The growing knowledge on several classes of non-coding RNAs (ncRNAs) and their different functional roles has aroused great interest in the scientific community. Beyond the Central Dogma of Biology, it is clearly known that not all RNAs code for protein products, and they exert a broader repertoire of biological functions. As described in this review, ncRNAs participate in gene expression regulation both at transcriptional and post-transcriptional levels and represent critical elements driving and controlling pathophysiological processes in multicellular organisms. For this reason, in recent years, a great boost was given to ncRNA-based strategies with potential therapeutic abilities, and nowadays, the use of RNA molecules is experimentally validated and actually exploited in clinics to counteract several diseases. In this review, we summarize the principal classes of therapeutic ncRNA molecules that are potentially implied in disease onset and progression, which are already used in clinics or under clinical trials, highlighting the advantages and the need for a targeted therapeutic strategy design. Furthermore, we discuss the benefits and the limits of RNA therapeutics and the ongoing development of delivery strategies to limit the off-target effects and to increase the translational application. In the last decades, growing attention pointed to the development of innovative therapeutic strategies that were based on new scientific findings. Specifically, the rapid development of RNA-based approaches that were used in basic research for scientific purposes inspired the application of these strategies to clinical research. More recently, thanks to the increased interest in ncRNAs  as regulators of gene expression in pathophysiology , the poGeneral concepts: miRNAs are a class of short non-coding RNAs that are characterized by a length of 20–22 nt, with a fundamental functional role in regulating gene expression at a post-transcriptional level. Their mechanism of action is exerted by their ability to interact with the RNA-induced silencing complex (RISC complex) and, in turn, to target a specific mRNA in order to block its translation and/or induce its degradation. miRNAs, natural short antisense RNAs, are essential for the correct gene expression program during development and organogenesis; indeed, a global decrease in miRNA expression is lethal [s lethal ,7,8,9.miRNAs are transcribed into the nucleus as precursor molecules (pri-miRNAs), which can be more than 1000 nt long and are processed in 60–120 nt-long pre-miRNAs by Drosha/DGCR8. Exported by exportin-5 into the cytoplasm, these molecules are processed via cleavage by Dicer in order to obtain a short double-strand miRNA, which in turn is recruited into the RISC complex, where a selection of one single strand occurs and interacts with Argonaute (Ago) proteins to target the 3′UTR mRNAs [In contrast to siRNA molecules (see below) and other non-coding RNAs, they are natural and naturally able to be processed and to function afterward. Depending on the degree of complementarity, miRNAs can induce mRNA degradation (perfect match) or translational inhibition (imperfect match). In addition, their activity is not limited to a single messenger RNA, but they usually target several mRNAs, often included in the same molecular pathway. This amplifies the miRNA function effects, since they repress the expression of several interconnected targets simultaneously; however, this also implies that miRNA activity is promiscuous.The therapeutic potential of miRNAs is based on the knowledge of specific miRNA families involved in several biological processes [rocesses  that arerocesses ,13,14,15rocesses ,17, metarocesses , diabeterocesses , stress-rocesses ,21,22 anrocesses . For thirocesses ,25. The Ongoing approaches: At present, the approval of miRNA therapeutics in clinical practice needs further steps of effectiveness validation; however, several miRNAs are under clinical trials for the treatment of different diseases. As examples, cobomarsen targets miR-155 for the treatment of blood cancers, remlarsen mimics miR-29 in keloids treatment and MRG110 is an antagomir for miR92 that is proposed for tissue repair [e repair .Conclusions and future perspectives: miRNA-based therapy is a well-recognized, exciting and significant approach that holds promise for next generation drugs than can reach specific targets and sites. Several efforts are incoming for the development of therapeutic miRNAs with advanced absorption, distribution, metabolism and elimination abilities. Moreover, great attention is needed for the delivery of specifically localized miRNAs to selected target sites. This delivery optimization should efficiently improve the effectiveness of therapeutic miRNAs.General concepts: Short interfering RNAs (siRNAs) are double-stranded short RNAs that are involved in the RNA interference pathway. These molecules are a product of evolution as a cellular defense from viral RNAs. As described for miRNAs, one strand of the siRNA is complexed with AGO2 in the formation of the RISC complex, and this interaction allows the cleavage of a specific transcript.In contrast to miRNA processing, prior to Dicer activity, siRNAs are double-stranded RNAs of 30–100 nucleotides, entirely complementary to the target mRNA.siRNAs, discovered in plants, invertebrates, and zebrafish, are not naturally produced in mammals, except in murine germline cells .Therapeutic potential: Concerning the biotechnological application, synthetic siRNAs can be designed to obtain a potent knockdown of a specific gene. This approach, routinely applied to the characterization of gene functions in basic biology, represents, at present, a translational strategy to selectively impair genes that are causal to several diseases.Ongoing approaches: As reported in Conclusions and future perspectives: one of the major issues regarding therapeutic siRNAs concerns their stability, safety and specificity. The accumulation of these molecules should reach a high level of biocompatibility in order to not compromise organ functionality. Moreover, the combination of several siRNAs may help in the targeting of complex pathways with the intent to sensitize the target site and allow the treatment of patients with similar but not identical clinical pictures. This is in line with a perspective of a personalized medicine.General concepts: Antisense oligonucleotides (ASOs) are short, single-stranded RNA or DNA oligonucleotides that also provide gene silencing. They can be naturally produced by the cells or artificially synthesized, and they have the ability to target a specific transcript through the Watson and Crick base complementarity. They can work by inducing transcript degradation and altering mRNA processing, blocking RNA translation, preventing ribosome attachment [tachment .Therapeutic potential: There are several ASOs in clinical trials, especially targeting mRNAs that are causal of several diseases [diseases ; howeverOngoing approaches: Among ASOs that are applied in clinical management  and to enhance their absorption.General concepts: lncRNAs (long non-coding RNAs) are non-coding RNAs with a length of more than 200 nt [n 200 nt . They arn 200 nt . In recen 200 nt ,35,36,37n 200 nt , mediaten 200 nt , act as n 200 nt  and behan 200 nt .The therapeutic potential: Based on their diversified roles, long non-coding RNAs have been considered as suitable targets to be used for therapy. An example of ncRNA-based therapeutic molecules that can recognize and antagonize lncRNA expression is represented by the use of siRNAs and antisense oligonucleotides (ASOs) [s (ASOs) .In addition, the potential of lncRNAs is exerted by their use as therapeutic molecules.For example, an approach to target specific mRNAs through lncRNAs is based on the use of SINEUPs, a new class of antisense lncRNAs that can increase the translation of specific mRNAs. These molecules are characterized by two main domains: one is the SINE element, which is the effector domain for the increase in translation activity; the other is the AS (AntiSense) region, which is able to target the specific mRNA through base pairing .With respect to RNA-based therapies to counteract lncRNA function, an approach was recently designed to counteract EMT  that was based on the use of a dominant-negative form of the lncRNA HOTAIR . This fiOngoing approaches: Concerning lncRNAs, at present, the principal application regards the modulation of their expression in order to obtain oncogenic lncRNAs knockdown (by RNA interference approach), or oncosuppressor lncRNA overexpression; therefore, the use of these molecules per se is a hot topic that requires development. With respect to SINEUPs, this class of both natural and synthetic lncRNAs shows a significant applicability in therapeutic approaches and has been described as a powerful strategy to treat haplo-insufficiencies.Conclusions and future perspectives: lncRNAs are certainly considered as pivotal elements in the development of different pathologies, including cancer. Based on these findings, the development of more effective therapeutics involving this class of molecules represents an emerging challenge. The development of more specific gene editing approaches, as well as the design and characterization of lncRNA-competing molecules, limiting the off-target effects, should be considered in the perspective of lncRNA-based therapeutic approaches.General concepts: circRNAs (circular RNAs) are non-coding RNAs that are obtained through a biogenesis mechanism, characterized by back splicing events. Back-splicing requires a flanking intron with a downstream 3′ donor site and upstream 5′ acceptor sites [or sites . They aror sites ,47,48,49The therapeutic potential of circRNAs to act as molecular sponges for miRNAs could be very interesting in order to delocalize oncogenic miRNAs or miRNAs that are causal to several diseases’ pathways. However, other interesting functions have been described for circRNAs, including their ability to bind and sponge proteins , proteins ,59.Finally, based on the described ability of circRNAs to be translated ,61, thisOngoing approaches: A recent application of this strategy concerns a coding circRNA that is used as a vaccine against Sars-CoV2 [ars-CoV2 . This apConclusions and future perspectives: Due to the wide spectra of activities that are attributable to circRNAs and their stability (due to their structure), circRNA targeting or impairment represent a potentially powerful strategy to impair several diseases’ onset and progression. CircRNAs are generally expressed in a tissue-specific pattern; however, limiting the off-target effects represent an open field of investigation. To this issue, the specific vehiculation of synthetic circRNAs or siRNAs targeting the junction sequence may provide substantial benefits for patients. Moreover, their modulation in vivo, based on the needed knowledge in this field, will advance the applicability of circRNA therapeutics.General concepts: Ribozymes are RNA molecules that are characterized by catalytic activity such as proteins. They are able to cleave target RNAs and thus impact gene expression [pression  and allopression . The bespression . Their fThe therapeutic potential of ribozymes