RNA-based vaccination strategies tailoring immune response to specific reactions have become an important pillar for a broad range of applications. Recently, the use of lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. Here, we have investigated whether small animal PET/MRI can be employed to image the biodistribution of RNA-encoded protein.For this purpose, a reporter RNA coding for the sodium-iodide-symporter (NIS) was in vitro transcribed in cell lines and evaluated for expression. RNA-lipoplex nanoparticles were then assembled by complexing RNA with liposomes at different charge ratios, and functional NIS protein translation was imaged and quantified in vivo and ex vivo by Iodine-124 PET upon intravenous administration in mice.NIS expression was detected on the membrane of two cell lines as early as 6 h after transfection and gradually decreased over 48 h. In vivo and ex vivo PET/MRI of anionic spleen-targeting or cationic lung-targeting NIS-RNA lipoplexes revealed a visually detectable rapid increase of Iodine-124 uptake in the spleen or lung compared to control-RNA-lipoplexes, respectively, with minimal background in other organs except from thyroid, stomach and salivary gland.The strong organ selectivity and high target-to-background acquisition of NIS-RNA lipoplexes indicate the feasibility of small animal PET/MRI to quantify organ-specific delivery of RNA.The online version contains supplementary material available at 10.1186/s13550-021-00753-2. Nanoparticles show promising potency as delivery vehicles for a variety of molecules, leading to fundamentally new applications and therapeutic strategies. Due to their complex chemical composition and relevant interaction with plasma proteins, the pharmacokinetic properties and delivery properties of nanoparticles are variable and remain challenging to adapt for optimal conditions . Additio−/CD31+) [In developing nanoparticulate delivery systems for RNA, it is important to understand how the physicochemical properties of the nanoparticles influence in vivo pharmacokinetic behavior . Then, t−/CD31+) . Within −/CD31+) . Thus, tIn evaluating the process of organ-specific, nanoparticle-mediated RNA delivery, in vivo reporter proteins are fundamentally important to prove feasibility and to quantify translated protein. The sodium-iodide-symporter (NIS), physiologically only expressed in thyroid tissue, gastric mucosa and salivary glands, is a widely used reporter-gene system offering the unique possibility of imaging radioactive iodine transport. NIS is a transporter protein normally located on the basolateral surface of thyroid follicular cells. After cellular uptake by thyroid cells, the iodine is oxidated and serves as a key atom in the synthesis of thyroid hormones. In the thyroid iodine uptake and metabolism are regulated by thyroid-stimulating hormone (TSH), typically leading to far higher physiological uptake than in other tissues. Given that in many organ systems, both expression of NIS and regulation of NIS activity by TSH are lacking, NIS reporter gene imaging is ideal for sensitive detection and possible quantification of protein translation from DNA or viral vectors for various purposes, including viral and cell therapies –12.Incorporating the positron emitter Iodine-124 in this reporter gene system allows imaging and quantification by high-resolution Positron Emission Tomography (PET). Radioactive iodine isotopes offer attractive advantages over other reporter systems in view of the rapid renal elimination of extracellular iodine and the high tissue penetration of emitted radiation. This results in high and rapid contrast, reflecting the extent of translated NIS . For smaIn this study, two RNA-lipoplex systems for systemic NIS-RNA delivery were compared by small animal PET/MRI of Iodine-124 uptake. One system with net anionic charge is known to mediate translation primarily within the spleen, and the other with net positive charge is known to yield translation primarily within the lungs. Following in vivo PET/MRI as well as ex vivo biodistribution analysis we also visualized NIS-RNA expression over time and quantified Iodine-124 uptake as a measure of effective targeting and translation.Female BALB/c mice  were housed under specific pathogen-free conditions in the animal care facility  according to the guidelines of the regional animal care committee. All experiments were performed after approval in accordance with federal guidelines.The murine tumor cell lines MC38 and TC-1 were kindly provided by Ferry Ossendorp (Leiden University) and T.C. Wu (Johns Hopkins University), respectively. Cells derived from working cell banks with low passage number were used in experiments.Codon-optimized human NIS (NIS) and firefly luciferase (LUC) coding sequences were cloned into the pST1 DNA vector optimized for stability as well as protein translation  mixture of the synthetic cationic lipid 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)  and the phospholipid 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) . To make the lung-targeting RNA-lipoplexes, liposomes consisting of DOTMA and cholesterol  at a 1:1 molar ratio were used.Lipoplexes were manufactured by mixing cationic liposomes with the RNA using dedicated protocols as described previously , 7. BrieThe charge ratio for particle formation was calculated using the molar mass of 670 Da for the positively charged DOTMA and a mean molar mass of 330 Da for each negatively charged nucleotide of the RNA. For particles targeting the lung the charge ratio was 4:1, resulting in particles with an average size of ~ 260 nm and a polydispersity index of ~ 0,23. For particles to target the spleen the charge ratio was 1.3:2, resulting in an average size of ~ 250 nm and a polydispersity index of ~ 0,25.Mice were injected intravenously with DOTMA/Chol:RNA (4:1) or DOTMA/DOPE:RNA 1.3:2) RNA-lipoplexes including 20 µg Luc-RNA, and in vivo luminescence imaging was performed as described previously [:2 RNA-li6 MC38 or TC-1 cells were washed twice in X-vivo medium (Lonza) and electroporated with 20 µg NIS-RNA in 4-mm cuvettes using ECM 830 electroporator (BTX Harvard Apparatus) . Cells were harvested 6, 24 and 48 h after electroporation and stained with AlexaFluor 647 labeled-human NIS antibody (1:100 dilution) for 30 min at 4 °C in the dark. Live-dead staining was performed using Fixable Viability Dye eFluor 780 (1:3000 dilution) (Thermofisher Scientific). Data were acquired with FACS Canto II flow cytometer (BD Biosciences) and analyzed by FlowJo Software ver 10.4 (Tree Star). % NIS + cells as well as mean fluorescence intensity (MFI) of NIS expression of live cells was presented. Cells electroporated without RNA served as mock control.2 × 10n = 3 animals were intravenously injected with RNA-lipoplexes containing 20 µg NIS RNA. Six hours later 6.64 ± 0.66 MBq Iodine-124  was injected intravenously. Three hours after Iodine-124 injection, mice were anesthetized with 2% isoflurane and static imaging was performed over 20 min by small animal PET/MRI . For anatomic imaging MRI measurements , Multi Field of View (FOV); slice thickness: 0.6 mm; TE: 2 ms; TR: 15 ms; flip angle: 25 deg) were performed afterward. Additionally, one animal per group was imaged dynamically for one hour. PET data were reconstructed with Teratomo 3D , co-registered to the MR and corrected for decay. Quantitative analysis was done with the PMOD software package (version 4.1). Volumes of interests were based on anatomical MRI, and counts/ml were derived for static and dynamic PET data sets. Mean activity concentrations were calculated based on a cross-calibration factor.Groups of All mice were killed at 3 h post Iodine-124 injection (9 h after NIS-RNA lipoplex injection) to measure ex vivo organ activity. Organs were collected and measured in a cross-calibrated gamma counter .In order to assess whether NIS can serve as a reporter for imaging the biodistribution of RNA-encoded protein upon intravenous administration of RNA-lipoplexes, we first cloned the human NIS gene into our DNA vector backbone and generated NIS-RNA by in vitro transcription. Transfection of this RNA into standard tumor cell lines MC38 and TC-1 via electroporation showed successful expression of NIS on the cell surface  RNA-delivering RNA-lipoplexes with spleen- or lung-targeting properties and imaged them with PET/MR. For this purpose, Iodine-124 was administered 6 h after RNA-lipoplexes as selected above and Iodine-124 uptake was imaged and quantified 3 h after Iodine-124 injection. PET/MRI of anionic NIS-RNA lipoplexes with spleen-targeting properties showed a visually detectable increase of Iodine-124 uptake in the spleen compared to control-RNA lipoplexes  which can be used as radioiodide therapy. Moreover, nanoparticulate delivery by polyplexes consisting of polymers (linear polyethylene imine-polyethylene glycol) and NIS-cDNA-targeting EGFR alone or together with cMET has been investigated , 17. By Systemic RNA delivery with RNA-lipoplex nanoparticles provides a versatile platform that can be utilized for a number of different prophylactic and therapeutic interventions. For example, it has already been used for cancer vaccination , 3 as weWe show highly specific targeting, delivery and expression of RNA to spleen and lung by anionic and cationic RNA-lipoplex nanoparticles, respectively, through the use of the NIS reporter gene system and Iodine-124 uptake as imaged by PET/MRI. Combining NIS reporter gene imaging with in vivo small animal PET/MRI thus represents a powerful tool to monitor the distribution and extent of expression of RNA targeted specifically to any tissue over time.Additional file 1.


