Viroids are non-coding circular RNA molecules with rod-like or branched structures. They are often ribozymes, characterized by catalytic RNA. They can perform many basic functions of life and may have played a role in evolution since the beginning of life on Earth. They can cleave, join, replicate, and undergo Darwinian evolution. Furthermore, ribozymes are the essential elements for protein synthesis of cellular organisms as parts of ribosomes. Thus, they must have preceded DNA and proteins during evolution. Here, we discuss the current evidence for viroids or viroid-like RNAs as a likely origin of life on Earth. As such, they may also be considered as models for life on other planets or moons in the solar system as well as on exoplanets. Viroids, which are known today as virus-like infectious agents of mainly plants, may be considered as remnants of the Ancient RNA World that is thought to have existed before the emergence of DNA and proteins . NaturalThere is evidence from sequence analyses that viroid-like RNAs have evolved into viruses  and thatViroids were first recognized as disease-causing agent in potatoes as potato spindle tuber viroid (PSTVd) . RibozymAvsun- or Pospiviroidae, respectively). The catalytic activity of the hammerhead structure is a common structural motif of the catalytically active viroids or ribozymes. The catalytic activity of the hammerhead in the Avsunviroides such as Avocado Sun Blotch Viroid (ASBVd) is involved in its replication and can lead to a self-cleavage mechanism to monomeric or dimeric forms, together with its own RNA ligase. A member of the other group is the Potato Spindle Tuber Viroid (PSTV), which is characterized by a conserved central region CCR without enzymatic activity or a hammerhead motif [The viroids consist of a self-complementary rod-like or branched structured covalently closed circular RNA. They comprise two families, but only one of them harbors an enzymatically active hammerhead motif, the other one relies on host factors. Their names refer to their hosts, avocados or potatoes  is: “Life is a self-sustaining chemical system capable of Darwinian evolution”. Two origins of life were proposed by theoretical physicist Freeman Dyson in cooperation with the mathematician John von Neumann. They were creating this concept together in Princeton . These t15 random RNA molecules, 220 nucleotides in length [10 random RNA molecules adopted a conformation that formed a binding site for a randomly chosen set of substrates [The Nobel Prize laureate Jack Szostak asked the question what kind of structures can be formed by RNA in vitro. He performed an experiment with stretches of RNA in a reaction vessel, starting with a large pool of 10n length . The RNAn length ,30. By ibstrates .“RNA can do it all” is a sentence attributed to Sir Francis Crick, one of the discoverers of the DNA double-helix structure . The smaThe RNAs exhibit structure-mediated functions. Ribozymes were tested by Gerald F. Joyce in a test tube, where they proved again to be multifunctional; they can cleave, join, chemically replicate other ribozymes, thus generating progeny, form peptide bonds, and mutate depending on environmental conditions ,34. It hThe question on energy sources is not answered, it is not even mentioned by the NASA definition of life above, yet any replication process would require an energy supply. The energy during early evolution was unlikely provided by the Sun, as sunlight can only reach to about 200 m below sea level. Life, however, likely started at hydrothermal vents deep in the ocean where sunlight cannot reach, where, for example, geothermal energy is available. In addition, ice-cold water allows cold-adapted ribozymes to catalyze rather long RNA in vitro .Some of the molecules which may have existed on the Early Earth can be identified by analyzing meteorites. A huge meteorite (over 100 kg) spread fragments over a large area in South-West Australia in 1969, the Murchison meteorite. Chemical analyses by various research groups indicated the presence of more than 80 amino acids—a surprising number considering the 20 amino acids used by biological entities (cellular organisms and viruses) on Earth ,38,39,40C, H, O, P, N, S), were present as simple precursors such as hydrogen cyanide, inorganic phosphate, and hydrogen sulfide in a so-called “one pot synthesis” vessel and indeed allowed for the synthesis of the necessary biomolecules for living matter, RNA, proteins, and lipids [The question of whether the building blocks that are detectable on meteorites are sufficient for the formation of life on Earth as we know it prompted the group of John D. Sutherland and colleagues in the United Kingdom to perform a synthesis of the major biomolecules for life on Earth: Amino acids, lipids, and ribonucleotides, the components for proteins, fat, and nucleic acids, respectively. The necessary elements, carbon, hydrogen, oxygen, phosphorous, nitrogen, and sulfur , methane (CH4), ammonia (NH3), and hydrogen (H2) under putative early Earth-like conditions, precursors which may have dominated the atmosphere of the early Earth. Artificial lightning served as a source of energy for chemical reactions. Among the many biomolecules, five different amino acids were generated [The one pot-synthesis experiment mentioned above had a precedent, the Miller–Urey experiment from 1959. It achieved the synthesis of organic compounds in the laboratory from simple inorganic precursors, such as water -like molecule that supported the transfer of the amino acid to a ribozyme, which fixed a peptide bond to another amino acid. This would be a primitive “pre-ribosome”. It is surprising that the protein synthesis machinery, the ribosomes as we know them today, use a catalytic RNA, a ribozyme, for peptide bond formation, even though RNA enzymes are typically less efficient than protein enzymes. Protein enzymes do not exist in the ribosomes. “Ribosomes are ribozymes” is the summary by Thomas Cech making them one of the most important factors for evolution and protein synthesis and precedents of cells .Ribozymes were found in the introns of RNA transcripts and can remove themselves, and are involved in the maturation of pre-tRNAs as component of the RNase P complex. They can catalyze their own synthesis as RNA polymerase ribozyme and they cleave themselves autocatalytically ,22.In today’s cells, the tRNA is processed from a pre-tRNA precursor by a ribozyme, the RNase P. It was Sidney Altman who discovered this ribozyme and shared the Nobel Prize in Chemistry in 1989 with Thomas Cech “for their discovery of catalytic properties of RNA”. Until then it was thought that all enzymes were proteins. The RNase P is closely associated with a protein, which however is enzymatically inactive. The RNA moiety alone harbors the catalytic activity.The first substrate of the RNase P was discovered by Altman, and he defined it as a very ancient structure: A tRNA precursor, pre-tRNA. He showed that the RNase P recognizes structures of this substrate and cleaves at a conserved site next to a loop of the molecule. Only later evolutionarily, in the protein world, was the RNase P associated with a protein. This is reminiscent of the catalytic ribozyme in the ribosomes—which finally accumulated about one hundred proteins as scaffold molecules .It is often discussed that RNA enzymes (ribozymes) evolved into proteins, which performed similar functions, but much more efficiently. For the RNase P, it was directly shown at the molecular level how the ribozyme evolved into a protein. Comparison of the role of metal ions and properties of the scissile bond indicated similarities between the ribozyme-based and a protein-based RNase. This indeed supports the notion that RNA can be a precursor of proteins. The protein’s catalytic activity is highly increased as demonstrated by Koutmos et al. ,47. ThusOther non-coding ribosomal RNAs exist, and about one hundred proteins serve some functions for structure and stability, but not for catalysis, resulting in the composition of today’s ribosomes. Thus, ribozymes, which originate from the Ancient RNA World, are even in the DNA and protein world that later evolved the essential components to produce proteins. Ribozymes must have been first .DNA could also have been produced without proteins by chemical deoxygenation of RNA. Thus, the transition from the RNA to the DNA and protein world may have taken place before the protein enzyme evolved, which catalyzes this conversion, the reverse transcriptase (RT) . The revWe happened to observe the effect of a protein on a ribozyme when trying to evaluate a ribozyme for gene therapy . We suppA few molecules are known, which appear to be a chimera of a protein with RNA, perhaps “left-overs” from the time of the transition of the RNA world to the RNA-protein world, as pointed out by Szostak in a university lecture, vitamin B12 and acetyl-CoA. They have RNA tails, as if removal of the RNA moiety had been incomplete, and now may serve a function such as promoting stability .It is worth mentioning that the first identified ribozyme was that of the satellite RNA of tobacco ringspot virus, consisting of a 359 nucleotide single-stranded covalently closed circular genome that is parasitic upon the tobacco ringspot virus . The catThere are still today other plant viruses with structures similar to ribozymes, the viroids, and others such as Narnaviruses or viruses with tRNA-like-structures ,54, whicKnown from history is Cadang-Cadang, a disease of trees caused by a viroid in the Philippines affecting coconut trees, where the viroid was transmitted by the workers during harvesting, possibly by using tools such as a knife to cut off the fruits of many trees, which might have become cross-contaminated ,57. TranAn interesting case is the effect of a viroid together with another virus on the patterns of carnation . CarnatiAlternatively, novel functions of viroids may be acquired by uptake of coding information from the host, as has likely been the case for the hepatitis delta virus (HDV). The HDV genome shares similarities with viroids and also has ribozyme activity . It has Structures of non-coding RNAs are abundant in all biological systems. They are reminiscent of viroid RNAs. They can act as regulators of gene expressions, such as siRNAs, or function as guardians of gene expression as described for circular RNAs (circRNAs) . These cThere is a chicken-and-egg dilemma, since present viruses are parasites that require cells to replicate. How then can viruses have been first ? The anc33 particles exist [If one assumes the ancestors of viruses and viroids were essential components at the beginning of life, how can they be today only parasites, unable to replicate autonomously? There is a tendency in evolution to increase genome sizes. However, the opposite exists as well and is often less obvious. Nature also reduces genes or gets rid of them to reduce the burden and energy requirements for their maintenance and replication. Viruses gain or lose genes to adapt to their environments. One experiment demonstrating the loss of genes was performed by Sol Spiegelman in 1965, who incubated the RNA of the phage Q beta, 4217 nucleotides long, in a test tube in the presence of an enzyme, an RNA replicase, in a rich milieu. Serial transfers of aliquots and regrowth in a new test tube were performed. The RNA replicated faster and faster and became smaller and smaller, ending up as non-coding RNA of 218 nucleotides after 74 generations ,71,72. Tes exist .This experiment is a model of how an autonomous biological entity can become dependent, how the ancestors of viruses and viroids may have been autonomous initially, but in a rich milieu they became parasitic. In line with these results, one may speculate that some of the viroids may have lost their intrinsic enzymatic activity and used cellular enzymes for replication. The rich cellular milieu may have allowed such a reduction of genes, which is also an energy-saving simplification. Typical viruses—today—do not have enough genes to live on their own. This parasitic lifestyle may have been adopted during evolution as a consequence of environmental luxury. Only