resides in their ability to selectively recognize a specific RNA  and to cleave it (with the consequent abrogation of RNA translation); these specific features make this class of molecules a promising, innovative and specific approach to be used in therapy as an RNA drug. Moreover, once the target RNA is cleaved, the ribozyme can detach from the substrate and can go on to bind and cleave other targets. Furthermore, ribozymes can be artificially engineered in order to have cleavage activity in cis or in trans, and to be allosterically activated by specific effector molecules [olecules .Ongoing approaches include Angiozyme, a synthetic 35-nucleotide-long ribozyme that is used in clinics to target the VEGFR mRNA to impair angiogenesis and tumor progression in renal carcinoma [arcinoma . Moreovearcinoma ,68,69. Warcinoma ,71.Conclusions and future perspectives: Ribozymes-based therapeutic approaches seem to be promising agents as alternative or adjuvant to chemotherapy. Moreover, their ability to catalytically inactivate many specific target RNAs and to maintain a higher-grade of stability with respect to ASOs, together with their negligible toxicity, confer them the potential to be applied to the patients’ management. At present, a challenge in the use of ribozymes as therapeutic molecules is centered on the need to improve their stability, activity and delivery to specific cells.General concepts: In recent years, the CRISPR-Cas9 system has been significantly improved as a strategy of genome editing, alongside the emergence of some concerns regarding clinics, as well as scientific controversies.Based on this technology, the first core member of the system, a non-coding RNA that is represented by the guide RNA (gRNA), specifically targets the portion of genomic DNA to be engineered, while the Cas9, the enzyme component, cuts the DNA at specific sites that are complementary to the gRNA sequence.A CRISPR-Cas system on RNA is also being developed. Differentially to Cas9, Cas13 only has target activity on RNA molecules, conferring to this strategy the possibility to manage and edit RNA for therapeutic purposes ,73. RNA The therapeutic potential of the use of a guide RNA represents, per se, a strategy to use an RNA molecule.This approach has been applied at present to Adenosine deaminase acting on RNA (ADAR)-mediated RNA editing and has inspired further programmable RNA-guided machines for potential translational applications. In this regard, ADAR-mediating RNA editing, such as CIRTS (CRISPR-Cas-inspired RNA targeting system); RESCUE (RNA editing for specific C to U exchange); RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing); and LEAPER (leveraging endogenous ADAR for programmable editing of RNA) have been developed and validated. The CIRTS system A is compCas13 editing can also be applied to add chemical modifications to specific RNAs for therapeutic purposes , in ordeOngoing approaches: The deadCas13 can edit RNAs at different levels, representing a powerful strategy to be applied to several diseases, including neurodegenerative diseases that are caused by the triplet repeat expansion. One of the most interesting aspects of this approach is that Cas13 and dCas13 can target numerous RNAs or different regions of the same RNA at the same time, representing an efficient tool for RNA-based approaches.Conclusions and future perspectives: The Cas13-based approach represents a knockdown strategy that is more specific and efficient than the RNA interference and with no detectable off-target effects. Moreover, by localizing the enzyme into the nucleus , it can significantly interfere with nuclear RNAs. Starting from the promising data that can be obtained from basic research, the development of these effective, specific and multitarget approaches to be applied to oncogenic RNAs could be desirable for complex diseases management, at first for in vitro study, and then for in vivo applications.General concepts: RNA aptamers are artificial structured sequences that can specifically bind to different classes of molecules and can be used in therapy for the treatment of pathological conditions, but also as diagnostic tools. They can be used to impact gene expression, and it is also possible to specifically design aptamers against specific ligands to be used as RNA antibodies [tibodies . The SELtibodies . The SELtibodies . Initialtibodies .The therapeutic potential of aptamer technology to impact gene expression is represented by the insertion of an RNA aptamer into the 5′UTR of mRNAs; upon ligand binding, there is a conformational switch that does not allow ribosome scanning at 5′UTR, with a consequent substantial decrease in translation activity [activity .Moreover, ligand-bound aptamers could be used to impact alternative splicing or exon skipping events through RNA conformational switches that impact the regulation of splicing machinery activity . RNA aptOngoing approaches: As for the other mentioned RNA-based approaches, some aptamers have also shown encouraging readouts in terms of cellular response to these therapeutic molecules  .Conclusions and future perspectives: The potentiality of aptamers in therapy is based on their specific features of low immunogenicity, safety, stability at room temperature, possibility to be conjugated to other small molecules, and customization. At present, the completion of the molecular mechanism-elucidating aptamers’ mechanism of action is ongoing, but the principal issue to be addressed remains their delivery. Thus, the development of carriers that can drive aptamers to specific sites remains an open field of investigation. The drug delivery of modified aptamers could allow to reach specific targets, and their synthetic origin warrants at first the reproducibility of the experimental data, and then of the clinical application in advanced stages of experimentation and clinical practice.Several companies are currently investing in the research and development of new RNA drugs to cure different diseases . The importance of the development of the two mRNA-based vaccines against COVID-19, mRNA-1273 (Moderna) , and BNTBeyond the use of messenger RNAs for therapeutic purposes, several non-coding RNA molecules have been approved by the FDA (Food and Drug Administration) or are currently in clinical trials.Great opportunities have been given to siRNAs and antisense oligonucleotides (ASOs). Approved siRNAs such as patisiran  in 2018 Moreover, it is worth mentioning the first clinical trial for the delivery in vivo of components of the CRISPR-Cas9 through viral vectors, with the challenge to use RNA-LNP (RNA-lipid nanoparticles) as delivery systems in animals. One example of this approach is represented by the correction of a splicing mutation of fumarylacetoacetate hydrolase .With respect to cancer, there are some RNA drugs in clinical trials targeting central genes that are responsible, among others, for cell-cycle regulation, cell proliferation, and apoptosis, as reported in However, it seems that there was more success in the development and translation into clinics of RNA drugs for the cure of other kinds of diseases with respect to cancer. This is probably justifiable by their nature; most of these disorders are monogenic, so it is simpler to obtain a successful response, while cancer is a multigenic disease, and several signaling pathways are dysregulated. Based on this evidence, it seems more challenging to find a drug that can counteract cancer, considering the different stages of tumorigenesis, the pleiotropic effects of the treatment, and the heterogeneity that is observed among cells and individuals.Among all these classes of RNAs and their applicability to translational approaches, one of the most stimulating topics in this field of investigation is represented by the development of strategies to deliver these RNAs for therapeutic purposes and to enhance their stability and efficiency for the on-target effects, limiting the off-target ones.One urgent need is represented by the development of strategies to stabilize or to enhance the half-life of these molecules. Due to the high sensitivity of RNAs to RNAses into the serum, chemical modifications seem to be very challenging. RNA is a hydrophilic and negatively charged molecule, so it has difficulties to enter in naked form into the cells because of electrostatic repulsion. This is why it needs some appropriate vehicles to penetrate target cells and gain function .Lipid nanovesicles are small vesicles that recently gained great success as vehicles for RNA vaccines  . They arWith respect to extracellular vesicles, these are natural membrane-enclosed particles that are released from the cells with a high impact on physiology and pathology through their important role in intercellular communication. Indeed, extracellular vesicles contain proteins, enzymes, DNA fragments, and RNA molecules, and they carry out a fundamental role in regulating gene expression in target cells . ExtraceExtracellular vesicles can be engineered to enclose specific RNA molecules, such as via electroporation. In this case, this method takes advantage of the porous structure of the vesicles, so the RNA drug is incubated with vesicles, and the application of an electric field allows the passage through ,103,104.In addition, it is possible to engineer vesicle-producing cells in order to load them with a specific RNA content and specific surface ligands and receptors ,106. In The successful output that was obtained during these years encouraged the scientific community more deeply explore the field of RNA. Looking to the past, we are beyond the doors of the RNA therapeutics era, and several goals have been met ,108.Specifically, at present, several RNA therapeutics have been designed and some of them are under clinical trials or being utilized in therapy  for different diseases .Nevertheless, there is an urgent need to take advantage of other biological properties of non-coding RNAs, in order to develop strategic alternatives to perturb pathological processes. The great advantages of RNA-based therapies seem to reside in their specificity, low toxicity and ability to act in combination to target complex pathways. Furthermore, some ncRNAs are characterized by a high safety and stability in naked or encapsulated form, representing added value to their applicability. These aspects hold promise for more effective therapeutics and personalized medicine.The new findings in the field of ncRNAs should further clarify their mechanism of action, may identify other classes of ncRNAs, and may propose new approaches for therapeutic applications of RNAs with a high grade of biocompatibility, absorption, distribution and metabolism.Furthermore, the future development of the RNA-based therapies must aim at limiting the off-target effects of these molecules and optimizing their low immunogenicity, safety, stability and customization.In conclusion, the most important points of investigation are dictated by the necessity to conceive novel methods to increase the efficiency, specificity and applicability in clinics of these RNA molecules’ delivery. With these intents, the interdisciplinarity among molecular biology, cell biology, chemistry, nanotechnology and immunology seems to be fundamental.


