Circular DNAs, such as most prokaryotic and phage genomes, are a frequent form of nucleic acids, whereas circular RNAs had been regarded as unusual macromolecules until very recently. The first reported RNA circles were the family of small infectious genomes of viroids and circular RNA (circRNA) satellites of plant viruses, some of which contain small self-cleaving RNA motifs, such as the hammerhead (HHR) and hairpin ribozymes. A similar infectious circRNA, the unique human hepatitis delta virus (HDV), is another viral satellite that also encodes self-cleaving motifs called HDV ribozymes. Very recently, different animals have been reported to contain HDV-like circRNAs with typical HDV ribozymes, but also conserved HHR motifs, as we describe here. On the other hand, eukaryotic and prokaryotic genomes encode sequences able to self-excise as circRNAs, like the autocatalytic Group I and II introns, which are widespread genomic mobile elements. In the 1990s, the first circRNAs encoded in a mammalian genome were anecdotally reported, but their abundance and importance have not been unveiled until recently. These gene-encoded circRNAs are produced by events of alternative splicing in a process generally known as backsplicing. However, we have found a second natural pathway of circRNA expression conserved in numerous plant and animal genomes, which efficiently promotes the accumulation of small non-coding RNA circles through the participation of HHRs. Most of these genome-encoded circRNAs with HHRs are the transposition intermediates of a novel family of non-autonomous retrotransposons called retrozymes, with intriguing potential as new forms of gene regulation. Covalently closed circular RNA molecules (circRNAs) have been historically regarded as a peculiar form of nucleic acids. However, circular DNAs, such as plasmids or many bacteriophage, viral, plastid and prokaryotic genomes, are ubiquitous molecules among all areas of life. This view has changed dramatically in recent years with the discovery of new examples of circRNAs of diverse origin, with intriguing regulatory and biotechnological capabilities. Now, we know the existence of several types of circRNAs, such as infectious agents with circRNA genomes, self-excising Group I and II introns, the circRNA intermediates of non-autonomous retrotransposons with ribozymes (retrozymes) or the intronic/exonic circRNAs . It is sPospiviroidae), these circRNAs encode small self-cleaving RNA motifs or ribozymes in either one or both of the genomic polarities  and the hairpin (HPR) ribozymes (see below), catalyze a transterification reaction in a specific point of the RNA sequence, which promotes the cleavage of the multimeric copies during their rolling circle RNA replication. A somewhat similar infectious agent with a circRNA genome is a satellite RNA associated with the human hepatitis B virus (HBV), the so-called hepatitis delta virus (HDV) B 8]. Th. Th8]. T mammals ,12,13,14 mammals . The HDV mammals , snakes Pospiviroids, the heterogeneous group of infectious circRNAs shares the presence of small self-cleaving motifs  as a common key feature among these mobile genetic elements. These data, together with the discovery of genome-encoded circRNAs with ribozymes in eukaryotes, open a more complex scenario than that previously imagined for the family of infectious circRNAs such as viroid-like and HDV-like agents. In this regard, the classical association of HDV with HBV would likely be specific to humans, whereas new metazoan HDV-like agents could be either examples of viral satellite RNAs or even a novel family of autonomous viroid-like agents in animals.Altogether, and with the sole exception of plant Among other open questions regarding these minimal infectious agents, an especially intriguing one relates to their evolutionary origins. Historically, they were reported almost 50 years ago as minimal replicating entities at the frontiers of life. This uniqueness, followed by the hypothesis of ribozymes as the most ancient biocatalysts, fueled the theory that infectious circRNAs could be “living fossils” from the prebiotic RNA world ,17,18,19Tetrahymena thermophila [The first example of a natural circRNA encoded in a DNA genome was the intervening sequence of the rRNA precursor of rmophila . The charmophila . Group Irmophila . Althougrmophila . Anotherrmophila ,27.In 1993, the first examples of circRNAs derived from protein-coding genes were reported in mammals ,29, whicThere is a second natural pathway of circRNA production in eukaryotes. In our lab, we have reported the expression of abundant circular RNAs encoded in the genomes of plants ,41 and mMore than 30 years ago, the laboratories of Tom Cech and Sydney Altman reported the ground-breaking discovery of catalytic RNAs or ribozymes ,43. ThisAmong natural ribozymes, the family of small self-cleaving RNAs 50–200 nt) is likely the most enigmatic group of catalytic RNAs. These motifs catalyze a sequence-specific intramolecular transesterification that promotes the breakage (or ligation) of the RNA chain. There are so far nine different classes of small self-cleaving ribozymes described, such as the HHR ,47, HPR –200 nt iMore recently, different labs have reported that HHR motifs are widespread from prokaryotic to eukaryotic genomes ,63,64,65Finally, classical type I and III HHRs , abundanA. thaliana [Met of the plant, and finishes with a poly-purine tract (PPT), which are both characteristic elements of LTR-retrotransposons [In 2005, Hammann’s group reported the existence of two canonical type III HHRs in the genome of the plant thaliana . Both mothaliana . Later othaliana . The ribthaliana . Comparansposons . Altogetnsposons  or smallnsposons . There ansposons   of circular and, to a lesser extent, linear RNAs corresponding to the sequence encompassed by the HHR motifs. These results confirmed that retrozymes are transcribed in most plant tissues, but also that the HHRs self-cleave in vivo, followed by RNA circularization. This step of circularization is efficiently carried out in vitro by a chloroplastic isoform of a plant tRNA ligase , in a siviroidae . MoreoveAcropora millepora), a mollusc  and an amphibian (the axolotl Ambystoma mexicanum), confirmed the accumulation of high levels of circRNAs in all the tissues analyzed [Type I HHRs  were orianalyzed . These cOn the other hand, previous bioinformatic searches reported that many metazoan genomes contain thousands of minimal type I HHRs , which uMet through their PBS motifs, which would prime the reverse transcriptases encoded by LTR-retrotransposons of the Ty3-Gypsy family. In the case of metazoan non-LTR retrozymes, the sequence repeats do not show any conserved motif that could help deduce the autonomous retrotransposon whose transpositional machinery they may utilize. In any case, the most likely pathway for both retrozyme families implies a reverse transcriptase that, properly primed and thanks to the circular nature of the RNA template, would produce cDNAs of more than one unit in length. Finally, the resulting cDNAs would be integrated in new genomic locations through the machinery of the autonomous retrotransposons (The family of plant LTR retrozymes is similar to other small non-autonomous LTR-retrotransposons of plants, such as TRIMs  and SMARnsposons .A last question to be addressed is related to the high levels of circRNAs with HHRs detected in most organisms analyzed, which suggest that these RNA circles may have other biological functions different from their own mobilization. The discovery of splicing-derived circRNAs involved in diverse forms of gene regulation  offers nSelfish mobile genetic elements, such as transposons, viruses and other subviral agents, can be found in virtually every cellular life form. These genetic parasites and their interaction with their hosts are a major feature of life evolution . On the Regarding the origin of this group of circRNAs with ribozymes in eukaryotes, multiple options are possible. We have already proposed a likely scenario where plant LTR retrozymes may have given rise to infectious circRNAs such as viroids and viral satellites with ribozymes . Regardi