today, viruses must rely on the machinery of the host, such as bacteria, human cells, or other organisms. One could envisage two arms of evolution, one with viruses increasing in numbers and not in size, and another one starting with viruses which by contrast evolved into larger entities, perhaps into giant viruses, and finally into subcellular structures and then into cells. That leads to today’s situation. Here, comes support from giant viruses.Giant viruses have only recently been discovered during the last decades. They are larger in diameter and genome size than many bacteria, often comprising more than 1000 genes and more than 1.5 million basepairs ,13. TheyGiant viruses harbor large genomes that encode components required for protein synthesis such as genes for ribosomal proteins, tRNAs, transferases, and translation factors. Yet, they do not have the complete set required for independence from cells. Protein synthesis is the definition of life in classical textbooks. This puts the giant viruses in the middle between living and inanimate matter and indicates that the transition between the two extremes is continuous. They may have been autonomously replicating at some stage of development. Are they “unfinished” bacteria on their evolutionary path to real bacteria, or regressed bacteria that lost genes during evolution? They can host viruses themselves, designated as virophages, because of their similarity to bacteriophages or phages that infect bacteria . This haBased on the endosymbiotic theory, or symbiogenesis, it is considered here that viruses may have undergone a reductive evolution. Viruses are parasites in today’s world, but they may have become intracellular parasites as a consequence of selection due to certain environmental conditions and energy-saving measures. In the prebiotic world, pre-cells or pre-viruses may have looked alike. They both may have evolved further. Thereby, the viruses starting from initially autonomous viroids or viroid-like RNAs and other viral species such as tRNA-like plant viruses or giant viruses, perhaps first became more complex and then evolved to host dependent parasitic viruses.Rickettsia species provide another example of loss of independence. They exert an obligatory intracellular lifestyle, yet, they have evolved from extracellular bacteria. The intracellular forms lost more than 20% of their genes [Non-coding RNA from coding RNA can be produced today experimentally depending on the conditions, as exemplified by Spiegelman’s Monster described above. A rich milieu, in this case the presence of a replicating enzyme, ribonucleotides, etc., can lead to loss of genetic information and result even in loss of autonomy. Examples found in nature are the endosymbionts of bacteria, which evolved to mitochondria or chloroplasts ,78. Theyir genes . Anotherir genes . Poxviruir genes . Endosymir genes . Endogenir genes .It is a hypothesis that the ancestors of viruses including viroids exerted an independent lifestyle and then lost their autonomy during evolution to become the host-dependent parasites as we know them today ,16. ThesThe message we give here is that the beginning of life must have been simple, and that viroid-like RNAs are good candidates for the first life-like biomolecules. Viroids are the smallest known entities that are able to replicate and undergo Darwinian evolution, one of the definitions of life. Today’s viroids require host cells for their replication, but their ancestors may have been capable of cell-independent replication and evolution before the emergence of cellular life. Their small size, their versatility, mutagenicity, and autonomy, solely based on structural information in the absence of the genetic code and protein synthesis, are features needed for other forms of life, as exemplified by the ribosomes whose catalytic activity depends on two ribozymes. One of them performs peptide synthesis, the other one, RNase P, processes the ribosomal RNA and trims the tRNA to its final structure. It is the essential transporter of amino acids as required by the genetic code of the mRNA. Consequently, we propose that viroids may serve as models for early forms of life on Earth. Analogous structural elements with catalytic functions required for replication may be present on other planets and celestial bodies as well as exoplanets. Terrestrial life-like entities with principle properties of the simple ribozymes or viroids could be envisaged to have evolved elsewhere. Thus, viroids may be considered as lead candidates or models in the search for analogous entities outside of the Earth in the universe.


Escherichia coli leads to an acceleration of a bistable RNA’s refolding kinetics through the selective destabilization of key base pairing interactions. We find in laser assisted real-time NMR experiments on photocaged bistable RNAs that the RNA chaperone leads to a two-fold increase in refolding rates at low temperatures due to reduced stability of ground state conformations. Further, we can show that upon interaction with StpA, base pairing interactions in the bistable RNA are modulated to favor refolding through the dominant pseudoknotted transition pathway. Our results shed light on the molecular mechanism of the interaction between RNA chaperones and bistable RNAs and are the first step into a functional classification of chaperones dependent on their biophysical mode of operation.In bacteria RNA gene regulatory elements refold dependent on environmental clues between two or more long-lived conformational states each associated with a distinct regulatory state. The refolding kinetics are strongly temperature-dependent and especially at lower temperatures they reach timescales that are biologically not accessible. To overcome this problem, RNA chaperones have evolved. However, the precise molecular mechanism of how these proteins accelerate RNA refolding reactions remains enigmatic. Here we show how the RNA chaperone StpA of  Modulation of gene expression in bacteria can be provided by RNA regulators. These RNAs functionally exploit their inherent structural plasticity that is founded in their natural ability to adopt more than a single stable conformation. Thereby they constitute a bi- or even multi-stable system. If the structural interchangeability within such a system takes place as an answer to changes of environmental clues, it can be exploited as a trigger for functional cellular processes such as transcription or translation. Within the regulation of gene expression, the conformational states of a bistable system are typically linked to two functional states (ON and OFF) ,2. Primein vitro  then all regulatory processes have to be faster in order to have a relevant functional consequence , refolding occurs at timescales of 1–1000 s ,15. Howesequence .in vivo bacteria exhibit a class of auxiliary proteins: RNA chaperones  T,12 TE. cE. coli  7). The. TheE. cE. coli . The lowE. coli . Such biE. coli . The swiBy NMR spectroscopy, we probe the influence of the RNA chaperone onto the equilibrium forming conformational states and the refolding mechanism. The binding of StpA to the RNA was investigated by isothermal titration calorimetry in order to establish the full thermodynamic cycle of the interaction. Further, the kinetics of their interconversion is studied by real-time NMR spectroscopy of photocaged RNAs. The results show the ability of StpA-CTD to accelerate RNA refolding kinetics by selective destabilization of base pair interactions.S)-NPE protected guanosine phosphoramidite was performed according to literature procedures  in 1 μmol or 16 μmol scales. Pac2O was used as capping reagent. The coupling time was 12 min. RNA was synthesized in DMTr-On mode. Deprotection was performed according to a protocol from Glen Research  from Waters (RP-HPLC conditions see SI). After evaporation of the solvent at 4°C the DMTr-group was cleaved by incubation of the RNA in 300 μl 80% acetic acid at room temperature for 20 min. The solvent was evaporated under vacuum and the resulting RNA was again purified by RP-HPLC using the same conditions as mentioned before. The solvent was removed under vacuum. Finally, all samples were coevaporated with ultrapure water several times and lyophilized (Sequence and ESI-MS results see SI). Buffer exchange to NMR buffer  was done with 2 ml 2 kDa centrifugal concentrator (Vivaspin from Sartorius) up to a factor of at least 1000. The NMR sample was in situ folded at 95°C for 5 min followed by cooling on ice.The synthesis of the  Acetonitril). The HPLC fractions were lyophilized and dissolved in H2O. Analytical denaturing 15%-PAA (polyacrylamide) gel verified corresponding fractions that were desalted with 2 ml 2 kDa centrifugal concentrator  at 4°C and 6000 g. After desalting LiClO4 (2% LiClO4 in acetone) precipitation followed. Buffer exchange to NMR buffer  was done with 2 ml 2 kDa centrifugal concentrator (Vivaspin from Sartorius) up to a factor of at least 1000. The NMR sample was in situ folded at 95°C for 5 min followed by cooling on ice. For sequences and sample conditions of all RNAs used see SI The unmodified bistable 20 nt RNA and the supplementary RNAs  were solid phase synthesized and purchased by Dharmacon . 2′-ACE Protection group was removed as described in the provided protocol by Dharmacon. After deprotection the RNA was purified via rp-HPLC (reversed-phase high-performance liquid chromatography) with a Kromasil RP18 100A 5 μm 10 × 250 mm column . The utilized plasmid carried the gene for ampicillin resistance. This vector provides a HISE. coli cells from NEB  following the instructions of the high efficiency transformation protocol of the manufacturer. Purification of the plasmid was done with QIAGEN plasmid Kits (Cat. No. 12145). The plasmids containing the protein sequences were transformed into T7-Express E. coli cells for subsequent expression as SUMO-fusion protein. Cells were grown in TB (Terrific Broth) medium supplemented with 100 μg/m. ampicillin at 37°C. Expression was induced when OD600 reached a value between 2 and 2.5 with 0.1 mM IPTG , the optimal concentration evaluated with test expressions. Expression took place at 24°C, 120 rpm for 16 h. After harvest the pellet was resuspended to homogeneity in 50 ml buffer A  supplemented with one protease-inhibitor tablet  per liter. The cells were mechanically lysed using Microfluidics M-110P. Purification by IMAC HisTrap HP column , connected to a FPLC  was done. Bound protein was washed with buffer A. To remove nucleic acid impurities, the protein was further washed with a high salt buffer . With a linear gradient of imidazole  the fusion proteins of interest were eluted. Cleavage of the Tag with SUMO protease took place during dialysis against imidazole in buffer A and removage with reverse Ni-NTA affinity chromatography After reverse Ni-NTA 0.015% (w/v) of polyethyleneimine (PEI) was added to precipitate bound nucleic acids Due to impurities additional size-exclusion chromatography  with an NMR running buffer . Further purification followed by Heparin column , to get rid of RNase activity. Therefore, binding buffer C containing the NMR buffer and 5% of glycerol was used  was used. Buffer exchange to NMR buffer without glycerol was achieved and purity was confirmed by SDS-PAGE (silver stained) and mass spectroscopy . StpA with a concentration of 400/ 800/ 920 μM was injected into the cell containing 40 μM RNA (cell volume 201.9 μl). The initial waiting time was 120 s, followed by a first injection of 0.2 μl. Afterwards 19 injections of 2 μl within 4 s, with a delay of 180 s after each injection, followed. In each experiment, a reference power of 11 μcal s–1 and stirring speed of 750 rpm were set with a high feedback mode. For each construct, three titrations were measured and the presented corresponding KD values and thermodynamic data represent the average with standard deviation, except the 5′-SS-OV that was measured once at 5°C and 25°C. Analysis of the thermograms was done with the software Origin7.0 (originLab) and were fitted to the following model  and the RNA-chaperone StpA-CTD were prepared in the same buffer stock . The RNA samples were in situ folded