Many surprising discoveries in the last few decades have radically changed our view of gene expression and its regulation, particularly concerning the role of RNA. Starting in the mid-1970s, we have witnessed the discovery of mosaic genes and splicing, the unearthing of RNA-mediated catalysis, the characterization of different classes of non-coding regulatory RNAs, and a plethora of RNA modifications and RNA-editing reactions. In parallel, the development of novel sequencing technologies has led to the sequence of the human genome and the genomes of many other organisms. It has also resulted in a more profound account of transcriptomes, expressed not only in different organs or at different developmental stages but also in individual cells. Together, such technological breakthroughs have provided many new insights into the organization, dynamics, and expression of complex genomes, and have also revealed the basis of organismal development and cell differentiation.https://www.routledge.com/RNA-the-Epicenter-of-Genetic-Information/Mattick-Amaral/p/book/9780367567781) is much more than what its title might suggest. It is not only a description of our current understanding of the role of RNA in cell and developmental biology but is also a useful history of molecular biology. The authors chronicle the major developments that established DNA as a hereditary material, that determined the principles of cellular protein synthesis (with ribosomes and transfer and messenger RNAs (!) involved), and that led to deciphering of the genetic code. They follow this with an account of how RNA gradually – despite the previous dominant protein-centric views – became recognized as the ‘Epicenter of Genetic Information’, where the transcription of non-protein-coding DNA into tens of thousands of regulatory RNAs plays a major role in the biology of complex organisms, their evolution, development, and function.The forthcoming book ‘RNA, the Epicenter of Genetic Information’ by John Mattick and Paulo Amaral  everything by simply reading Jim Watson’s 1965 textbook Halcyon Days, Worlds Apart, The Age of Aquarius, and All that Junk, reflect the authors’ very personal connection to the history  of RNA research. Their emotions are further shown in the way they deal with the early reluctance to accept new – often totally unexpected – findings that supported a central role of RNA in biology. I find it appropriate that the most important discoveries and technical breakthroughs are set into an historical context, where the main actors behind the findings are introduced together with the accompanying disputes. This is particularly relevant for those at the beginning of their careers who are overburdened with day-to-day duties at the bench. One can draw important lessons from accounts of how other scientists – many of them ‘Science Giants’ – have behaved.Each of the eighteen chapters is a brilliantly written semi-autonomous essay on a particular segment of the RNA odyssey. The poetic titles of several chapters, such as RNA Society, acting as its spokesman by advertising the centrality of RNA.John Mattick pioneered the concept that most of the genome in complex organisms, including introns, ‘intergenic’ regions and all sorts of repetitive elements  is dedicated to the production of regulatory RNAs with roles in epigenetic regulation, differentiation, and development. Many of Mattick’s predictions have been vindicated by discoveries of intron-encoded small nucleolar (sno) RNAs and micro (mi) RNAs, of the massive occurrence of RNA alternative splicing and polyadenylation, of the frequent transcription of both sense and anti-sense DNA strands, and – finally – of the existence of tens of thousands of long non-coding (lnc) RNAs, including enhancer RNAs (eRNAs) and transcripts from repetitive regions or pseudo-genes. I have frequently argued that John Mattick should be on the payroll of the Many pages of the book are devoted to controversies and paradoxes that have emerged as a corollary of new findings: the post-splicing-discovery ‘introns-early versus introns-late’ debates; the ‘junk or not junk’ disputes about non-protein-coding DNA that largely exceeds the coding DNA in complex organisms, and finally – when pervasive transcription of complex genomes was already evident – the question ‘do thousands of new lncRNAs reflect transcriptional noise or rather represent a new class of RNA regulators?’ These and other disputes are illustrated with interesting quotes from the adversaries that add much flesh to the discussion.trans when artificially expressed, is not clear whether they do so – as the 3’-UTR fragments – in more physiological settings. Likewise, I would welcome more evidence and also mechanistic insights that support a nuclear role of miRNAs or the involvement of exosome-contained regulatory RNAs in information transfer between distant cells or tissues in vertebrates.On some occasions, Mattick and Amaral seem to be rather annoyed by conservative views and the initial lack of acceptance of emerging new ideas. I think this is not necessary. Controversies and heated disputes are generally good for science as they drive its progress. By nature, I am also a rather conservative and sceptical person and I would argue that some concepts discussed in the book, not necessarily derived from the authors’ research, need caution and certainly more experimentation. For example, although evidence exists that independent RNAs originating from 3’-untranslated regions (UTRs) of vertebrate mRNAs can exert biological effects in As already discussed, this epic book by Mattick and Amaral superbly reflects the continuing excitement about RNA research. RNA is a molecule with many faces. It can carry genetic information, act as a catalyst, be a target of allosteric regulation , function as a scaffold in the assembly of ribonucleoproteins of different complexity, or guide itself or a cargo to a destination by base-pairing. This is not a complete list of RNA ‘capabilities’ by far and for more information you should read the book. I particularly recommend the last five chapters that discuss: the epigenome (Chapter 14), which also includes – as does Chapter 16 – an interesting section on enhancers and eRNAs; the programming of development (Chapter 15); the unusual complexity, and often uniqueness, of different RNA reactions in the brain (Chapter 17 ‘Plasticity’). Chapter 17 also contains an interesting discussion of transgenerational epigenetic inheritance, paramutations, and related phenomena, which generate a lot of interest despite being mechanistically ill-defined. Chapter 16 (‘RNA Rules') focuses to a large extent on the importance of RNA as a guiding molecule. The authors consider this as the key to exploiting the potential of thousands of regulatory RNAs to precisely direct generic effector proteins (e.g. chromatin modifying and remodelling enzymes or transcription factors) to related destinations, either chromatin components or even other RNAs. Given the huge developmental complexity of organisms and the even more sophisticated development and functioning of the human brain, the vast arrays and hierarchies of regulatory RNAs, combined with epigenetic control of gene expression, may reveal the source of the information that controls these tremendously complicated processes? The chapters listed above, as well as the concluding Chapter 18 (‘Beyond Jungle of Dogmas’), are more philosophical and understandably also more speculative than others. But they provide much food for thought about ‘dark matter’ areas in our understanding of evolution and the functioning of complex genomes.I would have liked to find a chapter in the book devoted to the continuously ‘expanding world’ of RNA-binding proteins (RPBs). Some are briefly discussed in Chapter 16 in connection with chromatin regulation by RNA, with their role in the formation of phase-separated domains , and with neurodegenerative pathologies associated with expansion of short intragenic repeats. It would be beneficial to have more information about the evolution and function of RBPs, and perhaps also about RNA helicases/ATPases, as both protein families intimately co-operate with RNA. Did the increase in the complexity of the two protein families occur in parallel to a gradual increase in the complexity of RNAs expressed from eukaryotic genomes? Did any new paradigms of RNA recognition or RBD and helicase gene organization appear at certain points in evolution?The publication of ‘RNA, the Epicenter of Genetic Information’ is very timely. We are witnessing a true, fully justified explosion of research on RNA. In principle, we have to understand the function of a new genome that encodes all sorts of regulatory RNAs. After decades spent on dissecting the protein-coding genes and the completion of the human genome project about 20 years ago, we still do not know the function of many human genes. Despite enormous technological progress, establishing specific functions of genes encoding regulatory RNAs will not be easy. These genes have no open reading frames to interrupt or AUG initiation codons to mutate. Moreover, the common occurrence of genes nested within other genes, of overlapping genes, and of frequent situations when RNA is expressed from both complementary strands of DNA make studies of the activity of any gene quite difficult. But do not panic. RNA afficionados like John Mattick and Paulo Amaral, joined by hundreds of others, will find a way to proceed. I wholeheartedly recommend this book to anybody interested in the biology of RNA, in evolution, and in the organization and function of complex genomes. As correctly stated in the publisher’s announcement, this book provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers in a broad area of biology.


Angiogenin is a pathologically relevant but little understood ribonuclease, the interactions of which with RNA are structurally unknown. Here, the first crystal structure of human angiogenin bound to RNA is presented. Angiogenin is an unusual member of the RNase A family and is of great interest in multiple pathological contexts. Although it has been assigned various regulatory roles, its core catalytic function is that of an RNA endonuclease. However, its catalytic efficiency is comparatively low and this has been linked to a unique C-terminal helix which partially blocks its RNA-binding site. Assuming that binding to its RNA substrate could trigger a conformational rearrangement, much speculation has arisen on the topic of the interaction of angiogenin with RNA. To date, no structural data on angiogenin–RNA interactions have been available. Here, the structure of angiogenin bound to a double-stranded RNA duplex is reported. The RNA does not reach the active site of angiogenin and no structural arrangement of the C-terminal domain is observed. However, angiogenin forms a previously unobserved crystallographic dimer that makes several backbone interactions with the major and minor grooves of the RNA double helix. Its common name refers to it first being identified as a factor that induces blood-vessel formation, a process known as angiogenesis . The protein exhibits the typical RNase A-like overall fold, although it lacks a fourth disulfide bond and instead features a cell receptor-binding site encompassing residues 58–70  and the two bases located at the 5′ and 3′ ends of the cleavage site (B1 and B2) . No structural data for angiogenin in an ‘activated state’ are available.Angiogenin from humans shares a high degree of sequence similarity with the eponymous member of the superfamily, and the catalytic triad consisting of a lysine (Lys40) and two histidines (His13 and His114) is conserved between the two enzymes  and thus is kept in an inactive state  and promotes the transcription of rRNA  plasmid . From this plasmid, an H114A mutant was generated by site-directed mutagenesis . For protein expression, E. coli BL21 (DE3) cells were transformed and grown in Terrific Broth medium with kanamycin (50 mg l−1) at 37°C. Once the culture had reached an OD600 of 0.8, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. Induced cultures were incubated for 3 h at 37°C with agitation and then harvested by centrifugation. The cells were washed once in 1× PBS, flash-frozen and stored at −20°C until further use. For purification, thawed cells from a 1 l culture volume were resuspended in lysis buffer  and ruptured using