The overhunting of wild species is a major threat to biodiversity in the Amazon; yet, managed, sustainable hunting is widely considered part of the solution to conserving wildlife populations. Hunting is both a culturally important activity for Indigenous people and provides an important food source. Mineral licks, a focal point of hunting in Amazonia, are naturally occurring areas in the forest where animals come to obtain essential minerals or clays that are thought to neutralize plant-based alkaloids. We sought to better understand the socio-cultural importance of mineral licks to the Maijuna Indigenous group to inform the sustainable management of this habitat and associated wildlife populations.Semi-structured interviews, focus groups, and participatory mapping were carried out with hunters to assess the significance of mineral licks and their associated animal resources as well as to determine how the relationship that the Maijuna have with mineral licks has changed over time.Mineral licks are culturally significant and useful to the Maijuna in a variety of ways. Hunters target these areas year-round both during the day and night, and animals killed are consumed for subsistence and sold to generate income. The spatial use of mineral licks across the landscape is determined on the generational family level, with families maintaining exclusive use of selected mineral licks and excluding access by other hunters. The Maijuna also have traditional beliefs for why animals visit mineral licks, which is linked to the traditional Maijuna story of the creation of the first tapir. The relationship that the Maijuna have with mineral licks has changed considerably over time, which is observed through changes in hunting technologies and methods as well as the loss of traditional knowledge and beliefs.Traditional and current Maijuna hunting conventions, in which families maintain exclusive use of selected mineral licks, likely reduce the probability of overexploitation of animal populations. Community-based management plans for mineral licks in Maijuna lands and beyond must incorporate and account for the multiple cultural and economic needs of local communities while also striving toward ecological sustainability. Country-wide strategies to conserving forests and using them sustainably should aim to ensure land tenure for rural peoples and encourage management that incorporates traditional sustainable hunting conventions. Tapirus terrestris), white-lipped peccary (Tayassu pecari), and red brocket deer (Mazama americana), and the large rodents, paca (Cuniculus paca), usually making up the largest proportion of kills [Hunting threatens wild mammal populations in the Amazon basin , but is of kills . The larof kills . Huntersof kills .Most large mammals in Amazonia visit mineral licks. Mineral licks are naturally occurring areas in the forest where animals exhibit geophagical behavior to obtain essential minerals lacking in their diet  or claysThe Maijuna-Kichwa Regional Conservation Area (MKRCA) is one of the Peruvian Amazon’s newest protected areas, comprising 391,039.82 ha of Maijuna Indigenous territory. The Maijuna are one of the smallest and most vulnerable Indigenous groups in Peru . Their aThe MKRCA is a powerful example of community-based conservation. The Maijuna originally conceived the idea for conserving this area, and they pushed relentlessly for its creation. After centuries of being marginalized and disempowered by outsiders, the Maijuna established an Indigenous federation in 2004 focused on conserving their lands and culture . AgainstUnderstanding the socio-cultural importance of mineral licks to the Maijuna, and the way they are currently and traditionally used are the first steps in understanding how wildlife populations are affected by hunting around mineral licks and whether mineral lick management might be a useful strategy in conserving mammal populations and safeguarding food security in rural Amazonia. Such understanding is also required to ensure that any management plans that are ultimately developed for the MKRCA incorporate and account for the multiple cultural and economic needs of the Maijuna while also striving toward ecological stability. Here we assess the significance of mineral licks and their associated animal resources to the Maijuna and document changing relationships between the Maijuna and mineral licks over time.The Maijuna, also known as the Orejón, are a western Tucanoan people of the northeastern Peruvian Amazon . Today, Data for this study was collected from 1999 to 2019 in the Maijuna community of Sucusari  was obtained from the Sucusari community as well as from individual research participants before beginning the interviews for this study. From 1999 to 2019, semi-structured interviews  were conAdditionally, in July 2017, a focus group was conducted with 16 hunters  to identify the locations of mineral licks in the Sucusari River basin using participatory mapping techniques , 25. Maptùàràónóbɨ (place of masato) in the Maijuna language and collpa in the local Spanish dialect. Mineral licks are one of dozens of different forest habitat types defined and classified by the Maijuna. They classify forest habitats based on geomorphology, physiognomy, disturbance, indicator plant species, and indicator animal species [Mineral licks are called  species . Accordi species , 28. In  species , 30.C. paca), collared peccary (T. tajacu), white-lipped peccary (T. pecari), red brocket deer (M. americana), and tapir (T. terrestris), all of which are well documented in the literature for visiting mineral licks [Mineral licks are culturally and economically important to the Maijuna because a number of game animals visit these sites and hunters target these areas year-round for hunting. These mammal and bird species are either diurnal or nocturnal; therefore, Maijuna hunters target mineral licks both day and night revealing an important temporal relationship with these areas Table . Hunted al licks , 9, 28. Hunting at mineral licks takes several forms. While walking trails on hunting trips during the day and night, hunters will quickly stop by mineral licks along their routes to see if they can opportunistically catch game species in the lick and to also check for signs of animal activity . Additionally, hunters will wait alone for several hours at a time at licks with signs of animal activity during both the day and night. They lie in wait at strategic spots above licks with good views and clear shots of the area. At these strategic spots, they will either sit on the ground, make a hunting platform, or string up a hammock between two trees. At night, they lie in wait with their flashlight off listening for animal activity and, according to hunters, they can tell the species entering the lick by the amount and type of noise that it makes as it walks through the mud and standing water to the lick “face.” If it is a desirable species, the hunter will then attempt to ambush the animal by turning on their flashlight when it has reached a desirable location in the lick with a clear shot.Eighty-four mineral licks were identified and had their locations fixed by GPS in the Sucusari River basin during this study. Of these, 43 were regularly visited by hunters during the 11-month period from August 2018 to June 2019. However, during this time period, all hunters did not regularly or equally visit each of these 43 licks. Instead, clear spatial use of 38 of these mineral licks is observed when analyzing the data by family, with each family having distinct mineral licks that they regularly hunt at that others do not enter  reA separate participatory mapping project carried out with the Maijuna between 2004 and 2009 see , 32–34) –34 32–34Out of the 84 mineral licks that were ultimately identified in the Sucusari River basin during this study, 21 have proper names in the Maijuna language. The Maijuna name mineral licks after plants found in or around the area, individuals who hunted there, birds or bats that frequent the