N6-methyladenosine (m6A) has been shown to be involved in post-transcriptional regulation processes including mRNA stability and promotion of translation, but the role of m6A in the hypoxia response is unknown. In this study, we investigate the effect of hypoxia on RNA modifications including m6A. Our results show hypoxia increases m6A content of poly(A)+ messenger RNA (mRNA), but not in total or ribosomal RNA in HEK293T cells. Using m6A mRNA immunoprecipitation, we identify specific hypoxia-modified mRNAs, including glucose transporter 1 (Glut1) and c-Myc, which show increased m6A levels under hypoxic conditions. Many of these mRNAs also exhibit increased stability, which was blocked by knockdown of m6A-specific methyltransferases METTL3/14. However, the increase in mRNA stability did not correlate with a change in translational efficiency or the steady-state amount of their proteins. Knockdown of METTL3/14 did reveal that m6A is involved in recovery of translational efficiency after hypoxic stress. Therefore, our results suggest that an increase in m6A mRNA during hypoxic exposure leads to post-transcriptional stabilization of specific mRNAs and contributes to the recovery of translational efficiency after hypoxic stress.Post-transcriptional regulation of mRNA during oxygen deprivation, or hypoxia, can affect the survivability of cells. Hypoxia has been shown to increase stability of a subset of ischemia-related mRNAs, including VEGF. RNA binding proteins and miRNAs have been identified as important for post-transcriptional regulation of individual mRNAs, but corresponding mechanisms that regulate global stability are not well understood. Recently, mRNA modification by  Hypoxia is a metabolic condition that occurs when oxygen levels are deficient in cells or tissues. This stress can occur during embryonic development, where crowding and rapid cell division causes a shortage of blood and oxygen supply . HypoxiaAlthough the transcriptional response to hypoxia is well established, the post-transcriptional response to oxygen deprivation is less understood. Post-transcriptional responses often regulate mRNA splicing and stability, and the stability of individual mRNAs, such as VEGF and Glut1, is increased with hypoxic exposure . We have6A mRNA modification has been shown to be important for the stability and translational efficiency of mRNA , as well as Wilms’ tumor associating protein (WTAP) (S-adenosyl methionine (SAM) binding domain, and utilizes SAM as a substrate to methylate target mRNAs that contain a DRACH m6A consensus sequence, often found in 3′ UTR's and around stop codons  or fat mass and obesity related protein (FTO)  . METTL3 p codons , while Mp codons ,b. m6A min (FTO) . Methylain (FTO) . While mA occurs . Additiol output .6A may alter the fate of RNA by potentially altering secondary structure, the ability of RNA binding proteins to bind, or the position of splicing events  conditions in the presence of 1 g/L glucose to mimic the nutrient deprivation encountered during ischemic events. Poly(A)+ mRNA was enriched by oligo(dT) selection followed by ribosomal RNA (rRNA) depletion (verified by qPCR) , and after fragmentation, liquid chromatography and tandem mass spectrometry (LC-MS/MS) were used to quantify various RNA modifications in the mRNA enriched samples. Surprisingly, in contrast to a recent report measuring m6A in total RNA and specific mRNAs in breast cancer cells . These data show that the mRNA modification, m6A, has a dynamic response to hypoxia.Changes in the mer cells , global 6A levels in total RNA were unchanged in hypoxia . Because rRNA makes up 80%–85% of the cellular RNA mass, we postulated that modifications with diminished content in total RNA derived chiefly from changes in rRNA modifications. Thus, 40 and 60S ribosomal subunits were isolated via differential centrifugation through sucrose gradients. rRNA was isolated from the fractions and subjected to qPCR to verify the composition of the fractions . Again, LC-MS/MS revealed no change in m6A content from either 18S or 28S rRNA in HEK293T cells exposed to hypoxia . Interestingly, pseudouridine also exhibited a decrease in content in the isolated 28S rRNA, but no changes were detected in total RNA . These results suggest that not only mRNA modifications, but also specific rRNA modifications, are dynamically altered after 24 h of hypoxic conditions. However, for this study we chose to focus on the effects of hypoxia on the mRNA m6A content, given its reported effects on mRNA regulation.As mRNA makes up <10% of the cellular RNA mass, we postulated that hypoxia may cause changes in modifications in other RNA species as well. We tested this hypothesis by first measuring RNA modifications in total RNA. Surprisingly, in HEK293T cells m hypoxia B. Howeve6A content of mRNA, we next wanted to determine whether m6A methylation of specific mRNA targets involved in the adaptive response was affected when cells were exposed to hypoxia. Using m6A RNA immunoprecipitation (MeRIP), target mRNAs related to hypoxia and tumorigenesis including Glut1, Jun, Myc, Dusp1, and Hes1 were investigated. HEK293T cells were exposed to 24 h of normoxia or hypoxia and poly(A) mRNA isolated via oligo(dT) selection and ribominus treatment. m6A containing mRNAs were then immunoprecipitated using an m6A-specific antibody. Following cDNA synthesis, the relative enrichment of the indicated mRNAs was determined using qPCR. The targets were quantified relative to input RNA and the negative IP. Interestingly, many of the hypoxia-associated and tumorigenic mRNAs from hypoxic cells, including Glut1 and Jun, increased more than twofold in the m6A captured fraction, as compared to the normoxic conditions (6A content of these mRNAs in response to hypoxic exposure. It is likely that these and other mRNAs contribute to the enhanced m6A content of poly(A)+ RNA (6A content (Given that we saw dynamic changes in the mnditions D. This s(A)+ RNA A. Import content D, sugges6A methylation plays a role in stabilization of these particular mRNAs as part of their post-transcriptional response to hypoxia, METTL3 and METTL14 of the m6A methyltransferase complex were knocked down via siRNA and the half-lives of our target mRNAs were determined. Knockdown of METTL3 and METTL14 was confirmed via Western blot analysis . After depletion of METTL3 and METTL14 for 48 h, HEK293T cells were transferred to either normoxic or hypoxic conditions for 24 h to simulate ischemia. During the last hour of treatment, newly transcribed RNA was metabolically labeled