size standard ROX ladder (CHIMERx) and 20 μl HiDi (Applied Biosystems). Samples were resolved on a SeqStudio instrument using the FragAnalysis run module.RNAs were analyzed by capillary electrophoresis  following the manufacturer's protocol. Data were analyzed and peaks were aligned in MATLAB. A final concentration of 10 ng/μL uncleaved ‘fresh’ P4–P6 RNA with 0.4 μM of FAM-labeled reverse transcription primer that binds to the 3’ end , 5 mM DTT, 1× first strand buffer (Invitrogen), 0.5 mM each dNTP, and 2 mM of either ddATP, ddCTP, ddGTP or ddTTP was reverse transcribed by SuperScript III (Invitrogen) to create fragments for sequencing. The P4–P6 RNA cleavage reactions containing 5, 10 and 15 mM Mg2+. The simplest model .We investigated RNA Goldilocks behavior for RNAs that fold in response to Mgl Figure  allows tKD modulates the position of the Goldilocks peak on the [Mg2+] axis; RNAs that fold at lower [Mg2+] show a Goldilocks peak at lower [Mg2+]. The Hill coefficient n modulates the sharpness of the Goldilocks peak without a substantial change in its position; a larger n gives a sharper peak. ku modifies the slope of the Goldilocks peak on the low [Mg2+] side, and kf modifies the slope on the high [Mg2+] side. The ratio of ku to kf modulates the intensity of the peak. Goldilocks peaks are absent for RNAs that (i) do not fold, (ii) are always folded, (iii) do not change cleavage rate constant upon folding, or (iv) transition very gradually between differential cleavage realms with varying [Mg2+]  or cleaved more rapidly than the unfolded state (ki > ku) can display especially intense Goldilocks peaks. If an intermediate state has intermediate protection, the net Goldilocks peak is less intense than in the absence of an intermediate.More realistic RNA folding mechanisms involve intermediate states . In a moi Figure . The sims Figure . When thn) or with increased extent of protection afforded by folding (decrease of kf relative to ku) .To experimentally investigate Goldilocks behavior, we assayed both fraction folded and lifetime of yeast tRNA of Mg2+ ,49–54. Cd Figure . The tRNPhe Goldilocks landscape is reasonably fit by a two-state model. The fit and observed Goldilocks peaks are centered at the same [Mg2+]. The cleavage rate constant of the unfolded tRNA is predicted to be 2.7 times greater than that of the folded tRNA. However, the observed Goldilocks peak is sharper and more intense than predicted by the two-state model. A three-state model provides a better fit to the data, especially in the center of the Goldilocks peak. In the three-state model the cleavage rate constant for the unfolded RNA is predicted to be 3.2 times that of the intermediate and 2.2 times that of the fully folded tRNA (ku > kf > ki). The statistical significance of the improved fit of the three-state versus the two-state lifetime and folding models is indicated by residual errors . Our results are consistent with previous observations of >2 states for folding of yeast tRNAPhe .2+  than for P4–P6 RNA (1.5). The fit parameters of both RNAs are shown in Table Both RNAs display Goldilocks peaks in experiment and in simulation. Both RNAs are best fit to models with more than two states. For the tRNA the Goldilocks peak is sharp, with a half-height peak width of around 1 mM Mgtructure ,63. Conv2+), on the partially folded shoulder of the peak (5 mM), and on the fully folded should of the peak (15 mM) . The average extent of cleavage is less under the conditions of the Goldilocks peak (0.18) than in regions flanking the peak (0.21 pre-peak and 0.20 post-peak). Subtracting the 10 mM Mg2+ Goldilocks peak condition as a baseline shows nucleotides that experience more cleavage off the peak than on the peak, with larger Δcleavage values indicating greater cleavage . These sites overwhelm the few nucleotides that are highly cleaved in the folded state (strong negative Δcleavage). In the partially folded realm, double-stranded RNA shows more uniform extent of cleavage than unpaired RNA  might beostasis . It seemRNA can transit between dangerous spaces and safe spaces. Finely controlled metastability, with access to Goldilocks peaks of protection, is most likely an imprint of evolutionary processes  during tAll data are available through NAR online.gkad124_Supplemental_FilesClick here for additional data file.