lick, general vegetative characteristics, soil type, biting ants present, and the size of the area. The extensive naming of mineral licks further highlights their significance to the Maijuna , 25, 35.Békɨtù was seeking revenge on his son-in-law. He tried to trick him three times, yet his son-in-law outsmarted him each time. After the third time, the son-in-law used his powers as a creator to turn Békɨtù into a tapir. During this slow transformation, Békɨtù began to take on more and more qualities of tapirs; he increased in size, made similar noises, began to eat Mauritia flexuosa fruits whole, and became skittish around people. Toward the end of this transformation, Békɨtù asked his daughter, who was unaware of what was happening, to make masato  and bring it to him in the forest. When his daughter returned, he grew increasingly skittish, and eventually refused to come close when she approached, despite her repeated assurances. The exasperated daughter threw her clay pot of masato on the forest floor, where it shattered. Békɨtù later came and licked up the masato, using his hooves to further mix up the brew and ultimately soften the moist earth, the very first tapir forming the very first mineral lick. In the Maijuna language, the word for masato is ónó and the word for place is bɨ. A mineral lick, ónóbɨ, literally means “place of masato,” which is why animals are attracted. The Maijuna believe that the different animal species that visit mineral licks are there specifically to drink masato.The Maijuna have a very different explanation for why animals visit mineral licks . AccordipatronesFirst contacted by Jesuit missionaries in 1682, the Maijuna’s population declined dramatically due to epidemics, fighting, and then enslavement during the rubber boom. Over the years, Maijuna traditional beliefs, language, and ways of life have been undermined by missionary teachings, Due to these realities, the relationship that the Maijuna have with mineral licks has changed considerably over time. For example, they traditionally hunted game animals at mineral licks with spears only during the day. Over the past 100 years, the introduction of shotguns in the Maijuna communities has drastically increased hunting efficiency over traditional methods . The acqThe Maijuna’s changing relationship with mineral licks is also evident in both the naming of and traditional beliefs about licks. Out of the 19 active hunters interviewed for this study in the Maijuna community of Sucusari, only four (21%) know the names of mineral licks in the Maijuna language. The rest use exclusively Spanish names for mineral licks in the Sucusari River basin, which are sometimes direct translations from the name in Maijuna but most of the time are not. This is not only due to the fact that the Maijuna language is highly endangered and in disuse by younger Maijuna generations , but alsAdditionally, although most of the 10 active hunters that are Maijuna know of or have heard the traditional Maijuna story of the creation of the first tapir, only two (ages 65 and 68) know the story in significant detail and are able to tell it in the Maijuna language. This loss of knowledge and traditional beliefs is significant given that traditional stories symbolize unity and the creation of shared bonds for Indigenous people . Most trThrough interviews, focus groups, and participatory mapping, we were able to gain a comprehensive understanding of the importance of mineral licks to the Maijuna. Mineral licks are important hunting areas for both subsistence and income generation, and their spatial use across the landscape is determined on the family level. Additionally, the classification and naming of licks as well as traditional beliefs about them further highlights their socio-cultural significance to the Maijuna. Learning all of this information is an essential first step for the development of community-based management plans for mineral licks in the MKRCA that incorporate and account for the multiple cultural and economic needs of the Maijuna while also striving toward ecological sustainability. Next steps could include large-scale monitoring of wildlife use of licks, examining the impacts of hunting at mineral licks on wildlife, and assessing the potential of working with local communities to protect licks as a strategy to manage hunting and conserve wildlife populations. Such management strategies are likely to be similarly useful across the wider Amazon basin, where mineral licks are also of considerable cultural and economic importance as hunting sites , 10.Traditional and current Maijuna hunting conventions, in which families maintain exclusive use of selected mineral licks, and exclude access by other hunters, likely reduce the probability of overexploitation, especially where hunting is for subsistence rather than commercial purposes. Where Indigenous or local people do not have ownership and control of their hunting grounds, more frequent use of mineral licks by more hunters may occur, increasing the chances of over extraction of wild species. Indigenous land title and the right to restrict access to hunting grounds are key to tackling overhunting in Amazonia.Although it was found that traditional knowledge and beliefs about mineral licks is in steep decline, the Maijuna still remain deeply connected to their ancestral lands and rely heavily on licks and their associated game species. The role and importance of Indigenous people in conservation is increasing –51, whic


International Journal of Molecular Sciences. In this Special Issue, the readers will find integrative information on transporters for organic cations. Besides reviews on physiology, pharmacology, and toxicology of these transporters [This editorial summarizes the 13 scientific papers published in the Special Issue “Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations” of the sporters ,3, whichSpecifically, the review “Organic Cation Transporters in Human Physiology, Pharmacology, and Toxicology” by Samodelov et al.  summarizIn the paper “Systems Biology Analysis Reveals Eight SLC22 Transporter Subgroups, Including OATs, OCTs, and OCTNs”, by performing a system biology analysis of SLC22 transporters, Engelhart et al.  suggest Drosophila melanogaster was performed [In the other paper by Engelhart et al. “Drosophila SLC22 Orthologs Related to OATs, OCTs, and OCTNs Regulate Development and Responsiveness to Oxidative Stress”, an evolutionary analysis of putative SLC22A transporter orthologs in erformed . At leasNeurotransmitters such as serotonin are important endogenous organic cations. Interestingly, the anesthetic drug ketamine has an antidepressant action. In the paper “Serotonin Transporter and Plasma Membrane Monoamine Transporter Are Necessary for the Antidepressant-Like Effects of Ketamine in Mice”, Bowman et al. investigated whether this effect of ketamine is due to an influence on extracellular serotonin concentration . They deSince organic cation transporters are normally expressed in well-differentiated cells and they can be involved in the cellular uptake and/or efflux of chemotherapeutic drugs, their expression level may be related to the prognosis of cancer clinical outcome. In the communication “Identification of Prognostic Organic Cation and Anion Transporters in Different Cancer Entities by In Silico Analysis”, Bayram Edemir  analyzedAcute regulation of transporter activity can change the exposure of the body to drugs. While the regulation of organic cation transporters is well known, there is only scarce information on Multidrug and Toxin Extrusion Transporters (MATE) regulation. This aspect of MATE function was analyzed in detail in the paper “Rapid Regulation of Human Multidrug and Extrusion Transporters hMATE1 and hMATE2K” by Kantauskaité et al . MATE acThe activity of renal OCT2 and MATE transporters was also regulated by the transcription factor Farnesoid X receptor (FXR), for which activation increased the expression and activity of the transporters, as demonstrated in the paper “Farnesoid X Receptor Activation Stimulates Organic Cations Transport in Human Renal Proximal Tubular Cells” by Wongwan et al. . On the Another mechanism by which transporter expression and function can be altered is by mutations due to the presence of single nucleotide polymorphisms (SNPs). The review “The Impact of Genetic Polymorphisms in Organic Cation Transporters on Renal Drug Disposition” by Zazuli et al.  illustraFocusing on the potential pharmacological role of OCT, in the paper “Tofacitinib and Baricitinib Are Taken up by Different Uptake Mechanisms Determining the Efficacy of Both Drugs in RA”, Amrhein et al.  demonstrRodents are used as a preclinical model to study the biological effects of drugs and xenobiotics. The paper “Functional and Pharmacological Comparison of Human, Mouse, and Rat Organic Cation Transporter 1 toward Drug and Pesticide Interaction” by Floerl et al.  investig