at 95°C for 5 min followed by cooling on ice. ITC experiments were carried out at 25°C and 5°C using a MicroCal iTCng model : \documeK Binding constant, n Hill coefficient, V0 active cell volume, Mt bulk concentration of macromolecule in V0, Xt bulk concentration of ligand, Q total heat content of the solution contained in V0, ΔH molar heat of ligand binding, ΔQ(i) heat released from the ith injection, Q(i) heat content at the end of the ith injection, ΔVi injection volume. As reference, the thermogram of the titration of StpA (921 μM) into buffer is given in SI With K3′/5′10°C = 5.22 ± 0.09 to K3′/5′40°C = 1.46 ± 0.04. This reports on a small energy separation of ΔG3′/5′25°C = −2.60 ± 0.13 kJ/mol, which is in good agreement with the prediction. However, the population ratio is strongly temperature-dependent with a steep gradient of ΔG/dT3′/5′ = 92 ± 5 J/mol K. Also, upon addition of the RNA chaperone StpA-CTD two sets of signals are observable. Comparison with the signals of the RNA alone shows that there is no significant change of the individual chemical shifts and therefore the pattern of signals remains virtually identical. However, for all signals an increase in linewidth is observable and some are even broadened beyond detectability  (SI G3′/5′+3eq. StpACTD25°C = −3.75 ± 0.62 kJ/mol). The effect of the chaperone on the RNA equilibrium interestingly levels off at three equivalents StpA-CTD per equivalent RNA  SI . The difA Figure . The freC Figure . This meKD values as well as the thermodynamic signatures of the binding at 5°C and 25°C. Supplementary to the bistable RNA sequence we utilize another five RNAs to obtain insight into the two single conformations and the role of the single strand overhangs, that are at least predicted to be hubs for the interaction between chaperones and RNA (KD) range from 12.3 to 24.7 μM at 25°C and from 8.3 to 16.3 μM at 5°C and show pronounced trends  × 10–3 s−1 and (240 ± 40) × 10–3 s−1 at 5°C and 25°C, respectively. The rate of the backward reaction k3′-5′ is determined to be (1.7 ± 0.3) × 10–3 s−1 to (84 ± 15) × 10–3 s−1 at 5°C and 25°C. In the presence of 3 eq. of StpA-CTD k5′-3′ ranges from (25 ± 3) × 10–3 s-1 to (190 ± 40) × 10-3 s−1 and k3′-5′ (3.7 ± 0.6) × 10–3 s−1 to (46 ± 10) × 10–3 s−1 (see SI ‡ of the refolding by 33 kJ/mol (for 5′-3′ transition) and 46 kJ/mol (for 3′-5′ transition)  (see SI k5′-3′) and 2.21 ± 0.44 (k3′-5′). The kinetic results at 25°C indicate, that the activity of the chaperone is much more temperature dependent, than the influence of the molecular crowder PEG-8000  Figure . This de) Figure ,49. As v) Figure  PEG-8000) see SI . In cont0 see SI . Consequdiss the overall trend within the helical stems remains the same . ,12. KD iC Figure  and 1.4 C Figure . Howeveraround 4 ,53, howeactivity ,26,54. Teratures . Furthereratures ,55.Summarizing, the acceleration of refolding is rather based on the selective destabilization of base pair interactions in the long-lived ground states as opposed to a stabilization of the transition state. The latter plays if at all only a minor role, in line with the expectation that the encounter of StpA-CTD with the short-lived transition state is not productive. It remains to be seen, if other RNA chaperones also act as selective ground state lubricants or act with a yet different mechanism.gkab876_Supplemental_FileClick here for additional data file.


The intrinsic cellular heterogeneity and molecular complexity of the mammalian nervous system relies substantially on the dynamic nature and spatiotemporal patterning of gene expression. These features of gene expression are achieved in part through mechanisms involving various epigenetic processes such as DNA methylation, post-translational histone modifications, and non-coding RNA activity, amongst others. In concert, another regulatory layer by which RNA bases and sugar residues are chemically modified enhances neuronal transcriptome complexity. Similar RNA modifications in other systems collectively constitute the cellular epitranscriptome that integrates and impacts various physiological processes. The epitranscriptome is dynamic and is reshaped constantly to regulate vital processes such as development, differentiation and stress responses. Perturbations of the epitranscriptome can lead to various pathogenic conditions, including cancer, cardiovascular abnormalities and neurological diseases. Recent advances in next-generation sequencing technologies have enabled us to identify and locate modified bases/sugars on different RNA species. These RNA modifications modulate the stability, transport and, most importantly, translation of RNA. In this review, we discuss the formation and functions of some frequently observed RNA modifications—including methylations of adenine and cytosine bases, and isomerization of uridine to pseudouridine—at various layers of RNA metabolism, together with their contributions to abnormal physiological conditions that can lead to various neurodevelopmental and neurological disorders. RNA is subjected to multifaceted regulatory processes to sustain diversity and complexity at the organismal and molecular levels. It has evolved to participate in diverse cellular processes owing to its capability to couple enzymatic activity with the storage and transfer of information. Upon being transcribed, the nascent RNA is subjected to various processing mechanisms, collectively termed post-transcriptional processing, that ultimately confer it with its information storage/transfer and regulatory functions. Post-transcriptional processing of eukaryotic RNA typically includes 5′ capping, intron removal or splicing, and addition of a 3′ polyadenylated tail. Another crucial mechanism of post-transcriptional RNA modification is the chemical modification of RNA bases and sugar residues on the RNA backbone. Similar to chemical modification of DNA cytosine residues that constitute part of the epigenome, chemical modifications of RNA, the “epitranscriptome”, adds another regulatory layer to organismal transcriptome-wide complexity. The functional impact of the epitranscriptome manifests in almost all tissues, but it is most apparent in regulating complex organs such as the brain. It is both transcriptomic and epitranscriptomic diversity that endows the nervous system with its complexity, with the latter altering various layers of RNA metabolism. RNA metabolism encompasses diverse processes including biogenesis, transport, splicing, stabilization, storage, and translation. Many recent studies have highlighted how dysregulation of RNA transport, splicing, stabilization, translation, or miRNA/tRNA biogenesis contributes to age-related neurodegenerative diseases  and neurAccording to the MODOMICS database, ~170 different RNA modifications have been identified to date, yet only a few have been well characterized and specifically linked to neurological disease . RNA modHowever, though RNA modification likely alters RNA metabolism, it remains enigmatic how such modifications contribute to neurodevelopmental and neurodegenerative diseases. In this review, we explore several exciting studies reporting the effects of different RNA modifications on various aspects of RNA metabolism and evaluate links between those modifications and a variety of neurological diseases.cis-acting elements and via formation of a multi-protein complex termed the spliceosome [cis-acting elements alters exon recognition by spliceosomes, giving rise to alternatively spliced mRNAs from the same mRNA transcript. Alternative splicing not only contributes to diversity among species, but also enables tissue-specific expression of differentially spliced products to perform different functions [cis-acting elements and splicing motifs result in splicing defects, potentially resulting in neurological disorders or neurodegenerative disease [Introns of pre-mRNAs are removed and exons are joined in a process called pre-mRNA splicing to form mature mRNAs, and this process is regulated by several iceosome . The invunctions . mRNA spunctions ,11,12. P disease .Effect on neurodevelopmental diseases: Approximately 1.4% of autism spectrum disorder (ASD) cases are caused by splicing defects . Changestau gene product MAPT [Effect on neurodegenerative diseases: Alternative splicing also regulates the expression of different isoforms of α-synuclein, the main component of Lewy bodies and a hallmark of Parkinson’s disease (PD) . Similaruct MAPT , namely uct MAPT . For insuct MAPT . Splicinuct MAPT .CAG repeuct MAPT .BDNF transcripts, where the short isoform is restricted to the cell body while the long isoform is predominantly found in the dendrites [CALCA) and immunoglobulin M heavy chain (IgM) [CstF-77) gene [The alternative polyadenylation (APA) of mRNAs is the use of multiple polyadenylation sites in primary transcripts and in conjunction with alternative splicing. APA expands cellular transcriptomic diversity by generating distinct mRNA isoforms . Dependiendrites . Lastly,endrites . CR-APA in (IgM) . CR-APA 77) gene .MeCP2, FMR1 to disorders with autistic phenotypes such as Rett syndrome, Fragile X-associated syndrome, autism, schizophrenia and other psychiatric diseases [NUDT21 gene that encodes a subunit of pre-mRNA cleavage factor Im is reported to regulate the length of MeCP2 transcript 3′-UTR [Effect of neurodevelopmental diseases: Neuronal commitment at the early stages of neurodevelopment is heavily influenced by the transcriptome repertoire of neural stem cells. During neurodevelopment, APA contributes significantly to the specification of neuronal lineage in association with other mechanisms such as microRNA networks, alternative splicing, non-sense mediated RNA decay, etc., that shape the transcriptome diversity of neural stem cells. APA events are known to be enriched in specific neuronal cell types ,35. Addidiseases ,40,41,42diseases . The efft 3′-UTR .huntingtin (Htt), involved in the development of HD. The huntingtin mRNA has two isoforms differing in the length of 3′-UTR. The abundance of Htt isoforms differs among cerebellum, motor cortex, fibroblasts and neural stem cells from patients and controls. Additionally, two isoforms differ with respect to their localization, the length of their poly-A tail, their half-lives and their binding sites for miRNA- and RNA-binding proteins. Moreover, the mRNA 3′-UTR isoform change is not restricted to Htt; 11% of alternatively polyadenylated genes in the HD motor cortex undergo changes with respect to the abundance of their mRNA isoforms [stathmin-2 gene in ALS and frontotemporal dementia (FTD). This results in reduced expression of the stathmin-2 gene, which is a hallmark of ALS-FTD [tau gene has been associated with binding of mir-34 family members of miRNAs and expression of tau mRNA isoforms [Effect of neurodegenerative diseases: The ability of APA events to generate transcripts with varying lengths of 3′-UTR gives rise to their intimate association with the regulation of gene expression. Since significant alterations of gene expression have been observed in neurodegenerative disorders ,46, APA isoforms . Reduced ALS-FTD . Finallyisoforms .Owing to the presence of extended neuronal processes, such as long axons and dendrites, it requires more energy and time to transport proteins on demand from the soma to distal parts of neurons. However, mRNAs are transported along neurites together with ribosomes and all the translation machineries, so mRNAs are ready to be translated in different parts of neurons . Recent Map1b, GluR1, Rac1, CamKII, Shank3, Gabrb1, among others) are targets of the RBP Fragile X mental retardation protein (FMRP) and are associated with synaptic structural anomalies and dysfunction, as well as impairments of long-term memory formation [https://gene.sfari.org/; accessed date July 2021). Further research is in progress to establish the molecular mechanisms underlying translational dysregulation of the mRNA targets of these proteins [Effect on neurodevelopmental diseases: An impressive body of work has uncovered how translational dysregulation of mRNAs is linked to ASD and Fragile X syndrome (FXS) . Most oformation ,62,63. Fproteins .c9orf72 gene