Caenorhabditis elegans, mutation in unc-78, encoding one of the two AIP1 isoforms, causes severe disorganization of sarcomeric actin filaments and near paralysis, but mutation in sup-13 suppresses the unc-78-mutant phenotypes to restore nearly normal sarcomeric actin organization and worm motility. Here, we identified that sup-13 is a nonsense allele of arrd-15 encoding an α-arrestin. The sup-13/arrd-15 mutation suppressed the phenotypes of unc-78 null mutant but required aipl-1 that encodes a second AIP1 isoform. aipl-1 was normally expressed highly in embryos and downregulated in mature muscle. However, in the sup-13/arrd-15 mutant, the AIPL-1 protein was maintained at high levels in adult muscle to compensate for the absence of the UNC-78 protein. The sup-13/arrd-15 mutation caused accumulation of ubiquitinated AIPL-1 protein, suggesting that a normal function of sup-13/arrd-15 is to enhance degradation of ubiquitinated AIPL-1, thereby promoting transition of AIP1 isoforms from AIPL-1 to UNC-78 in developing muscle. These results suggest that α-arrestin is a novel factor to promote isoform turnover by enhancing protein degradation.Precise arrangement of actin, myosin, and other regulatory components in a sarcomeric pattern is critical for producing contractile forces in striated muscles. Actin-interacting protein 1 (AIP1), also known as WD-repeat protein 1 (WDR1), is one of essential factors that regulate sarcomeric assembly of actin filaments. In the nematode  Caenorhabditis elegans, we found that the sup-13/arrd-15 gene, encoding an α-arrestin, promotes turnover of one of the two AIP1 isoforms in striated muscle by specifically targeting one isoform for ubiquitin-dependent degradation. Our study suggests that α-arrestin is a novel factor to promote isoform transition in striated muscle.Sarcomeric organization of contractile proteins is essential for efficient contractility of striated muscles. However, the mechanism of sarcomere assembly remains unclear. Actin-interacting protein 1 (AIP1), also known as WDR1, is a conserved protein that promotes disassembly of actin filaments and is essential for sarcomeric assembly of actin filaments. In the nematode Dynamic assembly and disassembly of actin filaments are critical for the function of the actin cytoskeleton that plays central roles in many cell biological processes including cell migration, cell division, and morphogenesis. Because actin filaments are relatively stable, severing and depolymerization of actin filaments are often promoted for cytoskeletal reorganization and recycling of actin monomers for new rounds of polymerization . Actin dAIP1 , also knWDR1, cause severe blood disorders, including reduction and impaired motility of neutrophils  in differentiated cells.In this study, we took advantage of cterized , 33. We radation . We founh unc-78 , 38. In unc-78, a sup-13 mutant allele, st210, was isolated from a screen for extragenic suppressors of unc-78 and mapped to chromosome III , a loss-of-function allele with a missense mutation (sup-13 mutation suppresses the phenotypes of unc-78(gk27), a null allele expressing no detectable UNC-78 protein (unc-78(gk27) single mutants moved much slower than wild type  double mutants moved slightly slower than wild type but significantly faster than the unc-78(gk27) single mutants  mutants, actin filaments were highly disorganized and accumulated in large aggregates  double mutants, striated organization of actin filaments were nearly indistinguishable from that in wild type  at the L3 larval stage and was nearly 100% penetrant after the L4 larval stage  at all developmental stages  was a nonsense mutation [W66 (TGG) to stop (TGA)] in the arrd-15 gene encoding an α-arrestin ::ARRD-15 fusion protein in the body wall muscle of sup-13(st210);unc-78(gk27) suppressed the suppressor phenotype and induced actin aggregate formation ;unc-78(gk27) using the myo-3 promoter from extrachromosomal arrays, which often cause transgenic expression in a mosaic manner  by CRISPR/Cas9-mediated gene editing  did not cause a detectable abnormality  mutants, aipl-1(RNAi) caused embryonic arrest  did not cause detectable alterations in the phenotypes in wild type    (in an unc-78 background) causes stronger phenotypes in the embryonic stages than in the adult stage (aipl-1(RNAi) in sup-13;unc-78(gk27), but not in unc-78(gk27), suggest that aipl-1 became functionally significant at the adult stage in the sup-13 mutant background.gulation , 38. We est Fig. , and thil stages    and sup-13, AIPL-1::GFP was significantly increased in the body wall muscle as compared with wild type and unc-78(gk27)  and sup-13;unc-78(gk27)  Fig. A and B. 27) Fig. A and B, ion Fig. C and D. ion Fig. , 30. Ind27) Fig. C and D. sup-13/arrd-15, we examined the ubiquitination levels of AIPL-1 and found that ubiquitinated AIPL-1 was accumulated in the sup-13/arrd-15 mutants  but detected at high levels in sup-13 and sup-13;unc-78(gk27)  suppressed the Sup-13 suppressor phenotype  in their C-termini as binding sites for the WW domains of Rsp5/Nedd4-like E3 Ub ligases . Howevere, arr-1 , and 28 ignaling . Howeverensation . ttm-2 il toxins . Either W domain  or be inOur study suggests that an α-arrestin is involved in isoform switching of muscle proteins during development. Many sarcomeric proteins undergo isoform transition during muscle differentiation, development, and under pathological conditions . Althougsup-13(st210);unc-78(e1217), and CB1217 unc-78(e1217) were obtained from the Caenorhabditis Genetics Center . ON5 unc-78(gk27) was reported previously (sup-13(st210);unc-78(gk27) was generated by crossing RW2337 and ON5 unc-78(gk27) and selecting sup-13(st210);unc-78(gk27) homozygotes in the F2 generation. ON341 sup-13(st210) was generated by crossing ON320 and N2 and selecting sup-13(st210) homozygotes in the F2 generation. Other transgenic strains are described below. All strains used in this study are listed in The worms were cultured following standard methods . N2 wildeviously . ON320 sWorm motility was quantified as described previously . Brieflysup-13(st210) III;unc-78(e1217) X and CB1217 unc-78(e1217) X as described previously (C. elegans reference genome (version WS249). SAMtools (sup-13 was previously mapped near the center of chromosome III by three-factor mapping using dpy-17 and lon-1 (arrd-15) within a 5-Mb interval in a protein-coding gene in the sup-13 mutant (RW2337), which is an expected mutation frequency for ethyl methanesulfonate mutagenesis in C. elegans as reported in the Million Mutation Project , and mutations in the F2 sup-13;unc-27(gk27) double homozygotes were analyzed. As a result, the nonsense mutation in the arrd-15 gene was linked with the Sup-13 suppressor phenotype.We sequenced RW2337 eviously . Genomiceviously  was usedSAMtools  was applSAMtools , 75 was SAMtools  was usednd lon-1 , 77. Themyo-3 promoter that is active in the body wall muscle (sup-13(st210) III;unc-78(gk27) X to generate transgenic animals with extrachromosomal arrays as described (sup-13(st210) III;unc-78(gk27) X;ktEx252.The cDNA for ARRD-15 (NCBI Reference Sequence: NP_001254970.1) was cloned in-frame at the 3′ end of the GFP sequence of pPD-118.20  containing the l muscle , and injescribed . The genaipl-1::gfp strain, the GFP sequence was inserted in the genome in-frame at the 3′-end of the aipl-1 coding region by CRISPR/Cas9-mediated genome editing using vectors and protocols described by Dickinson et al. (aipl-1(kt4[aipl-1::3xFLAG::gfp]) was established.For the n et al. . A singln et al. . Hygromyn et al. . In the arrd-15::gfp strain, the GFP sequence was inserted in the genome in-frame at the 3′-end of the arrd-15 coding region by CRISPR/Cas9-mediated genome editing at SunyBiotech , and PHX4137 arrd-15(syb4137[arrd-15::gfp]) was isolated.For the Escherichia coli expressing double-stranded RNA as described previously (aipl-1 (V-9L16) was obtained from MRC Geneservice  (RNAi experiments were performed by feeding with eviously . An RNAiKingdom) . An emptKingdom) . When L1Kingdom) .g for 10 min at 4°C. Protein concentrations were determined using a Pierce BCA Protein Assay Kit , and an equal amount of proteins (> 1 mg) was used for immunoprecipitation. The lysates were incubated for 1 h at 4°C with 10 μl suspension (binding capacity for 0.8–1.1 μg of GFP) of GFP-Trap Magnetic Particles M-270 , which was pre-equilibrated with the immunoprecipitation buffer. The magnetic particles were washed three times with the immunoprecipitation buffer, and bound proteins were eluted by adding 15 μl of sodium dodecyl sulfate (SDS) lysis buffer  and heating at 95°C for 5 min. Magnetic particles were separated from solution and the supernatant stored at −20°C for downstream application.Age-synchronized young adults were mixed with ice cold immunoprecipitation buffer  and mechanically lysed on ice with 20 passages through a Balch cell homogenizer with a 12 μm tungsten carbide ball bearing  . The lysWhole worm lysates were prepared from an equal number of young adults  antibody  , followed by horseradish peroxidase-conjugated goat anti-mouse IgG antibody  . The signals were detected by incubating with a SuperSignal West Pico Chemiluminescent Substrate  and exposing to X-ray films. The membranes were blocked again, and probed with rabbit anti-DYKDDDDK epitope polyclonal antibody  to detect AIPL-1::GFP with three copies of the FLAG epitope (DYKDDDDK), followed by incubation with IRDye 680RD goat anti-rabbit IgG polyclonal secondary antibody (1:7500). After washing with PBS-T, the signals were detected by scanning with an Odyssey Classic Infrared Imaging System. The signals for anti-Ub and anti-DYKDDDDK were quantified with ImageJ and Image Studio Lite, respectively, and the anti-Ub signals were normalized to the anti-DYKDDDDK signals.Staining of worms with tetramethylrhodamine-phalloidin  was performed as described , 89. SamTo measure fluorescence intensity of AIPL-1::GFP, live worms were mounted and immobilized with 25% Pluronic F-127  in M9 buffer containing 0.5 mM levamisole and 0.1% tricaine methanesulfonate. Fluorescence images were captured with the same settings for all samples in NIS-Elements using a Nikon Eclipse TE2000 inverted microscope with a CFI Plan Fluor ELWD 40 × (NA 0.60) objective. Fluorescence intensity of each worm was determined using ImageJ as average fluorescence intensity at 10 randomly selected points within the cytoplasm of the body wall muscle in the head region.t-test : one-way analysis of variance (ANOVA) and Tukey post hoc test Figs. C; unpairst Figs. C and 3C.pgad330_Supplementary_DataClick here for additional data file.


The majority of antivirals available target viral proteins; however, RNA is emerging as a new and promising antiviral target due to the presence of highly structured RNA in viral genomes fundamental for their replication cycle. Here, we discuss methods for the identification of RNA-targeting compounds, starting from the determination of RNA structures either from purified RNA or in living cells, followed by in silico screening on RNA and phenotypic assays to evaluate viral inhibition. Moreover, we review the small molecules known to target the programmed ribosomal frameshifting element of SARS-CoV-2, the internal ribosomal entry site of different viruses, and RNA elements of HIV. We lack antivirals for most viruses. The limitations in antiviral discovery are multiple. It is fundamental to find highly specific targets in order to avoid toxic effects on the host cell since viruses are intracellular parasites. Many viruses cause acute infections and, therefore, it is difficult to start the treatment at the right moment, as evidenced, for instance, by the limit of influenza therapies which have to be given within 48 h from symptom onset . FurtherOne possibility for overcoming these limitations is to change the approach and the target for antiviral molecules. So far, all the antivirals in commerce are targeting either viral proteins or, in rare cases, cellular proteins . HoweverRNA viruses can be targeted as well since they have multiple conserved secondary and tertiary structures in their genome to express superposed open reading frames (ORF) by inducing programmed ribosomal frameshifting (PRF) , as signAn important point to carefully monitor with the increase of RNA-targeting molecules will be the development of resistance. Despite the selection with some RNA-targeting antivirals targeting the IRES showed the possible insurgence of resistance ,13, withIn the last years, the growing interest in RNA as an antiviral target is partly linked to the development of techniques allowing for a better understanding of the structure of RNA in living cells, together with traditional techniques to reveal a purified RNA structure in vitro (reviewed in ). Since Different techniques have been optimized to assess RNA folding in vitro and in living cells. The 3D structure of viral RNA can be determined by crystallography , nuclearIn addition to the limitations of the previously described techniques, we need to consider that the viral RNA in the cell is associated with multiple proteins and in full length, which might result in a different folding. For this reason, it is important to investigate the structure of the RNA in the natural context of the infection and one of the possible ways is to probe the viral RNA with chemicals able to selectively modify nucleotides in loops and bulges but not paired ones. After adapted sequencing, it is then possible to predict secondary and tertiary structures. Different chemical probes can be used; one of the oldest is dimethyl sulfite (DMS), while more recently new probes were identified to perform selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) . More deThanks to DMS-MaP and SHAPE-MaP, entire viral genomes in infected cells have been studied. Several works have focused on