USH2A and ABCA4, correcting RNA offers an alternative to gene replacement. Genome editing of RNA rather than DNA may offer an improved safety profile, due to the transient and potentially reversible nature of edits made to RNA. This review considers the current site-directing RNA editing systems, and the potential to translate these to the clinic for the treatment of inherited retinal degeneration.RNA editing aims to treat genetic disease through altering gene expression at the transcript level. Pairing site-directed RNA-targeting mechanisms with engineered deaminase enzymes allows for the programmable correction of G>A and T>C mutations in RNA. This offers a promising therapeutic approach for a range of genetic diseases. For inherited retinal degenerations caused by point mutations in large genes not amenable to single-adeno-associated viral (AAV) gene therapy such as  Programmable editing of nucleic acids offers significant therapeutic potential for a wide range of genetic diseases. The development of the Clustered Regularly Interspaced Short Palindromic Repeat–CRISPR-associated genes (CRISPR-Cas) system for facile gene editing in mammalian cells has led to a wide range of approaches for gene manipulation including gene silencing, repair and editing . An areaRPE65 gene for Leber’s congenital amaurosis [Due to the retina having relative immune privilege, easy accessibility for vector delivery and non-invasive functional and structural endpoints for assessing treatment efficacy, retinal disorders are excellent candidates for genetic therapies . Gene remaurosis  and manymaurosis . Currentmaurosis . AAV vecABCA4 and USH2A do not fit in a single AAV, and in the USA mutations in these two genes account for 25% of families with inherited retinal degeneration [For the many patients who have a disease-causing mutation in genes too large for the ~4.7 kb packaging capacity of AAV, however, a different treatment strategy is required. The cDNA for genes such as neration . While aneration , a substneration ,10,11,12CEP290 gene, a strategy moving forward to clinical trial this year [Gene editing aims to correct the endogenous genetic sequence rather than delivering a replacement gene. The DNA editing capabilities of enzymes of the class II CRISPR systems such as Cas9 and Cas12 have been widely used for programmable RNA-guided DNA targeting ,14,15,16his year . Correcthis year ,21,22. Ahis year  or primehis year  can relahis year .Unlike DNA, messenger RNA (mRNA) molecules exist transiently within the cell and transmit the genetic information that encodes for the production of proteins. A strategy to edit RNA rather than DNA is, therefore, highly appealing, as this allows for the editing of pathogenic mutations at a transcript level, without the risk of creating permanent off-target mutations in the genome. Furthermore, the transient nature of RNA means that RNA editing is potentially reversible and controlled over time.Recent advances in RNA editing technologies have enabled the development of engineered enzymes capable of either adenosine-to-inosine (A-I) or cytosine-to-uracil (C-U) edits. Site-directed RNA editing uses molecular tools to recruit RNA editing enzymes to target sites of interest and enables the targeted correction of G>A and T>C mutations in coding sequences of RNA. Given that G>A and T>C mutations comprise up to 61% of all pathogenic point mutations in humans , these tHere, we review the potential for using RNA editing enzymes for site-directed RNA editing, and how this might be applied to correct mutations in large genes implicated in inherited retinal degenerations not amenable to AAV gene therapy.The majority of strategies to edit RNA utilize the activity of the naturally occurring human adenosine deaminase acting on RNA (ADAR) enzymes. ADARs act to convert adenosine residues to inosines (A>I editing) in double-stranded RNA. Inosine is structurally similar to guanosine and is read as a guanosine by most cellular machinery including during translation, splicing and reverse transcription, effectively creating an A>G edit in RNA  Figure . Two ADAR enzymes have been identified in humans to carry out A>I editing activity, ADAR1 and ADAR2. ADAR1 is expressed as two isoforms, a shorter 110kDa isoform (ADAR1 p110) and a larger 150 kDa isoform (ADAR1 p150). ADAR1 p110 is constitutively expressed and localizes to the nucleus, while ADAR1 p150 is inducible by activation of the innate immune sensing system and localizes to the cytoplasm . ADAR1 iGria2  in mice [Gria2 remains to be elucidated [A>I editing one of the most common post-transcriptional modifications in humans . Most A> in mice . Loss of in mice . Altered in mice . Howeverucidated .DD) catalyses hydrolytic deamination where an amino group is replaced by a hydroxyl group, converting adenosine to inosine  makes direct contact with double stranded RNA (dsRNA), while a C-terminal deaminase domain , which retains A-I editing capabilities. To develop the scope of RNA editing to other bases, Abudayyeh et al. developed a deaminase capable of cytosine (C) to uracil (U) RNA editing by undertaking mutagenesis of the ADAR2DD . Using iDD(C-U) mutant with a S375A mutation to yield a higher specificity variant with fewer A-I off-targets. This S375A substitution is at the same amino acid position as the higher specificity ADAR2DD mutant (T375G), supporting the role of this position in ADARDD A-I editing specificity [Further mutagenesis was used create a more specific ADARcificity . Unlike First proposed by Woolf and colleagues almost 25 years ago , the A-IDD). Strategies using the whole ADAR enzyme may utilize the naturally expressed endogenous ADAR or introduce this with exogenous expression. Strategies using the ADARDD typically fuse this to another protein effector that is capable of programmable RNA binding to enable site-directed RNA editing. To enable site-directed ADAR editing, it is necessary to create a double-stranded RNA structure at the target region and draw the ADAR to this site. Strategies, therefore, broadly comprise two parts: (1) a guide RNA (gRNA) sequence antisense that can hybridize to the target RNA, and (2) an effector mechanism that recognizes the gRNA and recruits the ADAR enzyme. These strategies may recruit the whole native ADAR enzyme including its dsRNA binding domains or express only the deaminase domain . First described in bacteriophage as an anti-terminator of transcription , the λN DD A. A guid protein . Subsequ protein . Mecp2 gene involved in Rett syndrome in primary murine neurons [DD (E488Q) together with a BoxB-gRNA expression cassette containing six copies of the guide, they demonstrated a 72% on-target correction of a missense Mecp2 mutation and partial restoration of MECP2 protein [Sinnamon et al. have also employed a similar BoxB-λN system delivered via AAV to edit the  neurons . Using a protein . DD (E488Q) [A significant drawback of λN-BoxB systems has been substantial off-target editing events, especially with the 4λN system with ADAR2 (E488Q) . Use of  (E488Q) . DD [DD fusion via the SNAP-BG reaction, and this guides the deaminase to the RNA target  is a naturally occurring substrate strongly recognized by the dsRBD of ADAR2. This was harnessed to engineer an adRNA  composed of