using 4-thiouridine (4sU). RNA was harvested, the 4sU labeled RNA was biotinylated, and streptavidin beads were used to separate the new labeled RNA from unlabeled RNA. RNA half-lives were determined from the ratio of the labeled to unlabeled RNA normalized to total RNA. As shown in 6A methylation in hypoxic conditions  also showed a significant increase in mRNA half-life. Interestingly, knockdown of METTL3/14 had no effect on the normoxic half-life of any of the mRNAs, but loss of the methyltransferases did significantly inhibit their hypoxic stabilization. eEFA1, which exhibited no change in m6A level (data not shown) also showed no loss of stabilization after METTL3/14 depletion. Therefore, mRNAs that were stabilized under hypoxic conditions lost that stabilization after an engineered decrease in m6A levels. This suggests that enhanced m6A modification of specific mRNAs can indeed affect their hypoxic post-trasnscriptional regulation.To address if manalysis B. Deplet6A content affected mRNA stabilization, we next examined how methylation might affect the translation of these messages. HEK293T cells were again depleted of METTL3/14 for 48 h, and transferred to either normoxic or hypoxic conditions for 24 h followed by 15 min of cyclohexamide treatment and fractionation of cellular extracts on sucrose gradients. Polysome profiles  were analyzed by qRT-PCR for specific mRNA targets. Sedimentation position as shown by polysome profiling allowed us to determine how efficiently each message was being translated. During sucrose-gradient resolution, mRNAs partition based on the number of ribosomes bound, which is a direct measure of their translational efficiency. For example, mRNA such as β-2-microglobulin (β2M), which is found to peak in fraction 8 in normoxic conditions, is considered to be moderately translated, while an mRNA that is primarily located in fractions 10 and 11, such as Glut1, is bound heavily by polysomes, indicating highly efficient translation  were again analyzed by qRT-PCR for our specific mRNA targets. After 4 h of reoxygenation, recovery of translational efficiency of Glut1, Jun, Dusp1, Hes1, and Myc was diminished by METTL3/14 knockdown compared to negative control siRNA transfected cells . These results suggest that m6A may be aiding the cellular response to recovery after stress.To determine whether increased mSupplemental Fig. 6E,F). EDTA chelates the magnesium necessary for large and small ribosomal subunit association, releasing all mRNAs to presumably sediment slower in their nontranslating state. Interestingly, upon EDTA release, normoxic β2M shifted completely from heavy-to-light fractions just as observed with hypoxia with or without EDTA treatment .We attempted to correlate the mRNA translational efficiency to the steady-state level of their protein products. If mnd Dusp1 B. There nd Dusp1 B. This s6A mRNA methylation of certain mRNAs is induced by hypoxia. Furthermore, increased m6A prolongs the half-life of specific mRNA targets. Although the increased stability of these messages did not correlate with translational efficiency or changes in protein levels during hypoxia, we found that loss of m6A through METTL3/14 knockdown decreased the cells’ ability to recover translational efficiency after reoxygenation following hypoxic stress. We also observed that under hypoxic conditions, some mRNAs may be associated with other ribonucleoprotein complexes instead of actively translating polysomes.Our findings suggest that m6A has received renewed attention over the past few years as a dynamic mRNA modification with many potential cellular functions. Although the factors involved in m6A mRNA methylation have been identified, the importance of mRNA methylation remains unclear. We now suggest a role for dynamic changes in mRNA m6A content in regulating mRNA stability in response to oxygen deprivation. In contrast to the total and rRNA, we saw a significant increase in the m6A content of mRNA levels in hypoxic cells, suggesting that the m6A modification may be important for regulating mRNAs in hypoxia. Our LC-MS/MS results did indicate the presence of other dynamic RNA modifications in total and ribosomal RNA after 24 h of hypoxia, but we have yet to identify what the dynamic regulation of these modifications may signify.m6A followed by qRT-PCR allowed us to determine the m6A methylation status of individual mRNAs. We observed an increase of m6A in specific mRNA targets after hypoxic exposure. However, this method cannot determine how hypoxia affects m6A methylation at specific sites. In follow-up studies, we will seek to determine whether hypoxia introduces m6A in new sites and if so, determine how specificity is regulated. It is possible that a hypoxic switch in methylation sites, for example, switching methylation from the 5′ end to the 3′ end of the mRNA, could also alter the regulation of the mRNA without affecting the overall m6A level.Immunoprecipitation of m6A can affect the stability of mRNAs  cells were obtained directly from ATCC and maintained in high glucose (4 g/L) DMEM (Corning/Mediatech) supplemented with 10% FBS , 2 mM glutamine (Corning/Mediatech), and 1× Pen/Strep (Corning/Mediatech) and passaged when ∼85%–90% confluent. Cells were tested for mycoplasma upon receipt. For experiments, cells were plated on 10 cm dishes  in high glucose (4.0 g/L) media and allowed to attach/recover for 18–24 h. The next day, the media were removed and replaced with media containing 1 g/L glucose. Hypoxic treatments were carried out in a Ruskin In Vivo 400 Hypoxia Hood (The Baker Company) maintained at 37°C, 5% COTRIzol (Life Technologies) was used for all RNA extractions according to the manufacturer's protocol. RNA was further purified and treated with RNase-Free DNase I (Life Technologies) using the PureLink RNA Mini Kit (Life Technologies). For RNA extraction from ribonucleoprotein immunoprecipitations (RNP-IP) and sucrose gradients, GlycoBlue (Life Technologies) was added as a carrier during the precipitation step. RNA quality and quantity were determined via NanoDrop 1000 (ThermoFisher Scientific).