a microfluidizer unit . hAng was prepared from inclusion bodies and refolded as described previously  Triton X-100 and once with only buffer, and was sonified and centrifuged in each step. Inclusion bodies were solubilized in 10 ml solubilization buffer  by gently stirring for 4 h or until fully dissolved. Refolding was achieved by dropwise dilution into 500 ml 0.5 Ml-arginine pH 8.0, 0.6 mM oxidized glutathione. In preparation for ion-exchange purification, the solution was filtered (0.45 µm), diluted fourfold with distilled H2O and applied onto a 10 ml SP Sepharose FF column equilibrated in 25 mM Tris–HCl pH 8.0, 0.2 M NaCl. The target protein was eluted in a gradient to 1.1 M NaCl in 50 ml. Unlike as described previously, fractions containing the target protein were pooled, concentrated using a 3 kDa molecular-weight cutoff Amicon device  and further purified by size-exclusion chromatography using a Superdex 75 16/600 column  in 20 mM Tris pH 8.0, 120 mM NaCl. The pure protein was concentrated to 9 mg ml−1 and stored at 4°C for several days until use.A synthetic gene encoding mature human angiogenin (hAng), without its N-terminal signal peptide and codon-optimized for expression in M Tris pH 8.0, 120 mM NaCl to a final concentration of 5 mg ml−1 protein and was incubated on ice for 30 min.N19-RNA with the sequence 5′-GCCCGCCUGUCACGCGGGC-3′ was synthesized by Axolabs . The RNA was heated to 95°C for 5 min and annealed by snap-cooling on ice. hAng H114A was slowly mixed with a 1.2-fold molar excess of annealed RNA in 20 m2.2.Crystals that diffracted well were obtained from high-throughput screening. Freshly prepared complex was mixed with screening condition in a 1:1 or 2:1 ratio in a 3 Lens 96-well sitting-drop vapour-diffusion plate  using a Mosquito pipetting robot . Sealed plates were incubated at 20°C for ten days and then placed at 4°C for eight days. Crystallization information is summarized in Table 12.3.XDS package –RNA complex was solved by molecular replacement with 2.5.APBS) as implemented in PyMOL  was used in molecular replacement, the density that emerged after initial refinement clearly showed a double-stranded RNA duplex structure. After the RNA model had been rebuilt as a duplex, Rfree improved substantially. Placing water and buffer molecules further improved the models and after further refinement Rwork and Rfree finally converged to 0.205 and 0.268, respectively.Attempts at molecular replacement using an angiogenin search model alone were unsuccessful. Only upon including an RNA model could a solution be found. Although the RNA sequence was designed to form a stem-loop structure and a search model representing the anticodon stem loop from a tRNA model (PDB entry 3.2.A and B) and one double-stranded RNA duplex (chains C and D). Since the RNA sequence was not designed to be self-complementary, the RNA complex, made up of two strands of identical sequence, contains three mismatches. However, all of them are pyrimidine–pyrimidine pairings which do not cause steric clashes. In both copies, the space left open between the Watson–Crick edges of the paired nucleotides C32 and C38 is bridged by the guanidium group of Arg24 of chain A and B, which forms hydrogen bonds to the keto groups of both cytidines. The single U35/U35 pairing is not bridged by an amino acid, but local distortion of the backbone geometry allows a stabilizing hydrogen bond between O4 (U35 in chain D) and H3 (U35 of chain C). The 3′- and 5′-terminal bases stack against the 5′- and 3′-terminal bases of the next unit cell, thereby forming an extended RNA helix that spans the whole crystal. Considering all symmetry-related copies of the two angiogenin chains, the extended RNA helix is fully covered by associated protein .The unit cell contains two molecules of angiogenin . All of these interactions are sequence-independent backbone contacts. For this reason, it is possible for the same interaction motif of the protein to bind to two different sections of the RNA duplex.Each chain c). Another arginine, Arg31, binds to a backbone phosphate in the vicinity . Both arginines are part of helix 2. Protein–nucleic acid contacts are typically dominated by electrostatic forces. Angiogenin is a strongly cationic protein with a theoretical pI of 9.7 . Lys17 and Lys60, while not part of helix 3, are located in the vicinity, although their distances to the closest backbone phosphates are greater than 5 Å. The arrangement of chains A and B is of optimal size to also occupy the major groove exactly one helix turn away, and the same residues are involved in contacts in both chains. The surface electrostatics of this contact reveal a large area of continuous, strong positive charge . This makes a strong interaction with the RNA backbone unsurprising, even though this represents the face located opposite to the active sites.The most extensive contacts are formed by helix 3, which reaches deep into the major groove of the RNA helix and contributes Lys50, Arg51 and Lys54 to the interaction site . The interaction is further strengthened through the nearby Arg66 also binding to the backbone. Interestingly, the strongest positive charge on this contact face is located in a central pocket which contains the active sites of both proteins .Another RNA contact is mainly mediated by the angiogenin-specific C-terminal helix 4, which is located close to the active site. In both angiogenin chains this helix contributes Arg121 and Arg122, which interact with opposite strands across the minor groove . The C-terminal helix which mediates the closest RNA contact at the same time acts as a gatekeeper and is positioned between the active site and the RNA. This helix is suspected to undergo a conformational change when binding to a physiological substrate to make the active site accessible. Here, the helix remains in an identical position in both chains  and the RNA binding that is observed in this structure does not trigger a structural rearrangement. Although the angiogenin molecules make extensive contacts with the RNA duplex, the structure is unlikely to resemble the arrangement during a cleavage event of a physiological substrate.However, the RNA duplex does not reach into the active site (3.3.a) has a larger surface area, its solvation free-energy term (ΔiG) is positive, with a ΔiGP-value above 0.5, which is indicative of a hydrophilic interaction and an artefact of crystal packing (Table 4b) is slightly smaller but has a negative solvation free-energy term and a P-value indicative of a largely hydrophobic interaction that is further stabilized by eight hydrogen bonds and four salt bridges.The observed crystal lattice has a fairly low solvent content of 41.61%. Neighbouring protein chains make very close contacts, which leads to the question whether they can provide insight into potential oligomerization sites in solution. The two contact types with the largest surfaces are symmetric and are mediated by the edges of β-sheets 2 and 5 of the protein and by its N-terminal α1 helices Table 4. Althougg Table 4. In contg Table 4b is sliet al., 20211b1j .While angiogenin is considered to be a primarily monomeric protein, the RNase A family as a whole is known to engage in various homodimeric configurations, including 3D domain-swapped arrangements. Recently, the first evidence of such a 3D-swapped dimer was reported for angiogenin (Fasoli 4.in vivo and are barely beginning to be understood. Even though the presented angiogenin–RNA complex does not occur in vivo, the observed RNA interactions might serve to derive the first structural models of more relevant interactions of angiogenin with known in vivo targets.The interactions of angiogenin are manifold, are crucial to its function human angiogenin bound to RNA duplex, 8af0PDB reference: 10.1107/S2053230X22008317/ek5029sup1.pdfSupplemerntary Figures. DOI:


Hepatitis E Virus (HEV) follows waterborne or zoonotic/foodborne transmission. Genotype 3 HEV infections are worldwide spread, especially in swine populations, representing an emerging threat for human health, both for farm workers and pork meat consumers. Unfortunately, HEV in vitro culture and analysis are still difficult, resulting in a poor understanding of its biology and hampering the implementation of counteracting strategies. Indeed, HEV encodes for only one non-structural multifunctional and multidomain protein (ORF1), which might be a good candidate for anti-HEV drugging strategies. In this context, an in silico molecular modelling approach that consisted in homology modelling to derive the 3D model target, docking study to simulate the binding event, and molecular dynamics to check complex stability over time was used. This workflow succeeded to describe ORF1 RNA Helicase domain from a molecular standpoint allowing the identification of potential inhibitory compounds among natural plant-based flavagline-related molecules such as silvestrol, rocaglamide and derivatives thereof. In the context of scouting potential anti-viral compounds and relying on the outcomes presented, further dedicated investigations on silvestrol, rocaglamide and a promising oxidized derivative have been suggested. For the sake of data reproducibility, the 3D model of HEV RNA Helicase has been made publicly available. Hepiviridae family (Orthohepevirus genus). It is the causative agent of the infamous Hepatitis E. As reported by the World Health Organization1, there are 20 million HEV estimated infections per year and 3.3 million symptomatic cases of Hepatitis E worldwide. The fatality rate is usually relatively low, ranging from 0.2 to 4%, although it significantly increases for pregnant women2.Hepatitis E virus (HEV) is a small positive sense single stranded RNA virus member of the 4 while in the latter it commonly gives waterborne outbreaks5. There are eight genotypes described so far (HEV-1 to -8), differing for host preferences and ways of transmission. Particularly, HEV-3, which is globally spread, and HEV-4, which is mostly limited to Asia, follow zoonotic and foodborne transmission7. Reservoirs of HEV include deer, wild boars, cows, sheep, and goats with evidence of human infection reported to be caused by contaminated milk11. Despite the wide range of potential hosts, the main reservoirs are pigs, and the related meat-based products are a major source of infection14. HEV was detected also in the berry fruit and leafy green vegetables supply chain because of irrigation with contaminated water16.The virus spreads following several routes with differences between high- and low-income countries. In the formers it is strictly related to zoonotic and foodborne transmission17. One of the critical knowledge gaps concerns the HEV ORF1 gene, which encodes for a 185 kDa polyprotein with no cleavage sites reported18. ORF 1 is a multifunctional, multi-domain and non-structural polyprotein with a crucial role in the viral diffusion and replication, which may be a possible druggable target to interfere with the viral mechanisms of infection19. However, its huge dimension and multi-domain architecture make its analysis challenging via canonical molecular biology and structural approaches. In addition, the lack of available crystallographic and NMR structures hampers the identification and characterization of its druggable sites using canonical medicinal chemistry approaches. This lack of information ultimately prevents the structure-based identification of molecules targeting ORF1 domains for their possible implementation in anti-HEV strategies. In this context, in silico approaches can efficiently overcome the lack of structural data either to analyze proteins mechanics and druggability, or to provide a useful tool to study chemical and biological aspects of small molecules, including antiviral compounds23. For this reason, an in silico procedure has been developed and applied to target HEV ORF1. Based on previous studies showing the inhibitory activity of the natural plant secondary metabolite silvestrol  investigate the underpinning mechanisms and viral target; and II) to estimate the activity of SLV analogues for further dedicated investigations. To do so, the HEV RNA Helicase domain was modelled and refined via a homology modelling approach based on an innovative, hybrid structure- and sequence-based big-data analysis targeting the whole set of crystallographic data available so far in the Protein Data Bank . Then, the interaction