the R/G motif fused to an antisense guide sequence. The dsRBD of a full-length ADAR protein recognizes the R/G motif and is recruited to the target site C. First LA cells . Recent LA cells . mdx mouse model of Duchenne’s muscular dystrophy required the editing of two adenosines to correct a termination codon (TAA) to tryptophan (TGG). Intramuscular AAV injection of GluR2-adRNA + ADAR2 (both with and without the E488Q mutation) resulted in TAA>TGG correction in ~0.8% of RNA transcripts and dystrophin partial protein restoration. In the second model, the authors targeted a G>A splice variant in the ashspf mouse model of ornithine transcarbamylase (OTC) deficiency. Systemic injection of GluR2-adRNA + ADAR2 resulted in 4.6–8.2% correction of the pre-mRNA and a reduction in the incorrectly spliced fraction of mRNA, confirmed by a 2.5–5% restoration of ornithine transcarbamylase protein. Correction rates were noticeably higher in those systemically injected with the E488Q mutant. However, the authors observed significant toxicity in these mice not observed in those injected with the wildtype ADAR. While the mechanism for this toxicity has not been established, it raises concerns for the systemic use of the E488Q mutant. As the full length ADAR2 protein is only 2.1 kb, it can be packaged in AAV together with the GluR2-adRNA sequences. Katrekar et al. attempted targeted correction of G>A mutations in two mouse models using AAV8-ADAR2-GluR2adRNA constructs . The corDD fusion protein to the editing site of interest , and deaminases with hyperactive mutations each demonstrated higher editing efficiencies. However, each of these factors also contributed to higher off-target editing rates. In an mdx muscular dystrophy mouse model, an intramuscularly delivered AAV8-MCP-ADAR1DD(E1008Q)-NLS construct delivered with an MS2 gRNA demonstrated 2% on-target efficiency and partial restoration of dystrophin expression [Recruitment of the ADAR deaminase domain has also been achieved using the bacteriophage-derived MS2-MCP tagging system. The MS2 bacteriophage coat protein (MCP) naturally binds the MS2 stem loop from its genome. This MS2 stem loop can be attached to a short guide sequence to create an MS2-adRNA, and this adRNA recruits an MCP-ADARinterest D. Katrekpression . DD system that used a 21 nucleotide gRNA with a single MS2 stem loop was found to have a 7% editing efficiency for a premature termination codon in an EGFP reporter system in HEK293 cells [DD fusion. Another group found that an MCP-ADAR193 cells . This paAlthough editing rates are noticeably improved by the overexpression of exogenous ADARs, evidence from a number of recent papers suggests that recruitment of endogenous ADAR to edit double-stranded RNA is a potentially viable strategy. Initial work by Fukuda and colleagues demonstrated that A to I editing of RNA molecules could be achieved using ADAR guiding RNAs (AD-RNA) and recombinant ADAR in an in vitro reaction . The cusPPIB, a chosen candidate endogenously expressed gene that encodes a cyclophilin enzyme. Long 111 nucleotide guides, chemically stabilized with 2′-O-methyl and phosphorothioate linkage modifications, were also electroporated into fibroblasts derived from patients with Hurler syndrome (a severe form of mucopolysaccharidosis type 1) with a Trp402Ter mutation. This restored previously deficient IDUA enzyme activity and was found to have higher A>I editing activity and enzyme activity restoration when targeting pre-mRNA (30%) compared to mRNA (10%). Lentiviral delivery of LEAPER guides was also attempted but only achieved 6% editing rates in the 5′UTR of PPIB in HEK293 cells [The LEAPER editing system (Leveraging Endogenous ADAR for Programmable Editing of RNA) developed by Qu et al. utilizes their observation that expression of long RNA (71–191 nucleotide) guides creates regions of dsRNA long enough to enable endogenous ADAR1 binding and editing . LEAPER GAPDH of 4–34% across a range of immortalized cell lines and 10–63% in primary cell lines were observed, but editing in the open reading frame was not possible. Further engineering and lengthening (40 nucleotide) of the ASO enabled a 10–20% correction of an α-1-antrypsin deficiency mutation in the open reading frame of the SERPINA1 gene in HeLa cells, and a 7–21% editing rate of tyrosine 701 in the STAT1 gene in primary fibroblasts and RPE. They noted all editing rates could be improved by induction of expression of the ADAR1p150 isoform by administration of interferon alpha [In contrast to the long guides of the LEAPER system, the RESTORE editing system (Recruiting Endogenous ADAR to Specific Transcripts for Oligonucleotide-mediated RNA Editing) recently published by Merkle and co-workers uses shorter 20–40 nucleotide chemically modified anti-sense oligonucleotides (ASO) with an R/G ADAR recruiting domain to edit transcripts with endogenous ADAR1 . Using 2on alpha . From these studies, it appears that the recruitment of endogenous ADAR is possible either with long gRNAs or with recruitment domains. Minimal editing activity is seen when using antisense guides without recruitment domains that are less than 60 nucleotide  or 70 nuThe type VI CRISPR nuclease Cas13 binds to single stranded RNA directed by a programmable CRISPR RNA (crRNA) guide ,59,60,61Prevotella sp. P5-125 (PspCas13b) fused to ADAR2DD to create a programmable RNA editor termed REPAIR (RNA Editing for Programmable A to I Replacement) [Using the capacity of dCas13 to create targeted double-stranded RNA substrates, Cox and colleagues used a Cas13b orthologue derived from acement) . A guideA key advantage of Cas13 is that, unlike Cas9 and Cas12 nucleases, Cas13 does not require a protospacer adjacent motif (PAM) sequence local to the gRNA binding site ,63. AlthDD(E488Q) (dPspCas13b-ADAR2DD(E488Q)) and displayed editing rates of 10–40% across a range of overexpressed gene fragments and up to 29% of two endogenously expressed RNA transcripts [REPAIRv1 uses the hyperactive ADAR2nscripts . The REPDD(E488Q) can produce numerous off-target editing events, so further rational mutagenesis was undertaken to generate a more specific ADAR2DD(E488Q/T375G) deaminase fused with dPspCas13b (termed REPAIRv2). This modification produced a 900-fold decrease in off-target events, however, with an approximate 2-fold loss of on-target efficiency [DD has not been evaluated in many other systems but was reported to have low efficiency in the SNAP-tag system [As previously observed, the hyperactive ADAR2ficiency . This dog system .DD-E488Q mutant editing at dsRNA sites. It is likely, however, that a majority of the RNA editing rates observed using Cas13-ADAR editing are due to recruitment by the guide RNA. The direct repeat hairpin of the gRNA is proposed to recruit the Cas13-ADAR fusion to the target site. Vogel and colleagues found that they could achieve low editing rates with a 50 nucleotide guide without the 35 nucleotide direct repeat (DR) region using the REPAIRv1 construct . RNA ediDD-E488Q/T375G) was compared to the λN-BoxB-ADARDD (E488Q) and similar on-target efficiencies were observed but as expected when using the E488Q ADAR, λN-BoxB had substantially more off-targets [RNA editing with REPAIR constructs has been directly compared to other genetically encodable RNA editing systems relevant for clinical use. REPAIRv2 (dCas13b-ADAR-targets . Direct -targets .DD to create the ADARDD(C-U) described earlier. Fused with a different Cas13 orthologue, deactivated Cas13 from Riemerella anatipestifer (dRanCas13b), the resultant dRanCas13b-ADAR2DD(C-U) was named “RESCUE”. They reported C-U editing efficiencies of up to 30% of endogenous transcripts and up to 42% of synthetic reporter constructs in HEK293FT cells. RESCUE editing was also used to activate STAT and Wnt/β-catenin pathways and alter cell proliferation phenotypes through C-U editing of key phosphorylation sites in transcripts in HEK293FT and human umbilical vein endothelial cells (HUVECs). The RESCUE editor still maintains A-I editing activity and is capable of multiplexed editing of the same transcript with different guides to