+ RNA was first purified from total RNA through oligo(dT) selection using a poly(A) Purist-MAG magnetic mRNA Purification Kit (Life Technologies), followed by ribosomal RNA depletion using a RiboMinus Eukaryote Kit (Life Technologies) according to the manufacturer's protocols.Poly(A)+ RNA was hydrolyzed enzymatically by first denaturing the RNA at 95°C followed by immediate placement on ice. Poly(A)+ RNA was incubated with S1 nuclease buffer and 1 unit of S1 nuclease (Life Technologies) per 300 ng of RNA for 30 min at 37°C. Alkaline phosphatase buffer, 1 unit of alkaline phosphatase per 300 ng of RNA (Life Technologies), and 0.00025 units of venom phosphodiesterase I (Sigma-Aldrich) were added to incubate for 30 min at 37°C. Fragmented RNA was purified through two rounds of chloroform extraction. LC-MS/MS quantification of m6A and adenine was performed by Craft Technologies. Separations and identification by LC-MS/MS were performed using a Thermo Finnigan Linear Ion Trap Quadrapole (LTQ) mass spectrometer utilizing an electrospray ionization interface in selected reaction monitoring mode connected to the Agilent 1100 autosampler and Agilent 1100 HPLC pump system (Agilent). Detection was performed using an electrospray ionization source operated in positive ion mode.Poly(A)2 for 2 h at 37°C, followed by incubation with Antarctic Phosphatase  for an additional 2 h at 37°C. Nucleosides were then separated and quantified at the Duke Molecular Physiology Institute using UPLC-MS/MS as previously described  and layered on a 12 mL 15%–30% sucrose gradient in buffer B. The gradient was centrifuged at 86,000g for 14 h in a Beckman SW-41 Ti swinging bucket rotor. Of note, 1 mL fractions were collected from the top of the gradient and the positions of the 40S and 60S ribosomal subunits were found by measuring each fraction at an absorbance at 254 nm. RNA was isolated from each sample via TRIzol.Cells grown in normoxic or hypoxic conditions were harvested in “Buffer A” . Cell lysate was centrifuged for 15 min at 15,0006A ribonucleoprotein immunoprecipitation reactions were performed by first isolating poly(A)+ RNA from normoxic and hypoxic cells. Protein G Dynabeads (Thermo Fisher Scientific) were washed 3× in 1 mL of IPP buffer . Twenty-five microliters of beads were required per IP. Anti-N6-methyladenosine mouse monoclonal antibody  was added to the beads (5 µg/IP) and brought up to 1 mL with IPP buffer. As a negative control, beads without antibody were used as well. Bead mixture was tumbled for 16 h at 4°C. Beads were washed 5× with IPP buffer and 100 ng of poly(A)+ RNA was added to the beads along with 1 mM DTT and RNase out. The mixture was brought up to 500 µL with IPP buffer. Bead mixture was tumbled at 4°C for 4 h. Beads were washed 2× in IPP buffer, placed into a fresh tube, and washed 3× more in IPP buffer. m6A RNA was eluted off the beads by tumbling 2× with 125 µL of 25 mg/mL N6-methyladenosine-5′-monophosphate sodium salt . Supernatant was added to TRIzol-LS followed by RNA isolation as per manufacturer's protocol. The final RNA sample was brought up in 10 µL of water.m6A poly(A)+ RNA from the MeRIP with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). After diluting cDNA twofold, quantitative real-time PCR was performed using a Roche Lightcycler 96 with Fast Start Essential DNA Green (Roche Diagnostics Corporation) and primers from Integrated DNA Technologies, Inc. Primers used are listed in Supplemental Table 1. Primer efficiency was verified to be over 95% for all primer sets used. Quantification of mRNA from the MeRIP was carried out via ΔΔCT analysis against nonimmunoprecipitated input RNA and RNA pulled down from non-antibody bound beads. All real-time PCR primer sets were designed so the products would span at least one intron (>1 kb when possible), and amplification of a single product was confirmed by agarose gel visualization and/or melting curve analysis.Reverse transcription was performed on 10 µL mSupplemental Table 2. Cells were incubated for 48–72 h post-transfection with the last 24 h in either normoxic or hypoxic conditions.Either a negative siRNA  or METTL3 and METTL14 siRNAs (Qiagen) transfected together using a Lipofectamine RNAi Max 54 µL/plate as per manufacturer's protocol (Life Technologies) using a 180 pM siRNA/10 cm dish. siRNAs used can be found in 2O brought to 400 µL. The mixture was incubated in the dark with rotation for 1.5 h. Biotinylated RNA was extracted using an equal volume of Chloroform/Isoamyl alcohol (24:1) 2× in phase lock gel heavy tubes (5 Prime) followed by RNA precipitation with isopropanol. RNA was heated to 65°C for 10 min and placed immediately on ice. RNA was added to Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific) that had been thoroughly washed and resuspended in 2× streptavidin binding buffer . The RNA bead mixture was incubated with rotation for 30 min. Beads were washed 5× with 65°C wash buffer  and the supernatant was kept containing the nonlabeled RNA. Three rounds of 100 mM dithiothreitol (DTT) elution followed by one round of TE eluted labeled RNA from the beads. RNA was isolated via Isopropanol and resuspended in 40 µL of water.mRNA half-life determinations using 4sU were performed as per established protocol . Cells wmRNA levels were determined by real-time quantitative PCR using 1 µL of RNA from both labeled and unlabeled 4sU samples. Decay rates were calculated by the natural log of one minus the RNA input normalized ratio of labeled over unlabeled RNA. All half-lives were normalized to GAPDH half-life which was defined as 8 h based on previous publications and our unpublished data .2, 10 mM Hepes, 0.5% NP40, 200 µM cyclohexamide, 1mM DTT, 1× protease inhibitors, RNase out) and incubated on ice for 30 min. Lysates were precleared by centrifugation for 8 min at 5000g. Supernatant was layered on a 10%–45% sucrose gradient in polysome profile buffer . The gradient was centrifuged at 38,000g for 1 h 45 min in a Beckman SW-41 Ti swinging bucket rotor. Twelve 1 mL fractions were collected from the top of the gradient and the polysomes were measured from lightest to heaviest at an absorbance at 254 nm. RNA was isolated from each sample using TRIzol.Cells were treated with 200 µM cyclohexamide for 15 min prior to harvest. Cells were harvested in PLB buffer . Equal amounts of protein (30–50 μg) were electrophoresed on a mini-PROTEAN any KD acrylamide gel (Bio-Rad Laboratories) and transferred to Hybond ECL nitrocellulose . The transfer was verified via Ponceau S staining, then the blot was blocked with 5% nonfat dry milk (LabScientific) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature, followed by primary antibody in blocking buffer overnight at 4°C. After washing extensively with TBST, blots were incubated for 1–2 h at room temperature with appropriate HRP-linked secondary antibody , washed again with TBST, developed using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific), and exposed to film for detection. Primary Antibodies used and their concentrations can be found in t-test comparing experimental to control conditions. A P-value below 0.05 was defined as statistically significant.All experiments were performed on at least three separate occasions to generate biological replicates unless otherwise indicated. qPCR was performed at least twice on each cDNA for technical verification of data. Half-lives were calculated for each biological replicate and then averaged together to determine final value and standard error of the mean. Statistical significance was calculated by a two-tailed, paired Student's Supplemental material is available for this article.