Intense positive staining for POWV RNA was frequently detected in dermal foci and occasionally detected in hypodermal foci after 24 h of POWV-infected tick feeding. Additionally, duplex chromogenic RNA ISH staining demonstrated co-localization of POWV RNA with Mus musculus F4/80 RNA, CD11c RNA, vimentin RNA, Krt14 RNA, and CD3ε RNA at the feeding site of POWV-infected ticks. In future studies, RNA ISH can be used to validate transcriptomic analyses conducted at the tick-virus-host cutaneous interface and will provide cellular resolution for specific gene signatures temporally expressed during infected tick feeding. Such a systems biology approach will help create a more refined understanding of the cellular and molecular interactions influencing virus transmission at the cutaneous interface.Skin is the interface between an attached, feeding tick and a host; consequently, it is the first line of defense against invading pathogenic microorganisms that are delivered to a vertebrate host together with tick saliva. Central to the successful transmission of a tick-borne pathogen are complex interactions between the host immune response and early tick-mediated immunomodulation, all of which initially occur at the skin interface. The focus of this work was to demonstrate the use of RNA  Mammalian skin serves as a mechanical and immunological barrier to protect the host from injury and infection  are valuable tools for studying the early host immune response to infected tick feeding. Using such models, transcriptional profiling and histopathological analyses conducted at the tick feeding site comprehensively illustrate how TBFV-infected tick feeding temporally influences the host cutaneous immune response. Comparative transcriptional analyses of TBFV-infected vs. uninfected tick feeding sites reveal significant up-regulation of transcripts related to the recruitment, migration, and accumulation of immune cells after the first 1–3 h of infected tick feeding  antigen was detected in fibroblasts and macrophages via immunofluorescence  for precise localization of Mus musculus-specific F4/80, CD11c, vimentin, keratin 14, CD3ε, and CD49b RNAs at the tick feeding site, and to detect the distribution of POWV RNA at the skin site of POWV-infected Ixodes scapularis feeding. Simultaneous visualization POWV RNA with Mus musculus-specific mRNA targets is also demonstrated. Ultimately, a systems biology approach enables analyses of the complex interplay between tick-host-virus at the skin interface, and RNA ISH can be incorporated as a tool to visualize and validate cellular and molecular interactions occurring within the infected tick feeding site.Skin is the first mammalian organ that a tick-borne pathogen and tick salivary factors encounter during their journey from the infected tick salivary glands to the host. With respect to several tick-borne flaviviruses (TBFVs), an infected 2. The World Reference Center for Emerging Viruses and Arboviruses at UTMB provided stock of the POWV (LB strain), which had previously been passaged 7 times in suckling mice brains. The stock was then passaged 5 times on VeroE6 cells. Stock virus titers were determined by focus-forming immunoassay as described previously  cells were purchased from the American Type Culture Collection (ATCC) and maintained in culture with Modified Eagle's Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, and a 1% antibiotic mixture of penicillin/streptomycin incubated at 37°C with 5% COI. scapularis nymphs were generated in the laboratory via synchronous infection, a technique that has been previously validated in our laboratory for virus infection of I. scapularis nymphs , mice were euthanized by CO2 inhalation followed by cervical dislocation. Immediately following euthanasia, 4 mm skin punch biopsies were harvested, including the attached ticks.One day after capsule attachment, a single Each skin plus tick biopsy was formalin-fixed for a minimum of 48 h in 10% neutral buffered formalin at room temperature. Tick legs were snipped prior to fixation to expedite formalin penetration into the tick body cavity. The biopsy samples were treated with Decal (StatLab) for 2 h at room temperature, thoroughly washed with DI water, and returned to formalin. The biopsies were then dehydrated with a standard ethanol series followed by xylene. Paraffin embedding of each biopsy was performed at an orientation that, upon sectioning, would produce a cross-section of the mouse skin and a longitudinal section of the tick mouthparts and body , Mus musculus F4/80 RNA (Cat. # 460651-C2), M. musculus CD11c RNA (Cat. # 311501-C2), M. musculus vimentin RNA (Cat. # 457961-C2), M. musculus keratin 14 RNA (Cat. # 422521-C2), M. musculus CD3ε RNA (Cat. # 314721-C2), M. musculus CD49b RNA (Cat. # 441081-C2), positive control M. musculus ubiquitin C (Ubc) RNA (Cat. # 310771), positive control M. musculus Polr2a RNA (Cat. # 312471-C2), and positive control M. musculus Ppib RNA (Cat. # 321651) were hybridized to the tissues for 2 h at 40°C. All remaining steps of signal amplification and detection were conducted as recommended by the RNAscope 2.5 HD duplex detection kit user manual. Horseradish peroxidase (HRP)-based green and alkaline phosphatase (AP)-based red chromogenic substrates were used for signal detection. Slides were counterstained with Gill's Hematoxylin I (diluted 1:1 in DI water) for 30 s, briefly washed in tap water, air dried for 45 min at 60°C, and cover-slipped using VectaMount (Vector Laboratories) reagent. Tissue sections were examined under a standard light microscope.RNA ISH was performed with the RNAscope™ platform (Advanced Cell Diagnostics), including the RNAscope 2.5 Duplex reagent kit, RNAscope probes, and the RNAscope HybEZ hybridization system. RNA ISH was performed manually with a RNAscope 2.5 HD duplex chromogenic assay (Advanced Cell Diagnostics) in accordance with the manufacturer's recommendations for FFPE tissue. The FFPE tissue sections were incubated at 60°C for 1 h and deparaffinized in xylene. Endogenous peroxidases were quenched with HM. musculus housekeeping genes, ubiquitin C (Ubc), Ppib, and Polr2a, were used as positive controls to assess mouse skin biopsies with attached ticks for RNA integrity and to serve as technical assay controls for the RNA ISH procedures. Endogenously expressed Ubc RNA was detected with HRP-based green chromogen in a punctate staining pattern and was moderately to highly expressed (>20 transcript copies per cell) in many M. musculus skin cells  used in this study reveals positive RNA signals as punctate staining, and previous work demonstrated that each punctate dot represents a single mRNA transcript  RNA was characterized at the tick feeding site via RNA ISH. Krt14 is expressed in the basal layer of the epidermis and in hair follicles, and it is a prototypic marker of proliferative basal keratinocytes  facilities in strict accordance with an animal use protocol approved by the University of Texas Medical Branch (UTMB) Institutional Animal Care and Use Committee (IACUC: # 0907054).MH and ST designed the experiments and analyzed the data. ST provided reagents and materials, and critically read and revised the manuscript. MH performed the experiments and drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alphavirus which causes millions of human infections every year. Outbreaks have been reported in Africa and Asia since the early 1950s, from three CHIKV lineages: West African, East Central South African, and Asian Urban. As new outbreaks occurred in the Americas, individual strains from the known lineages have evolved, creating new monophyletic groups that generated novel geographic-based lineages. Building on a recently updated phylogeny of CHIKV, we report here the availability of an interactive CHIKV phylodynamics dataset, which is based on more than 900 publicly available CHIKV genomes. We provide an interactive view of CHIKV molecular epidemiology built on Nextstrain, a web-based visualization framework for real-time tracking of pathogen evolution. CHIKV molecular epidemiology reveals single nucleotide variants that change the stability and fold of locally stable RNA structures. We propose alternative RNA structure formation in different CHIKV lineages by predicting more than a dozen RNA elements that are subject to perturbation of the structure ensemble upon variation of a single nucleotide.Chikungunya virus (CHIKV) is an emerging  Alphavirus of the family Togaviridae that causes millions of human infections every year, particularly in tropic and subtropic regions. CHIKV is the etiological agent of chikungunya fever, an acute febrile illness associated with joint pain, rash, and, rarely, neurological manifestations [Aedes aegypti and Aedes albopictus [Chikungunya virus (CHIKV) is an arthropod-borne stations . CHIKV ibopictus . In Afribopictus . The absbopictus .A. albopictus. As A. albopictus is present in temperate regions, this adaptation also has implications on the geographical range of transmission, with CHIKV no longer being bound to tropical and subtropical latitudes [A. albopictus, facilitated by climate change, raises public health concerns worldwide [The first documented outbreak of CHIKV was in 1952 on the Makonde Plateau in the Southern Province of Tanganyika (present-day Tanzania) . Since tatitudes . Reporteorldwide ,12,13.Early phylogenetic analyses suggested that CHIKV can be separated into three geographically disjoint lineages: West African (WA), East Central South African (ECSA) and Asian Urban Lineage (AUL) . FollowiCHIKV is a small, spherical, enveloped virus with a single-stranded, (+)-sense RNA genome of approximately 11.8 kb ,26 that  cleaved . The non cleaved . This diisolates . It is pisolates . Specifistraints .Like many other RNA viruses, CHIKV encodes not only viral proteins but also functional RNAs that mediate the viral life cycle. These structured RNAs are found in coding and non-coding regions of the viral genome. A specific fold is often a prerequisite for functional RNAs, and there are selective evolutionary pressures on maintaining these folds, both in coding and non-coding regions . As RNA Alphavirus, knowledge of functional RNA elements and their specific association to viral pathogenesis and replication remains elusive. Unlike other RNA viruses that are characterized by structural conservation of a critical amount of functional RNAs in their UTRs, such as flaviviruses [Although CHIKV is one of the best-studied viruses within the genus iviruses  or coroniviruses , genus-wiviruses , probabliviruses .Likewise, the association between primary sequence and secondary structure in the terminal regions of CHIKV genomes has raised considerable research interest over the last years, mainly motivated by the observation that different CHIKV lineages maintain variable-length 3aviruses , recombiaviruses . In a reelements . CurrentAn aspect related to the formation and specificity of RNA structure is the effect of single nucleotide variants (SNVs). These are mutations that can alter the RNA structural ensemble by mediating the base-pairing pattern, potentially resulting in an alternative fold and disrupted functionality. SNVs are sometimes associated with so-called riboSNitches, i.e., RNA elements that are subject to perturbation of the structural ensemble resulting in large conformational changes ,38. Examhttps://www.nextstrain.org (accessed on 20 January 2021)) [With the availability of large numbers of next-generation sequencing data in public databases, multiple efforts to analyze and visualize the spread of infectious diseases have been made ,44,45. Ny 2021))  is an opy 2021)) , and shoy 2021)) . We repoTo better understand the evolutionary traits associated with functional RNA conservation among different lineages, we set out to use the CHIKV molecular epidemiology data to study the impact of lineage-associated sequence variability on viral RNA structure. We were particularly interested in the structural divergence induced by fixed SNVs that are specific to particular lineages, and predict the existence of more than a dozen locally stable RNA elements in the coding regions of the CHIKV genome, whose structural ensemble is substantially altered by lineage-associated SNVs.We downloaded viral genome and annotation data from the public National Center for Biotechnology Information (NCBI) Genbank database  on 23 OcMEGA X under default settings [To calculate the genetic distance within and between Chikungunya lineages, we estimated the evolutionary divergence over sequence pairs between groups as implemented in settings ,51. The settings .Snakemake [Snakemake workflow, we retrieved metadata including collection date, country, and place of isolation (if available) from the Genbank records of all available CHIKV isolates. Each country was then assigned to one of the following regions: South East Asia, East Asia, South Asia, West Asia, Caribbean, Northern America, Central America, Southern America, Europe, Oceania, Eastern Africa, Middle Africa, Southern Africa, or Western Africa. In the next step, a file with lineage association for each isolate was created. Isolates with unknown lineage association were assigned to lineages via the the time-resolved phylogenetic tree produced by Nextstrain iteratively. The standard augur pipeline was then applied to construct all relevant data for Nextstrain visualization [We employed the workflow management system nakemake  to buildlization .RNALfold [RNAdistance from the ViennaRNA Package [MutaRNA web server [For characterizing SNVs that affect CHIKV RNA structure formation, we performed local RNA structure prediction with RNALfold  in the r Package , employib server  using dehttps://nextstrain.org/community/ViennaRNA/CHIKV (accessed on 20 January 2021). The data can be downloaded by scrolling down to the bottom of the page and clicking on the “Download Data” link.The CHIKV Nexstrain instance is available at Nucleotide divergence analyses based on the average number of base substitutions per site can highlight the proximity among groups of taxa. To get an updated view of the distances within and between CHIKV lineages at the level of nucleotides, we computed the average evolutionary divergence over sequence pairs. Our results show that the nucleotide divergence within each individual lineage is relatively low (<0.01), with an average of 0.006 . The evohttps://nextstrain.org/community/ViennaRNA/CHIKV (accessed on 20 January 2021) and comprises 924 genomes, which represents a substantial increase compared to the 590 genomes considered in the previous most comprehensive study of CHIKV phylogeny [In an attempt to provide a publicly available epidemiological and phylogeographical interactive visualization of CHIKV spread, we created a custom Nextstrain build that encompasses all currently available CHIKV genome data. Our community build is available via hylogeny . The Nexhylogeny  , aiming at characterizing structural elements that show increased thermodynamic stability, as expressed by z-score statistics. We intersected loci that fold into locally stable RNA structures with genome diversity data from Nextstrain to obtain regions of the CHIKV genome that both fold into stable RNA structures and contain one or more single-nucleotide mutations. Each SNV in this set was then characterized in terms of geographic spread and association to specific CHIKV lineages, as well as their base-pair distance between non-mutated wild-type MFE structure and mutant MFE structure. Using base-pair distance to pre-filter variants that induce a substantial change in the global fold of the locally stable RNAs, we identified 12 candidate RNAs within the CHIKV coding regions that overlap one SNV, and two candidates that overlap two SNVs each. In total, we have 14 candidate RNAs and 16 fixed SNVs, as shown in MutaRNA webserver [For each candidate RNA, we quantified the effect of mutation-induced changes on the RNA structure ensemble with the ebserver . In addiWe selected two examples that exhibit interesting structural traits: The first example is a variant at nucleotide position 1653 , which hAnother example encompasses multiple lineage-specific mutations at position 11,246, as depicted in A. albopictus. Although the C10651U mutation overlaps a locally stable region , the overall fold of this structure is not altered by the mutation. This suggests that the biological effect is mediated by the protein level rather than the RNA level.An interesting observation relates to a C-U mutation at position 10,651, which is responsible for the E1-A226V mutation that has been associated with increased CHIKV transmissibility by In this contribution, we address the question as to what can be learned about CHIKV genotype/phenotype associations by comparative approaches, bringing together different concepts of molecular epidemiology, phylogeny reconstruction, and computational RNA biology. To this end, we build on the Nextstrain  frameworWhile the nucleotide divergence within the observed CHIKV lineages is relatively low, this can be explained by the geographical constraint and the reduced collection period for novel lineages. Conversely, the divergence between lineages varies considerably. The highest divergence of the West African lineage when compared to the other CHIKV lineages can be explained by the observation that WA has been an isolated lineage that emerged decades before the more recent outbreaks .A. albopictus [However, the divergence between major clades and geographically delineated lineages does raise the question of whether the current nomenclature, as canonized by experts from the field when first identified, is not misguiding. IOL is an example of a well-established lineage in the literature that presents low divergence from its African origin, but it does introduce the A226V mutation in E1, which mediates the capacity of the virus to replicate in bopictus .Most viral genotypes/subgenotypes/subclades are based on whole-genome nucleotide divergence of a specific percentage, usually determined by phylogeny showing clades/lineages of a defined ‘high’ statistical support (>70% bootstrap) ,61. HepaIn the most recent comprehensive phylogeny of CHIKV to date, de Bernardi Schneider et al.  analyzedAlthough we were not aiming to discuss a reclassification of CHIKV into a system independent of geography, we find the urge to bring to attention that a new coherent system should replace the current taxa classification. Such a new system could assist drug and vaccine development researchers to target specific genotypes or subgenotypes.In an epidemiological context, the current lineage system, or, respectively, the way the strains are currently grouped, allows the inspection of major outbreak instances and looking at TMRCAs as a way to gauge when a lineage has been introduced in a region, causing outbreaks. From this perspective, our Nextstrain instance provides a reasonable amount of data to conduct a more in-depth investigation of the outbreaks that have recently occurred. Importantly, the calculated TMRCA of the major clades is in accordance with previous studies . While bMolecular epidemiology provides a detailed picture of the geographical spread and fixation of RNA variants in viruses and can be used in combination with in silico RNA structure prediction to study the structural divergence of different lineages. Owing to the error-prone replication machinery inherent to many RNA viruses, SNVs are created continuously and represent the constitutive driving force behind viral quasispecies . AlthougWe asked whether Nextstrain can be utilized to infer novel insight into RNA structure-association of individual clades. As Nextstrain is particularly convenient for discriminating characteristics of viral clades/lineages, we set out to expand the sequence-centric approach to computation of lineage-associated structural traits. By combining Nextstrain genome diversity data with RNA structure prediction methods, we could associate sequence variants observed in different CHIKV lineages with alternative RNA structure formation. Although we cannot associate lineage-specific SNVs with particular biological functionality at this point, the fixation of nucleotide mutations in certain CHIKV lineages suggests that they are either neutral or they confer an adaptive advantage in an evolutionary setting.Building on a thermodynamic model for RNA structure formation, we present here 14 RNA candidates in the CHIKV coding region that exhibit an alternative fold upon mutation of individual nucleotides. While we were focusing on mutations that induce the most obvious changes to the RNA structural ensemble, there are many more lineage-specific mutations that may have only a subtle effect on local RNA folding or may be involved in long-range RNA structure formation. Importantly, phenotypic traits result from the complex interplay of all lineage-specific mutations, and in this line, our approach can help by defining target RNAs for experimental verification.In summary, we show here that the combination of molecular epidemiology data with RNA structure prediction can help to gain insight into hitherto unresolved aspects of genotype–phenotype associations within viral species. On a broader scale, specifically when applied to different viruses, this can augment our understanding of RNA structure evolution.