achieve both A-I and C-U editing. As seen with REPAIR, RESCUE also demonstrates A-I off-targeting, as well as C-U off-targeting although to a lesser extent. RNA Editing for Specific C to U Exchange (RESCUE) is a cytosine (C) to uracil (U) RNA editing platform based on the work of Abudayyeh et al., created through serial mutagenesis of the ADARDD(C-U), and this system was termed RESCUE-S. This yielded an approximately 12-fold reduction in A-I off-targets and ~1.8-fold reduction in C-U off-targets while maintaining specificity. Further mutagenesis was used to create a S375A mutation in the ADARDD(E488Q) was delivered via plasmid transfection to HEK293T cells and demonstrated 47% efficiency repairing a premature termination codon in a luciferase reporter. While more work is required to validate this in other cells lines, on endogenous targets and in vivo, due to its small size (<3kb), the CIRTS system could easily be delivered with AAV [Recent work has also been undertaken to engineer a synthetic RNA targeting system made from human-derived proteins that mimic the gRNA binding, ssRNA recognition and effector functions of Cas13 proteins . The CRIwith AAV . In theory, base editing might be used to correct any gene implicated in inherited retinal degeneration with G>A or T>C single-base mutations. Some diseases may be more amenable to RNA editing than others, however. Dominant diseases require knockdown or correction of mutant alleles. It is likely that for dominant diseases, in vivo editing rates higher than those that are currently observed will be required for a therapeutic effect, while small recessive genes are probably more effectively treated with AAV gene replacement. The most immediate application, therefore, is for recessive genes larger than ~4.2 kb, which are unlikely to be treatable with AAV-mediated gene replacement. ABCA4 and USH2A mutations accounting for the vast majority. The most common large genes implicated in recessive inherited retinal degeneration and their relative frequencies in the cohort of 1000 consecutive patients diagnosed with inherited retinal degeneration by Stone et al. are shown in CEP290 to 32% in ABCA4. Many of the mutations within ABCA4 are missense mutations (18%), while donor/acceptor splice mutations (8%) and the creation of premature stop codons (6%) are more common unique pathogenic mutations in USH2A. Overall, there are a greater proportion of G>A than T>C mutations. In both ABCA4 and USH2A, there are at least 75 unique pathogenic G>A mutations. Of all mutations in these genes, a proportion are amenable to single-nucleotide base editing with the currently available tools. G>A mutations are among the most common single-nucleotide mutations, with over 20,000 in the genome . An evolOf all targetable mutations, G>A mutations in tryptophan codons that produce premature stop codons such as TGG>TGA/TAG/TAA (p.Trp>Ter) are the most obvious initial targets as they are more easily defined as pathogenic, and restoration of the full-length mRNA should restore protein expression. In theory, the spliceosome will recognize inosine at the same strength and manner as guanosine if donor/acceptor splice sites are repaired, but this remains to be further investigated. Missense mutations will need to be carefully targeted on a case by case basis. USH2A: the common c.2299delG mutation and the c.11864G>A which result in a codon change from tryptophan (TGG) to a premature termination codon (TAG). A 50-year-old female presented for further investigation of her deteriorating vision. Diagnosed with congenital bilateral moderate deafness aged four, she subsequently developed increasing nyctalopia and was diagnosed with syndromic retinitis pigmentosa at age 15. Her parents and children are unaffected, but her brother experienced deafness and vision loss from an early age. On assessment, she was bilaterally pseudophakic, and her best corrected visual acuity was reduced to 6/24 bilaterally. Fundus examination revealed moderate pigmentary retinopathy and narrowing of the retinal vasculature  and optiUSH2A cDNA encoding the c.11864G>A mutation into a plasmid driven by a ubiquitous promoter. A 50 bp guide RNA with a 32 bp distance between the direct repeat and a mismatched cytosine was designed to target the mutation. HEK293T cells were transfected with the USH2A plasmid, guide RNA and the REPAIRv2 expression construct dPspCas13b-hDAR2DD(E488Q/T375G). At 48 h post transfection, cells were harvested, and isolated RNA was sent for Sanger sequencing. Analysis of the Sanger sequencing peak heights with trace decomposition revealed 43% correction of the target adenosine, repairing the termination codon to tryptophan. No off-target editing of adjacent adenosines was observed 100 bp either side of the mutation. Given that this was a targetable mutation with RNA editing, we cloned a synthetic sequence of exon 60 of As RNA editing rates are incomplete, ranging from 1% to 80% editing across different targets, cell lines and expression systems, diseases where partial restoration of a functional protein are required are ideal targets. While the level of correction required for the adequate expression of Usherin protein expression remains unknown, given the large size and stability of the Usherin protein in the photoreceptor cilium, it is possible that only a small amount of protein is required to improve or slow down degeneration.The potential for RNA editing has now been demonstrated in vitro and in vivo for pathogenic mutations in genes related to cystic fibrosis, Duchenne’s muscular dystrophy, Hurler’s syndrome, and Ornithine transcarbamylase (OTC) deficiency, among others ,49,54,74RNA editing may be advantageous compared to genome editing when moving towards clinical translation. Due to the transient nature of RNA transcripts, RNA editing is potentially reversible or titratable depending on the formulation or delivery of the editing system. For example, inducible RNA editors, systems with a built in “off” switch, or systems that require continual dosing such as using ASOs, have inbuilt safety mechanisms that allow a finer degree of control than DNA editing. This is balanced, however, against the attractiveness of a single-treatment DNA editing approach, and different approaches may suit different clinical scenarios. As ADAR proteins are of human origin, there are less concerns for their immunogenicity, although this may still be a factor with construct containing elements such as the viral origin MS2-MCP and bacterial origin CRISPR-Cas systems. Immune reactions to editing proteins may neutralize their editing efficiency or cause potential harmful immune responses. For therapeutic efficacy and safety, sufficient and persistent on-target editing will need to be balanced against rates of off-target editing. Although off-target effect will occur in transitory transcripts, further work is required to understand where and how often these off-targets are likely to occur and what phenotypic effects these may have. While off-targeting editing often occurs in non-coding regions or causes a silent variant that results in a synonymous amino acid, editing of off-target sites may cause undesired changes in the transcriptome.Using endogenous ADAR strategies are attractive as this limits concerns for immunogenicity and appear to have lower off-target editing events compared to strategies using exogenous ADAR ,57. It rFor the correction of pathogenic mutations, editing of the coding sequence is required. As natural ADAR editing predominantly occurs in the UTR, it is not surprising that strategies using endogenous ADAR editing demonstrate higher efficiency in the UTRs and editing is limited in the coding sequence. Editing in the coding sequence may be reduced by translation, as inhibition of translation with puromycin has been noted to increase A-I editing . FurtherConstructs expressing and recruiting exogenous ADAR or its deaminase domain with shorter gRNAs offer a number of advantages compared to endogenous strategies. Exogenously delivered ADAR may overcome reduced targeting scope of endogenous ADAR due to the