SARS-CoV-2 ,33,34, sIn many of these works, the identification of structures is just described or investigated by deleting the entire secondary structure identified and seeing the effect on the viral life cycle. To date, we are missing a comprehensive evaluation, from the identification of the secondary structure to finding small molecules targeting it. To do so, druggable pockets need to be selected, an in silico screening performed, and, subsequently, the compounds tested in biological assays. Otherwise, the sequence forming the secondary structure can be cloned upstream of a reporter gene and a phenotypic screen can be directly performed. The steps to perform these experiments are described in the following paragraphs.In silico screening allows for the reduction of the time and cost of in vitro assays for the identification of antivirals. The starting point is the choice of the RNA model. In the Protein Data Bank, X-ray, electron microscopy, or even solution NMR structures are present. However, only 280 structures of viral RNA are available , at the time of writing. They might have a high resolution but these structures were generated from crystal, frozen, or in-buffer samples. X-ray or cryo-EM structures could then be refined through classical molecular dynamics, as done by our group . HoweverIn case of the appearance of a new virus or a lack of a 3D structure available, the model can be created from the sequence of the virus. Park et al. have built directly their model using the sequence of SARS-CoV . PSEUDOVIn the past years, in silico software was developed and largely used for proteins. Recently, since RNA became an interesting target, some existing tools were modified or validated , and new tools were developed for RNA  . In the RNA, LigandRNA), or even machine-learning based  [After obtaining the binding poses, they can be rescored. Several scoring functions are known for proteins and, nowadays, also for RNA. Unfortunately, scoring functions are algorithms ranking binding poses based on different criteria, such as physic based , knowledge based (such as DrugScoreNAmigos) . To overNAmigos) , helpingAnother critical point in the virtual screening against viral RNA is the selection of the pocket. In RNA, pockets were considered too polar and solvent exposed, in opposition to the pocket present in proteins. However, a study showed that the properties of RNA pockets are similar to those of proteins using all ligand–RNA structures available .To avoid off-target effects, the antiviral should target a specific pocket. To increase the selectivity of the compound, screening should be done on complex structures . The virOnce the hits of the screening have been selected, a filtering step to exclude molecules binding to host RNA can be performed, thanks to the presence of tools and databases of predicted human RNA structures ,49,50. OAfter the in silico screening, it is important to validate the results with phenotypic assays, such as dual luciferase assays and, more importantly, to verify the inhibition of the compound on wild-type virus to truly validate the screening results.Even without understanding the exact folding of a viral RNA structure, it is possible to find inhibitors with phenotypic assays. Chemical assays in which the affinity of a small molecule for a structure of interest, such as fluorescent dye displacement assays, can be used . HoweverThe alteration of the expression of the reporter can be evaluated directly upon transfection of plasmids ,12, throAll the alternatives have advantages and disadvantages. For instance, the transfection of plasmids in cells is linked to nuclear expression of the transcript with possible mistakes or unwanted splicing events, especially for long constructs, resulting in altered expression of the reporter independent of the action of the compounds. In the in vitro transcribed RNA, the transfection itself can play a role in altering the RNA; while for in vitro experiments, we lack the protein interactors naturally present in the cells that might prevent the binding of a molecule that is found in these conditions, or there might be compounds that interact with complexes of RNA and proteins and, therefore, will not be identified in experiments performed in vitro .Moreover, it is important to consider a general limitation of the dual luciferase approach: viral RNA can have, as well, long distance interaction. For instance, the interaction between the 3′ untranslated region (UTR) and the 5′ UTR of different flaviviruses is fundamental for viral replication , or distIn the design of the experiment and of the construct, it is important to take into consideration cloning a consensus sequence rather than a sequence of a specific isolate, to assess the effect of the drug on a sequence that is likely to be shared among different circulating strains of the virus of interest.Considering all these limitations, while recognizing the importance of these high-throughput assays for initial screening, the evaluation of the selected compounds with wild-type viruses is a fundamental step in the evaluation of the identified molecules; however, in numerous works on RNA targeting antivirals, this crucial experiment is missing.The evaluation of the antiviral activity of the compound against the wild-type (WT) virus in infected cells is a fundamental condition for finding true viral RNA interactors. The identification of potent inhibitors with other techniques can result in complete failure of activity with a WT virus for two main reasons, either the compound has poor cell permeability or the targeted RNA cannot be bound due to the presence in a discrete zone of the cell not being accessible to the compound. For instance, viral RNA could be associated with nucleoproteins, and this might mask the interaction site with the small molecule.The other important point to verify with the natural infection is the possible insurgence of resistance. The selection of resistance in vitro could reveal if the site targeted can be easily mutated, therefore hampering the further development of the molecule as an antiviral. However, the eventual selection of resistance is an important verification of the binding site that can be then re-engineered in dual luciferase assays or modeled to understand the change of the binding pocket of the molecule, as was previously done for inhibitors of the RNA of enterovirus 71 . ImportaThe genome of coronaviruses has two superposed ORFs, named ORF1a and ORF1b. At the end of ORF1a, a stop codon is present; however, upon a −1 PRF, the ribosome can change the reading frame, skip the stop codon, and allow the translation of the full ORF1ab with the expression of the replication machinery encoded in the ORF1b. The −1 PRF is allowed by the presence of a slippery sequence on viral RNA and of a pseudoknot. The secondary structure of the pseudoknot has been investigated with multiple methods ,23,24,58Since the importance