harboring G4C2 repeat expansion mutations at intron 1 has been established as the main cause of ALS and FTLD diseases [Mfsd10 and Ppbp) that contribute to HD progression [Map1b, Nefl) or with FUS  contributes to ALS and frontotemporal lobar degeneration (FTLD) [Effect on neurodegenerative diseases: Patients suffering spinal muscular atrophy (SMA) exhibit reduced binding of survival motor neurons (SMN) to small nuclear RNA (snRNAs) because of genetic mutation-driven impairment of SMN protein stability, resulting in abnormal snRNA trafficking and maturation . In contdiseases . In SCA3diseases . RNA focdiseases ,71. Recen (FTLD) ,74. Inten (FTLD) ,76. Togecis-acting elements. Exonucleases and endonucleases contribute to these decay processes [cis-acting elements or enable alternative polyadenylation, thereby regulating the stability of the mRNA [To maintain RNA homeostasis, mRNAs transcribed inside the nucleus decay through various biological processes directed by rocesses . Methylarocesses . mRNA harocesses . Alternathe mRNA .Bdnf and Nf1, thus regulating neuronal differentiation and function [Effect on neurodevelopmental diseases: The Hu/Elav group of proteins exert an important role in exon inclusion and differential polyadenylation to alter the stability of mRNAs such as function . HuD-nulfunction . Moreovefunction . Reducedfunction . Recent function .Effect on neurodegenerative diseases: nELAVL-mediated changes in mRNA stability have also been implicated in neurodegenerative diseases such as AD and PD . A recenMicro-RNAs (miRNAs) are small non-coding regulatory RNAs that post-transcriptionally silence specific mRNAs, representing another form of temporal gene expression control. These miRNAs are involved in fine-tuning gene expression required for neural development, structure and function, so aberrant miRNA activity can induce neurological disease . miRNA pEffect on neurodevelopmental disease: miRNA biogenesis has been implicated in synaptic plasticity and long-term memory formation . DysreguEffect on neurodegenerative diseases: Interestingly, the progression of neurodegenerative diseases also appears to be dependent on the differential expression of miRNAs. Post-mortem AD brains display significantly different miRNA expression profiles compared to age-matched controls ,94. SpecDifferent RNA metabolisms described above are also shown in Various mechanisms of RNA metabolism temporally regulate the protein expression responsible for brain development, structure and function. RBPs fine-tune RNA metabolism, resulting in further complexities of gene expression in different neuronal parts . ExpressDespite hundreds of RNA modifications on coding and non-coding RNAs having been identified to date, only a few have been studied extensively or linked to disease. Well-studied RNA modifications include the methylation of adenosine at position 6 , N1-methyladenosine (m1A), 5-methyl cytosine (m5C), pseudouridine, and RNA editing, e.g., A-to-I see  113,114114.m6A is the most studied RNA modification in humans. It is a dynamic and reversible modification of RNA. m6A marks have been identified on mRNAs, transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), non-coding RNAs (ncRNAs), circular RNAs (circRNAs), and miRNAs. On mRNAs, most m6A marks are found at the beginning of the last exons, in the 3′-UTR or near the stop codons. Methyltransferase complexes, such as METTLE3, METTLE4 and WTAP, act as writers to methylate adenosine at position 6. Demethylases such as FTO and ALKBH5 are the known erasers of m6A modification marks ,116. Them6A modifications impact dendritic structure, spinogenesis, learning memory, neurogenesis, axon regeneration and brain development ,120. NeuMethylation of the N1 position of adenosine is another dynamic RNA modification in mammalian systems. It stalls translation elongation and prevents misincorporation of nucleotides during reverse transcription (RT) by inhibiting the formation of Watson–Crick base pairs . M1A modCytosine carbon-5 methylation (m5C) of DNA has long been considered a highly important epigenetic modification impacting diverse physiological processes such as cellular differentiation and organism development, and it has been implicated in cancers and cardiovascular abnormalities. In more recent years, m5C modification of RNAs has gained increasing attention owing to its roles in RNA transport, translation and stability ,143,144.Leu(CAA) [Arabidopsis thaliana and zebrafish have shown that mRNA stability is regulated by m5C modification [m5C RNA methylation impacts diverse RNA metabolic processes. For instance, eukaryotic rRNA stability is affected by the presence of m5C marks, influencing folding of essential ribosomal regions and thereby regulating translation . m5C modLeu(CAA) . MoreoveLeu(CAA) ,156. Recfication ,157. In fication . Interesfication ,157,158,fication .NSUN2 gene that causes a non-synonymous substitution of glycine at position 679 with arginine results in mislocalization of the protein in the nucleolus, which has been linked to the development of autosomal-recessive intellectual disability [NSUN2 exon 6 that reduces mRNA stability and NSUN2 protein levels, resulting in diminished methylation of target RNAs [NSUN2 in the prefrontal cortex of mice observed a bidirectional change in depression-related behaviors. NSUN2 deficiency resulted in changes in 1488 proteins in the prefrontal cortex, together with a decline in translation efficiency associated with a glycine-codon defect. Consequently, the mice displayed impaired synaptic signaling of pyramidal neurons in the prefrontal cortex and defective contextual fear memory [NSUN3 has been identified in a patient suffering severe mitochondrial respiratory chain complex deficiency, characterized by combined developmental disability, microcephaly, recurrent increased lactate levels in plasma, and muscular weakness [The crucial functional roles played by m5C methyltransferases in RNA metabolism imply that impaired expression of these genes may be responsible for various diseases. For example, several neurodevelopmental disorders have been linked to mutations in genes encoding NSUN proteins . Deletiosability ,164. Simget RNAs . A recenr memory . Disruptr memory . Furtherweakness .2O2 treatment and heat shock.Pseudouridine is a uridine isomer, and it is mostly found in non-coding RNAs such as rRNAs, tRNAs, and snRNAs. High-throughput sequencing has revealed the Ψ modification in yeast and human mRNAs . Two disRluA-1 and RluA-2, which encode Drosophila PUS enzymes, causes hypersensitive nociception phenotypes such as thermal hyperalgesia [CNBP, expression of which is associated with type 2 myotonic dystrophy (DM2) in humans, results in reduced binding of muscleblind-like 1 protein to CCUG repeat-expanded CNBP RNA linked to DM2 pathogenicity [PSU genes are known to cause neuronal dysfunction. For example, two point mutations in PUS1 were identified in a patient presenting with mild cognitive impairment and sideroblastic anemia since childhood, together with adult-onset hepatopathy, cardiomyopathy and insulin-dependent diabetes, all of which are typical clinical signs of myopathy-lactic acidosis-sideroblastic anemia (MLASA) syndrome [Pus3 mRNA expression in the nervous system of mouse embryos indicates a possible role for it in neuronal development, which is corroborated by a report linking PUS3 truncation to intellectual disability in human [PUS7 have been identified in human patients displaying intellectual disability, microcephaly, speech delay and aggressive behavior [PUS7 knockout [Dyskerin 1 (Dkc1) coding for a PUS component of the H/ACA-box snoRNP complex were observed in mouse embryonic neural tissues and specific neurons of the cerebellum and olfactory bulb of adult mouse brain, implying a potential role for it in neurodevelopment [Several studies have illuminated the impact of pseudouridylation on neuronal functions. For instance, nociceptor neuron-specific loss of ralgesia . Elevateralgesia . deLonimgenicity . Thus, psyndrome . Furtherin human ,177. Mutbehavior ,179. Theknockout . Moreoveelopment .cytochrome c oxidase subunit II (COX-2) of trypanosomes. In mammalian systems, it was first reported in the mRNA of hepatic apolipoprotein B, where it alters glutamate residue 2153 to a stop codon and results in formation of a truncated apolipoprotein B isoform known as Apo-B48 [Adenosine Deaminases Acting on RNA). Mammals have four ADARs—ADAR1p150, ADAR1p110, ADAR2 and ADAR3—generated from three ADAR genes (ADAR1-3), whereas Caenorhabditis elegans has two ADAR genes and Drosophila has only one [Adenosine Deaminases Acting on tRNA). Eukaryotes are reported to have three ADAT genes (ADAT1-3) [Escherichia coli too has a tRNA-specific deaminase [RNA editing is a type of modification whereby adenosine (A) or cytosine (C) residues are converted to inosine (I) or uridine (U) residues, respectively. The C-to-U conversion was first noted in the mRNAs of mitochondrial  Apo-B48 . RNA edi Apo-B48 ,184,185. Apo-B48 . Besides Apo-B48 ,188,189.only one ,191. DeaADAT1-3) . Escherieaminase . C-to-U eaminase ,195. Theeaminase ,197.RNA editing exerts multiple functional impacts on RNA metabolism. A-to-I and C-to-U editing both influence RNA secondary structure ,199. RNA2+ impermeability and has been linked to the formation of long-term potentiation in synaptic plasticity. Absence of GluA2-Q/R editing results in epileptic seizures and death in mice due to uncontrolled Ca2+ influx [2C serotonin receptor transcripts in human and rodents reduced the efficiency of G-protein coupling [A receptor [RNA editing has a myriad of effects on brain functioning. A prominent role for RNA editing has been observed in the case of AMPA and Kainate glutamate receptors, whereby the glutamines (Q) of Q/R sites are replaced by arginine (R). This change leads to Ca+ influx . A role + influx . Moreovecoupling . Evidenccoupling . This foreceptor , and it receptor ,209.KCNA1, which encodes Kv1.1, cause episodic ataxia type 1 (EA1), typically characterized by seizures, myokymia and ataxia. GluA2-Q/R site editing was found to be reduced in the motor neurons of ALS patients, which enhances Ca2+ influx and thereby elevates calpain activation to cleave more TDP-43, which is associated with mis-localization and cytoplasmic aggregation of TDP-43, a hallmark of ALS [Htr2c mRNA (encoding serotonin receptor subtype 5-HT2CR) may increase the propensity for suicide [In humans, approximately 85% of all pre-mRNAs undergo A-to-I RNA editing ,210,211.k of ALS . GluA2-Qk of ALS . Moreovek of ALS . Psychiak of ALS  (see Tab suicide . Notably suicide , zebrafi suicide , mice [2 suicide  and in h suicide . Lastly  suicide . TherefoWith the rise of epitranscriptomics, another level of gene expression control beyond transcription, translation, and epigenetic regulation has emerged as one of the most interesting fields of research. Several studies have now shown that post-transcriptional RNA modifications play a significant role in the spatio-temporal expression of diverse proteins in the brain. Neural development from embryonic to adult stages, neurogenesis, and the maintenance of proper neuronal structure and functioning necessitate tightly controlled protein expression. RNA modification writers and erasers control the presence or absence of epitranscriptional marks such as m6A, m1A, m5C, Ψ and RNA editing on diverse RNAs. Readers interpret these modifications to recruit RBPs that modulate RNA metabolic pathways, including splicing, mRNA decay, miRNA biogenesis, alternative polyadenylation, transport, and translation. In this way, RNA-modifying regulatory proteins and RBPs orchestrate the finely-tuned gene expression landscape to ensure appropriate brain development, structure and functioning. In neurodevelopmentally- and neurodegeneratively-diseased brains, these control mechanisms fail, likely due to disease-specific mutations or abnormal aggregates that trap epitranscriptomic proteins. More extensive studies are now being undertaken in this field that will enrich our understanding of how RNA modifications and their regulatory proteins change under conditions of neurological/neurodegenerative disease.