Eggplant latent viroid (ELVd) derived RNAs in Escherichia coli together with the eggplant tRNA ligase, this being the enzyme involved in viroid circularization in the infected plant, RNAs of interest like aptamers, extended hairpins, or other structured RNAs are produced in amounts of tens of milligrams per liter of culture. Although ELVd fails to replicate in E. coli, ELVd precursors self-cleave through the embedded hammerhead ribozymes and the resulting monomers are, in part, circularized by the co-expressed enzyme. The mature viroid forms and the protein likely form a ribonucleoprotein complex that transitorily accumulates in E. coli cells at extraordinarily amounts.Viruses have been engineered into useful biotechnological tools for gene therapy or to induce the synthesis of products of interest, such as therapeutic proteins and vaccines, in animal and fungal cells, bacteria or plants. Viroids are a particular class of infectious agents of higher plants that exclusively consist of a small non-protein-coding circular RNA molecule. In the same way as viruses have been transformed into useful biotechnological devices, can viroids be converted into beneficial tools? We show herein that, by expressing  Viroid replication is mediated by host enzymes, such as RNA polymerases6, RNases7 and ligases9, but also by the activity of the hammerhead ribozymes embedded in the RNAs of some viroid species11. More than 30 viroid species have been characterized to date and classified into two families, Pospiviroidae and Avsunviroidae1. Most viroids, like Potato spindle tuber viroid (PSTVd), contain a central conserved region (CCR), approximately in the middle of the folded molecule, and replicate in the nucleus of infected cells through the asymmetric version of the rolling circle mechanism. They belong to the family Pospiviroidae. In contrast, some viroids, like Avocado sunblotch viroid  or Eggplant latent viroid (ELVd)12, lack CCR, contain hammerhead ribozymes in the strands of both polarities, and replicate through the symmetric variant of the rolling circle mechanism in the chloroplasts of infected cells. They are classified into the family Avsunviroidae.The relatively small genomes of viroids, which range between 246 and 401 nucleotides (nt) in presently known species8. For the viroids of the family Avsunviroidae, we also showed that the ELVd monomeric linear RNAs that result from ribozyme processing and contain 5′-hydroxyl and 2′,3′-cyclic phosphodiester termini are efficiently circularized in vitro by the chloroplastic isoform of eggplant tRNA ligase, and also that this reaction most probably occurs in vivo9. To further investigate the interaction between eggplant tRNA ligase and ELVd RNA, we co-expressed both molecules in Escherichia coli. Interestingly, when we analyzed the RNAs that accumulated in transformed bacteria, we observed that some ELVd RNAs accumulated at levels higher than the E. coli ribosomal RNAs did. This occurred despite the analysis of the ELVd-derived RNAs that accumulated in E. coli ruled out viroid replication in bacterial cells, because no replication intermediates were detected. This observation suggested that, in the presence of eggplant tRNA ligase, ELVd longer-than-unit transcripts are efficiently transcribed, cleaved and circularized, and that extraordinary levels of the resulting monomers transitorily accumulate in E. coli cells. Based on this observation, we aimed to develop a viroid-derived system to produce large amounts of recombinant RNAs in E. coli. This system is herein described and used to produce tens of milligrams of RNAs of interest per liter of E. coli culture, such as the RNA aptamer Spinach, which mimics fluorescent proteins13, hairpin RNAs like those that efficiently induce RNA silencing14, or the direct repeat-containing precursor of a CRISPR RNA (crRNA) like those that guide Cpf1 nuclease for genome editing15.We recently demonstrated that, in the last step of PSTVd replication, the host DNA ligase 1 is redirected to accept RNA templates to circularize monomeric linear PSTVd RNAsE. coli clones transformed with plasmids to co-express both molecules  terminator. A recombinant version of eggplant tRNA ligase, which included the amino-terminal transit peptide that mediates translocation to chloroplasts16, but also a carboxy-terminal hexahistidine tag9, was expressed in a compatible plasmid of p15A replication origin under the control of the T7 bacteriophage promoter and terminator. The tRNA ligase expression was induced in cultures of E. coli strain BL21(DE3) by adding isopropyl β-D-1-thiogalactopyranoside (IPTG), which activated the expression of a copy of bacteriophage T7 RNA polymerase set under the control of the lacUV5 promoter. E. coli cultures were grown at 25 °C to an optic density at 600 nm (OD600) of 0.6 and the tRNA ligase expression was induced by adding IPTG to 0.4 mM. Cells were harvested at 10 h post-induction and total RNA was recovered by phenol:chloroform extraction. Aliquots of the RNA preparations were separated by denaturing (8 M urea) polyacrylamide gel electrophoresis (PAGE). Gels were first stained with ethidium bromide, and then RNA was blotted to membranes and analyzed by northern blot hybridization using an ELVd 32P-labeled RNA probe of complementary polarity. This analysis showed that the bacteria co-transformed with plasmids to express ELVd RNA and eggplant tRNA ligase accumulated substantial amounts of the viroid monomeric circular and linear forms, which were clearly detected in both the stained gel  showed that the amount of monomeric circular and linear ELVd RNAs per culture volume increased with time and reached a maximum at around 12 h post-induction, and then decreased to essentially disappear at 24 h  was co-transformed with pLELVd, and with either the original p15tRnlSm or new p15LtRnlSm. Recombinant clones were grown to OD600 0.1 and were induced, or not, with IPTG depending