of the −1 PRF was known for other coronaviruses, research on this target was already started before the COVID-19 pandemic, especially on SARS-CoV-1. In particular, a compound, 2-{[4-(2-methylthiazol-4-ylmethyl)-. The comSeveral compounds proposed to act on the −1 PRF have been identified through high throughput screening or specific design. In particular, the compound merafloxacin was identified with a high throughput screening: a dual fluorescence assay, based on a plasmid containing mCherry, the sequence of the junction between ORF1a and ORF1b, and a GFP coding sequence in a −1 frame . The actOur study, instead, evidenced the activity of one aminoglycoside, geneticin, against the −1 PRF . AminoglAdditional compounds were identified with dual luciferase/fluorescence assays ,61 Tabl and otheHowever, further optimization is required to obtain compounds endowed with stronger antiviral activity and drug-like properties against this target, also considering that the molecules were not tested in vivo. However, these preliminary results evidence that the target is valuable in cells and human-derived tissues without major issues of toxicity. This is possibly due to the presence of few human genes known or predicted to have pseudoknots .Additionally, the highly conserved structure of the −1 PRF among beta-coronaviruses suggests that optimization of compounds on this target might be useful for newly emerging coronaviruses.Picornaviridae and in the Hepacivirus, Pegivirus, and Pestivirus genera of the Flaviviridae. Moreover, they are present in the Dicistroviridae, a family of viruses infecting arthropods and insects.Different viruses rely on secondary or tertiary structures in their 5′ UTR in order to mediate multiple functions for the viral life cycle: the most important, for viruses with uncapped RNA, is the IRES, which allows viral protein translation. IRES are present in the The IRES presents different structures in different viral families , and eveFew viral RNA inhibitors have been tested in vivo; however, to the best of our knowledge, the majority have as a target the IRES of enterovirus 71 (EV71). The flavonoid prunin was identified with a screening in which the IRES of EV71 was cloned upstream of a luciferase gene. The compound was then further studied and revealed potent antiviral activity from in vitro to in vivo ,70, and Picornaviridae ) [Picornaviridae, with dual luciferase assays in which the compound inhibited only the IRES-mediated translation of HAV, EV71, and Cardiovirus A [Another interesting example of IRES targeting compound is amantadine. This compound is well known to be an inhibitor of the ion channel of the Influenza A virus, necessary for viral entry. However, several reports showed its activity on IRES-mediated translation of different members of s (HAV)) ,76 and dovirus A ; subsequovirus A . Howeverovirus A .Picornaviridae, or the ones shown in Nevertheless, the IRES of HCV was successfully targeted by a series of different compounds ,80,81,82Picornaviridae family, it will be important to continue the research of small molecules active on the IRES of other members, for instance, enterovirus D68 for which mutations in the IRES have been linked to increased pathogenicity [In the genicity  or for wgenicity .In addition to the known viruses bearing IRES, additional viruses have been proposed to have IRES, for instance, hepatitis E virus to express the ORF4  or flaviIn parallel with the research on new targets, a focus must be as well the research of new molecules specifically acting on viral IRES in order not to risk targeting human IRES, which are involved in the translation of genes involved in stress and apoptosis , processA largely studied field of viral RNA targeting structures has been HIV. Several RNA structures were identified as targets for antivirals: frameshift site Gag:Pol, TAR, RRE, and Psi Stem Loop 3.Gag is a polyprotein encoding for the matrix, capsid, nucleoprotein, and p6. After expression, it is involved in the assembly, release, and maturation of HIV particles. However, a polyprotein named GagPol containing the domains of Gag and three additional ones  can be translated after a −1 PRF . The fraTo replicate efficiently, HIV uses TAT protein. TAT recognizes the TAR element on the nascent 5′ end viral RNA and recruits host factors to enhance transcription ,97. ModiRRE, an RNA structure containing multiple bulges, internal loops, and hairpins, is present on unspliced and partially spliced viral RNA ,99. In tDuring viral assembly, Gag interacts with viral RNA and, in particular, with the Psi packaging domain located in the 5′UTR of HIV . DestabiMost of the structures targeted on HIV elements are hairpins highlighting that targeting simpler structures of RNA can also be successful. Unfortunately, to our knowledge, none was studied in vivo. This limitation is possibly due to the presence of many effective conventional antivirals, or to the availability of small animal models.The targets described in this review are the ones for which more literature is available; however, other viral RNA targets were investigated such as the 5′ and 3′ conserved nucleotides on the segmented genome of influenza  or the f


Plasma BLVRB levels were also measured in a mouse vein graft model of IPH at baseline and following antiangiogenic therapy targeting vascular endothelial growth factor receptor 2 (VEGFR-2). Results: Plasma BLVRB levels were significantly higher in patients with IPH (737.32 ± 693.21 vs. 520.94 ± 499.43 mean fluorescent intensity (MFI), p = 0.033), but had no association with baseline clinical and biological parameters. Plasma BLVRB levels were also significantly higher in patients who developed recurrent ischemic stroke . Plasma BLVRB levels were significantly reduced following prevention of IPH by anti-VEGFR-2 therapy in mouse vein grafts . Conclusions: Plasma BLVRB was associated with IPH and increased risk of recurrent ischemic stroke in patients with symptomatic low- to moderate-grade carotid stenosis, indicating the capacity to monitor the efficacy of IPH-preventive pharmacotherapy in an animal model. Together, these results suggest the utility of plasma BLVRB as a biomarker for atherosclerotic plaque instability. Background: Intraplaque hemorrhage (IPH) is a hallmark of atherosclerotic plaque instability. Biliverdin reductase B (BLVRB) is enriched in plasma and plaques from patients with symptomatic carotid atherosclerosis and functionally associated with IPH. Objective: We explored the biomarker potential of plasma BLVRB through (1) its correlation with IPH in carotid plaques assessed by magnetic resonance imaging (MRI), and with recurrent ischemic stroke, and (2) its use for monitoring pharmacotherapy targeting IPH in a preclinical setting. Methods: Plasma BLVRB levels were measured in patients with symptomatic carotid atherosclerosis from the PARISK study ( Atheroembolism from atherosclerotic plaques in