Xist (inactivated X chromosome specific transcript) in live mouse cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb  was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female mouse embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain plays critical roles in XCI. In Ssb knockdown cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged  Xist RNA is a prototype long non-coding RNA (lncRNA) involved in X chromosome inactivation (XCI), a mammalian dosage compensation mechanism, in which one female X chromosome is transcriptionally silenced to balance the X-linked gene dosage between males and females (Xist RNA transcripts on the chromosome territory of the chosen inactive X chromosome (Xi) recruits epigenetic factors for heterochromatinization and establishes the chromosome-wide gene silencing , proteins involved in N6-adenosine (m6A) RNA methylation (RBM15 and WTAP), heterogeneous nuclear ribonucleoproteins (hnRNP K and hnRNP U), histone modifiers (polycomb proteins)  . It is bXist interactome. We have previously shown that the Xist RNA can be efficiently tagged in live mouse cells by the programmable RNA-sequence binding domain of the Pumilio protein   as an Xist-binding protein functionally critical for XCI.Here, we devised a different system for profiling the ng site) . A transs Figure  10). We. WeXist k Figure . Meanwhik Figure . We nameMouse ES cells were cultured in medium containing 1000 unit/ml LIF. Feeder cells used were Drug Resistant 4 Mouse Embryonic Fibroblasts (DR4-MEF) . Feeder-free ES cells were cultured on culture dishes pre-treated with 0.2% gelatin .in vitro differentiation, cells were cultured in differentiation medium containing 50 μg/ml L-ascorbic acid (Sigma). For the first 4 days of in vitro differentiation, embryoid bodies (EBs) were cultured in suspension. On day 4, EBs were transferred to a gelatinized T75 tissue culture flask. On day 5, EBs failed to attach to the tissue culture flask were washed away. On day 6, surviving cells were harvested for subsequent experiments.For 2 of UV254 nm. Cells were then harvested by trypsin treatment and lysed in 3 ml of lysis buffer . Cell lysates were first digested with TURBO DNase (Ambion). After centrifugation, supernatants were collected for co-IP experiment using anti-Flag M2 beads (Sigma). Elution of IPed proteins from anti-Flag M2 beads was achieved by five rounds of RNA/DNA digestion using 250 U of benzonase  for 2 h at 37°C. The eluted proteins were concentrated in a speed-vacuum to a final volume about 60 μl. One third of the elusions were separated on a 10% SDS-PAGE gel and stained by colloidal blue. The rest of the elusions were run on another 10% SDS-PAGE gel shortly, and the unresolved gel slices were subjected to in-gel trypsin digestion and liquid chromatograph-tandem mass spectrometry (MS/MS) using Orbitrap FusionTM (Thermo) . For proteins identified in both i-FLAG-Xist and i-Empty samples, ranking numbers were assigned according to the ranks of their protein scores  in each sample  treatment was carried out at 250 μg/ml. If ES cells were cultured as undifferentiated ES cells, Alkaline phosphatase (AP) staining (Vector Laboratories) was used at the end of the assay for counting the cells. After AP staining, milk is added to the well to provide a white background for picture taking, then the whole tissue culture plate is put onto a scanner to scan the image. ImageJ  was usedMybbp1a (5′-CCGGAGTGTATTTGGTCATAT-3′), Tardbp (5′-GAATATGAAACCCAAGTGAAA-3′), Ssb-1(5′-GAGAAGATTTACACTTCCTTT-3′), and Ssb-2 (5′-GAACAGATCAAATTGGATGAA-3′). 300 μg/ml Hygromycin B  was used to select stably transfected cell lines. Single surviving ES cell colonies were picked and the knockdown efficiency was validated using RT-qPCR.An shRNA system  was used. The following shRNA sequences were designed against Total RNA was isolated by TRIzol (Life technologies). cDNA was synthesized using iScript reverse transcription kit . The real-time PCR was carried out on the CFX Connect real-time PCR system (Bio-Rad) using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). The following PCR primers were used: Actb ; Gapdh ; Mybbp1a ; Tardbp ; Ssb ; Xist E1 ; Xist E7 ; GFP .The padlock probe library design and padlock SNP capture procedure were performed as previously described . BrieflyThe multiplexed sequencing libraries were PCR amplified in a real-time PCR system  using the following primers: CA-2-RA.Miseq (5′-AATGATACGGCGACCACCGAGATCTATCGGCTACACGCCTATCGGGAAGCTGAAG-3′); CA-2-FA.Indx7Sol (5′-CAAGCAGAAGACGGCATACGAGATGATCTGCGGTCTGCCATCCGACGGTAGTGT-3′); CA-2-FA.Indx45Sol (5′-CAAGCAGAAGACGGCATACGAGATCGTAGTCGGTCTGCCATCCGACGGTAGTGT-3′); CA-2-FA.Indx76Sol (5′-CAAGCAGAAGACGGCATACGAGATAATAGGCGGTCTGCCATCCGACGGTAGTGT-3′); CA-2-FA.Indx91Sol (5′-CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCATCCGACGGTAGTGT-3′); CA-2-FA.Indx92Sol (5′-CAAGCAGAAGACGGCATACGAGATTCAAGTCGGTCTGCCATCCGACGGTAGTGT-3′); CA-2-FA.Indx93Sol (5′-CAAGCAGAAGACGGCATACGAGATATTGGCCGGTCTGCCATCCGACGGTAGTGT-3′). The following sequencing primers were used: Read1.Miseq (5′-ATCGGCTACACGCCTATCGGGAAGCTGAAG-3′); IndexRead (5′-ACACTACCGTCGGATGGCAGACCG-3′). Sequencing was carried out on an Illumina NextSeq system.2, 0.1% (v/v) Igepal CA-630) for 3 mins and centrifuged at 500 × g at 4°C for 10 min. Pellets were resuspended with transposition reaction mix   and incubated in Thermomixer at 37°C for 30 min with 1000 rpm. Transposed DNA samples were purified with Qiagen MinElute PCR purification kit (Cat. No.: 28006) and eluted with 20 μl elution buffer from Qiagen MinElute PCR purification kit. The purified transposed DNA fragments were amplified by PCR. Each reaction was carried out in 50 μl volume containing 20 μl of transposed DNA, 2.5 μl of 25 μM Ad1_noMX primer , 5 μl of NEBNext High-Fidelity 2× PCR Master Mix (Cat. No. M0541) with the following cycles: 1 cycle: 30 s at 98 °C and 20 cycles: 10 s at 98 °C, 30 s at 63 °C, 1 min at 72 °C. The additional number of PCR cycles (N) is the cycle that corresponds to one-third of the maximum fluorescent intensity. After determining the additional PCR cycles required, the remaining 45 μl of transposed DNA was further amplified with the following cycles: 1 cycle: 30 s at 98 °C and N cycles: 10 s at 98 °C, 30 s at 63 °C, 1 min at 72 °C. ATAC-seq libraries were two-stage purified using AMPure XP beads: 0.5× and 1.3×. Libraries are eluted in 20 μl nuclease-free water. The size and pattern of the ATAC-seq libraries were assessed by 6% TBE-PAGE. The concentration of each library sample was measured by KAPA Library Quantification kit (Cat. No.: kk4824). All libraries were pooled and a final quality check was performed using Bioanalyzer High-Sensitivity DNA Analysis kit. Sequencing was carried out on Illumina HiSeq 4000 system.Trypsinized cells were pelleted and washed with PBS. A total of 55,000 cells were lysed using cold lysis buffer ; Ad2.1_TAAGGCGA(5′-CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGT-3′); Ad2.2_CGTACTAG (5′-CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGT-3′); Ad2.3_AGGCAGAA (5′-CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGT-3′) Ad2.4_TCCTGAGC (5′-CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGT-3′) Ad2.5_GGACTCCT (5′-CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAGATGT-3′) Ad2.6_TAGGCATG (5′-CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAGATGT-3′); Ad2.7_CTCTCTAC (5′-CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGGAGATGT-3′); Ad2.8_CAGAGAGG (5′-CAAGCAGAAGACGGCATACGAGATCCTCTCTGGTCTCGTGGGCTCGGAGATGT-3′); Ad2.9_GCTACGCT (5′-CAAGCAGAAGACGGCATACGAGATAGCGTAGCGTCTCGTGGGCTCGGAGATGT-3′) Ad2.10_CGAGGCTG (5′-CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGGAGATGT-3′).Xist inducible male ES cells. And stable cell lines were established after puromycin selection. Three 15-cm dishes of ES cells were harvested, crosslinked with 1% formaldehyde for 10 min at room temperature, and quenched with 0.125 M glycine for 5 min. After centrifugation, cell pellets were resuspended in lysis buffer . Incubate the cells for 30–60 min on a rotor at 4°C. Treat the lysate with RQ1 DNase (Promega M6101) at 37°C for 10 min. Insoluble material was removed by centrifugation, and the supernatant was incubated with anti-FLAG M2 beads (Sigma M8823) overnight at 4°C. The beads were washed with IP 200 buffer  for five times. RNA-protein complexes were eluted from FLAG-beads by adding Proteinase K digestion buffer ), and incubated at 55°C for 1 h. Then RNA was extracted using Trizol (Invitrogen). Quantitative RT-PCR was performed to quantify the amount of Xist, 7SK and Actin RNA. The following PCR primers were used: Xist-1 ; Xist-2 ; Xist-3 ; Xist-4 ; Xist-5 ; Actb ; 7SK .Empty pCAG-IPuro vector or pCAG-IPuro expressing FLAG-Ssb was transfected into 2 of UV254 nm, collected and resuspended in 1 ml lysis buffer . After a 15-min incubation on ice, the lysate was treated with 2 μl RQ1 DNase and 10 μl RNase I (0.001 U/μl), and incubated for 5 min at 37°C on the mixer. Insoluble material was removed by centrifugation, and the supernatant was incubated with anti-FLAG M2 beads overnight at 4°C. The beads were washed with 1 ml high salt wash buffer  for two times; 500 μl wash buffer  for one time; 1 ml IP 200 buffer  for three times. RNA-protein complexes were eluted from FLAG-beads by adding 200 μl Proteinase K digestion buffer , and incubated 20 min at 37°C. Next, 200 μl PK urea buffer  was added and incubated 20 min at 37°C. Then RNA was extracted using Trizol. Quantitative RT-PCR was performed to quantify the amount of Xist, 7SK, and Actin RNA. Primers were described in the RIP section. Additional PCR primers were used: Xist-1a ; Xist-1b .Three 15 cm dishes of WT ES cells or ES cells stably expressing FLAG-Ssb were treated with Dox for 24 h. Medium was removed and replaced by ice-cold PBS. Then the cells were irradiated with 0.4 J/cm2, 10 mM DTT and 0.5 mM dNTPs), using random primers. The cDNAs were amplified by PCR using Xist-specific primers and Q5 high-fidelity DNA polymerase (NEB). The amplified DNA fragments were gel purified and pooled together. Construction of high-throughput sequencing libraries and sequencing were carried out by Novogene. The following primers were used for PCR amplification of Xist cDNA: Xist_-3 ; Xist_-1 ; Xist_3 ; Xist_4 ; Xist_6 ; Xist_8 ; Xist_10 ; Xist_11 ; Xist_12 ; Xist_14 ; Xist_15 ; Xist_16 ; Xist_18 ; Xist_19 ; Xist_20 ; Xist_21 .SHAPE-MaP was performed as described elsewhere ,15. BrieXist RNA was detected with Sx9 probe, a P1 DNA construct containing a 40 kb genomic fragment covering the Xist gene. The Usp9x probe was prepared with a BAC DNA (RP24-306P3). Nucleotide analogs used in probe labeling were Cy3-dUTP .RNA FISH, immunostaining and immuno-RNA FISH were carried out as previously described . ImmunosSsb cDNA clone was purchased from OriGene (MG206549) and the sequence of the cDNA was confirmed by Sanger sequencing (data not shown). A series of in-frame deletions of various functional domains of Ssb were generated by PCR amplification using Herculase II Fusion Enzyme (Agilent Technologies) followed by Gibson Assembly (NEB). The sequences of the cloned cDNA fragments were confirmed by sequencing (data not shown).A mouse Ssb stably knockdown. G418 (ThermoFisher) was used at 250 μg/ml to select for stably transfected cells. Single surviving colonies were picked and examined using RT-qPCR. The shRNA construct, which worked efficiently against Ssb, was designed against the LAM domain, but not against the 3′ UTR of the RNA. Therefore, the shRNA is against some of the rescue plasmid constructs, except ΔLAM. Nonetheless, transfecting the Ssb knockdown cells with the rescue plasmids should compensate the effect of Ssb knockdown and serve as a rescue assay to study the functional domains of La.For rescue experiments, the plasmid constructs were transfected into a female 3F1 ES cells with Wide-field fluorescent microscopy work was carried out on an Eclipse Ti microscope (Nikon) with a digital camera .Xist expression. For live-cell time-lapse video recording, cells were placed into the microscope cell culture chamber heated to 37°C at least 1 hr before imaging. Imaging was carried out in a closed chamber maintained at 37°C with 5% CO2 and 90% humidity. A 488-nm laser line (iChrome MLE-LFA) was set at 100% laser power. Images were acquired using a PlanApo 63×/1.4 N.A. oil-immersion objective (Zeiss) (heated to 37°C) with standard filter sets. The exposure time was set at 200 ms. All live-cell time-lapse video recording, unless explicitly stated otherwise, was carried out in a 2-hr time span with a 2-min time interval. For each time point, a 10-μm thick Z-stack with a 1-μm interval was collected. Autofocus system (Focus Clamp) was used to minimize out-of-focus throughout the recordings. Time-lapse imaging was started 1 h after the addition of doxycycline for differentiating cells. All acquired images were processed and analyzed using ImageJ  equipped with an Orca R2 CCD camera (Hamamatsu). 1 day before imaging, 800K feeder-free ES cells were seeded on fibronectin-coated glass-bottom dishes (MatTec Corp). Prior to live-cell imaging, cells were washed with 1× PBS and replaced with imaging medium composing complete medium for differentiating ES cells with DMEM substituted with FluoroBrite DMEM (ThermoFisher). 1 μg/ml of doxycycline was supplemented to the imaging medium to induce g ImageJ . Drift cXist signals in live cells were captured with an 800-ms exposure time at 100% laser power in 10-μm Z-stacks at 1-μm intervals.In the ‘sunset’ experiments, the snap-shot images of For padlock SNP capture, we only selected the SNPs which were allelotyped in all six samples. A nucleotide position with a read count less than 10 was considered as undetected and its read count was set to 0. Read counts of all the SNPs from one gene were combined to calculate the allelotype of the gene. Genes known to escape XCI were removed. The allelotype of each gene is calculated as Log ((129 count + 10)/(cast count + 10)). To avoid division by 0, we added a pseudocount of 10 to each read count. The sequencing data is available in sequence read archive .http://broadinstitute.github.io/picard/). Genome feature annotation was done using ‘annotatePeak’ from ChIPseeker package  using bowtie2 . The seq package . The seqhttps://weeks.chem.unc.edu/software.html) (https://weeks.chem.unc.edu/software.html) was also downloaded for software test runs. The source code written in Python 2 was revised for Python 3 updates. The source code was also modified to calculate SHAPE reactivity using one control (MutationRate1M7 – MutationRateDMSO) instead of two controls ((MutationRate1M7 – MutationRateDMSO)/MutationRateDenatured). The following parameters were customly adjusted for the data analysis of this study: primerLength = 30; minDepth = 5000; trimBothEnds = on. The rest parameters were kept the same as the default setting of the software. The software was used for counting mutation and generating the parameters used for RNA structure analysis. Other data analysis, such as profiled nucleotides, mutation rate profiles and principle component analysis, was performed using Perl and Python scripts built in-house. The in-cell SHAPE sequencing data is available in SRA .For SHAPE-MaP, the data analysis software ‘ShapeMapper_v1.2’ was downloaded from Dr Kevin Weeks lab website (re.html) . The recXist induction, we fixed the differentiating mouse ES cells by UV-crosslinking and performed FLAG affinity pull-down  was selected because systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a strikingly high female to male ratios of 9:1  and TAR DNA-binding protein 43  were selected because they are known transcription repressors ,21. The s of 9:1 . Moreoves of 9:1 . All theon stage . Mybbp1aormation ,26. EarlXist transgene. Inducible Xist expression in this cell line causes cell death due to the inactivation of the single X chromosome in male cells. About 60% of cells die after 4–5 days of doxycycline (Dox) treatment, which can be used as a convenient assay to assess the functionalities of XCI , was calculated to quantitatively assess XCI  and UV-crosslinking and immunoprecipitation (CLIP)  . Even thol cells , implyinMeanwhile, our results do not confirm the functional roles of Mybbp1a and Tardbp in XCI Figure  and E. HSsb in female mouse ES cells  to examine the chromatin compaction along X chromosome. We also performed padlock SNP capture to directly assess the XCI status by allelotyping the X-linked gene expression chromosome-wide.As Ssb is an RNA-binding protein involved in various house-keeping functions, the massive cell death, which occurred in Xist transgene so that the sequencing reads mapped onto X chromosome only reflect the chromatin status of one X chromosome, the inactive X. In ATAC-seq, the density of transposon insertion reveals the status of chromatin compaction. We analyzed the transposon insertion sites genome-wide and defined ‘Cut Count’ as the number of transposon insertion sites identified within a 1-Mb region. The chromatin compaction status during the induced XCI was then quantitatively measured by the ratio of the Cut Counts between the uninduced undifferentiated cells and the differentiated cells treated with Dox  failed to generate chromatin compaction  and the Mus musculus castaneus (CAST/Ei) mouse strain (XCast). The ‘preemptive choice’ mutant phenotype of 3F1 cells causes non-random inactivation of the X129 allele . Padlock SNP capture, a high-throughput and high-resolution RNA allelotyping method  enveloping the Xi chromosome territory. We performed Xist RNA FISH in differentiating ES cells. After 6 days of in vitro differentiation, Xist clouds could be detected in >80% of the wild type cells. However, in Ssb knockdown cells, Xist clouds were detected in a significant lower percentage of cells , an RNA chaperone domain (RCD) and a nuclear localization signal (NLS) ,29. We gs Figure . GFP fuss Figure . We firsrichment . Meanwhis Figure . For thes Figure  and E. TXist-binding protein important for folding of the RNA, possibly as an RNA chaperone. To assess the folding of Xist RNA, we performed SHAPE-MaP  , Ssb knockdown cells (duplicate samples) and DMSO control . Along the ∼18kb Xist RNA, we only analyzed the 3800 nucleotides which were covered by >10 000 reads in each sample  . The RNA of Xist . Consistwn cells . These dXist transcripts are subjected to degradation. To assess the stability of Xist RNA on Ssb knockdown background, we studied the disappearance of the Xist cloud signals after Dox removal (the ‘sunset’ process). Cells were cultured in differentiating conditions and treated with Dox overnight before Dox removal. The rate of the sunset process reflects the stability of the Xist RNA. We observed a significantly faster sunset rate on Ssb knockdown background (Figure Xist RNA, we performed quantitative RT-PCR to quantify the Xist RNA during sunset. The cell lines used were male ES cell lines carrying the inducible Xist transgene (Figure Xist transcription was blocked by Dox removal and actinomycin D treatment. The data was best fitted using a first-order exponential decay model (Figure Xist RNA was then calculated (Figure Xist is significantly shorter on Ssb knockdown background. Interestingly, it seems the decay reaction kinetics of Xist in Ssb knockdown cells can be separated into two phases (Figure Xist RNA estimated from the data of the second phase is shorter and with more statistical significance (Figure We further investigated whether the misfolded d Figure  and B, ee Figure  and D. Xl Figure . Half-lid Figure . Consists Figure . The fire Figure . We provXist RNA transcripts are misfolded on Ssb knockdown background, and the shorter half-life of Xist RNA suggests that the misfolded Xist RNA transcripts are subjected to faster degradation.Taken together, SHAPE results show that the Xist, a position close to the most critical functional domain of Xist. In our previous study, we have shown that the 5′ PBSb fusion does not affect the Xist functionality (Xist functionality, we believe that the fusion of PBSb to Xist and PUFb-FLAG binding may not alter the RNA’s structure and its association with protein partners. It is notable that only 9 out of 138 proteins identified by FLAG-out were identified as Xist-binding proteins in previous studies (Xist binding proteins identified in other studies did not have a good overlap either. It is very likely that none of these studies, including ours, comprehensively identified Xist binding proteins. A variety of technical issues limit the precision and sensitivity of the current methods.Here, we show that FLAG-out is a useful method for profiling the interactome of lncRNAs. The advantage of FLAG-out is