on the harbored tRNA ligase plasmid. A time-course analysis of the RNA produced in both cultures showed no substantial difference in circular and linear ELVd RNA production  fluorophore. We constructed plasmid pLELVd-Spinach  was co-transformed with pLELVd-Spinach and p15tRnlSm. A recombinant clone was grown and the tRNA ligase expression was induced. Aliquots of the culture were taken at different time points after induction. Interestingly, chimeric ELVd-Spinach RNA also accumulated in the recombinant E. coli cells in vast amounts  failed  that lacked RNase III21. Finally, we also produced a 65-nt-long structured RNA moiety consisting of the precursor of a crRNA with two repeated hairpins, like those that guide a Cpf1 nuclease for genome editing15  and assembled27 into plasmid by ligation with T4 DNA ligase (Thermo Scientific). The sequences of the resulting plasmids were experimentally confirmed .All plasmids were constructed using standard molecular cloning techniques. DNAs were amplified by the polymerase chain reaction (PCR) using the Phusion High-Fidelity DNA polymerase (Thermo Scientific). DNAs were digested with the type-IIS restriction enzyme 21 of E. coli were electroporated or co-electroporated  with the different plasmids and recombinant clones selected at 37 °C in plates of LB solid medium  that included the appropriate antibiotics . E. coli was grown in liquid cultures in LB or TB medium , containing the appropriate antibiotics (see above), at the indicated temperature  with vigorous shaking (225 revolutions per min –rpm–). Cell densities were measured at 600 nm with a colorimeter . When needed, induction of eggplant tRNA ligase expression was carried out by adding the appropriate amount of 0.25 M IPTG to the culture.The strains BL21(DE3) (Novagen) and HT115(DE3)At the desired time points, 2-ml aliquots of the liquid cultures were taken and cells were sedimented by centrifuging at 13,000 rpm for 2 min. In general, cells were resuspended in 50 µl of TE buffer  by vortexing (40-fold concentration). One volume (50 µl) of a 1:1 (v/v) mix of phenol  and chloroform was added and the cells broken by vigorous vortexing. The aqueous and organic phases were separated by centrifugation for 5 min at 13,000 rpm. The aqueous phases were recovered and re-extracted with one volume (50 µl) of chloroform. The aqueous phases containing total bacterial nucleic acids were finally recovered by pipetting and either subjected directly to further analyses or stored frozen at −20 °C.E. coli was analyzed by denaturing PAGE. In general, 20 µl of RNA preparations were mixed with 1 volume (20 µl) of loading buffer , heated for 1.5 min at 95 °C and snap cooled on ice. Electrophoresis was run for 2 h and 30 min at 200 V in 5% polyacrylamide gels  of 140 × 130 × 2 mm in TBE buffer  including 8 M urea. Electrophoresis buffer was TBE without urea. Gels were stained by shaking for 15 min in 200 ml of 1 µg/ml ethidium bromide. After washing three times with water, gels were photographed under UV light . Image analyses were performed using the UVIDoc 1D software (UVITEC Cambridge). In some experiments . Hybridization was performed overnight at 70 °C in 50% formamide, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 100 ng/ml salmon sperm DNA, 1% sodium dodecyl sulfate –SDS–, 0.75 M NaCl, 75 mM sodium citrate, pH 7.0, with approximately 1 million counts per minute of 32P-labelled ELVd RNA of complementary polarity. Hybridized membranes were washed three times for 10 min with 2 × SSC, 0.1% SDS at room temperature and once for 15 min at 55 °C with 0.1 × SSC, 0.1% SDS . Results were registered by autoradiography using X-ray films (Fujifilm). The radioactive probes were produced by in vitro transcription of a linearized plasmids containing a dimeric ELVd (sequence variant AJ536613) cDNA in the proper orientation. One µg of linearized plasmid (Hind III or Xba I) was transcribed with 50 U of T3 bacteriophage RNA polymerase (Epicentre) in a 20-µl reaction containing 40 mM Tris-HCl, pH 8.0, 6 mM MgCl2, 20 mM DTT, 2 mM spermidine, 0.5 mM each of ATP, CTP, and GTP, and 50 μCi of [α-32P]UTP (800 Ci/mmol), 20 U RNase inhibitor  and 0.1 U yeast inorganic pyrophosphatase (Thermo Scientific). Reactions were incubated for 1 h at 37 °C. After transcription, the DNA template was digested with 20 U DNase I (Thermo Scientific) for 10 min at 37 °C, and the probes were purified by chromatography using a Sephadex G-50 column .For northern blot hybridization analysis, RNAs separated by electrophoresis were electroblotted to positively charged nylon membranes  and cross-linked by irradiation with 1.2 J/cmE. coli culture in TB medium distributed in four 1 l baffled Erlenmeyer flasks was grown at 37 °C with intense shaking (180 rpm). Protein expression was induced at an OD600 of 0.1 by adding IPTG to 0.1 mM. Cells were recovered at 14 h post-induction by centrifugation at 10,000 rpm for 10 min. Sedimented cells were resuspended in water and pelleted again in the same conditions. Cells were resuspended in 50 ml of chromatography buffer  and RNA extracted by adding 50 ml phenol:chloroform  and vortexing. The aqueous phase, recovered after centrifugation for 10 min at 10,000 rpm, was re-extracted with 50 ml of chloroform. RNA in the second aqueous phase was filtered  and chromatographed through a DEAE Sepharose column  of 5 ml at 5 ml/min using an ÄKTA Prime Plus liquid chromatography system . The column was equilibrated with 50 ml of chromatography buffer and the sample (35 ml at this point) loaded. The column was washed with 50 ml of chromatography buffer and the RNA eluted with 100 ml of chromatography buffer plus 1 M NaCl. Fractions (5 ml) were collected during elution and analyzed by denaturing PAGE. Chromatography fraction 2, corresponding to the RNA elution peak, was mixed with 1 volume of formamide loading buffer and the RNA denatured (see above). RNA was separated by two consecutive electrophoreses. First by PAGE in a 5% polyacrylamide, 8 M urea, TBE gel, as described above. After staining with ethidium bromide, the band of the gel containing the monomeric circular ELVd RNA was cut and loaded on top of a second non-denaturing gel cystamine–, TAE −40 mM Tris, 20 mM sodium acetate, 1 mM EDTA, pH 7.2–). After staining, the band of gel containing the monomeric circular ELVd RNA was cut and solubilized by adding 0.1 volumes of 2-mercaptoethanol. RNA was purified from the solution by chromatography with a DEAE Sepharose column as explained above.For preparative purposes  with UV illumination and a GFP2 filter (Leica). In the case of the RNA extracts, DFHBI was directly added to 20 µM and photographed under the same conditions.To assay the fluorescence of RNA aptamer Spinach, aliquots of the Supplementary Information