the carotid bifurcation is an important cause of ischemic strokes . IntraplNevertheless, identifying IPH in clinical practice could aid risk stratification and individualized therapy for patients with carotid stenosis. IPH can be identified by carotid imaging using innate biological properties to guide detection of tissue features ,12. In pUsing an inductive approach for the identification of plasma biomarkers causally linked to unstable carotid atherosclerosis based on a biobank of carotid endarterectomies , we recently discovered biliverdin reductase B (BLVRB) as a candidate biomarker associated with IPH. BLVR is a key enzyme in Hb metabolism, catalyzing the final conversion of biliverdin to bilirubin downstream from heme oxygenase, with the BLVRB isoform typically expressed during fetal development, while BLVRA is predominantly expressed in adult tissues . BLVRB wThe aim of this study was, therefore, to explore the biomarker potential of BLVRB by evaluating the relationship between plasma BLVRB levels and the clinical presence of IPH, as well as the incidence of recurrent ischemic stroke in patients with symptomatic carotid stenosis. Furthermore, to evaluate the capacity of BLVRB to monitor pharmacotherapeutic effects specifically targeting IPH, plasma BLVRB levels were assessed in a preclinical atherosclerotic mouse vein graft model of unstable atherosclerosis subject to antiangiogenic treatment targeting IPH.The Plaque At RISK (PARISK) study  is a prospective multicenter cohort study that included patients with recent (<3 months) neurological symptoms due to ischemia in the carotid artery territory with a ≥2–3 mm carotid plaque and <70% ipsilateral carotid artery stenosis (based on NASCET criteria)  who were1-weighted inversion recovery turbo field echo or 3D T1-weighted (T1w) spoiled gradient echo images and was delineated to calculate the volume [1w MRI that did not enhance on the post-contrast T1w MRI. In addition, IPH volume was always considered as part of the lipid-rich necrotic core [In the PARISK study, MRI was performed at 3 T using a whole-body scanner with a dedicated carotid coil (four or eight channels), as described previously ,21. Six e volume . The liptic core  and was tic core ,21.ApoE3Leiden mice, 10–16 weeks old, were fed a hypercholesterolemic diet  from 3 weeks prior to surgery until sacrifice with resulting plasma cholesterol levels between 12 and 24 mmol/L . Before surgery, mice were anesthetized with midazolam , medetomidine , and fentanyl . After surgery, sedation was reversed with atipamezole  and fluminasenil . Buprenorphine  was given for analgesia. Vein graft surgery was performed by interposition of the inferior vena cava from donor littermate mice in the carotid artery of recipient mice as described before [The animal study was performed in compliance with Dutch government guidelines and European regulations, with all experiments approved by the animal welfare committee of the Leiden University Medical Center . Male d before . −1 DC101; Bio X cell, Lebanon, NH, USA) (n = 14) or control rat anti-mouse IgG antibodies  on days 14, 17, 21, and 25. Two mice in this group were excluded due to vein graft thrombosis [2. Plasma samples were collected at T0 before the surgery and randomization and at T28.For IPH inhibition, mice were treated with IP injections of rat anti-mouse VEGFR-2 IgG monoclonal blocking antibodies  of the vein grafts were processed for Masson’s trichrome and immunohistochemistry staining. In brief, tissue sections were deparaffinized in Histolab clear  and rehydrated in graded ethanol. For antigen retrieval, slides were subjected to high-pressure boiling in DIVA buffer (pH 6.0). After blocking with Rodent block M, (RBM961), primary antibodies BLVRB  and HMOX1  were diluted in Da Vinci Green solution , applied on slides, and incubated at room temperature for 1 h. For colocalizations, antibodies for cell-specific markers were used: von Willebrand factor , CD68 , and smooth muscle α-actin . A double-stain probe-polymer system containing alkaline phosphatase and horseradish peroxidase was applied, with subsequent detection using Warp Red  and Vina Green . Slides were counterstained with Hematoxylin QS , dehydrated, and mounted in Pertex . The presence of ferric iron deposits was assessed by Perl’s Prussian blue stain on adjacent serial plaque sections or fixed cells, performed using Perl’s stain kit,  according to the manufacturers’ instructions, followed by counterstaining with nuclear Fast Red. Images were taken using an Olympus VS200 slide scanner and processed with OlyVIA V3.3 software. ®, Luminex Corp, Austin, TX, USA). Beads coupled with rabbit IgG from nonimmunized rabbits (Bethyl) and beads without proteins immobilized served as negative controls. EDTA plasma samples were centrifuged and diluted (1:50) in assay buffer (PVXcasein + 10% rabbit IgG) and heated for 30 min at 56 °C, followed by incubation with antibody-coupled and negative control beads overnight. The beads were then washed (PBS/0.05% Tween-20), and bound proteins were crosslinked through the addition of 0.4% paraformaldehyde (PFA). Lastly, streptavidin-conjugated fluorophore  was added as detection reagent. The relative abundance of proteins was measured in FlexMap3D instrument (Luminex Corp.), and the resulting median fluorescence intensity (MFI) per bead identity was used for data processing. Samples were randomized and run in duplicate in a 96-well plate, and measurements were averaged for each sample. Samples with a high reactivity with rabbit IgG beads were excluded from the analysis. BLVRB levels are expressed as the mean fluorescence intensity using arbitrary units. Optimization of this method was performed in our previous publication in the supplementary files  [Plasma BLVRB levels were analyzed from both human and mouse blood samples. The affinity proteomic reagent targeting BLVRB (HPA041937) was obtained from the Human Protein Atlas (HPA) project and was used to detect BLVRB levels as previously described . In briegure IV) .