that once the target RNA is FLAG-tagged, the high specificity and high sensitivity of anti-FLAG antibody can be applied for the subsequent protein isolation. It should be noticed that selection of the PBS insertion site along the target RNA affects the outcome of FLAG-out. In this study, the PBSb sites are fused at the 5′ end of ionality . Given t studies . In factXist-binding protein involved in Xist RNA folding and spreading (Figure Ssb knockdown cells, the misfolded Xist RNA transcripts are unstable and unable to spread and form the Xist clouds. These defects eventually lead to failure of Xist cloud formation and collapse of XCI. However, we still cannot rule out the possibility that the indirect effect of Ssb regulates XCI. Ssb might be required for the proper expression of a specific factor in the process of XCI. Without this specific factor, Xist RNA unfolds and degrades, leading to a defect in Xist cloud formation.Ssb is known as an RNA chaperone . Based og Figure . In the Xist-binding protein identified in previous studies, only showed the earliest mutant phenotype around embryonic day E12.5 in the gene knockout animals (vitro differentiation of ES cells, the live-cell imaging results and many other experimental results of this study clearly reveal the critical roles of Ssb in XCI.Spen, the critical  animals ,31. No f animals . NonetheSsb may be a potential genetic factor of autoimmune diseases. However, identifying Ssb, as a protein involved in XCI does not help to further explain the sex ratio distortion. Rather, we believe it is a consequence of autoimmune disorders. Nuclear RNPs are immunogenic (Xist RNA and its associated proteins is a large piece of nuclear RNP in female cells, which can trigger autoimmune responses when exposed to the immune system under abnormal situations.Ssb is an autoimmune antigen found in the serum of patients with autoimmune diseases. More than 80% of the patients with autoimmune disorders are female . It is aunogenic . The inaXist RNA structure have been reported, but significant inconsistency exists among the results of the initial studies (ex-vivo structure of the RNA purified with mild conditions. Second, subregions of Xist may form dynamic structures in living cells. SHAPE analysis suggests that the dynamic structures function as loading pads for protein recruitment (Xist structure is highly dynamic in living cells (Xist folding provides an important piece to the puzzle. Interestingly, Ssb possesses three RRMs, which may function in a synergistic manner in regulating Xist structure in living cells. The mechanistic details await future research. Here, we discuss three possible ways to imagine the mechanistic roles of Ssb in XCI. The three possibilities are not mutually exclusive to one another. Firstly, it is possible that Ssb may function as an Xist chaperone. Indeed, the RCD domain of the human Ssb protein has been characterized as an RNA chaperone domain (Xist, a lncRNA with poor sequence homology among mammalian species (Xist in living cells (Xist. Misfolding of Xist observed in Ssb knockdown cells may be a consequence of protein loading failures.Understanding the structure of lncRNAs is critical for understanding lncRNAs’ functionality. Initial efforts to elucidate the  studies ,34–36. Nruitment . In PARIng cells . In our e domain . Ssb proe domain . Interese domain . Thus, t species . Secondlng cells ,37. ThesXist and its associated proteins forms a unique RNP complex in female cells. The fact that Ssb is an Xist-binding RNA chaperone may be one reason behind the frequent association of the Xi with the nucleolus (As a protein involved in tRNA maturation, a significant fraction of Ssb is located in the nucleolus ,39. The ucleolus ,40.Xist in Ssb knockdown cells seems to consist of two phases. The first hour of the reaction is a transient phase in which the decay reaction shows a slow kinetics. The rest of the data points form the second phase of the reaction which shows a significantly faster kinetics. To interpret the data, we assume that the decay reaction of Xist is catalyzed by both endonucleases and exonucleases. Endonuclease creates nicks along the RNA, which further serve as the starting points for exonucleases. Without the RNA chaperone activity of Ssb, misfolded Xist RNA may be depleted for protein-binding and the RNA may be randomly folded in order to ‘protect’ the hydrophobic bases from the hydrophilic environment. Therefore, compared to the protein-binding biologically active form, the misfolded structures may be more compact, which prevents the initial endonuclease reaction. Once the endonuclease reaction generates enough nicks to trigger the action of exonucleases, the degradation reaction quickly speeds up. However, we do not have enough data to study the transient first phase in detail, therefore, this interpretation may be speculative at this point. The details of the decay reaction and, more importantly, the structural details of Xist await future studies.It is an interesting observation that the decay reaction of All data are available in the main text or the Supplementary Data.gkab1003_Supplemental_FilesClick here for additional data file.


Hepatitis delta virus (HDV) is a defective human virus that lacks the ability to produce its own envelope proteins and is thus dependent on the presence of a helper virus, which provides its surface proteins to produce infectious particles. Hepatitis B virus (HBV) was so far thought to be the only helper virus described to be associated with HDV. However, recent studies showed that divergent HDV-like viruses could be detected in fishes, birds, amphibians, and invertebrates, without evidence of any HBV-like agent supporting infection. Another recent study demonstrated that HDV can be transmitted and propagated in experimental infections ex vivo and in vivo by different enveloped viruses unrelated to HBV, including hepatitis C virus (HCV) and flaviviruses such as Dengue and West Nile virus. All this new evidence, in addition to the identification of novel virus species within a large range of hosts in absence of HBV, suggests that deltaviruses may take advantage of a large spectrum of helper viruses and raises questions about HDV origins and evolution. Deltavirus genus. HDV forms enveloped particles with an average diameter of 36 nm that consists of cell-derived lipid vesicles harboring HBV surface proteins coating an inner ribonucleoprotein (RNP) [Hepatitis D virus (HDV) was discovered 40 years ago in the liver of individuals chronically infected with hepatitis B virus (HBV) , a liverin (RNP) . This RNin (RNP)  that is in (RNP) . Althougin (RNP) .It is noteworthy that the origin of HDV is currently unknown. The HDV genome is unique and the smallest among animal viruses but shares some properties with some plant agents called viroids . Indeed,A cellular protein termed delta-interacting protein A (DIPA), initially identified as an HDAg ligand in a yeast two-hybrid screen , was proAnother hypothesis was that HDV RNA might have emerged as a consequence of an aberrant splicing event. A process known as mis-splicing, which produces RNA circles, was proposed; this occurs when a downstream (5′) splice donor is joined with an upstream (3′) splice acceptor site. However, an event of RNA-primed hairpin formation needs to occur in order to explain the base pairing of the HDV RNA ,17.Thus, it is theoretically possible that HDV could have emerged in HBV-infected hepatocytes subsequent to the evolution of cellular circRNA forms becoming autonomously replicative  and for Proechimys semispinosus ; and in a liver transcriptome from a lesser dog-like bat Peropteryx macrotis (PmacDeV) [Odocoileus virginianus; OvirDeV) and from captive-born Eastern woodchucks  [Taeniopygia guttata deltavirus (tgDeV), Serinus canaria-associated deltavirus (scDeV), and Erythrura gouldiae deltavirus (egDeV) [For almost 40 years, HDV formed and was the sole member of the genus Deltavirus. Recently, however, virus-prospecting studies have discovered hepatitis D relatives (known as “deltaviruses”) in a diverse range of animals . Mining s ; in comPmacDeV) . Additio; mmDeV) ,26. Thre (egDeV) .In addition to the discovery of several novel deltavirus sequences in a broad host range spanning several taxonomic hosts, these metatranscriptomic approaches across different tissues and organs stressed important features of these novel deltaviruses infections; in particular, they highlighted tissue tropism differences among them and HDV. SDeV genome reads were found in NGS libraries prepared from the brain, blood, and liver . SimilarIncreased viral reads from the predicted transcribed regions, i.e., delta-like antigens coding region, have been used as a marker of active deltavirus replication/transcription , even thUsing conventional RT-PCR, SDeV was found in the liver of four out seven individuals of the offspring of the SDeV positive parental founder snakes as well in a water python that lived in close proximity. Additionally, 3 out of 20 blood samples from snakes from a different breeder turned to be positive for SDeV, suggesting that SDeV can be transmitted among animal populations . HoweverAnother unresolved question in the field is whether the newly reported deltaviruses can establish chronic infection like for the human HDV. Most likely the helper viruses involved and the efficacy of the host response combined shall determine the natural course of non-HDV deltaviruses infections. For tgDeV, mmDeV, and RDeV, it has been suggested that a host immune response can be mounted to suppress and clear deltavirus infections. For instance, the number tgDeV reads in birds correlated with the immunosuppressive effect of testosterone . For oneDespite being highly divergent at the level of primary sequence, these non-human deltaviruses share many features with HDV. All characterized deltaviruses have a negative-sense self-complementary circular RNA genome folded into the classical unbranched rod-like structure, including ribozymes ,25,26,27Nevertheless, all of the newly identified sequences include a single open reading frame (ORF) representing the delta antigen encoding for proteins with sizes ranging from 180 to 225 amino acids (DAgs). It is noteworthy that a second ORF was identified in the genome of SDeV , though The most important and intriguing observation from these virus