Structured RNA elements may control virus replication, transcription and translation, and their distinct features are being exploited by novel antiviral strategies. Viral RNA elements continue to be discovered using combinations of experimental and computational analyses. However, the wealth of sequence data, notably from deep viral RNA sequencing, viromes, and metagenomes, necessitates computational approaches being used as an essential discovery tool. In this review, we describe practical approaches being used to discover functional RNA elements in viral genomes. In addition to success stories in new and emerging viruses, these approaches have revealed some surprising new features of well-studied viruses e.g., human immunodeficiency virus, hepatitis C virus, influenza, and dengue viruses. Some notable discoveries were facilitated by new comparative analyses of diverse viral genome alignments. Importantly, comparative approaches for finding RNA elements embedded in coding and non-coding regions differ. With the exponential growth of computer power we have progressed from stem-loop prediction on single sequences to cutting edge 3D prediction, and from command line to user friendly web interfaces. Despite these advances, many powerful, user friendly prediction tools and resources are underutilized by the virology community. This review illustrates the key concepts and strategies used for prediction of RNA structural elements in RNA viral sequences. A range of RNA structure prediction software and relevant resources are available, but most are underutilized by virologists. Here the concepts and strength of these methods are introduced using examples of successful approaches in viruses, with the intention of bridging the gap. The roles of RNA elements in viral biology is illustrated using well-studied viruses, flaviviruses, influenza, and barley yellow dwarf virus (BYDV). We further review the structures and functions of well-characterized types of RNA elements with the emphasis on prediction approaches and their limitations.There have been several excellent recent reviews on generally predicting RNA structures, in particular relating to integrating experimental data and on 3D predictions . Stockholm format also stores some metadata e.g., the description of the aligned RNA sequences. R2R uses Stockholm file as the input to generate a novel stem-loop diagram of the consensus RNA secondary structure annotated with sequence conservation and covarying base-pairs . However, many RNA structure prediction tools and RNA-RNA interaction prediction tools have been compared for use in different applications . Notably, these are beginning to include newer webservers which predict RNA 2D and 3D structures with high confidence.Many of these powerful applications have been underused by virologists. For example, Infernal  RNA genome Figure . This coViral RNA elements have been identified as antiviral targets due to conservation of sequence and functions that are distinguishable from the host   can be predicted, in at least the three forward reading frames. This is an important step prior to prediction of RNA structures located in the coding sequence  and/or contain RNA structures, alignment algorithms that consider this should be used .An example of useful resource that is not frequently cited by virus research articles is Rfam, the database of RNA structure families  contains 252 virus records (accessed in June 2017). IRESite contains 44 viral IRES entries  by Olsthoorn and Bol  using mfMore importantly, many independent experiments have successfully uncovered their complex roles which have clinical implications. For example, deletion of the dengue virus 5′ dumbbell structure attenuates the virus, generating vaccine candidates that have been used for clinical testing . This frameshifting was first discovered in a retrovirus, Rous sarcoma virus . This has a consensus sequence of “X XXY YYZ,” the zero frame codons are separated by spaces, X is an identical nucleotide, Y is either A or U, and Z is not G  are formed from the base-pairing between the loop of two stem-loops Table . Many kiThe first viral kissing hairpins were discovered in enteroviruses (plus strand viruses), namely poliovirus and coxsackievirus B3 . This structure is involved in dimerization of virus genomic RNAs, which is a critical step in retroviral replication , and in viral structures may also contain additional pseudoknots Figure , Table 1Many positive strand plant viruses have such tRNA-like structures in the 3′UTRs of genomic RNAs Dreher, . Most viThe elements considered above form mainly local structures. These local structures may form in nascent RNA Meyer,  or be stLong-range interactions from over a few hundred bases to >26 kb do occur in RNA viruses, but are difficult to predict accurately. Challenges include: there are many possible interactions; likely complex structures ; structures will form co-transcriptionally limiting interactions; and small molecules, proteins, RNAs and complexes  will bind and affect folding  will tend to predict large structures with a large number of long-distance interactions—which should be viewed with caution. Indeed, experimentally determined structures of full length genomes show more local than long-range interactions e.g., HIV RNA has many local structures , may require long-range interactions , co-packaging elements, or interactions during translation with the rRNA in the ribosome . The sORF(s) precede the large RNA structure is involved, whereas the following upstream AUGs are folded up in a large RNA structure. This RNA structure has a stable base-pairing at the stem base. The shunt take-off site (sequence around the sORF termination codon) and landing site are expected to be conserved between closely related viruses or co-evolved viruses. For example, the shunt cis-elements are remarkably similar between a pair of co-evolved viruses, a RNA picorna-like virus, rice tungro spherical virus and a DNA pararetrovirus, rice tungro bacilliform virus , or programmed ribosomal frameshifting  and a stop/restart site. TURBS consists of a motif 1 (18S rRNA complementary site), and motif 2 and 2* (likely base-pairing and structured). Motif 1 is loosely structural to allow tethering of small ribosomal subunit for reinitiation whereas motif 2 and 2* could form an RNA structure that