of the model with the already known RNA Helicase inhibitor rocaglamide (RCG) and a set of 9 natural-related compounds27 was calculated through docking and molecular dynamics (MD) simulations.Although this virus is widespread, in vitro culture and analysis are still difficult and consequently its molecular biology has not been fully understood yet28.The HEV RNA Helicase domain of the HEV ORF1 polyprotein was meant to be modelled via homology modelling (HM), as reported in the "29 who succeeded to recombinantly express the putative HEV1 ORF1 RNA Helicase region (from amino acid 960 to 1204) proving its activity. The focus on HEV3 (UniProt AC Q6J8G2) was due to its prevalent foodborne/zoonotic transmission and its worldwide spread30. The localization of the RNA Helicase domain on the HEV3 ORF1 was achieved based on the alignment with the HEV1 RNA Helicase sequence proved as active by Karpe and Lole. Particularly, the target sequence (Tar-Seq) HEV3 ORF1 region ranging from amino acid 975–1219 shared 90.6% identity and 95.5% similarity with the HEV1 RNA Helicase domain  were downloaded from PDB. Then, 3D structure files were converted to FASTA files via an in-house script setting up a database which included the “crystallome” of the prokaryotic RNA Helicase available at the time of analysis. This database was finally searched for Tar-Seq homologous to identify a proper template for HM.Concerning the template selection for HM, it is typically obtained identifying homologous sequences with available 3D structure via the BLAST UniProt web-interface, which is a gold benchmark standard in this kind of studies33, which showed identity and similarity percentage to HEV RNA Helicase of 24.5% and 39.2%, respectively, and with alignment score and e-value of 63.5 bits and 1e-12, respectively. Of note, the identity percentage was high enough with respect to the alignment length to ensure a reliable modeling procedure in agreement with previous evidence34. Such protein belongs to the Tomato mosaic virus (Tmv), a positive sense single stranded RNA virus belonging to the same HEV class. Before using it as template to model the HEV RNA Helicase domain, the last 160 N-terminal residues, which were organized in a self-standing sub-domain, were removed being not covered by our Tar-Seq. The model and its refinement process, reported in the "35 further confirming the model reliability. The last check was the comparison of the model with the one produced by the blind deep learning-based web resource trRosetta36: their structural alignment resulted in an RMSD lower than 2.6 Å, confirming the reliability of the entire procedure described above  keep the stacking interaction with the RNA stably; and II) preserve the stability of the RNA–protein complex avoiding RNA detachment.Once obtained the docking pose for each ligand, 25 ns long MD simulations were run to investigate the evolution of the built complexes. Based both on the 5ZC9 crystal structure and other evidence from the literature claiming the RCG stabilization of the protein-RNA complexRCG and SLV were both engaged in a base–base stacking interaction, also stabilizing the protein-RNA complex with no appreciable RNA detachment Fig. .Figure 227. RCG2 caused a self-collapse of the RNA, promoting its detachment from the protein, and no stacking interactions were observed. This also suggested a low inhibitory potential, in line with the inactivity reported by Pan et al.27 against human RNA Helicase eIF4A1. RCG3, a less cytotoxic RCG analogue27, showed a single base interaction, rather than a base–base stacking interaction, and promoted the RNA detachment. Therefore, it was not deemed an efficient inhibitor. RCG4 and RCG6 both favoured the RNA detachment, with the latter reported as less active than the former, although RCG4 was found in an RCG-like stacking interaction with the RNA. They were not considered able to appreciably inhibit the HEV RNA Helicase activity.RCG1 did not show a proper stacking interaction and promoted the RNA detachment, pointing to its limited theoretical inhibitory activity against the viral RNA Helicase. Interestingly, this result is in line with the Pan et al. study reporting more than 500-fold activity decrease with respect to RCG against the human eIF4A1 ATP-dependent RNA helicase44. Concerning the two decoys, they both promoted RNA detaching, particularly ZINC8584442. This, along the lack of base–base stacking, was expected for inactive compounds and confirmed the procedural performances.S–Ag did not interact at all with the RNA. Shortly after the beginning of the MD simulation it slipped out the RNA chain and kept a likely unspecific surface interaction with a near protein portion Fig. . This waLastly, a novel RCG analogue, i.e. RCG5, was rationally designed and evaluated based on the calculated pose of other RCG analogues included in this study. Particularly, starting from docking poses it was noticed there was a hydrogen bond between the hydroxyl group in position R4 of RCG2 and an RNA base Fig. . Moreove45, in silico approaches may ensure a useful framework of analysis to advance HEV understanding and design counteracting strategies. In this context, the present work provided a useful prioritization of compounds, most of which are not commercially available, supporting a knowledge-based and informed selection either for their synthesis or purification in future dedicated works. Overall, this study: (i) presented mechanistic insights on HEV RNA Helicases and its SLV/RCG-dependent inhibition; (ii) expanded the current understanding of the structure–activity relationship for SLV and RCG-related compounds; (iii) provided a blueprint for further analysis targeting HEV RNA Helicases. Moreover, this study described for the first time the inhibition of HEV RNA Helicase as a plausible mechanism of action of SLV, which was already described reducing HEV capability to penetrate cells. This ancillary mechanism, which deserves further investigation with high priority, was found plausible also for other RCG/SLV-like compounds and may complement the already reported activity of this HEV impairing agents on host RNA Helicases. Furthermore, there are evidence reporting SLV as well-tolerated in animals40 making it an interesting candidate as a feed additive. The described methodology, starting from the template selection moving to the actual modelling and testing, succeeded to build a reliable model able to qualitatively discriminate several ligands. In addition to this, the pipeline is highly versatile, flexible and it can be replicated on other HEV ORF1 domains, such as the RNA Dependent RNA Polymerase which has already been proved as a suitable target to inhibit virus replication41.Keeping in mind the intrinsic issues in running experimental trials on HEV related to its biology and to the shortage of a robust cell-culture system supporting its life cyclerd August 2022), might suggest a broad inter-genotype activity for RCG, SLV and RCG6.As a general remark, the high identity percentage among the HEV RNA Helicases of genotypes 1 to 4 .For the sake of data reproducibility and to support further studies, the 3D model of HEV RNA Helicases used in this work has been made publicly available  at the time of analysis (last database access January 2022) a HEV RNA Helicase model was obtained via HM. T47To do so, a hybrid structure- and sequence-based strategy was developed as the search of publicly available PDB structures via the webserver BLAST interface 43, which is a goldstandard in this kind of study, did not gave useful structures for HM. Specifically, a set of non-eukaryotic proteins annotated as RNA Helicases  with available 3D structure in the Protein Data Bank was collected . To retrieve the 3D structure of this set of proteins, their UniProt ACs were downloaded and mapped towards the Protein Data Bank using the “Retrieve/ID Mapping” tool available on UniProt. Starting from their PDB IDs their 3D structures were iteratively downloaded in the .pdb format using a PyMol script developed in-house (available upon request). Each chain belonging to a .pdb file was converted to a fasta formatted file using an ad hoc script developed in-house (available upon request) and subsequentially concatenated in a unique multi.fasta file. This multi.fasta file was converted into a local database of sequences via the makeblastdb command set with default parameters 47 selecting the tabular output format. The best hit in terms of alignment score, e-value and identity percentage was considered to develop the homology model of HEV RNA Helicase.The primary protein sequence used to model the 3D structure of HEV RNA Helicase was stored in the UniProt database (release 2021_04) with the Accession Code (AC) Q6J8G248 was used to generate the model of HEV RNA Helicase. The chosen template was the crystallographic structure with PDB code 3WRY . Fifty models were generated, setting the inclusion of non-water heteroatoms, using a thorough optimization and choosing the best scored model according to zDOPE for subsequent analysis.Modeller version 10.0 interfaced to Chimera (version 1.15)49, regions with improper dihedrals  were stepwise refined with the Modeller loop-refinement tool (version 10.0 interfaced to Chimera50 version 1.15) using the DOPE modelling protocol, generating 5 models and carrying forth to the analysis the best model according to the zDOPE scoring.After computing a tentative model and checking its Ramachandran Plot with PROCHECK v3.535 to verify the proper topology.Before using the model, a final assessment was performed by re-building its Ramachandran Plot and checking its Z-score on the ProSa-web Server38 set with default parameters. The input RNA structure was chosen based on the following protocol. First, all the structures of RNA Helicase containing RNA were download from Protein Data Bank and aligned to the model previously obtained. The RNA of the structure with the most similar organization  was chosen (PDB ID 6JIM). In the last step, the RNA of 6JIM and the HEV RNA Helicase model were uploaded on ClusPro 2.038. Out of the obtained complexes the analysis of one of the mostly hydrophobic-favoured poses, showing the RNA molecules arranged in the same area as 6JIM structure, was carried forth. The sequence was than edited to a poly-purine fragment in agreement with previous studies reporting its suitability to interact with SLV-related compounds51.The HEV RNA Helicase-RNA complex was obtained docking the RNA sequence to the previously built RNA Helicase model via ClusPro 2.052 to provide a plausible binding architecture for a set of flavagline compounds. The 3D structure of RCG 53 in the .sdf format. The other RCG analogues .Docking studies were performed with GOLD The RNA Helicase model was used as input structure and the space to arrange ligands was set based on the architecture of binding of the RCG in the human eIF4A1 ATP-dependent RNA helicase having PDB code 5CZ9. The structure was visually aligned to the HEV RNA Helicase model, and the binding site was defined in a 10 Å-radius sphere around the centroid of the inter-bases space occupied by RCG in 5CZ9 structure 54. The 50 decoys generated were ranked according to chemical similarities to SLV using LiSiCA algorithm55 and the two extremes  58. Input structures were solvated with SPC/E waters in a rhombic dodecahedron periodic boundary condition, and counter ions (Na+ or Cl−) were added to neutralize the system. Prior to perform molecular dynamic simulations, the systems were energetically minimized to avoid steric clashes and to correct improper geometries using the steepest descent algorithm with a maximum of 50,000 steps. Afterwards, all the systems underwent isothermal  and isobaric  100 ps simulations before running a 25 ns MD simulation.Molecular dynamics (MD) simulations were performed to investigate the overall geometrical stability of HEV RNA Helicase-ligands complexes over time. The adopted software was GROMACS (version 2019.4)rd August 2022). We performed the analysis using Clustal Omega Web Server  with default parameters59.A multiple sequence alignment has been performed to infer the activity of RCG, SLV and RCG6 over the HEV RNA Helicases of different HEV genotypes. The analysis focused on genotypes 1 to 4 as they were those publicly available on the manually curated and reviewed section of UniProt database (last accessed 3Supplementary Information.