® SPSS software version 28.0.0.0 . Patients with baseline blood samples taken no more than 3 months following their neurological symptoms were included. Patient plasma samples with cross-reactivity with the rabbit IgG antibody were also excluded from the analysis (t-test) was conducted to compare BLVRB levels between patient groups. Logistic regression analysis was applied to evaluate the association of BLVRB levels with the presence of IPH adjusted for age and sex. Pearson’s correlation analysis was performed to explore associations between BLVRB levels and continuous clinical parameters measured in this study. Cox regression was used to evaluate the association between BLVRB levels and the occurrence of a recurrent clinical ipsilateral ischemic stroke during 5 years follow-up (primary endpoint), as well as the occurrence of recurrent clinical ipsilateral ischemic stroke, TIA, or AFX during 5 years follow-up (secondary endpoint). Kaplan–Meier curves and life tables were generated to describe survival probabilities for the primary and secondary endpoints. In the mouse study, a paired t-test was used to compare plasma BLVRB levels between baseline and the end of the study separately within the VEGFR-2- and IgG-treated groups. Statistical significance was defined at p < 0.05 (two-tailed).Statistical analysis was performed using IBManalysis . BLVRB pp < 0.001). There was a nonsignificant trend for more IPH in patients admitted with stroke compared to patients with TIA or patients with AFX  (p = 0.022), s well as in patients with stroke compared to those with TIA or AFX combined   . However= 0.035) . p = 0.027) and had a significantly higher degree of stenosis estimated by the NASCET criteria  compared to patients with no IPH. Of note, LDL-C levels were lower in patients with IPH compared to patients without IPH   . n = 161), 20 blood samples showing high reactivity with the detection antibody (rabbit IgG) were excluded from the plasma analysis (n = 141), plasma BLVRB levels were significantly higher in patients with IPH compared to those without IPH (737.3 ± 693.2 vs. 520.9 ± 499.4 mean fluorescence intensity (MFI), p = 0.033) (p = 0.057) due to the higher presence of IPH in men vs. women . However, plasma BLVRB levels were not significantly different between males and females  . In cont= 0.033) . In addi3  or other parameters assessed by MRI including the lipid-rich necrotic core size , volume of fibrous tissue , or calcification volume .Of note, plasma BLRVB levels did not correlate with IPH volume in mmp = 0.223) (n = 126) compared to patients admitted with AFX (n = 15), this did not infer statistical significance . However, for patients admitted with stroke or TIA upon admission only (excluding those admitted with AFX), patients with IPH had significantly higher levels of plasma BLVRB levels compared to those without IPH .We found no association between the severity of symptoms upon admission  and plasma BLVRB levels (= 0.223) . Likewisn = 11) of the patients developed a recurrent ischemic stroke. These patients had significantly higher levels of plasma BLVRB on admission compared to those without recurrent ischemic stroke . This association remained statistically significant following adjustment for age and sex  (p = 0.042); however, this association did not remain statistically significant once adjusted for age and sex (p = 0.075)  B,C. Of n= 0.075) D,E.p = 0.016) but not recurrent TIA (p = 0.213) or recurrent AFX (p = 0.225). However, plasma BLVRB levels significantly associated with the combined endpoint of recurrent ischemic stroke and TIA after excluding AFX . Stratification of the secondary endpoint to associate plasma BLVRB levels with recurrent ischemic stroke, TIA, or AFX separately showed that plasma BLVRB levels were significantly predictive of recurrent ischemic stroke , BLVRB levels significantly predicted the secondary composite outcome  , but not in those admitted with AFX (n = 15), (p = 0.503). Of note, no patients admitted with AFX experienced recurrent ischemic stroke upon follow-up.Next, we investigated if the severity of symptoms upon admission was associated with plasma BLVRB levels and recurrent events. In patients admitted with a stroke or TIA (p = 0.004), whereas no significant difference was seen in the IgG-treated group  compared to baseline (p = 0.051) (p = 0.272), where plasma samples were collected before the surgery and before randomization.In the mouse vein graft model, plasma BLVRB levels were significantly lower in the VEGFR-2 inhibitor group compared to baseline  . Of notein a clinical setting, we demonstrated that plasma BLVRB levels were associated with the presence of IPH in patients with symptomatic, low- to moderate-grade carotid stenosis and with recurrent ischemic stroke during a 5 year follow-up. Second, in a preclinical setting, we showed reduced plasma BLVRB levels following VEGFR-2 inhibition of IPH, highlighting the ability to monitor response to pharmacotherapy targeting plaque neovessel formation and IPH. Together, these results strengthen the potential of plasma BLVRB as a future biomarker for plaque instability.In this study, we evaluated the potential of plasma BLVRB as a biomarker for IPH, plaque instability, and recurrent ischemic cerebral events across multiple platforms. First, First, we found an association between plasma BLVRB levels and the presence of IPH, as assessed by MRI in patients with low- to moderate-grade symptomatic carotid stenosis. This finding suggests that plasma BLVRB measurement can be used for identification of high-risk patients, as the detection of IPH by MRI was previously shown to predict stroke in patients with carotid stenosis ,26, as wPreviously, we reported elevated plasma BLVRB levels in patients with symptomatic vs. asymptomatic carotid stenosis, as well as an association with increasing coronary atherosclerosis as stratified by coronary artery calcification scoring . Here, wIn a dedicated preclinical setup, we showed how plasma BLVRB levels can be successfully used to monitor pharmacotherapy geared toward modulation of IPH through antiangiogenic therapy, a strategy previously shown to inhibit atherosclerotic plaque neovessel formation and IPH in animal models ,32. HereA few limitations should be pointed out. First, a comparably small cohort size of symptomatic patients was utilized, along with a low number of recurrent ischemic events recorded, warranting replication of our findings in larger cohorts. Additionally, as seen in many other CVD trials , our cohThis study strengthens the potential value of plasma BLVRB as a biomarker for unstable carotid atherosclerosis in multiple ways: first, by demonstrating an association between plasma BLVRB and IPH in a clinical cohort of patients with symptomatic carotid atherosclerosis; second, by indicating an independent association with the incidence of recurrent ischemic stroke in the same cohort; third, by showing reduced plasma BLVRB levels following inhibition of plaque neovascularization and IPH development in a preclinical mouse model. Together, these results strengthen the potential clinical utility of plasma BLVRB measurement in ischemic stroke risk prediction of patients with carotid atherosclerosis, as well as in monitoring of drug response during the development of pharmacotherapy for plaque stabilization and prevention of IPH. IPH is a vulnerable atherosclerotic plaque feature and a major predictor of cardiovascular events. IPH detection is possible by MRI but remains expensive. Plasma BLVRB measurement could offer added value for IPH detection and ischemic stroke risk prediction of patients with carotid atherosclerosis. Moreover, during the development of antiangiogenic pharmacotherapies for prevention of IPH and plaque stabilization, plasma BLVRB could potentially be used to monitor drug response.