Subsequently, we engage in a discussion about the RNA structure-mediated mechanisms of alternative splicing regulation. On one hand, the impairment of formation of optimal RNA structures may have critical consequences for the splicing outcome and further contribute to understanding the pathomechanism of severe disorders. On the other hand, the structural aspects of RNA became significant features taken into consideration in the endeavor of finding potential therapeutic treatments. Both aspects have been addressed by us emphasizing the importance of ongoing studies in both fields. Alternative splicing is a highly sophisticated process, playing a significant role in posttranscriptional gene expression and underlying the diversity and complexity of organisms. Its regulation is multilayered, including an intrinsic role of RNA structural arrangement which undergoes time- and tissue-specific alterations. In this review, we describe the principles of RNA structural arrangement and briefly decipher its  MBNL1) splicing factor is spliced to several mRNA isoforms and the majority of them encode the MBNL1 protein isoforms of distinct cellular localization, splicing activity, stability, and propensity for dimerization [CLCN1) pre-mRNA by regulating splicing of exon 7a which carries an in-frame premature termination codon (PTC) [CLCN1 transcript which manifests in reduced chloride ion conduction and myotonia affecting skeletal muscles in myotonic dystrophy type 1 (DM1) [Splicing is an essential process of gene expression in eukaryotes, leading to the production of mature RNA species including messenger RNAs (mRNAs) , long norization . Moreoverization ,6. Inter 1 (DM1) . cis-acting elements and trans-acting factors, the availability and activity of which may vary at different developmental stages and between tissues [cis-acting exonic and intronic splicing silencers and enhancers  [cis-acting silencers and enhancers comprise an interaction platform for auxiliary trans-acting factors recognizing strictly defined or degenerated motifs and represented by various families of RNA-binding proteins (RBPs) whose expression is spatially and developmentally regulated [cis-acting sites promotes the alternative exon inclusion, whereas the exclusion occurs due to MBNL1 binding within an alternative exon and/or upstream intron  ,10,11 Fa. AS com tissues . The spl tissues ,21,22. I tissues . In loweSE, ISE) ,25,26,27SE, ISE) ,28,29. Megulated ,31,32. Aegulated . MBNL1 eegulated ,35. Addiegulated . Its assm intron b. ReminiIn addition to the RNA primary structure (nucleotide sequence), the RNA secondary and tertiary conformation emerged as a source of functionality and a significant layer in AS regulation ,42,43,44The aim of this review is to highlight recent findings exploring the phenomena of RNA structural arrangement, its cellular modulators and biological functionality linked to AS regulation and pathomechanism of splicing-associated diseases.+, Na+, Mg2+), pH, and temperature [The first discoveries of consensus motifs at exon-intron junctions were made in late 70′s . Only a perature ,64, the perature . 15N relaxation dispersion nuclear magnetic resonance (NMR) combined with chemical probing to capture such intermediates of p5abc subdomain of the Tetrahymena group I intron ribozyme [Recent advances including high-throughput chemical footprinting combined with next-generation sequencing enabled to define remarkably detailed structural features of RNA in association with their biological role. Gracia and others provided a substantial body of evidence for incremental and concerted cooperativity between RNA structural motifs leading to folding of preferential RNA secondary and tertiary structures ,67. Thisribozyme . This suribozyme .Considering given opportunities to form many energetically favored RNA structures exclusively to the native one, albeit at the expense of high energy- and time-consuming processes of RNA structural rearrangements, it is profound the way cells employ and manage factors introducing effective kinetics of functional RNA folding . Therefo3-kethoxal  [The development of high-throughput RNA structure probing on a whole-transcriptome level brought an immense revelation in gaining a deeper insight into biologically relevant RNA structure information in vivo ,72. It wesidues) ,49,76. Iesidues) ,78,79. Most research reflects the RNA structurome as the average RNA structures at steady state obtained in a whole-cell analysis that may limit our insight into the functional implications of RNA folding. This limitation has been partially overcome by achieving the mRNA structure dynamics during zebrafish development using DMS-seq analysis  or durinIt needs to be considered that methodological aspects of experiments my underlie discrepancies between obtained results as some reagents show stronger bias towards sampling kinetically more stable or unfolded states of RNA structure or particular nucleotides during the time course of the experiment, whereas RNA structural arrangement undergoes transient and dynamic changes. Adding to the complexity, an important discovery was recently published showing much higher recruitment of RBPs to structured regions of transcripts , and thuThe complexity of the cellular environment provides a great variety of dynamic modulators of RNA structure orchestrated by a spatiotemporal network of interactions. Their interplay results in a heterogeneity of the RNA structurome where RNAs are at different stages of the life cycle from transcription through translation and decay. Here, we will decipher selected modulators and their impact on RNA folding, whereas their involvement in mechanism of AS regulation will be extended in the next chapter.Cellular environment is characterized by molecular crowding underlain by a heterogeneous composition of inorganic components  and organic molecules  of defined sizes and charge which entrain a steric hindrance due to their high accumulation and impenetrability ,83. Thiscis-acting elements and the kinetics of RBPs’ recruitment. Nevertheless, a growing body of evidence considers the significance of cotranscriptional RNA folding in RNA processing. Due to such limitations as the speed of Pol II (~1–4.6 kb min−1) [Cotranscriptional RNA folding has long been given a biological relevance ,97,98. Ab min−1)  and the b min−1) . The autb min−1) ,68 and tb min−1) . Contrarb min−1) . They obb min−1) . On the b min−1) . An impob min−1) . The newN6-methylation of adenosines (m6A) is deposited at RRA*CH  consensus sites by the activity of methyltransferases like 3 and 4  [6A-imposed RNA structural regularities associated with destabilization of RNA duplexes [6A-induced location-dependent switch of RNA secondary structure may recruit m6A ‘reader’ proteins or increase the accessibility of the adjacent RNA motifs for splicing factors or, in contrast, m6A may be recognized and removed by ‘eraser’ proteins; all these mechanisms are substantial in coordination of several aspects of RNA metabolism predominantly AS [6A deposition is most likely driven before or soon after the occurrence of exon definition in nascent pre-mRNA consistently with recent findings showing a strong correlation between AS regulation and enrichment of m6A signals within introns or their reduction at splice junction exonic boundaries [6A-driven RNA structural arrangement is further extended in the next chapter. Among dozens of internal RNA modifications shape-shifting RNA structural arrangement, methylation of adenosines and isomerization of uridines to pseudouridines are the most ubiquitous in a nucleus, albeit still perplexing researchers with their role in gene expression regulation. The ctively) ,115. Botduplexes ,116,117 antly AS ,124,125.undaries . The mecPseudouridines (Ψ) are widely abundant modifications in eukaryotes added primarily by the activity of standalone pseudouridine synthases (PUS) and Box H/ACA ribonucleoprotein enzymes in the course of transcription ,128. Notcis-acting sites and splicing machinery or auxiliary factors [Adenosine-to-inosine (A-to-I) RNA editing conducted by adenosine deaminase acting on RNA (ADAR) is another RNA shape-shifting factor whose activity leads to unwinding of RNA duplexes as A-U Watson-Crick base pairs are converted to I-U wobble pairs ,136. Its factors ,139,140. factors . In contDue to the fact that the fidelity of adenosine methylation, pseudouridylation, and RNA editing depends on linear and/or structural RNA motifs and that they are engaged in gene expression, the dynamics of RNA modification stoichiometry and their kinetic timing during RNA maturation comprise another crucial layer of regulation which remains to be deeper characterized.Alu short transposed elements which may be distinguished from a linear sequence and perceived as specific cis-acting modulators of RNA structure. Alus appear in tandem inverse orientation and tend to form intermolecular dsRNA structures subjected to ADAR editing [Alu elements, short complementary runs of nucleotide repeats are reported to play an essential regulatory role [Intriguingly, ADAR activity is tightly associated with primate-specific  editing ,143. The editing , especia editing ,146,147. editing , which m editing ,145. Anaory role . Lin andory role . The autory role ,150,151.Alterations in RNA structure in response to environmental and cellular signals constitute one of the signaling pathways and executors of gene expression regulation. These signals in a form of particular metabolites are recognized by aforementioned riboswitches ,46. TheyIn case of the majority of eukaryotes the protein- and nucleic acid-directed RNA structural switches predominate. They enable stabilization or formation of functional RNA secondary structures through transient or stable interactions and, in turn, lead to a functional response. Their thorough characterization has been captured in several reviews ,158,159.cis-acting elements altering RNA-RNA interactions and remodeling RNA-protein complexes [Helicases comprise the largest group of transient remodelers of DNA and RNA structural arrangement, categorized into superfamilies and families and involved in virtually every aspect of DNA and RNA metabolism at the expense of ATP . The actomplexes ,170,171.Another largely heterogeneous class of RNA structureswitches, RBPs, binds and forms a stable but not always functional complexes with RNA in an energetically independent manner. Upon binding, the RBPs exert local RNA structural alterations as well as changes of structural context of adjacent regions due to torsional stress and thermodynamic compensation of local alterations. A recent whole-transcriptome analyses of RNA structurome enabled to decipher this issue in a broader context. The authors noticed that the occupancy of many RBPs is linked to RNA structural arrangement either in favor of stabilization or destabilization of RNA structures. For instance, the chromatin-associated proteins tend to interact with less structured RNA regions which undergo folding once they dissociate from the chromatin and the ribonucleoprotein (RNP) complexes . A doublThe class of RNA-mediated RNA structure switches engaged in RNA processing is rather scant, but it is worth mentioning C/D box small nucleolar RNAs (SNORDs) which are 60- to 300 nt-long non-coding RNA species derived from excited introns and accumulated in a nucleolus . One of cis- and trans-acting factors underlying dynamic and intrinsic alterations of RNA structural arrangement in a cellular environment. The same factors may utilize distinct mechanisms to coordinate AS, which overlap or complement one another with a vague borderline between them. Below, we discuss the main RNA structure-mediated mechanisms of AS including bridging or looping out cis-acting elements, blocking or promoting interaction with splicing factors or their allosteric activation/inhibition, modulating the splicing kinetics  [Drosophila melanogaster pre-mRNAs [cis-acting elements presenting only one exon from the cluster of MXEs to the spliceosome at a time while others are looped out. A leading example comprises Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) transcript carrying four exon clusters, including multiple MXEs which give rise to nearly 40,000 isoforms [Apart from bridging splice sites and other decisive regulatory elements, the RNA duplex-formation may lead to their looping out which subsequently excludes these elements from a splicing process. One of the first observations displayed the hnRNP A1-induced RNA structure formation which triggered looping out of internal 5′ss; however, an equivalent result was obtained in a protein-independent approach by inserting RNA duplex-forming inverted nucleotide repeats into the minigene replacing