Arabidopsis thaliana PBs during Cauliflower mosaic virus (CaMV) infection. We found that several PB components are co-opted into viral factories that support virus multiplication. This pro-viral role was not associated with RNA decay pathways but instead, we established that PB components are helpers in viral RNA translation. While CaMV is normally resilient to RNA silencing, dysfunctions in PB components expose the virus to this pathway, which is similar to previous observations for transgenes. Transgenes, however, undergo RNA quality control-dependent RNA degradation and transcriptional silencing, whereas CaMV RNA remains stable but becomes translationally repressed through decreased ribosome association, revealing a unique dependence among PBs, RNA silencing, and translational repression. Together, our study shows that PB components are co-opted by the virus to maintain efficient translation, a mechanism not associated with canonical PB functions.Viral infections impose extraordinary RNA stress, triggering cellular RNA surveillance pathways such as RNA decapping, nonsense-mediated decay, and RNA silencing. Viruses need to maneuver among these pathways to establish infection and succeed in producing high amounts of viral proteins. Processing bodies (PBs) are integral to RNA triage in eukaryotic cells, with several distinct RNA quality control pathways converging for selective RNA regulation. In this study, we investigated the role of  Cauliflower mosaic virus to maintain efficient virus translation in the presence of RNA DEPENDENT POLYMERASE6-governed silencing.Arabidopsis RNA processing body components LSM1 and DCP5 are co-opted by  IN A NUTSHELLBackground: Viruses are unique in their ability to reuse and recycle host proteins and other components for their own benefit. Cauliflower mosaic virus (CaMV) forms special structures inside the host cells known as viral factories (VFs) to facilitate efficient replication and escape defense. VFs consist of viral proteins, as well as particles and nucleic acids, but also numerous host proteins and ribosomes that are co-opted into these structures. Building on knowledge from the animal field, RNA granules, including stress granules and processing bodies (PBs), are at the forefront of viral disease regulation. Several granule-localized proteins directly interact and influence virus replication.Question: We investigated the role of PB components in CaMV infection. We wanted to elucidate the interplay from two sides: What is the effect of CaMV infection on the localization and abundance of PB components, but also how do these proteins influence CaMV replication and especially viral protein production?Findings: Decapping proteins DCP5 and LSM1 localize to VFs during CaMV infection. CaMV DNA and protein accumulation, but not RNA levels, are reduced in Arabidopsis dcp5 and lsm1 mutants. We found that viral RNA is not a target of LSM1-mediated decapping and that RNA stability is not affected in either mutant. We examined dcp5 and lsm1 single mutants as well as double mutants with RNA-dependent RNA polymerase 6 (rdr6), finding that less viral RNA was associated with ribosomes in the single but not double mutants. Thus, PB proteins help the virus evade translational repression by RDR6.Next steps: We do not yet know how RDR6 mediates translational repression of viral RNA in the absence of DCP5 or LSM1. Elucidating the exact mechanism and which roles the VF and viral proteins play in this interaction will help further our understanding of plant virus infections.Eukaryotic gene expression is tightly regulated from RNA transcription to translation and decay. The importance of posttranscriptional control, especially during stress-induced cellular reprogramming, is becoming increasingly evident, as several studies have revealed extensive uncoupling between transcriptomes and translatomes .Due to the high energy cost and possible detrimental effects of uncontrolled protein translation, eukaryotic cells have evolved a network of pathways to govern and regulate mRNA translation, including the “mRNA cycle” . Here, cArabidopsis thaliana (Arabidopsis), decapping is carried out by the nudix hydrolase DECAPPING2 (DCP2) and its co-factors DCP1 and VARICOSE  and is essential for subsequent 5′- to 3′-end mRNA degradation. In OSE VCS; . SeveralOSE VCS; . UncappeOSE VCS; . The decOSE VCS; . NMD is OSE VCS; . siRNA bOSE VCS; . These bOSE VCS; .dcp2 mutants  machinery and PTGS have been discovered in plants . An init mutants . Subsequand dcp2  makes thViruses challenge the RQC and PTGS machineries through their massive production of RNAs during replication, and the targeting of viral RNAs by RNA silencing is one of the major defense pathway plants employ against viruses. In turn, viruses have frequently evolved RNA silencing suppressors to overcome this silencing . The rolCauliflower mosaic virus  and the model plant Arabidopsis. CaMV is a double-stranded DNA virus that harbors seven open-reading frames in two mRNAs transcribed from two promotors (19s and 35s). While 35s RNA encodes all viral proteins, 19s RNA only encodes the viral transactivator protein P6. P6 is a highly abundant, essential protein that assembles in large cytoplasmic aggregates termed viral factories (VFs) that are the site of viral translation, reverse transcription, and particle packaging  . Under mUpon infection with two CaMV strains (CM1841 and Cabb B-JI), the PB marker proteins formed two morphologically distinct classes of visible structures in systemic leaves 21-day postinfection dpi; , while fDCP1, DCP2, and more strongly for VCS with both CaMV strains, while DCP5 expression was only induced during Cabb B-JI infection, and LSM1a expression was not responsive to either strain  leads to the disassembly of canonical PBs . Under olsm1a/b (hereafter referred to as lsm1) and knockdown mutant dcp5 were chosen for this study, because both mutations cause a reduction in PB formation and PB size, as well as an over-accumulation of capped mRNAs  confirmed reduced P6 and P3 accumulation in lsm1 and dcp5 for CM1841 and also Cabb B-JI, albeit the effect was weaker (lsm1 and dcp5), but not in exonucleolytic RNA decay (xrn4). Owing to the similarities between the two strains, we continued our subsequent analysis with the milder CM1841 strain.Immunoblot analysis showed that less of the viral inclusion protein P6, the coat protein P4, and the virion-associated protein P3 accumulated in both to Col-0 . Viral ps weaker . In comblsm1 and dcp5 mutants  reads to the TAIR10 Arabidopsis reference genome and against the CaMV genome (GenBank V00140.1). In agreement with previous observations, most viral sRNAs mapped against the highly abundant noncoding 8s RNA  against endogenous targets  in noninfected Col-0 and infected Col-0, lsm1, and dcp5. CaMV infection led to a decrease in sRNA counts on TAS-loci and tasiRNA targets  to counteract RNA silencing. For CaMV, the VSR protein P6 inhibits the generation of secondary RDR6-dependent trans-acting siRNAs tasiRNA; . To asse targets . The rednfection , furtherd growth . Whetherlsm1 and dcp5 have a general activation of virus-induced gene silencing (VIGS), as reported for other RQC mutants, we used the tobacco rattle virus-Phytoene Desaturase (TRV-PDS) system, which leads to leaf whitening through VIGS of PDS  do not show altered viral sRNA quantities or profiles (2), do not show elevated VIGS, and (3) do not compromise the capacity of the viral silencing suppressor P6 to target the RDR6-dependent tasiRNA pathway, despite full rescue of virus DNA and protein accumulation by rdr6 in double mutants. Importantly, based on the unaltered viral RNA levels and decay rates in lsm1, we propose that the RDR6-dependent suppression in the lsm1 and dcp5 backgrounds does not involve viral RNA degradation or transcriptional silencing.Taken together, lsm1 and dcp5. First, we performed polysomal fractionation of CaMV-infected Col-0, lsm1 and dcp5 samples from three independent infection experiments. Notably, CaMV-infected tissue showed markedly increased polysome abundance compared to the mock controls for both Col-0 and the mutants    and Arabtivation . The vir infection  There were no evident changes in viral sRNA quantity or profiles in the mutants; (2) viral RNA levels remained largely unaffected, unlike transgenes, which are degraded and transcriptionally silenced; (3) xrn4 did not weaken CaMV infection; and (4) viral RNA does not qualify as a substrate, as it showed no detectable levels of LSM1-dependent decapping and decay.Our results suggest that the RNA silencing component RDR6, likely in conjunction with DCLs, mediates translational repression of the viral RNA in oth xrn4  and dcp2n4  background, which was used as a control for all experiments  at 22°C day temperature (20°C night temperature) and 65% relative humidity for crossing, propagation, and transient expression assays. For infection experiments, plants were grown under short-day conditions  at 22°C day temperature (19°C night temperature) and 65% relative humidity. Light spectra in both conditions ranged from 400 to 720 nm.All mutants used in this study were in the eriments . Mutants35s promoter  0.2, 10-mM MgCl2, 10-mM MES pH 5.6, 150-µM acetosyringone) and the constructs analyzed after 48 h.The pENTRY clone containing the full-length Cabb B-JI P6 coding sequence  was clonpromoter ; all linp method . For traAgrobacterium tumefaciens strain C58C1 carrying CaMV strain CM1841 or TRV RNA1 and 2  and expression levels determined as described by Arabidopsis plants were infected with CaMV or TRV 18 days after germination. The first true leaves were infiltrated with A1 and 2   supplemented with 2% SDS. Samples were incubated at 95°C for 5 min in 1× Laemmli sample buffer and cleared by centrifugation. The protein extracts were separated by SDS–PAGE, transferred to polyvinylidene difluoride (PVDF) membranes , and blocked with 8% (w:v) skimmed milk in 1× PBS, supplemented with 0.05% Tween-20. Blots were incubated with 1:2,000 diluted primary antibodies α-P3 , α-P4 C, α-P6 S, or α-GF7G)-capped RNA was performed as described by 7G-Cap mAb (clone 150-15) was purchased from MBL International Corporation. For exonucleolytic digestion, total RNA was extracted from rosettes 21 dpi and incubated at 37°C with 1U XRN1 enzyme (Thermo Fischer) or in reaction buffer (mock) .Rosettes of CaMV-infected plants (21 dpi) were vacuum infiltrated with 1-mM Cordycepin  in buffer  and placed in a damp chamber. Two plants were harvested per sample corresponding to 0, 15, 30, 60, and 120 min after transcriptional inhibition. Total RNA was extracted using TRIzol reagent, followed by cDNA synthesis and qRT-PCR as described in the previous section. RNA levels were normalized to sRNA libraries were prepared from 500-ng total RNA with a NEBNext Multiplex Small RNA Library Prep Set for Illumina  according to the manufacturer’s protocol. The amplification step was set to 12 cycles, and amplified libraries were cleaned using SPRIselect beads . Size selection was performed on a 6% polyacrylamide gel according to the manufacturer’s protocol and eluted in 10-µL TE buffer. Size range and library concentrations were confirmed using the Bioanalyzer 2100 . Libraries were sequenced on an Illumina Nextseq2000 system in paired-end 50-bp mode at the SciLifeLab facility, Solna, Sweden.https://github.com/ExpressionAnalysis/ea-utils/blob/wiki/FastqJoin.md) and the option-v for illumina reads. Joint fastq files were size trimmed to obtain only sizes of 18–26 nt by utilizing cutadapt (version 1.9.1) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Size trimmed fastq files were further processed as described in https://github.com/AlexSaraz1/paramut_bot). To create sRNA