enhances translation of the second ORF . In addition, specialized workshops and training may facilitate the use of these RNA tools e.g., The EMBO Practical Course on Computational RNA Biology course material available online (https://bibiserv.cebitec.uni-bielefeld.de/EMBO-RNACourse/).However, some challenges remain for the application of newer RNA structure tools in virology Table . Some ofBoth authors have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs  in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells. Many aptamer-fluorophore complexes suffer from low quantum yield and low extinction coefficients limiting their usability. Here the authors isolate new Mango aptamers with improved fluorescent properties for both fixed and live-cell imaging by using competitive ligand binding microfluidic selection. However, developing high contrast aptamer-fluorophore systems with brightness comparable to existing fluorescent proteins has posed a significant experimental challenge. In an ideal system, unbound fluorophores with high extinction coefficients and low quantum yields become highly fluorescent when bound by an RNA aptamer whose tertiary structure correctly positions the fluorophore into an orientation that maximizes its brightness7. While reported aptamer-fluorophore complexes make use of fluorophores with high extinction coefficients, notably RNA Mango8 and the cytotoxic Malachite Green binding aptamer5, these systems suffer from low quantum yields. Conversely, systems with high quantum yields such as the GFP-mimic aptamers10 have intrinsically low extinction coefficients. As a consequence, such complexes are all significantly less bright than enhanced GFP11, diminishing their utilitySince their introduction, fluorogenic RNA aptamers that enhance the fluorescence of an unbound fluorophore have sparked significant interest and hold great potential to enable the visualization of RNA molecules within a cell12. Despite the inability to simultaneously optimize aptamer-fluorophore brightness and binding affinity, existing fluorogenic systems have achieved some notable successes in bacteria, yeast and mammalian cells15. This suggests that using newly developed screening methodologies to select brighter fluorogenic RNA aptamers either by FACS9 or droplet-based microfluidics platforms10 can provide powerful and easy to use fluorescent RNA imaging tags to study cellular RNAs.High-affinity aptamers, with the notable exception of RNA Mango, have also been difficult to develop. While not important for a perfect fluorophore with zero unbound quantum yield, high-affinity fluorophore aptamer complexes allow lower free fluorophore concentrations to be used during imaging, effectively decreasing background fluorescent signal8. These aptamers all contain a closing RNA stem, which isolates a small fluorophore-binding core from external sequence, making them easy to insert into arbitrary biological RNA. Unexpectedly several of these constructs are resistant to formaldehyde, allowing their use in live-cell imaging and also in conventional fixed cell methodologies. Stepwise photobleaching in fixed cell images indicate that as few as 4–17  molecules can be detected in each foci, and photobleaching rates in live cells or in vitro were at least an order of magnitude slower than found for Broccoli. These new aptamers work well with existing fluorescence microscopy techniques and we demonstrate their applicability by imaging the correct localization of 5S, U6 and the box C/D scaRNA (mgU2-47) in fixed and live mammalian cells. Together, these findings indicate that the new Mango aptamers offer an interesting alternative to existing fluorogenic aptamers12.Here, we have used a competitive ligand binding microfluidic selection to isolate three new aptamers  with markedly improved fluorescent properties, binding affinities, and salt dependencies compared to the original Mango I aptamer8. Its structure consists of a three-tiered G-quadruplex with mixed parallel and anti-parallel connectivity  using microfluidic-assisted in vitro compartmentalisation  binding affinity rather than for its fluorescent properties, which may have precluded the enrichment of the brightest aptamers in the libraryo I Fig. .Fig. 1RNKD (~3 nM) and could potentially prevent the selection of high-affinity aptamers. To mitigate this, we supplemented the in vitro transcription (IVT) mixture with TO1-B competitors NMM (N-methyl mesoporphyrin IX)18 and TO3-Biotin8  during the screening. Such high values would greatly exceed the Mango I In a second set of screenings, we increased the selection stringency by using the Mango I specific competitor TO3-Biotin, which differs from TO1-B by having two additional carbons in the methine bridge of TO1-B  a common benchmark for the characterization of improved fluorescent proteins11.Based on the parental sequence isolates R2-1, R5-NMM-20 R10-17), and R5-NMM-5 , lacked N-7 protection of residue G15, but like Mango II has strong DMS protection of G17 after A16 , whereas Mango I fluorescence was strongly inhibited at such high concentrations . This protocol is based on the observation that, in vitro, Mango I, II and IV-fluorophore complexes are substantially resistant to formaldehyde at room temperature  is expected to associate with snRNP (ribonuclear protein) complexes in the nucleus. We tagged U6 snRNA by incorporating Mango IV directly into an internal stem loop  that mediates the 2′-O-methylation of the U2 snRNARNA Fig. . The nucRNA Fig. . Taken t30.To be of broad utility, fluorescent RNA aptamers should be bright, bind their ligands with high affinity, and be compatible with existing methodologies to image proteins in live and fixed cells. To achieve this, we have developed a novel, competition-based, ultrahigh-throughput fluorescent screening approach that takes advantage of microfluidic-assisted in vitro compartmentalization to select three new and highly effective RNA Mango fluorogenic aptamers. The broad range of novel photophysical and biochemical properties in the new Mango aptamers promises to make them highly competitive with existing aptamer-fluorophore systems31. Mango III and IV, when bound to TO1-B, have quantum yields of ~0.56 and 0.42, respectively. Such high quantum yields are comparable or exceed that of TO1-activating proteins32. When combined with the high absorbance of thiazole orange, these quantum yields result in fluorescence as bright or brighter than EGFP, a significant milestone for aptamer-fluorophore systems.The new Mango aptamers when bound to TO1-B are very