the natural hnRNP A1 binding site . Anotherve exons . Miriamive exons . Accordi[CCDC15)  , its derivatives or a RNA Bind-and-Seq (RBNS) assay ,199,200.cTNT) exon 5 mediated by two mutually exclusive RNA structures. The binding region for these splicing factors folds into either a single strand or a stem-loop structure enabling only one of these proteins to bind. Correspondingly, Sun and others have recently described RNA structure-mediated control of AS of programmed cell death 1 (PDCD1) alternative exon 3 [An interesting example of RNA structure-mediated interaction between splicing factors was described by Warf and others . The aute exon 3 . In thisPAX9) [FMR1) [AS control by G-rich elements residing nearby alternative events has been well covered in research ,235,236.PAX9) , p53 [23PAX9)  or fragi) [FMR1) . In the 6A modification may orchestrate the access to regulatory elements through the impact of the modification itself on RNA-substrate recognition by RBPs [6A-imposed RNA structure switch affecting the recognition of RNA regulatory elements by RBPs [The m by RBPs ,250 or t by RBPs . The str by RBPs , and hnR by RBPs . Recent  by RBPs ,140. Alu elements could also act accordingly. Indeed, there are several lines of experimental evidence that ADAR-associated Alu elements, due to their propensity to form long-distance structures, are also capable of occluding the splice sites or preventing their recognition by splicing machinery [Alus comprises a substantial determinant of splicing profile of adjacent alternative exons which could suggest a great importance of long-distance interactions within RNA in regulation of AS [SMN2) transcript which is critical for development of spinal muscular atrophy (SMA), a genetic disease fatal at early age [achinery . Consiston of AS ,146,147.arly age . A uniquarly age ,255,256.arly age . Among oarly age . These aarly age .Regardless of the extensive pseudouridylation of snRNAs and its role in spliceosome assembly, little is known about the mechanism of splicing through pseudouridylation of pre-mRNA ,134. Potnmt1 pre-mRNA comprises an intronic TPP binding cassette which, according to in-line probing experiments, partially base pairs with and occludes an alternative 5′ss, acquiring the “ON” state and allows for translation of nmt1 protein [nmt1 expression. Intriguingly, Gong and others provided even deeper elucidation of the kinetics of TPP-riboswitch structural response by exploiting a systematic helix-based computational method [In fungi and plants, the RNA structural rearrangement of splicing sites modulating their availability for the spliceosome was found to occur in the context of TPP-sensing riboswitches ,152,154.l method . The autThe diversity of RNA structures and their coexistence driven by multiple cellular factors, constitute a regulatory layer of RNA-protein interactions similarly to optimal and suboptimal linear motifs with varying degrees of their effect on AS ,201,203.Even though there is no strong or direct evidence, the model of RNA structure interference into splicing kinetics has been proposed to mediate AS outcomes. In yeast, the cotranscriptional RNA folding was predicted to substantially influence transcriptional elongation rate, which, in turn, determines inclusion or skipping of alternative events ,269,270.Alu-mediated structure disturbance occurring close to splice sites may further diminish their recognition by splicing components and affect kinetics of their binding leading to slower splicing kinetics or suboptimal exon selection [6A modification has been recently correlated with splicing kinetics of alternative exons either through direct recognition of m6A by readers or indirectly through m6A-imposed RNA structural rearrangements [6A by METTL enzymes at exon-intron splice junctions was strongly linked to fast splicing kinetics and constitutive splicing  and a class of frontotemporal lobar degeneration (FTLD) [MAPT) transcript, which causes MAPT pre-mRNA missplicing, were found in patients and might contribute to the pathomechanism [On the other hand, the SNV-based structural alterations of regulatory elements may also change their accessibility for n (FTLD) . They arn (FTLD) . Howeverechanism . Particuechanism . Disruptechanism ,283. Varechanism ,284. Sucechanism . SMN1 and SMN2 genes differ from each other by the presence of deletions and substitutions leading to SMN2 exon 7 exclusion and expression of a truncated and only partially functional SMN2 protein due to in-frame occurrence of PTC within exon 8 [SMN2 splicing regulation [SMN2 exon 7 splicing, culminated in the design of antisense oligonucleotide-based therapeutics. SpinrazaTM is a drug approved in 2016 by Medical Drug Association (MDA), which is complementary to a cis-acting element within intron 7 called Intronic Splicing Silencer N1 (ISS-N1), and abrogates its negative effect [An interesting example of a disease in which SNVs and RNA structural arrangement play a critical role in therapeutic strategy development is SMA, a recessive genetic neurological disease. In general, the pathomechanism of SMA is underlain by disease-associated deficiency of SMN1 protein, while the production level of its paralog, SMN2, is naturally low and insufficient to compensate for SMN1 ,286. In n exon 8 ,287. Sevn exon 8 ,288. In gulation . Until ngulation . A greate effect ,291,292.e effect .Microsatellites are 2–10 bp-long repetitive DNA sequences which are abundant in human genome and, due to their structural properties, they sporadically undergo pathogenic expansions or contractions as a result of aberrant replication or DNA repair . They exA common feature of the majority of these mutations within transcripts is their propensity to form intrinsic secondary structures of different stability and with sequence- and position-dependent functional relevance in pathomechanism of the diseases . For exaDMPK) transcript or intron 1 of cellular nucleic acid-binding protein (CNBP) transcript, respectively [exp, which impair nuclear export of its host transcript, leading to the formation of nuclear RNP inclusions [RX, was the first potential DM1-specific drug which underwent clinical research [Myotonic dystrophy type 1 and type 2  constitute illustrative examples of neuromuscular diseases with an interplay between RNA secondary structure and splicing playing a crucial role in their pathomechanism. DM1 and DM2 manifestation relies on microsatellite mutations occurring either within 3′UTR of dystrophia myotonica protein kinase  [cis-regulatory elements conveyed by structural conformation of the repeats and created RNP complexes. Interestingly the IR-based pathomechanism turned out to be relevant for other diseases with intronic GC-rich but not A/AT-rich microsatellite mutations including C9orf72-linked amyotrophic lateral sclerosis with frontotemporal dementia (C9-ALS/FTD) and Fuchs endothelial corneal dystrophy (FECD). This difference may arise from structural stability of GC-rich mutations compared to those enriched in A/ATs [exp-based pathomechanism seems to differ from DM1. Although MBNLs exert higher affinity to CCUG than CUG repeats [Contrary to DM1, the DM2-associated CCUG expansion (CCUG)n 1 (IR) . The mecin A/ATs . In addi repeats ,172, the repeats . The lat repeats  but also repeats .cis-regulatory elements are greatly correlated with AS regulation of proto-oncogenes and tumor suppressors [RELL2) is only subtly regulated by dsRNA-dependent activity of ADAR [RELL2 exon 3. In consequence, an alternative isoform prone to NMD is produced, promoting tumorigenesis. Revealed mechanism shows that ADAR binds dsRNA formed between GA-rich regions at exon 3 and an upstream Py-tract which impedes U2AF65 association with 3′ss for exon recognition [The range of deregulated processes in cancer cells make it impossible to emerge early molecular alterations leading the cell to a tumorigenic pathway. AS is one of these processes which is vastly disturbed and contributes to nearly all deleterious cancer cell phenotypes including metastasis, angiogenesis, or proliferation ,320,321.pressors ,323. Duepressors . Two stupressors ,140. In  of ADAR . Howeverognition . CD44) transcript due to binding hnRNP F and mediating the production of epithelial-specific CD44 isoform. Conversely, hnRNP F depletion contributed to epithelial-to-mesenchymal transition (EMT) which associates with tumor invasive and survival properties, whereas breast cancer patients with hnRNP F lowly expressing tumors exhibited a lower survival rate [G-rich elements with a propensity to form stable RNA- and DNA-G4s, also correlate with tumorigenesis underlying many abnormalities during gene expression , whereasval rate .Here, we provided an overview of several issues in relation to the role of RNA structural arrangement in regulation of alternative splicing in eukaryotic genes. We mainly focused on cellular modulators of RNA structure and the mechanisms undertaken by RNA structure to govern AS in physiological and pathological conditions. Increasing lines of evidence indicate RNA structure as a significant regulatory layer in the control of gene expression. It becomes more rational to perceive RNA structures as executors of gene-encoded information exploiting RBPs remodelers and a network of interactions to direct AS. Thus, the role of RNA conformation in AS is expected to be wide-ranging, but its experimental confirmation sometimes remains out of reach. Its complex nature including heterogeneity, dynamics as well as its interaction network with cellular environment, biomolecules and ongoing biological processes make it challenging to decipher RNA structurome and its functional relevance. The direction of future approaches has been already initiated by a few studies, bringing closer to our understanding the functional aspects of RNA structure and its dynamics relevant to a crucial context of cellular biomolecules, compartmentalization, development, and kinetics of RNA folding ,80,113. cis-acting motifs usually densely residing near alternative exons or within them, as it was shown for SMN2 [Large-scale technological advances, including in vitro structural probing or transcriptome-wide methods coupled with sequencing, have definitely brought deeper understanding in a global pattern of RNA conformation in cells, underscored its importance in association with biological processes and emerged particular structural elements alongside their potential regulatory function. However, detail-oriented analysis should always be appreciated as they deliver an intrinsic insight into the biological mechanism and enable to emerge single nucleotide details of great value for therapeutic interventions. This thorough knowledge on the functional structural elements within introns in alternative splicing regulation of disease-associated transcripts may vastly facilitate the development of potential therapeutics. First, targeting distant intronic regions potentially less abounding in regulatory elements could prevent from unintended deregulation of overlapping for SMN2 . Second,Eventually, future studies are envisioned to bring great discoveries of novel functions of RNA conformation which could, for example, serve as a binding platform for splicing factors to increase their local concentration and facilitate pre-mRNA processing or to temporarily and locally coordinate highly packed condensates.