codon preferences and potentially are better able to edit the open reading frame. Furthermore, exogenous strategies may benefit from future enzyme engineering work to expand the base targeting possibilities from solely A-I editing, as demonstrated by the recently developed C-U editors. The robust on-target editing rates observed with all of these exogenous systems are counterbalanced by relatively high off-target rates both within the guide region and throughout the transcriptome. Ongoing optimization will likely reduce this, as seen with the development of the E488Q/T375G variant. Protein localization may also play an important role in the balance between on-target and off-target editing. Natural ADAR editing likely occurs in the nucleus or nucleolus. In experiments investigating active Cas13 knockdown of RNA without a fused ADAR construct, higher RNA cleavage rates are observed when Cas13b is fused to a NES rather than a NLS , and a sDD construct packaged with appropriate tissue specific promoters targeting natively expressed transcripts has yet to be demonstrated. Cas13b orthologues identified to date for RNA targeting in mammalian cells are already relatively small (~3.2–3.5 kb) and appear to tolerate C-terminal truncations with little loss in RNA editing activity. Fused with the ADARDD (1.15 kb), CRISPR-RNA editors delivered via a single AAV are a viable option, unlike the large DNA base editor constructs. Although the full-length REPAIR fusion (dPspCas13b-ADAR2DD) is 4.47 kb and unable to fit in the AAV coding capacity together with the required regulatory elements, the dCas13bΔ984-1090-ADAR2DD variant (4.15 kb) has relatively preserved editing efficiency compared to the full length construct and is theoretically small enough to package in AAV with short regulatory sequences. The C-terminal truncated mutant tested with the RESCUE system, dRanCas13b-del892-1095 is the smallest identified Cas13b-ADAR used for RNA editing to date (3.88 kb) and maintained similar editing efficiency to the full-length protein. Efforts to identify smaller Cas13 orthologues are ongoing. For methods requiring chemical stabilization of the RNA guide [Delivery of both DNA and RNA base-editing constructs continues to be a challenge for therapeutic use. AAV-mediated delivery is the most readily clinically translatable delivery route, and low rates of editing using AAV-MCP-MS2 and GluR2 systems have been achieved in muscle . The MCPNA guide ,53, AAV RNA editing is an exciting therapeutic prospect for the treatment of point mutations in genes implicated in inherited retinal degeneration that are too large for gene replacement therapy with AAV. The growing armament of tools for RNA editing will allow the interrogation of a range of approaches for safety and efficacy in vivo. As this requires a mutation-specific approach, it will be important to elucidate design principles for guide RNAs in order to expand this to a wide range of individual mutations in their individual sequence context.


The frontiers of our knowledge about RNA structure are rapidly moving. Twenty-five years ago, there were fewer RNA structures in total than there are generally today within a single family of RNAs . The longest RNA ever solved had 160 nucleotides [Constantly pushing technical boundaries is what enabled us to generate, analyze and make sense of this sheer amount of structural information. In turn, the triumph of methods such as single-molecule studies or cryo-electron microscopy (cryo-EM) has offered glimpses into the function of large RNP assemblies. We have reached a point where we have three-dimensional visuals of the various stages along core cellular processes like transcription and translation ,3.However, we are not done. The field of RNA structural biology has never been so vibrant. The current COVID-19 pandemic naturally brought an impetus to advance the field even further, as vaccines may be available one day that will be RNA-based, or drugs might be developed that target an RNA genome. In some way, tackling RNA viruses epitomizes where the field is at: the study of long RNAs that adopt transient structures, each having distinct regulatory roles through binding small molecules, proteins, DNA, or other RNAs.Molecules lies precisely at the intersection between the biology of RNAs acting as sensors or regulators, and the methods we are developing to study them.This Special Issue of Zappulla at Lehigh University and Bou-Nader and Zhang at NIH propose timely reviews on long RNAs, how they may fold as “flexible scaffolds” to spatially but loosely constrain binding partners , and howLong RNAs nonetheless comprise independently folded modules. Using X-ray crystallography, the Batey lab at the University of Colorado delivers a thought-provoking story that will help shift the paradigm of ligand binding to riboswitches located upstream of messenger RNA in bacteria . The KonFleeting RNPs may be characterized by single-molecule fluorescence approaches, as reviewed by the Hengesbach lab at Goethe University, Frankfurt . They ilI am very excited about the collection of articles in this Special Issue for two reasons. First, constantly updating our perception of RNA folding and structure would not be possible without concomitant advances in biophysical approaches, as this collection of reviews and original work exemplifies. Second, the various labs that have deemed it worth their precious time to submit a manuscript are esteemed colleagues, ranging from long-time collaborators to serendipitous encounters. The majority represent young principal investigators.


Interferon (IFN) signaling induces the expression of a wide array of genes, collectively referred to as IFN-stimulated genes (ISGs) that generally function to inhibit viral replication. RNA viruses are frequently targeted by ISGs through recognition of viral replicative intermediates and molecular features associated with viral genomes, or the lack of molecular features associated with host mRNAs. The ISGs reviewed here primarily inhibit viral replication in an RNA-centric manner, working to sense, degrade, or repress expression of viral RNA. This review focuses on dissecting how these ISGs exhibit multiple antiviral mechanisms, often through use of varied co-factors, highlighting the complexity of the type I IFN response. Specifically, these ISGs can mediate antiviral effects through viral RNA degradation, viral translation inhibition, or both. While the OAS/RNase L pathway globally degrades RNA and arrests translation, ISG20 and ZAP employ targeted RNA degradation and translation inhibition to block viral replication. Meanwhile, SHFL targets translation by inhibiting -1 ribosomal frameshifting, which is required by many RNA viruses. Finally, a number of E3 ligases inhibit viral transcription, an attractive antiviral target during the lifecycle of negative-sense RNA viruses which must transcribe their genome prior to translation. Through this review, we aim to provide an updated perspective on how these ISGs work together to form a complex network of antiviral arsenals targeting viral RNA processes. Organisms must constantly defend themselves against viral pathogens. In order to stem viral spread, cells must both signal the presence of viral infection and hinder their replication. One key first line of cellular defense in vertebrates is the type I interferon (IFN) response. Hosts possess sensors which recognize pathogen-associated molecular patterns (PAMPs) of invading viruses such as the viral replicative intermediate double-stranded RNA (dsRNA) and activate transcription factors such as IFN-regulatory factors 3 or 7 (IRF3/7) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). As a result, these transcription factors translocate to the nucleus to activate expression of type I IFN and other proinflammatory cytokines.The type I IFN receptor is expressed ubiquitously on almost all cell types, allowing for IFN signaling in both infected and neighboring cells that are uninfected. Janus kinase-signal transducer and activator of transcription (JAK-STAT) is the predominant, canonical