profiles along the CaMV sequence, the hygromycin phosphotransferase transgenic sequence from V00140.1) by setting the start position to the beginning of the 35s promoter. Reads were aligned to the TAIR10 genome including an extra contig containing the above-mentioned CaMV sequence. Alignment was done using Bowtie2  was used with the options -t gene -s 1 -M on the aligned bam files  and then 1.9.1)  with theam files . Graphic2, 25-mM EGTA, 1-mM DTT, 1-mM phenylmethanesulfonylfluoride, 100-μg/mL CHX, 1% (vol/vol) detergent mix (20% (w/v) Brij-35, 20% (v/v) Triton X-100, 20% (v/v) Igepal CA630, and 20% Tween-20), 1% (v/v) polyoxyethylene 10 tridecyl ether), resuspended, and kept on ice for 10 min. The plant debris was removed by centrifuging at 16,000 g for 15 min at 4°C in a JA-25.50 rotor and AvantiPolysome extraction was performed based on 2, 5-mM EGTA, 1-mM DTT, 100-μg/mL CHX in 60% sucrose) in a 26-mL polycarbonate tube (Beckman Coulter). After proper balancing, the samples were centrifuged at 35,000 RPM for 18 h at 4°C in a 70Ti rotor and L8-M ultracentrifuge (Beckman Coulter). The ribosome pellets were gently washed with RNase-free water and resuspended in 300 μL of resuspension buffer (100-mM Tris–HCl (pH 8.0), 40-mM KCl, 20-mM MgCl2, 100-μg/mL CHX). The resuspended samples were kept on ice for 30 min, followed by centrifugation at 16,000g at 4°C to remove any debris. The RNA content was measured for each sample using a Qubit BR RNA assay kit (Thermo Fisher Scientific). The resuspended ribosome samples were loaded on 15%–60% sucrose gradients and centrifuged at 50,000 RPM in a SW55.1 rotor and L8-M ultracentrifuge (Beckman Coulter). The gradient samples were fractionated using an ISCO absorbance detector  to obtain fractions of ∼250 µL. The fractions were pooled before RNA extraction with TRIzol to obtain samples from free RNA as well as monosome-bound RNA and three pools of light, medium, and heavy polysome-bound RNA. RNA levels were normalized to SAND (AT2G28390) in each fraction and depicted as fractions of the sum of ribosome-bound RNA in Col-0  or rdr6 (for lsm1 rdr6 and dcp5 rdr6) to enable the comparison of total abundance on ribosomes, as well as relative abundance along the gradient, after testing for comparable RNA input.J-20 XP centrifuge (Beckman Coulter). The clear supernatant was gently poured on top of an 8-mL sucrose cushion (100-mM Tris–HCl (pH 8.0), 40-mM KCl, 20-mM MgClMicrographs from leaf abaxial epidermal cells were taken under a Zeiss LSM 780 microscope. GFP and RFP signals were detected at 488 nm/490–552 nm and 561 nm/569–652 nm, respectively. Co-visualization was achieved through sequential scanning mode. For HS conditions, leaves were kept in water at 38°C for 30 min (1 h for LSM1-GFP) before imaging. Translational inhibition treatment was achieved by infiltrating young leaves with 200 µM CHX (Sigma-Aldrich), followed by incubation for 1 h before imaging. Images were processed with ZEN black software (Zeiss) and ImageJ version 1.53b. For quantification, Z-stacks were Brightness increased and a median filter of 2 pixels applied. Stomata were manually deleted from micrographs, and a mask was generated through thresholding. Foci were counted using the “Analyze Particles” tool.https://cran.rproject.org/web/packages/agricolae/index.html). Test statistics are shown in Statistical comparisons of two groups were performed by Student’s t test in Excel. One-way analysis of variance (ANOVA) followed by a post-hoc Tukey HSD test (α = 0.05) was performed with R version 4.0.02 and the R-package “agricolae” , DCP2 (AT5G13570), LSM1a (AT1G19120), LSM1b (AT3G14080), DCP5 (AT1G26110), XRN4 (AT1G54490), VCS (AT3G13300), RDR2 (AT4G11130), RDR6 (AT3G49500), DCL2 (AT3G03300), DCL3 (AT3G43920), DCL4 (AT5G20320), UPF1 (AT5G47010), TAS1a (AT2G27400), TAS1b (AT1G50055), TAS1c (AT2G39675), TAS2 (AT2G39681), RPS6 (AT5G4670), and SMG7 (AT5G19400).Sequence data from this article can be found in the EMBL/GenBank data libraries under the following accession numbers: DCP1 (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE194186, and raw data were deposited in the Sequencing Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accession number SRP356192.sRNA sequencing data from this study were deposited in the Gene Expression Omnibus database (The following materials are available in the online version of this article.. Description of PB markers during CaMV infection.. PB double marker lines show co-assembly after HS and during CaMV infection.. Co-expression of PB components with CaMV proteins in N. benthamiana.. Co-localization of P6 with PB components under mock conditions.. Viral DNA accumulation in additional PB mutants. 35s RNA decay after transcriptional arrest with cordycepin.. CaMV disease in combinatorial mutants with NMD and RNA silencing.. Expression of NMD targets in dcp5 and upf1 during CaMV infection.. sRNA profiles in lsm1 and combinatorial mutants.. Translational profiling during CaMV infection. Total number of sRNA reads in each sample and their mapping to the 8,031-bp CaMV genome. Plant material used and generated in this study. DNA oligonucleotides used in this study for genotyping. DNA oligonucleotides used in this study for molecular cloning. DNA oligonucleotides used in this study for expression analysis. ANOVA tables.koac132_Supplementary_DataClick here for additional data file.


Tombusviridae and another (the 3′TSS) found in numerous umbraviruses and several carmoviruses. In addition, three 3′ terminal replication elements are found in nearly every umbravirus and carmovirus. For this report, we have identified a set of three hairpins and a putative pseudoknot, collectively termed “Trio”, that are exclusively found in a subset of umbraviruses and are located just upstream of the 3′TSS. Modification of these elements had no impact on viral translation in wheat germ extracts or in translation of luciferase reporter constructs in vivo. In contrast, Trio hairpins were critical for viral RNA accumulation in Arabidopsis thaliana protoplasts and for replication of a non-autonomously replicating replicon using a trans-replication system in Nicotiana benthamiana leaves. Trio and other 3′ terminal elements involved in viral replication are highly conserved in umbraviruses possessing different classes of upstream 3′CITEs, suggesting conservation of replication mechanisms among umbraviruses despite variation in mechanisms for translation enhancement.The 3′ untranslated regions (UTRs) of positive-strand RNA plant viruses commonly contain elements that promote viral replication and translation. The ~700 nt 3′UTR of umbravirus pea enation mosaic virus 2 (PEMV2) contains three 3′ cap-independent translation enhancers (3′CITEs), including one (PTE) found in members of several genera in the family  The 3′ untranslated regions (UTRs) of positive-strand RNA [(+)RNA] viruses commonly contain elements that promote minus (−)-strand synthesis and translation ,2,3. TheTombusviridae have been intensively studied using turnip crinkle virus (TCV) and its satellite satC as models for the genus Carmovirus ,1817,18]2 and Ψ3 . These hnd Ψ3 . SuperScript III reverse transcriptase (Invitrogen) was used for reverse transcription reactions as described previously  methionine in a 10 μL reaction and incubated at 25 °C for 30 min. Translation reaction mixtures were resolved by 10% SDS-PAGE. Gels were dried, exposed to a phosphor screen, and visualized by autoradiography. Quantitation of band intensities was performed using ImageJ software.A. thaliana callus cultures were transfected with 30 μg of in vitro transcribed luciferase reporter construct RNA. Samples were also co-transfected with 10 μg of in vitro transcribed Renilla luciferase reporter construct RNA as a transfection control. Transfected protoplasts were incubated for 18 h at 22 °C under light. Luciferase levels were measured using a Modulus microplate multimode reader  as described previously [To assay for translation of PEMV2 gRNA in protoplasts, a luciferase reporter construct was used containing the 3′UTR (positions 3550 to 4252) and 5′ 89 nt of the gRNA . For traA. tumefaciens GV3101 by electroporation [A. tumefaciens C58C1. A. tumefaciens cultures were grown in Luria-Bertani liquid media with 50 μg/mL kanamycin and 20 μg/mL rifampicin at 28 °C in a shaker. Cultures carrying the viral replicon were mixed with separate cultures carrying pCB301-p33, pCB301-94, and pBIN61S-p14. The final concentrations of the cultures at OD600 after mixing were: 0.1 for the viral replicon, 0.15 for p33, 0.015 for p94, and 0.1 for p14. Culture mixtures were infiltrated into the 6th true leaf of Nicotiana benthamiana plants. Control plants were infiltrated with only infiltration solution . After infiltration, plants were grown for 5 to 7 days. Total RNA was extracted from infiltrated leaves using phenol-chloroform and RNA extraction buffer , followed by ethanol precipitation. RNA gel blots were performed to assess viral RNA accumulation and p14 mRNA levels. Quantitation of viral replicon band intensities was performed using ImageJ software.PEMV2 gRNA cDNA containing mutations in the p33 start codon (AUG to ACG) and the slippery site of the −1 programed ribosomal frameshift signal just upstream of the p33 stop codon (GGAUUUU to GGAUUCU) , was insporation , except https://rna2drawer.app (accessed in February through November 2022) [All two-dimensional drawings of RNA structures were produced using the on-line version of RNA2Drawer at er 2022) .4) with nearby upstream sequences. SL2, which is located 0 to 4 A-rich residues upstream of SL1, and SL3, located 5 to 10 G-rich residues upstream of SL2, have high sequence conservation in their apical loops that can extend into the stems. An additional stem-loop (SL4) is located 4 to 6 G/U-rich residues upstream of the SL3 supporting stem. The SL4 apical loop may form a pseudoknot (Ψ5) with the bulge loop created by the SL3 supporting stem. Five of the remaining 9 umbraviruses also have SL1/Ψ4 in the identical location upstream of the 3′TSS, but are missing SL2, SL3 and SL4  were highly detrimental and reduced gRNA levels to 14% and 7% of WT, respectively  did not affect gRNA accumulation E. Consid7% of WT G. MutatiPEMV2 gRNA and sgRNA translation in wheat germ extract (WGE) was previously shown to be negatively affected by deletion of the PTE or kl-TSS 3′CITEs, or by point mutations that eliminated the long-distance interaction between the kl-TSS and the 5′end of the genome . AdditioArabidopsis thaliana seed callus cultures and luciferase activity was assayed 18 h later. The SL2 mutations caused no significant effects on luciferase levels in any of the constructs -strand synthesis . To testa leaves  along wia leaves . Total R1. These elements are also found near the 3′end of TCV satellite satC, which shares its 3′terminal region with TCV, and their importance for replication of satC has been well established [1 are also found in the 3′UTR of TBSV, where they were first characterized (and where H5 is referred to as “SL3”) [Information on umbravirus replication has relied on identifying structures similar to known replication elements present in other tombusvirids. In particular, the high degree of structural similarity between 3′UTR terminal regions of PEMV2 and TCV suggested that overlapping mechanisms of replication would be employed by the two viruses . Structus “SL3”) , and app4 in PEMV2, TBTV, RCUV, OPMV and ETBTV significantly extends our knowledge of umbravirus-specific replication elements. SHAPE data supported the presence of all Trio elements within in vitro synthesized transcripts with the exception of SL1, suggesting that this hairpin may require a conformational rearrangement for formation. We recently found a conserved umbravirus hairpin  just upstream of all 3′CITEs located in the middle portion of the 3′UTR  that suppresses a conformational change in OPMV affecting SL1 and formation of the 3′TSS . Alterations in PEMV2 CAS did not elicit a similar conformational change, suggesting that despite conservation of hairpins from SL4 to the 3′end in PEMV2 and OPMV, interactions of this region with upstream elements differs markedly.Our discovery of Trio stem-loops and phylogenetically conserved ΨN. benthamiana  by which Trio elements support PEMV2 replication  also remain to be identified.Trio hairpins were critical for both PEMV2 accumulation in Arabidopsis protoplasts  and for thamiana . In contthamiana . Althougutations G, its asutations G may be A strong correlation was found between relatedness of RdRp nt sequences among umbraviruses and the presence of all Trio elements A, as the