Termini often determine the fate of RNA molecules. In recent years, 3′ ends of almost all classes of RNA species have been shown to acquire nontemplated nucleotides that are added by terminal nucleotidyltransferases (TENTs). The best‐described role of 3′ tailing is the bulk polyadenylation of messenger RNAs in the cell nucleus that is catalyzed by canonical poly(A) polymerases (PAPs). However, many other enzymes that add adenosines, uridines, or even more complex combinations of nucleotides have recently been described. This review focuses on metazoan TENTs, which are either noncanonical PAPs or terminal uridylyltransferases with varying processivity. These enzymes regulate RNA stability and RNA functions and are crucial in early development, gamete production, and somatic tissues. TENTs regulate gene expression at the posttranscriptional level, participate in the maturation of many transcripts, and protect cells against viral invasion and the transposition of repetitive sequences.RNA Interactions with Proteins and Other Molecules > Protein‐RNA RecognitionRNA Processing > 3′ End ProcessingRNA Turnover and Surveillance > Regulation of RNA StabilityThis article is categorized under: Terminal nucleotidyltransferases are a diverse group of enzymes that regulate many processes in metazoan cells. Here, we comprehensively summarize recent work on the role of posttranscriptional 3′ end RNA extensions in metabolism, organism development, and disease progression. A substantial part of such regulation is provided by tight coordination between transcriptional and posttranscriptional mechanisms. The posttranscriptional addition of nontemplated nucleotides to the 3′ end of various types of RNA molecules, also known as RNA tailing, is a highly conserved process that modulates their stability and activity in multiple organisms and plays a crucial role in regulating gene expression. The 3′ ends of RNA molecules, especially their tails, are a powerful regulatory location that can determine the fate of target RNAs. Almost all classes of RNA species can be subjected to some kind of 3′ end tailing at various steps of their existence. Thus, enzymes that are responsible for these extensions, namely terminal nucleotidyltransferases (TENTs), play an especially important role among RNA metabolism machinery.Caenorhabditis elegans and Drosophila melanogaster.The best‐described role of 3′ tailing is the bulk polyadenylation of messenger RNAs (mRNAs) in the cell nucleus that is catalyzed by canonical poly(A) polymerases (PAPs), which also belong to the TENT superfamily  catalytic domain with the signature helix‐turn motif, a central domain, and a C‐terminal RNA‐binding domain (RBD) with a nuclear localization signal and multiple regulatory sites  tail length, and capture noncanonical tail modifications, such as uridylation and guanylation , splint ligation of the 3′ hairpin adaptor is used to significantly increase sensitivity of the method to mRNAs and decrease costs  tail length profiling by sequencing) accurately measures the length of poly(A) tails  and mRNA sequencing) and PAIso‐seq (poly(A) inclusive RNA isoform sequencing) enable the global and accurate determination of the full‐length sequence of endogenous mRNAs, including their poly(A) tails along with non‐adenosine residues at all positions within the tails  tail length without a need for RNA fragmentation or amplification, thus significantly reducing bias that can be introduced by the PCR amplification of long adenosine tracts within tails  is a screening approach to analyze nontemplated 3′ end extensions that are added to reporter RNA by a tethered known or putative TENT  tails that are added by canonical nuclear PAP is well‐described. They are crucial for regulating mRNA stability, export to the cytoplasm, the regulation of mRNA translation, and decay. Noncanonical nuclear and cytoplasmic polyadenylation may have varying effects on target RNA molecules, depending on the particular enzyme or subcellular localization. This section summarizes our current knowledge about all metazoan families of ncPAPs.2.1C. elegans as a gene that controls the germline mitosis‐to‐meiosis decision, progression through meiosis, and gamete production  was first identified in C. elegans, GLD‐2 influences multiple aspects of germline development. High‐throughput analysis and case‐specific studies identified over 500 germline‐enriched mRNAs that are potential substrates for GLD‐2, the expression levels of which are significantly downregulated in gld‐2‐defective worms  tails and a decrease in mRNA abundance, suggesting that GLD‐2 promotes the stability and expression of translationally repressed germline mRNAs , is highly enriched in the testes and acts during postmeiotic spermatogenesis  tail lengths are shortened during late oogenesis , but its role in cytoplasmic polyadenylation is independent of RNAi machinery  tail elongation in mouse oocytes or on oocyte maturation , p27, and β‐catenin mRNAs for TENT2 polyadenylation in human embryonic kidney 293 (HEK293) cells  and modulate its translational competence. In the proposed model, TENT4B is recruited to its target mRNA through its interaction with CPEB , an ortholog of human ZCCHC14  tail length , is a family of four highly similar  proteins—TENT5A, TENT5B, TENT5C, and TENT5D—that are differentially expressed in tissues and organs  and human induced pluripotent stem cells, and its level is sharply downregulated during differentiation , which occurs in approximately 20% of all cases. MM is a malignant proliferation of bone marrow plasma cells, and the loss of TENT5C is associated with limited survival  light chain and genes that are involved in the unfolded protein response ‐targeted secreted proteins . Despite only 30% homology between human and fly proteins, their functions and some properties are similar  have been shown to carry poly(A) tails. However, their functional relevance is not entirely clear  and localizes exclusively to this organelle where it contributes to the regulation of gene expression via the polyadenylation of mitochondrial RNAs  or endonuclease RNase Z removes the excessive nucleotides, producing a substrate for the addition of CCA  have incomplete stop codons with either a terminal U or UA because full termination codons are not encoded in the mitochondrial genome. For these mt‐mRNAs, adenylation by MTPAP is essential to generate a complete UAA stop codon and thus functional ORFs Figure  Anderso. Importadmmtpap gene leads to a severe reduction of mitochondrial polyadenylation, with all mt‐mRNAs having trimmed 3′ ends , and these ends are also protected at their 3′ termini. Intriguingly, the identity of the protective moiety/modification remains unknown, in which U6 snRNAs in worms do not carry 2′,3′‐cyclic‐, 3′‐monophosphate, or 2′‐O‐methylation blocking groups , which is involved in regulating U6 snRNA stability and function   that are incorporated into the Argonaute (Ago) protein CSR‐1. CDE‐1 uridylates these siRNAs to limit their accumulation  is a TENT that is homologous to GLD‐2, MTPAP, and CDE‐1. It was first identified in a screen of mutants with a higher frequency of transposon Tc1 excision in the 4As summarized in the present review, metazoan TENTs are crucial for homeostasis in animals through the regulation of gene expression at the posttranscriptional level. RNA tailing clearly participates in many biological processes, although we are only at the beginning of exploring its significance. Thus, much more needs to be discovered.Drosophila, Wispy relies on the CPEB homolog Orb and other RNA‐binding proteins, such as Dicer‐2 and Aubergine, for target RNA recognition  RNA‐binding proteins and polyadenosine RNA: New members and novel functions.Exonucleases and endonucleases involved in polyadenylation‐assisted RNA decayControl of poly(A) tail length


Effective DNA repair is essential for cell survival: a failure to correctly repair damage leads to the accumulation of mutations and is the driving force for carcinogenesis. Multiple pathways have evolved to protect against both intrinsic and extrinsic genotoxic events, and recent developments have highlighted an unforeseen critical role for RNA in ensuring genome stability. It is currently unclear exactly how RNA molecules participate in the repair pathways, although many models have been proposed and it is possible that RNA acts in diverse ways to facilitate DNA repair. A number of well-documented DNA repair factors have been described to have RNA-binding capacities and, moreover, screens investigating DNA-damage repair mechanisms have identified RNA-binding proteins as a major group of novel factors involved in DNA repair. In this review, we integrate some of these datasets to identify commonalities that might highlight novel and interesting factors for future investigations. This emerging role for RNA opens up a new dimension in the field of DNA repair; we discuss its impact on our current understanding of DNA repair processes and consider how it might influence cancer progression. DNA damage occurs regularly in response to both exogenous and endogenous queues, and a lack of, or ineffective, DNA repair might lead to genomic alterations – point mutations, insertions, deletions, expansions/contractions of repetitive sequences and translocations across the genome – which can ultimately result in cell-cycle arrest and eventual cell death if they accumulate.8 and double-strand breaks (DSBs), resolved by pathways such as non-homologous end-joining (NHEJ) and homologous recombination (HR).10 The various pathways involved in the DDR have been well studied at the molecular level, resulting in comprehensive mechanistic understanding behind their modes of action. However, a growing body of evidence suggests that RNA plays a significant role in the repair of DNA damage through currently unresolved mechanisms.18 The emerging role of transcription, RNA-interacting proteins, RNA-processing enzymes, and RNA itself, in the repair of DNA damage is becoming increasingly apparent,22 and understanding the contribution that RNA makes to the DDR will provide a new level of insight into genome maintenance. The concept of RNA-dependent DNA repair (RDDR) has the potential to alter our understanding of, and prospects for, cancer research and therapy, especially considering the recent emergence of RNA therapies.24 The development of RNA therapeutics is providing a series of novel treatment targets and strategies for a variety of conditions, including cancer.28 RDDR has the potential to allow these therapies to augment DNA repair through manipulation of the RNA substrates it relies on, possibly providing novel, sequence-dependent treatment targets. Here we discuss the new, pivotal research in this area and attempt to shed light on the possible mechanisms for the involvement of RNA in the DDR, with a focus on DSB repair due to its clinical significance and the breadth of data available.The DDR comprises multiple pathways, each of which recognises and resolves specific types of DNA damage. Such damage includes large nucleotide adducts, which are resolved by nucleotide excision repair; small lesions, resolved by base excision repair;10 NHEJ and HR are the two major DSB repair pathways, with NHEJ being a fast, non-specific ligation and HR being a slower, higher fidelity pathway.10 HR utilises the sister-chromatid as a template for repair and therefore primarily occurs in S- and G2-phase of the cell cycle when the sister-chromatid is available, whereas NHEJ occurs throughout the cell cycle. Both pathways utilise the same signalling propagation cascade to drive the cellular responses to the DSB, initiated by activation of the phosphatidylinositol 3-kinase (PI3K)-like kinases (PIKKs): ataxia telangiectasia mutated (ATM), ataxia telangiectasia and rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) in response to induction of a break , which acts as a central marker in DDR signalling. PIKK-dependent phosphorylation of Chk1/2 and p53 also mediates overall cellular responses, leading to cell-cycle arrest and the upregulation of repair factor gene expression.33 The recruitment of MDC1, another PIKK substrate, to γH2AX facilitates the recruitment of the E3 ubiquitin ligases, such as RNF8 and RNF168, which then mediate polyubiquitylation of the H1-linker histone and H2A.36 P53-binding protein 1 (53BP1) is then recruited to ubiquitin modifications on H2A. The retention of 53BP1 at the break then facilitates NHEJ; however, 53BP1 can be removed from the break site by BRCA1–CtIP binding to promote HR  overhang. NHEJ provides a mechanism that rapidly repairs DSBs and can occur during any phase of the cell cycle. However, the lack of any fidelity checks means that NHEJ is prone to the insertion or deletion of bases – indels – and translocations.10 By contrast, HR requires the long-range resection ssDNA overhangs up to kilobases from the break. This ssDNA gets coated in a RAD51 protein filament to allow the invasion of the ssDNA overhang into the double-stranded sister-chromatid , XRCC4, Ligase IV and Artemis, that process and ligate the ends to resolve the break.6 This is highlighted by the association of cancer-prone syndromes with the inheritance of defective DSB repair genes, such as BRCA1/2 and ATM/ATR.47 However, DDR pathway components can also be therapeutically targeted in an attempt to induce damage and death in cancer cells via chemotherapeutics that induce DSBs, such as cisplatin,49 as well as targeted therapeutics such as Olaparib.51 Olaparib inhibits the poly-ADP ribose polymerase (PARP) family of proteins, including the DNA repair enzyme, PARP1, and is used to treat patients with BRCA1/2 mutations whose cells are more reliant on PARP1 for DNA repair.51Defects in components of the DDR pathway can lead to the rapid accumulation of genomic instability, consequently promoting cancer development and progression; mutations in genes that encode DSB repair factors are therefore common in cancer cells.53 Canonical components of microRNA (miRNA) biogenesis, splicing and transcriptional regulation machineries have been shown to have a variety of critical roles in the repair of DNA damage.58 In addition, a number of well-studied DNA repair proteins have been identified to have RNA-binding motifs and to interact with RNA-processing enzymes in a manner that facilitates DNA repair.59Our understanding of the contribution of RNA to the DDR and genome maintenance is expanding rapidly with well-known RNA-binding/processing enzymes being identified as novel players in these processes.17 Depletion of Drosha and DICER results in deficient recruitment of repair factors to the damaged site and reporter assays show a significant reduction in both HR and NHEJ repair efficiency, comparable with that seen after BRCA1 and 53BP1 depletion, respectively.17 This role in the DDR is distinct from Drosha and Dicer’s roles in miRNA biogenesis, as the depletion of other factors in this biogenic pathway does not significantly hinder DNA repair.17 Immunofluorescence studies found that Drosha acts at the DDR ubiquitin cascade, as Drosha depletion significantly hindered the recruitment of factors from RNF168 onwards and impaired damage-induced ubiquitylation.17 In addition, Drosha has been shown to localise to break sites, and to be required for processing of RNA in response to DSBs to generate DNA–RNA hybrids around the damaged loci.17The RNA endonucleases Drosha and DICER are core components of the miRNA biogenesis machinery, but have been shown to be required for propagation of the DDR.56 have also been implicated in DNA repair and might represent a sub-family of RNA-processing proteins involved in the DDR.60 In addition, research from the Farnebo lab has identified WRAP53β, the small Cajal body-specific RNA (scaRNA)-regulating protein, as being essential for DDR signalling.61 Similar to the phenotype shown following Drosha depletion, recruitment of repair factors from RNF8 in the DDR ubiquitin cascade onwards is abrogated following WRAP53β depletion, while upstream factor recruitment remains unperturbed.61 With growing evidence that the DDR ubiquitin cascade is a complex and critical signalling step of histone modifications,62 this could represent a central point for DDR co-ordination directed by RNA-related processes.RNA-splicing factors such as NONO and THRAP352 DEAD-box proteins traditionally unwind RNA, although some have been identified to also unwind DNA and DNA–RNA hybrids, or R-loops.64 DDX1 is phosphorylated by ATM and co-localises with γH2AX rapidly in response to irradiation to facilitate HR.66 DHX9 interacts directly with PARP1 and is required for the formation of R-loops.67 Both DHX9 and Senataxin have been shown to reduce chromosomal instability and promote cell survival.58 Senataxin was suggested to facilitate HR, but not NHEJ, and was found to suppress DSB-induced translocation events.18 Importantly, all of these DEAD-box helicases have been shown to directly interact with R-loops, which have recently been shown to be generated around break sites and are currently thought to play a pivotal role in the repair process.71Numerous members of the DEAD-box helicase family, including DHX9, DDX1 and Senataxin, comprise yet another growing sub-family of RNA-processing proteins implicated in DNA repair.72 and RBM7,73 and intrinsically disordered proteins, such as RBM1474 and the FET family .77 These intrinsically disordered proteins are characterised by disordered Gly-Arg-Rich (GAR) regions known as RGG boxes.78 Despite this similarity, they have a diverse range of biological functions related to RNA processes, and appear to act in a transcription-coupled manner to facilitate DNA repair.79These gene families are not an exhaustive list of RNA-interacting proteins involved in the DDR. On the contrary, several other groups of proteins have also been implicated: exosome complex-associated proteins, including EXOSC1086 often uncovering an abundance of RNA-interacting proteins associated with DNA repair. However, this group of proteins has not been examined in depth, and integrating these studies could uncover novel aspects of the DDR. To gain a wide perspective on the proteins involved in the damage response, we aggregated a number of studies that utilised a variety of different techniques – a general mass-spectrometry approach to identify proteins associated with damaged chromatin,80 a screen of tagged proteins for co-localisation with γH2AX in response to UV micro-irradiation77 and an invitro-based experiment to investigate interactors of replication protein A (RPA)-coated ssDNA81 – each therefore identifying a slightly different subset of DDR factors. Gene ontology analysis on the resulting group of DNA-damage-associated proteins showed that RNA-interacting gene groups were significantly enriched for among the proteins identified, with the top five gene groups all having roles in transcription and transcript processing 82 changes in response to DNA damage. Again, gene ontology analysis identified various RNA-related gene groups as being significantly enriched for , which then has a role in facilitating efficient repair.14To assess the contribution of cellular RNA to repair factor recruitment, a number of laboratories have used RNase treatment of cells followed by immunofluorescence of repair factors. Treating cells with RNase A as soon as 20 min after irradiation significantly impaired 53BP1 focus formation, while the formation of γH2AX foci was unperturbed. This phenotype can then be reverted by incubation of cells treated with RNase A with nuclear RNA extracted from other cells.D-ribofuranoside (DRB), alongside damage induction to observe the effect on repair factor recruitment.90 To prevent compensatory alterations in gene expression from confounding these experiments, inhibition of transcription is often done for short periods of time prior to damage induction. Inhibition of transcription reduces the recruitment of NHEJ- and HR-specific factors, suggesting a direct role for transcription in their recruitment.68 In addition, the reformation of repair foci following the addition of exogenous nuclear RNA described above was shown to be dependent on transcription, implicating nascent transcripts in this mechanism.13Another technique being employed to examine the role of RNA in the response to DNA damage is the use of transcription inhibitors, such as α-amanitin and 5,6-dichlorobenzimidazole 1-β-58 and the fact that DNA–RNA hybrids directly connect RNA- and DNA-related processes, the contribution of these RNA species to DNA repair is now being extensively investigated.71 The generation of R-loops is tightly linked to active transcription by RNA polymerases, due to the physical proximity of single-stranded DNA to complementary RNA  and interactome studies to identify R-loop-binding proteins within the proteome.58 DRIP-Seq studies using inducible DSB cell systems found that R-loops are generated around endogenous DSBs early on during the repair process. Transcription is thought to be shut down in response to DSBs102 and since canonical R-loops form behind transcription bubbles, in theory, this should reduce the formation of R-loops at break sites. Consistent with this, R-loop levels are reduced across coding regions in response to breaks; however, in close proximity to the breaks, R-loops were generated in response to damage in a manner dependent on transcriptional activity of the locus.18The use of RNase H is still a relatively indirect method of investigating R-loops. A more direct and quantitative method is the use of the S9.6 antibody, which has high specificity for DNA17 These findings suggest that not only is RNA acting directly at the break site, in the form of R-loops, but that RNA-processing enzymes might be acting to facilitate the generation, and possibly also the removal, of these structures in response to damage. This provides invaluable insight into the mechanisms at work here and allows us to begin to paint a picture of how this fascinating pathway functions. Interestingly, an S9.6 interactome study identified both DHX9 and DDX1 as being among the strongest interactors of R-loops along with multiple other RNA-binding proteins that have been implicated in DNA repair.105 Most notable, however, is the identification of DNA-PK and PARP1 as significant R-loop interactors, both of which are core components of the DDR; their knockdown also resulted in significant alterations to global R-loop levels.58 This association of R-loops with canonical DNA repair factors not only implicates R-loops further in DNA repair, but also suggests that these structures might contribute to canonical DNA repair processes, rather than simply being a part of a distinct RDDR pathway.The appearance of these R-loops was also found to be dependent on Drosha and their removal dependent on Senataxin, consistent with previous reports identifying DDRNAs as products of Drosha and DICER.68 In vitro assays found that RAD52 binds DNA–RNA hybrids and single-stranded RNA (ssRNA) as well as double-stranded DNA (dsDNA) and ssDNA. In addition, Mazina et al.59 have characterised an additional function of RAD52 in inverse strand exchange between dsDNA and ssRNA. This process allows the formation of a DNA–RNA hybrid via the exchange of one of the dsDNA strands with ssRNA and has been shown to facilitate RDDR in yeast cells.68 In addition, RAD52 was shown to facilitate transcription-associated homologous recombination repair via the processing of R-loops at DSBs.106 More recently, BRCA1 and the BRCA1–BARD1 heteroduplex was shown to interact with hybrids, while BRCA2 recruits RNase H2 to facilitate hybrid removal at break sites.71 These experiments provide direct evidence of canonical DDR factors not only interacting with R-loops, but also being able to enzymatically utilise RNA in the same way as DNA. Combined with the loss of the recruitment of repair factors upon depletion of RNA-processing enzymes, this suggests that RNA and RNA-processing enzymes co-operate with canonical DNA repair factors to facilitate repair, and that the process of RDDR is strongly linked to canonical DNA repair.Other canonical DDR factors have also been implicated in processes related to RDDR. RAD52 is required for the assembly of the RAD51 filament around resected ssDNA during HR and is recruited to sites of damage in a manner dependent on transcriptional activity in neuronal cells.