Camu camu (CC) is a prebiotic that selectively stimulates growth and activity of beneficial gut microbiota. Work in murine models demonstrated that castalagin, the active compound in CC, preferentially binds to beneficial gut microbiome bacteria, promoting a stronger CD8+T cell anti-cancer response. We present two patients with metastatic melanoma whose cancer progressed on immune checkpoint inhibitors (ICIs) and developed clinically significant immune-related adverse events (irAEs). They were rechallenged with ICIs in combination with CC. The first patient is a 71-year-old woman with metastatic melanoma, whose ICI treatment was complicated by immune-related pneumonitis and colitis. Upon progression on maintenance nivolumab, CC was added to nivolumab, leading to a near complete response (CR). The second patient is a 90-year-old man with recurrent unresectable melanoma, treated with nivolumab, complicated by immune-related rash and diabetes. He developed new subcutaneous calf lesions and a metastatic popliteal lymph node. CC was added to nivolumab. One month later, the patient experienced a CR. Both patients have been on nivolumab and CC with durable responses for more than a year, with minimal irAEs. These two cases suggest that CC may modulate the microbiome, synergizing with ICIs to produce deep, durable responses with minimal irAEs. Ruminococcus bromii) and increases their abundance. This promotes an increase in the cluster of differentiation 8 (CD8+)/cluster of differentiation 4 (CD4+)/forkhead box P3 (FOXP3+) T cell ratio and enhances the anti-cancer response to anti-programmed cell death protein 1 (anti-PD-1) agents in solid tumours in mice .RCC NCT052546 [p =cal data . MET4 wasiveness ,14,15. The results of our patients in this case series support a potential novel, easily accessible strategy of microbiome modulation for immunotherapy-refractory patients, which has been confirmed in prospective studies NCT05303493). We presented two cases of patients with metastatic melanoma who had disease progression on ICIs and then durable deep responses with the continuation of ICIs and the addition of CC. These cases support preclinical models of CC or its active component, castalagin, enabling re-sensitization to ICIs. Both patients had previous responses to ICIs , and it is unlikely that the tumour response with addition of CC was due to pseudoprogression, given that both patients were treated with ICIs for at least 5 months prior to progression. It is possible that the gut microbiome had been altered unfavourably in these patients, thus leading to progression on ICIs. For example, during her previous lines of therapy, patient #1 received five courses of antibiotics  for cellulitis and respiratory infections, which may have caused significant alterations in her gut microbiome. This is consistent with extensive published data demonstrating an association of antibiotic use with ICIs and worse survival outcomes, particularly in melanoma and renal cell carcinoma, which was confirmed in systematic reviews and meta-analyses ,8,9.3. We preRuminococcus bromii, which is associated with improved anti-tumour responses, by preferentially binding to the cellular membrane of Ruminococcus bromii and increasing its abundance. Abundance of this bacteria has been correlated with the dose-dependent anti-tumour effect of castalagin [In mouse models, CC supplementation is associated with antitumour activity that is microbiome-dependent and circumvents the anti-PD-1 resistance found in mice that received fecal matter transplant (FMT) from non-responsive patients with non-small cell lung cancer (NSCLC) . Castalastalagin . This case report supports further study of CC as a less expensive, less cumbersome, and more easily scalable alternative to FMT for immunomodulation of the microbiome to achieve improved responses to ICIs. It may also have a lower risk of adverse effects, compared to FMT, which is known to be associated with diarrhea, risk of transmitted infections, and hypophosphatemia ,4. CurreOf note, the rapidity of progressive disease may be a limiting factor for successful use of prebiotic modulation of the microbiome for patients with ICI-refractory cancer. Both of our patients had progression, but not rapidly progressive disease. Therefore, we were able to continue with immunotherapy while waiting for the effects of re-sensitization to ICIs by microbiome modulation.Given that our two patients had histories of significant irAEs prior to ICIs, their minimal irAEs on ICIs with CC raise interesting questions about irAEs and microbiome modulation with CC. In vitro and in vivo models show a correlation of CC with downregulated expression of proinflammatory cytokines and chemokines, such as those associated with the mitogen-activated protein kinases/activator protein-1 pathway, the nuclear factor kappa-light-chain-enhancer of activated B cells pathway, and the nuclear factor of activated T cells pathway ,17. DecrCC is a prebiotic that has demonstrated an additive effect to anti-PD-1 activity in mouse tumour models. There is evidence that the active component of CC, castalagin, shifts the microbiota composition and the systemic metabolite profile, and it has the potential to circumvent anti-PD-1 resistance in mouse models. Here, we presented two patients with ICI-refractory metastatic melanoma, who experienced durable CR/near CR after CC was added to their ICI regimen, with minimal irAEs. A phase I clinical trial is underway to further evaluate the therapeutic role of CC for ICI-refractory advanced melanoma and CC added to standard-of-care first line ICIs for advanced melanoma and NSCLC (NCT05303493). CC, or the active ingredient, castalagin, may prove to be a cost-effective, easily administered microbiome-modulating agent that synergizes with ICIs to induce and maintain durable tumour responses to ICIs.