prospecting studies, based on epidemiological and serological evidence supported by metagenomic and transcriptomic findings, is the concept that deltaviruses infections and transmission seem to be independent of coinfection with an HBV-related hepadnavirus. With the exception of mmDeV, all of the other HDV-like agents were found in the absence of hepadnaviruses . In addiBased on the percentage of nucleotide identity of the genome, HDV was classified into eight genotypes, with two to four subtypes per genotype characterized by >90% similarity over the entire genome sequence ,35,36. TThe discovery of divergent HDV-like viruses from different hosts raises questions about the origin and evolution of HDV. Consequently, phylogenetic inferences and sequence analysis have been conducted each time a new deltavirus species was discovered. One of the first phylogenetic analyses estimated an average nucleotide identity of 29 to 46% and amino acid identity of 21 to 54% among all HDV genotypes and 7 of the newly reported deltaviruses . RDeV waMore recent phylogenetic analyses based on full-length protein sequences of DAg, including all HDV genotypes and the 12 HDV-like viruses described so far, revealed, however, different relationships among deltaviruses ,26. The With the discovery of new HDV-like viruses, new phylogenetic analyzes will allow a better understanding and reconstruction of the evolutionary history of animal deltaviruses and their relationships with their respective hosts.HDV virion encloses a circular, single-stranded negative-sense RNA molecule associated with an internal core delta antigen surrounded by envelope glycoproteins. The full-length circular genome for HDV is approximately 1680 nucleotides, whereas for HDV-like agents, it ranges between 1592 and 1735 nucleotides. The GC contents of these novel HDV-like agents range between 46 and 58 percent, which is slightly lower than the human HDV with a GC content of around 60 percent ,24,25,26A ribozyme, a self-cleaving RNA sequence, resides in the viroid-like sequence of the HDV genome that cleaves a linear form of multiple-copy length of the viral genome or antigenome into monomeric units that are then circularized to complete the replication cycle. All described HDV-like agents contain self-cleaving motifs from either the HDV or the hammerhead class ,26,27,29Many cell culture model systems have been developed to study HDV genome replication and regulation of viral gene expression. One of the most simplified systems relies on the transfection of cultured cell lines with larger than unit-length cDNA clones  of HDV sequences . HDV cDNUsing an analog approach, it was shown that RDeV could initiate replication in Huh7 cells transfected with a construct carrying a head-to-tail dimer of the full RDeV genome in genomic orientation as demonstrated by the presence of the marker of on-going RNA-directed RNA replication, i.e., viral RNA of antigenomic polarity and S-RDAg as early as 4 days post-transfection .HDV uses RNA editing as a mechanism to switch from genome replication to packaging. During replication, a fraction of the antigenome RNA is edited by ADAR1 such that the adenosine within the amber stop codon that terminates the S-HDAg ORF is deaminated to inosine . The stoSimilar to S-HDAg, the RDAg ORF also terminates with a UAG amber stop codon . A similIn contrast to RDeV, two species of NGS reads were detected for OvirDeV at the second position of the stop of the S-OvirDAg (UAG). The base-editing frequency measured was 0.4% with all edited sequences showing a G instead of the consensus A. While this A-to-I nucleotide variation may suggest RNA editing by ADAR1, the generated ORF would be extended by two amino acids only due to the presence of a stop codon immediately after . HoweverA putative 28-amino acid carboxyl-terminal extension was also identified in the bat deltavirus (DrDeV) genome , althougSequence analysis of the SDeV genome revealed a potential editing site at the UAG amber stop codon of the S-SDAg. RNA editing would increase the ORF by 22 additional amino acids, and thus, two bands with estimated molecular weights of 22.7 kDa and 25.6 kDa should be detected by Western blotting in the homogenates of tissue samples from infected snakes. Indeed, an anti-S-SDAg antiserum detected two bands with molecular weights that are approximate to the theoretical estimates for S- and L-SDAg in liver samples of infected snakes but mainly only the large form was detected in the brain samples . WhetherIn contrast to HDV, RNA editing seems not to be the mechanism regulating the switch from replication to viral RNPs packaging for the newly identified deltaviruses . AdditioHDV RNA and HDAg proteins interact to form the HDV ribonucleoprotein (RNP), the delta antigens form multimers, and the HDV RNA wraps around such multimers. It has been suggested that the multimers may be predominantly octamers of the delta antigen and that the genomic RNA, which is partially double-stranded, wraps around the octamers in a way analogous to how double-stranded host DNA wraps around histone octamers ,54. The The envelope proteins of HBV are the only contribution of the helper virus to the HDV life cycle. The determinants governing HBV tissue and species tropism also dictate HDV tropism to human hepatocytes. HBV and HDV entry requires the specific interaction with the preS1 domain of viral L-HBsAg. Despite sharing the same envelope and using the same host receptor, HBV and HDV show different requirements of host factors to establish productive infections. In contrast to HBV, HDV infection is predominantly determined at the receptor level . EctopicFor budding HDV takes advantage of the quite inefficient HBV assembly process that produces relatively huge amounts of empty particles . The budSimilar to HDV, the newly identified deltaviruses RNA can self-replicate in a wide variety of cell types and species ,23,27,33While the potential candidate helper viruses for these novel deltaviruses are currently unknown, the lack of a farnesylation motif (CXXQ), required for HDV assembly and release in the C-terminal region of the novel deltaviruses antigens , highligHDV transmission among the human population requires HBV envelope proteins. Likely, the transmission of the newly reported deltaviruses in animal populations might also be dependent on coinfecting helper viruses to provide envelope proteins to copackage deltaviruses RNPs and assist dissemination.Orthohepadnavirus.The final stage of the HDV life cycle, that is, the incorporation of the newly replicated RNA genome associated with delta antigens into enveloped virions, can be easily accomplished by coinfection with a helper virus or by cotransfection with constructs encoding the envelope proteins of hepadnavirus belonging to the genus Orthohepadnavirus, which infects mammalian species, and genus Avihepadnavirus, which infects avian species. Since 2019, however, five hepadnavirus genera are recognized [Herpetohepadnavirus), which infect amphibians and reptiles, as well as two highly distinct groups that infect fishes—the metahepadnaviruses (genus Metahepadnavirus) and the parahepadnaviruses (genus Parahepadnavirus) [For decades, only two hepadnavirus genera were known: genus cognized . The thrnavirus) ,63,64.Since HDV has to coexist with HBV for propagation, a first assumption would be that HDV should have evolved in animals that were susceptible to infection by hepadnaviruses, namely, two of the newly discovered deltaviruses, aDeV and mmDeV, were retrieved from RNA-seq libraries of ducks and eastern woodchucks that are natural hosts for hepadnaviruses ,25 and cSDeV isolated from brain homogenates was shown to be able to establish persistent infection in boid kidney cells (I/1Ki cells). The same metatranscriptomic analyses that led to SDeV discovery revealed that the infected snakes had no traces of coinfection with HBV-like viruses. Instead, NGS analyses retrieved sequences of the L and S RNA segments of reptarena- and hartmanviruses , suggestMarmota monax) experimentally infected with WHV and in two sequence read archive (SRA) data derived from PMBCs of animals negative for antibodies against WHV as well as WHV DNA [Many in vitro and in vivo infection studies demonstrated that woodchuck hepatitis virus (WHV), a hepadnavirus related to HBV, could provide helper activity for HDV ,65. As o WHV DNA . The liv WHV DNA . In line WHV DNA .Despite the lack of experimental evidence, the libraries dominated with WHV also contained reads matching viral sequences of Herpesvirus, Flavivirus, Retrovirus, and Poxvirus, representing potential sources of helper activity. Interestingly, Poxvirus hits were identified in libraries where four other deltaviruses have been identified, i.e., DrDeV-A, DrDeV-B, PmacDeV, and OvirDeV, which was hence considered as a potential ecological association worth to be evaluated in future studies .As hepacivirus infections are highly prevalent in rodents and since HCV, in particular, can provide envelope proteins for HDV and support its transmission in vivo ,21, the Undoubtedly, the discovery of HDV-like viruses in distantly related species other than humans and their detection in the absence of HBV or hepadnavirus infection, as well as the evidence that HDV and HDV-like virus can indeed use envelope glycoproteins from viruses of diverse genera for transmission, open numerous questions over the origin of deltaviruses and the nature of the relationship between HDV and HBV. This suggests that HDV-like viruses have been associated with animals long before their first appearance in humans  and duriIt is noteworthy that three clinical studies have now been performed to verify the possibility of HDV infection in patients only coinfected with HCV ,70,71. AAltogether, these studies pave the way to a better understanding of the origin of HDV and enlarge the deltaviruses research field. They also raise several questions concerning the immunological and virologic relations between HDV and HBV or alternative helper viruses, and the exact pathogenesis and tissue tropism of HDV. Perhaps one of the most important questions is if HDV can be transmitted in humans by other helper viruses than HBV. Additionally, if HDV has a wider range of potential coinfections than previously thought, what could be the clinical relevance and pathobiological implications?