pathway that regulates ISG transcription. IFN binding to its cell surface receptor, comprised of IFN-α receptor 1 (IFNAR1) and IFN-α receptor 2 (IFNAR2), leads to phosphorylation of pre-associated JAK1. Phosphorylated JAK1 and tyrosine kinase 2 (TYK2) then phosphorylate the IFN receptor, which recruits STAT1/2 to be phosphorylated themselves. Phosphorylated STAT1/2 recruit IRF9 to form the transcription factor complex IFN-stimulated gene factor 3 (ISGF3). ISGF3 translocates to the nucleus where STAT1 is further phosphorylated for full activation. Within the nucleus, ISGF3 binds to IFN-stimulated response elements present in the promoters of IFN-stimulated genes (ISGs), which then effect an antiviral cellular environment . FoRather than provide a comprehensive, surface-level view of myriad ISGs with their arrayed antiviral functions, we have chosen to focus on a subset of ISGs that interfere with viral RNA processes. Recent studies have provided an emerging view on the diversity and complexity of RNA-based mechanisms by which different ISGs inhibit viruses with an RNA genome. With the exception of retroviruses which replicate through a DNA intermediate, single-stranded RNA (ssRNA) viruses can generally be categorized as positive-sense and negative-sense. Positive-sense (+) ssRNA viruses possess genomes that generally resemble mRNA, in that it can be translated directly by host translation machinery. However, negative-sense (-) ssRNA viruses code their proteins in the reverse orientation. Therefore, they must package their own RNA-dependent RNA polymerases and transcribe their genomes before viral protein synthesis can occur. While there are many more ISGs that inhibit viral RNA processes 2, this Degrading viral genomes presents one potent method of antiviral activity; digesting viral genetic material ensures that no further steps in replication can occur. However, the challenge lies within being able to control RNA degradation to ensure cellular survival or limit destruction within the host. The OAS/RNase L pathway is activated upon sensing the PAMP of dsRNA, serving two functions: sensing viral intruders and inhibiting viral replication by degrading RNA almost indiscriminately, inducing global translational arrest.The OAS/RNase L pathway was one of the first ISG antiviral mechanisms to be identified and elucidated in the 1970s [reviewed in –64]. BinIn the past 10 years, great strides have been made in clarifying dsRNA substrate specificity and 2-5A synthesis activity of individual OAS isoforms due to the advent of CRISPR-Cas9 gene-editing techniques and generation of OAS knockout cell lines and mouse models. Moreover, sweeping improvements in genome-wide RNA-seq and in-depth proteomics have illuminated new intricacies of OAS/RNase L-mediated inhibition of viral replication, which we will review here.Recent years have not only seen a flurry of biochemical and structural studies on individual OAS paralogs, which have advanced understanding of their RNA substrate specificity and activation, but also of their individual splice variants. Surprisingly, OAS3 has been identified as the main contributor to the OAS/RNase L pathway, as OAS3-mediated 2-5A synthesis has been demonstrated as necessary and sufficient for RNase L pathway .in vitro after incubation with poly(I:C), a dsRNA mimetic, with isoforms p42 and p46 expressed more highly in HEK293 cells  are highly enriched for ISGs such as IFIT2, OAS2, MDA5, and RIG-I, suggesting that antiviral mRNAs are resistant to RNase L turnover , Flaviviridae (yellow fever virus (YFV), WNV, HCV), Picornaviridae , and (-) ssRNA viruses from Rhabdoviridae (vesicular stomatitis virus (VSV)), Orthomyxoviridae (IAV), and Bunyaviridae  pathway has been linked to ISG20 antiviral activity in the context of HBV infection. Briefly, m6A is the most common reversible post-transcriptional modification that occurs on cellular RNAs. The m6A pathway involves three primary types of proteins—writers, erasers, and readers—which respectively add, remove, and bind m6A (6A mRNA (6A readers is the YT521-B homology (YTH) domain-containing proteins. The cytoplasmic YTH members (YTHDF1-3) were recently found to play redundant roles in mediating degradation of m6A-mRNAs , HBV (Hepadnaviridae), the (-) ssRNA viruses Ebola virus  and IAV (Orthomyxoviridae), and (+) ssRNA viruses such as alphaviruses (Togaviridae), coxsackievirus B3 , and Japanese encephalitis virus   –132. ZAPviridae) , 131. Itviridae)  and restviridae) , 135. ZAviridae)  that dirviridae) , 137–139viridae) . Alternaviridae) .ZAP targets viruses primarily by two distinct antiviral mechanisms, namely viral translation inhibition (for the (+) ssRNA viruses SINV and JEV) and viral RNA degradation  ssRNA virus EBOV, and the (+) ssRNA viruses CVB3 and JEV). These disparate mechanisms can be explained in part by recruitment of co-factors and differing viral contexts. ZAP inhibition of SINV translation has been linked to its disruption of the interaction between translation initiation factors eIF4A and eIF4G . This diCG) but not wild-type HIV-1  ssRNA family, Picornaviridae, inhibiting replication of encephalomyocarditis virus but not poliovirus or enterovirus 71 , but not HSV-2  which is crucial for many viruses to control protein expression levels . SHFL inpression . Noticeapression . SHFL wapression . It is ipression , supportpression .However, SHFL may also act on some viruses independent of its direct effects on viral translation. For example, SHFL associates with the flavivirus replication complex in both DENV and ZIKV infections and interacts specifically with nonstructural protein 3 (NS3) in an RNA-independent manner , 168. ByTaken together, SHFL inhibits viral replication by regulating translation through -1PRF-dependent and independent mechanisms and by specific antagonism of viral proteins and structures. SHFL displays both RNA-independent and -dependent interactions with other proteins, relying on RNA for its interaction with cellular RNA helicases MOV10 and UPF1 while interacting with flavivirus NS3 in the absence of RNA. These varied requirements for protein-protein interactions may reflect SHFL’s diverse antiviral mechanisms.The really interesting new gene (RING) proteins are the most abundant family of E3 ligases, characterized by their N-terminal catalytic RING domain. E3 ligases occupy the final step in cellular ubiquitination. Ubiquitination of a protein can alter its cellular fate depending on the type of linkage, ranging from proteasomal degradation to scaffold formation for assembly of cellular signaling complexes . In ordeFiloviridae) (Retinoblastoma binding protein 6 (RBBP6) is a RING E3 ligase that inhibits transcription of the (-) ssRNA virus EBOV (viridae) . RBBP6 wviridae) . RBBP6 cviridae) . Howeverviridae) . Curiousviridae) . KnockdoThe tripartite motif containing proteins (TRIM) are the largest group of RING E3 ligases and constitute an important family of proteins in the type I IFN response , 172. Thvia both ligase-dependent and -independent mechanisms. TRIM22 does both, though its ligase activity is required to inhibit HBV transcription, it inhibits HIV-1 transcription independent of its ligase activity , a (-) ssRNA virus, in ligase-independent manner (NJ) or other negative-strand RNA viruses such as SeV, rabies virus, or IAV  circumvent RNA degradation by enzymatically degrading 2-5A, thus preventing RNase L activation . MeanwhiThe perpetual arms race between antiviral effectors and viruses has likely driven the development of multilayered mechanisms of viral inhibition. Some individual ISGs have acquired multiple antiviral mechanisms, enabling them to circumvent viral evasion. For example, ZAP is still able to inhibit replication of SARS-CoV-2, a positive-sense ssRNA virus with highly suppressed CG content . This suTogaviridae and Flaviviridae family members to elucidate new host-virus interactions , UCLA AIDS Institute and Charity Treks 2019 Seed Grant (ML), and Ruth L. Kirschstein Multidisciplinary Training Grant in Microbial Pathogenesis .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.