UCU, CCUG, CAG, and CUG repeats cause spinocerebellar ataxia type 10, myotonic dystrophy type 2, Huntington’s disease, and myotonic dystrophy type 1, respectively. By performing extensive molecular dynamic simulations, we investigated the bending propensities and conformational landscapes adopted by 3×3, 2×2, and 1×1 internal loops observed in RNA AUUCU, CCUG, CAG, and CUG repeat expansions using model systems having biologically relevant repeat sizes. We show that the conformational variability experienced by these loops is more complex than previous reports where a variety of unconventional hydrogen bonds are formed. At the global scale, strong bending propensity was observed in r(AUUCU)10, r(CCUG)15, r(CAG)20, and r(CUG)20, and, to a lesser extent, in r(AUUCU)4, r(CCUG)10, r(CAG)10, and r(CUG)10. Furthermore, RNA CAG repeats exhibit a tendency toward bent states with more than 50% of observed conformations having bending angles greater than 50°, while RNA CUG repeats display relatively linear-like conformations with extremely bent conformations accounting for less than 25% of the observed structures. Conformations experienced by RNA AUUCU repeats are a combination of strongly bent and kinked structures. The bent states in RNA CCUG repeats mostly fall into the moderately bent category with a marginal ensemble experiencing extreme bending. The general pattern observed in all the bent structures indicates the collapse of the major groove width as the mechanical trigger for bending, which is caused by alteration of base pair step parameters at multiple locations along the RNA due to local distortions at the loop sites. Overextension is also observed in all the RNA repeats that is attributed to widening of the major groove width as well as undertwisting phenomenon. This information and the rich structural repository could be applied for structure based small molecule design targeting disease-causing RNAs. The bending propensities of these constructs, at the global level, could also have implications on how expanded RNA repeats interact with proteins.Expansions of RNA AU UCU repeats, r(AUUCU)exp, within intron 9 of the ATXN10 pre-mRNA causes SCA10, a disease with no cures available (CUG), r(CUCG)exp, residing within the intron 1 of the zinc finger 9 (ZNF9) precursor mRNA (pre-mRNA) cause DM2. Expansion of RNA CUG, r(CUG)exp, and CA repeats, r(CAG)exp, cause DM1 and HD, respectively. These expanded RNA repeats fold into structures, which can strongly interact with proteins like heterogeneous nuclear ribonucleoprotein K (hnRNP K) exp and r(CAG)exp have repeating 1×1 UU and 1×1 AA internal loops, respectively, r(AUUCU)exp and r(CCUG)exp possess continuous 3×3 UCU/UCU and 2×2 CU/UC internal loops, respectively, with non-canonical UU, CC, and UC base pairs. As a result, they are highly dynamic and impart far more instability compared with non-canonical 1×1 internal loops.Expanded RNA repeats are responsible for a wide range of neuromuscular diseases such as spinocerebellar ataxia type 10 (SCA10) , myotonivailable . ExpandehnRNP K)  in SCA10hnRNP K) . hnRNP Kpomyosin  and its Inhibition of RNA-protein complexes causing neuromuscular diseases, and hence, restoring cell regulation is a new therapeutic strategy, which has attracted a lot of attention . Among dUCU) and r(CCUG) repeats create challenges while studying the conformational landscapes experienced by these RNA repeats. In the meantime, however, due to their distinct nature, 3×3 UCU/UCU and 2×2 CU/UC internal loop motifs provide unique druggable targets, which could be exploited for therapeutic use. Despite the recent advancements in studying RNA dynamics using NMR exp, r(CCUG)exp, r(CAG)exp, and r(CUG)exp using biologically relevant RNA models  were applied on the RNA molecule in the first step of minimization with 5,000 steps of steepest-descent algorithm and subsequently followed with the second round of minimization with 5,000 steps of conjugate-gradient algorithm and no restraints. Minimization was followed by an equilibration protocol first in constant volume with restraints on the RNA molecule  and gradually increasing the temperature up to 300 K for several nanoseconds using the Langevin thermostat. The second round of equilibration was performed at constant pressure with constant temperature at 300 K and pressure coupling  with a 2 fs time step was performed for each system with isotropic positional scaling. The reference pressure was set to 1 atm with a pressure relaxation time of 2 fs. SHAKE  adopting unique values varying between 10 Å and 18 Å depending on the sequence and the type of the internal loop (see details below).Base pair step parameters, groove widths as well as bending angles, and curvilinear helical axis, were measured using Curves+  and 3DNA+ . The fir+ . Thus, wUCU, and performed 2.9 µs MD simulation to investigate the global structural behavior of AUUCU repeats responsible for SCA10. The 10×AUUCU includes 10 copies of 3×3 UCU/UCU internal loop motifs, which makes it a realistic system to investigate AUUCU repeats. MD trajectory of 10×AUUCU exhibits structures having both bent exp. We performed 2.6-µs-long MD simulation on this system to determine the bending properties of r(CUG)exp. To identify the bending, cluster analyses were conducted on the MD trajectory, average structures for each cluster were calculated, and structural analyses were performed on the average structures using the Curves+. Visual inspection of average structures, which correspond to states with different bending angles  base-paired RNA helixes for 20×CUCU repeats, three RNA constructs each having two, four, and ten copies of AUUCU loops were utilized   . Populatectively . Moreoveectively . It was  minimum  but with minimum . In 4×AUbending. . ResultsCUG repeats, where three models with 4, 10, and 15 copies of CCUG loops were investigated   . Similar limited . RNA CCUnd state . Anotherectively . The casectively . CytidinUG by 8% . Furtherted form . For exase pairs  start toAG and CUG repeat expansions. Again, we used three different model systems in each case having 2, 10, and 20 copies of CAG  and CUG loops   . Previoud states . We obseectively . In one-ectively . Furtherectively , while 0ectively . Similarectively , one of ectively . Analogo systems . This isd states , and samd states .UCU, CCUG, CAG, and CUG repeats have tendencies to bend dramatically with the increase in repeat size. The native form of RNA is A-form, which is a linear structure stabilized by hydrogen bonds formed by Watson-Crick GC, AU, and GU base pairs. Defects in RNA structure such as formation of internal loops can distort the global RNA structure and form states, which are bent or display kink-like structures. RNA repeat expansions have regularly spaced internal loops such as 3×3 UCU/UCU in AUUCU repeats, 2×2 CU/UC in CCUG repeats, 1×1 A/A in CAG repeats, and 1×1 U/U in CUG repeats, which can have global structural implications due to the dynamic behavior of the internal loops exp, r(CUCG)exp, r(CAG)exp, and r(CUG)exp have repeating units with unique internal loops. At atomic level, electrostatics, stacking, and hydrogen bonding interactions are directing the structural preferences of bases in each internal loop, which will create local distortions. The combined effect of all these distortions generates the bending phenomenon discussed above. We already discovered that collapse of Mgw is coordinated with bending phenomenon. Thus, we investigated the connection of Mgw with minor groove width (mgw) and base pair step parameters    in ordernd rise) . One thiUCU, 4×AUUCU, and 10×AUUCU  in 4×AUUCU, and around   and  in 10×AUUCU profiles, where Mgw and θ stand for major groove width and angular change of base pair step orientation , respect10×AUUCU . Results 10×AUUCU. No genu 10×AUUCU.CUG and 15×CCUG display slight differences , , and  profiles calculated for 10×Cferences . For exa 10×CCUG .AG and CUG models are particularly interesting because the 1×1 AA and 1×1 UU pairs in each RNA construct are well defined, which allows base pair step data to be more physical compared with AUUCU and CCUG. For example, analyses display that angular change as well as twist and slide are all important in analyzing properties of RNA CAG repeats    , and   profiles  , and  in 10×CUG and 20×CUG compared with 2×CUG    , and  (UCU)exp, r(CUCG)exp, r(CAG)exp, and r(CUG)exp.The base pair step data of RNA C repeats . Compare −2.5 Å) . Increasth 2×CUG . As befoe = 3 Å) . ResultsUCU, CCUG, CAG, and CUG repeat expansions have repetitive 3×3 UCU/UCU, 2×2 CU/UC, 1×1 A/A, and 1×1 U/U internal loops, respectively, which are dynamic. We found that these RNA repeats have strong bending propensities due to the local structural changes observed in their repetitive internal loop motifs, which collapse the major groove width at different locations in RNA causing global structural change called bending phenomenon. Furthermore, we discovered that there is a link between the magnitude of bending and the system size as well as the RNA internal loop motifs in these RNA repeat expansions, where extreme bending is observed in sequentially long RNA repeats.Bending in nucleic acids, especially in DNA having A-tracts , is a weCUG and AUUCU repeats are alike, we found that latter system exhibited extreme bending regimes as well as kinked conformations. Furthermore, studies on RNA CAG revealed the role of bulky 1×1 A/A mismatches in creating extreme curvatures in RNA structure. We also studied the effect of RNA length on its global behavior in these repeat expansions. We found that extreme bending regimes are only observed in long systems, although bending was also observed in small systems. Analysis of structural parameters revealed that changes in major groove width were the general outcome for observed bent states in all the studied systems. Changes in major groove width are due to weak pairings observed in RNA loop motifs, which can either distort the closing base pairs or the loop structures to provide enough energetics to create local distortions in multiple locations along the RNA resulting bent geometries. Finally, all the model RNA repeats displayed overextension, which is attributed to widening of the major groove widths as well as undertwisting phenomenon. The combined results can provide an understanding on how proteins would interact with RNA repeat expansions. Furthermore, results might be utilized in structure-based drug designs specifically targeting RNA repeat expansions.In summary, we utilized model RNA systems having biologically relevant repeat sizes to provide a glimpse on the behavior of RNA repeats both at global and local levels. Presence of multiple pyrimidine–pyrimidine mismatches in internal loops was found to impose structural stress in RNA resulting in bent or kinked conformations. While RNA C