–RNA hybrids,107 further investigations have shown that the antibody also exhibits significant affinity towards double-stranded RNA108 and variable affinity towards different R-loop sequences.109 Off-target binding of S9.6 could therefore be a potential pitfall of these experiments, especially given the complex nucleic acid structures formed during DSB repair.110 RNase-H treated samples act as an effective negative control for these experiments; however, these results are not always published. The use of RNase-H in laser micro-irradiation and immunofluorescence experiments does support the formation of R-loops at DSBs; however, much of the data regarding their profile across break sites, protein interactions and dependence on transcriptional activity is dependent on the S9.6 antibody. Thus, although there is convincing evidence for the formation of R-loops at sites of DNA damage, some of these data should be viewed with caution.Although some studies on R-loops use RNase-H as a tool for their investigation, most of the data discussed here utilised the S9.6 antibody. This is problematic, as any potential biases from this antibody could skew the results of these experiments. Whereas in vitro experiments found S9.6 to be highly specific towards DNAThere are two prevailing theories for the origin of RNA involved in DNA repair: transcription events that occur after break induction and produce RNA from the broken DNA; or the utilisation of a transcript produced prior to break induction that remains in the vicinity of the break. Both theories have extensive supporting evidence, however, identifying the RNA experimentally is challenging as it is difficult to distinguish from background RNA, especially at highly transcribed loci.111 Until recently, it was thought that all transcription around DSB sites is shut down to prevent transcription over broken DNA thus to avoid collisions/interference of the transcriptional and repair machineries.112 However, it has been hypothesised that this similarity in chromatin conformation between DSB sites and transcriptionally active loci allows the recruitment of RNA polymerase at broken DNA ends and the bi-directional transcription of damage-induced long non-coding RNAs (dilncRNAs).115 A recent study has shown that, whereas promoter proximal transcription was silenced upon DSB induction, this was then followed by DSB-induced transcription events that occur from the region of the broken DNA ends.102 This suggests that dilncRNA transcription at DSBs may be independent of promoter activity, and is instead a distinct mechanism of transcriptional induction regulated by the DDR. It has also been corroborated in multiple organisms that Drosha- and DICER-like enzymes can then digest these dilncRNAs into mature DDRNAs via a mechanism dependent on the transcriptional activity of the damaged locus (Fig. 115 These DDRNAs could then be used for targeted degradation of potentially damaged mRNAs, a mechanism that would explain the strong dependence of these small RNAs on RNA-interference enzymes.116 However, an mRNA silencing mechanism would not explain the dependence of the DDR on these enzymes, since removing these transcripts would not impact the recruitment of repair factors to damage sites or effect repair efficiency. dilncRNAs and DDRNAs have now been shown to form R-loops at break sites,115 and it has also been suggested that DDRNA can be used as a sequence-specific signal for downstream DDR events (Fig. 117 With RNA-processing enzymes commonly functioning in the ubiquitin cascade of the DDR, it could be that DDRNA is not only used as a sequence-specific target for factor recruitment, but also to aid the chromatin remodelling events that occur at this point in the response. R-loops have already been implicated in controlling chromatin conformation, and have even been shown to recruit chromatin-regulatory complexes that are involved in DSB repair.120 This hypothesis provides a well-rounded explanation for the involvement of RNA in the DDR by linking the canonical roles of implicated enzymes with the RNA-related phenotype. However, the evidence for damage-induced transcription is based on the use of highly expressed reporter systems or break sites flanked by repetitive sequences, which have previously been suggested to confound the results.116 In addition, RNA sequencing and a variety of next-generation transcription profiling techniques using inducible DSB systems have failed to identify nascent, bi-directional transcription around endogenous break sites in mammalian cells,114 and only recent data have suggested that this scenario might occur at endogenous mammalian loci.115 Further research using more endogenous methods is therefore required to support this theory, and to provide a more detailed mechanism for this complex series of events.Upon DSB, the surrounding chromatin gets remodelled via modifications, which causes it to decondense and thereby facilitates the recruitment of repair factors and processing of the DNA. Notably, this chromatin state resembles that of transcriptionally active loci and even harbours some of the same histone modifications.cus Fig. .12,69,11nts Fig. .69,115,190 This theory has gained popularity as a transcript produced prior to the DSB has the potential to maintain the fidelity of the repair by acting as a template for repair.An alternative theory for the involvement of RNA in the DDR is the possibility that a transcript produced prior to damage induction is processed and incorporated into the repair process in response to DSBs. This has been demonstrated to occur in yeast in an NHEJ-dependent manner using a plasmid reporter, and a recombination-based repair mechanism using RNA was also shown to be dependent on RAD52.121 This mechanism would in principle be similar to HR, but importantly could be utilised throughout the cell cycle (Fig. 88 Furthermore, previous research has indicated that transcriptionally active loci are preferentially targeted for templated repair101 and, with R-loop generation and the recruitment of RNA-processing enzymes being dependent on transcriptional activity of the loci, it is possible that these phenotypes both occur as a result of the increased levels of potential template RNA in the local environment of highly transcribed genomic regions. RNA-templated repair could therefore provide a direct mechanism to explain how highly transcribed sites have increased rates of repair factor recruitment and templated repair, acting as a safeguarding mechanism for the transcribed, and therefore most active, regions of the genome.The possible mechanism of RNA-templated repair is based on enzymes processing transcripts produced prior to break induction to facilitate their hybridisation with the broken DNA, forming an R-loop that can be used as a template.cle Fig. . RNA-tem122 found that NHEJ can occur through a resection-dependent mechanism during the G1-phase of the cell cycle that is dependent on a cohort of HR-related factors such as MRE11, CtIP and BRCA1. This was speculated to be an RNA-templated repair mechanism that utilises NHEJ machinery for a rapid response to the break, but in which recruitment of an RNA template initiates a resection-dependent templated repair facilitated by HR-related machinery.123 This crossover of factors from both NHEJ and HR is also consistent with the loss of recruitment of factors for both pathways upon knockdown of RNA-processing enzymes.59 An RNA-templated repair mechanism has also been proposed as the method for high-fidelity DSB repair in terminally differentiated cells, especially considering that cells such as neurons are not rapidly replaced and will therefore accumulate mutations over time.90Interestingly, Biehs et al.121 As it stands, the current data do not provide direct evidence for RNA-templated repair, and this phenomenon is yet to be shown to occur in mammalian cells.88 In addition, it has recently been suggested that R-loops form at DSBs independent of transcriptional activity, instead suggesting damage-induced transcription generates the RNA component of these R-loops.71 Significant breakthroughs that demonstrate this mechanism in mammalian cells and that link together the observed phenotypes of G1-phase resection-dependent repair and the seemingly critical role of RAD52 are therefore still required.One problem associated with this model, though, is the lack of a mammalian RNA-dependent DNA polymerase other than telomerase; however, there is evidence that DNA replication polymerases, such as polymerases α and δ, can function in this capacity.125 It is likely that there are further roles for RNA in the DDR that currently remain unexplored, especially given the prevalence of RNA-interacting proteins in proteomic studies of the DDR (Fig. Both damage-induced transcription and RNA-templated repair provide interesting and logical explanations for the observations made in the discussed studies, and it is, in fact, possible that both mechanisms exist. They might serve separate functions within the DDR, as damaged-induced transcription could provide an early signalling response and a gene-silencing mechanism, whereas RNA-templated repair would act as a downstream pathway similar to NHEJ and HR. However, these are simply two theories for RDDR based on our current knowledge and understanding. Given the importance of RNA in many other processes, it is not unlikely that the role of RNA in the DDR is multifactorial and that RNA acts at multiple points within the pathway to fine-tune the repair process. Although we have focused on DSB repair here, there is also substantial evidence implicating RNA in other branches of DNA repair, such as nucleotide excision repair and the UV response.127 Hyper-transcription leads to highly varied levels of mRNA, miRNA and lncRNA, which vary drastically even between cells from the same cancer subtype due to their heterogeneity.128 In addition, cancer cells display extremely high levels of DNA damage from processes such as increased rates of transcription and replication.129 Given the substantial role of the DDR in carcinogenesis, further understanding the contribution of RNA to the DDR is essential. Theoretically, mutations in components of RDDR could result in an increased mutation rate, thereby promoting carcinogenesis and cancer progression. Mutations in these repair factors could be linked to altered prognosis, such as reduced survival or resistance to certain therapeutic strategies, and therefore could provide potential prognostic markers aiding our ability to treat patients.130We have discussed here the evidence that RNA is a vital component of the DDR and that it is required to maintain the fidelity of repair. The issue of how transcriptional landscapes influence DNA repair still needs further investigation, especially given the dynamic nature of the cellular RNA repertoire. This presents many possibilities with regards to how altered cellular conditions, differentiation states and even extracellular signals can influence DNA repair through RNA landscape changes. Moreover, RNA expression is grossly altered in cancer cells.Alternatively, RDDR could act as a critical mechanism that prevents the accumulation of toxic levels of genome instability and mutations in highly expressed genes that are essential for cancer cell survival. Accordingly, further investigation could reveal a host of novel targets for selective therapies that hinder RDDR, resulting in a build-up of mutations at these highly expressed loci that cannot be tolerated by the transformed cells. The possibilities for cancer biology in general are also interesting, as one could imagine an auto-feedback loop of damage and repair at highly expressed gene loci: high levels of transcription would be correlated with higher rates of DSBs, the repair of which can introduce mutations that further increase transcription rates. This feedback loop would be enhanced by mutations in the RDDR machinery and could greatly enhance cancer cell progression. Of course, these are just a few intriguing possibilities and a far deeper understanding of the mechanisms involved here is required to support them.–RNA hybrids.131 The discovery of a repair mechanism that occurs in the G1-phase of the cell cycle and that utilises factors from both HR and NHEJ repair pathways is consistent with phenotypic data for RDDR, such as immunofluorescence61 and DRIP-seq,17 and suggests that RNA is a critical molecule in canonical DSB repair pathways.It is becoming clear that RNA contributes to the repair of DSBs via a direct mechanism that is distinct from its role in mediating the expression of DNA repair factors. A broad range of enzymes is required to process RNA in response to DSB induction to facilitate the formation of R-loops at break sites, which are key for the progression of the DDR and probably serve multiple roles. Furthermore, multiple canonical DSB repair factors, including RAD52, PARP1, DNA-PK, BRCA1 and 53BP1, interact with RNA and DNA133 in which life originally evolved from RNA molecules, it becomes almost obvious that RNA would be closely entwined in DNA repair from its conception.134 Assuming that DNA evolved from RNA as a stable storage platform for genetic information, RNA could have been required for repair as the original source of genetic information in conjunction with its vital contribution to catalytic processes. Finally, in the burgeoning field of RNA therapeutics24 it is important to consider how RNA is involved in the repair of DNA. This will allow us to avoid possible issues with RNA therapeutics, such as off-target interactions with DNA repair processes, and, more excitingly, potentially harness the activities of the RDDR in cancer cells to develop novel RNA therapeutic strategies.When viewing RNA as part of the process of DNA repair, it is common to perceive it as an ‘optional extra’. However, if we consider the possibility of an RNA world,