bright. The original Mango I construct when bound to TO1-B exhibits a quantum yield of ~0.14. This quantum yield is similar to that typically observed when thiazole orange is rigidified by intercalation into a duplex nucleic acid29, the high brightness of the Mango aptamers combined with their extremely high binding affinity toward TO1-B and the very low unbound fluorescence of TO1-B strongly enables high contrast imaging. The nanomolar binding affinity of these aptamers allows very low concentrations of fluorophore ligand to be used . Indeed, transfecting RNA bound to stoichiometric amounts of TO1-B resulted in fluorescent RNA foci that could be tracked in cells, implicating that fluorophore-bound RNA Mango complexes are both stable and robustly fluorescent in living cells, even when at very low effective concentrations. Likewise, staining fixed cells with low concentrations of TO1-B (200 nM) readily generated Mango-tagged fluorescence with only modest levels of nonspecific nucleolar staining being observed, strongly suggesting that nonspecific fluorescence induced by intercalation of TO1-B into nonspecific nucleic acids can be effectively controlled using the Mango system. As modifying thiazole orange to TO1-B considerably destabilizes the weak intercalation of thiazole orange into dsDNA31 and RNA17, further optimization of the imaging approaches presented here appears likely to enable higher contrast RNA Mango imaging in the future.While a brighter aptamer system has been recently discovered33. Furthermore, pulsed illumination dramatically enhances the imaging time possible with RNA Mango, opening up the interesting possibility to track biological RNAs for 10 min or longer. Just as critical, our results with three small non-coding RNAs demonstrate that Mango tags can be incorporated either into non-essential stems or as 3′-tags, without significantly interfering with RNA subcellular localization and we do not believe a folding scaffold is required for the Mango system as evidenced by the success of the U6 and scaRNA constructs. Indeed, using either genetically encoded Mango-tagged RNA or direct RNA transfections correctly recapitulates the expected localization patterns.Equally important for RNA imaging is the photostability and biological compatibility of the Mango system. The RNA Mango aptamers are at least an order of magnitude more photostable than the Broccoli systems and comparable to the recently published Corn aptamer, albeit Corn’s requirement for dimerization seems less compatible with its simple biological utilization8 strongly suggests, at least for highly expressed biological RNAs, that the RNA Mango system can be used to enable RNA-based FACs experiments. Third, the high affinity of Mango I-based tags to TO1-desthiobiotin has been used to recover native RNP complexes from streptavidin beads34. Such pulldowns should be readily applicable with our new Mango constructs potentially enabling a unique combination of RNA imaging and RNP pulldown experiments via the insertion of a single Mango tag into a biological RNA of interest. Finally and while not utilized here, the broad salt tolerance of the new Mango aptamers in contrast to that of Mango I and other fluorophore aptamer systems, makes the Mango system compatible with a range of enzymatic reactions. This implies that in addition to the in-cell demonstrations given here, in the future, in vitro fluorescent applications can be developed that make use of the Mango systems high brightness and fluorophore-binding properties.There are many other applications of the Mango systems that appear likely based on the established biochemistry and photophysics of these aptamer complexes. First, as we have demonstrated, via the observation of quantized photobleaching of RNA foci, as few as four RNA Mango can be robustly imaged within one puncta. In the future, tagging biological RNAs with a small number of Mango repeats could, therefore, enable robust single-molecule RNA imaging. Second, our ability to sort the latest Mango constructs with microfluidics, together with our observation of Mango I dependent fluorescence in bacteria via FACs16, combined with their unusual ability to withstand formaldehyde fixation all promise to be very useful for investigating biological systems in the future.While the full biological compatibility of the Mango aptamers is still not completely explored, their small size relative to other fluorogenic RNA aptamers, their ability to fold correctly into monomers at physiological temperaturesHigh-throughput screening proceeds in three major stages:35 to have a final average number of DNA molecule per droplet (λ) of ~0.2–1  carried by HFE 7500 fluorinated oil (3 M) supplemented with 3% of a fluorosurfactant35. Droplet production frequency was monitored and used to determine droplet volume by adjusting pumps flow rates . Emulsions were collected in 0.2 µL tubes as described35 and subjected to an initial denaturation step of 2 min at 95 °C followed by 30 PCR cycles of: 30 s at 95 °C, 30 s at 55 °C, 1 min 30 s at 72 °C.(i) Digital droplet PCR: DNA libraries were diluted in 200 µg/mL yeast total RNA solution (Ambion) as described before35 at a rate of ~1500 droplets per s, spaced by a stream of HFE 7500 fluorinated oil supplemented with 2% fluorosurfactant. Each PCR droplet was synchronized with a 16 pL IVT droplet containing 2.2 mM of each NTP (Larova), 24 mM MgCl2, 44 mM Tris-HCl pH 8.0, 50 mM KCl, 5 mM DTT, 1 mM Spermidine, 35 µg/mL of Dextran-Texas Red 70 kDa (Molecular Probes), 0.1% Pluronic F68, 3500 U T7 RNA polymerase , 100 nM TO1-B8, 5 ng/µL inorganic pyrophosphatase (Roche) supplemented with the desired concentration of NMM. For the screenings performed in the presence of TO3-Biotin8, the T7 RNA polymerase (New England Biolabs) concentration we reduced to 70 U per reaction. The IVT mixture was loaded in a length of PTFE tubing  that was kept on ice during all the experiment. IVT droplets of 16 pL were produced at a rate of ~1500 droplets per s and paired to one PCR droplet. Pairwise droplets were then fused by electrocoalescence while passing between a pair of electrodes subjected to an AC electric field of 400 V (30 kHz) via high-voltage amplifier 10. The resulting emulsion was collected off-chip and incubated for 120 min  or 30 min  at 37 °C.(ii) Droplet fusion: PCR droplets were then injected into a fusion device10. Droplets were re-injected at a frequency of ~200 droplets per s, spaced with a stream of surfactant-free HFE 7500 fluorinated oil. The green fluorescence (TO1-B in complex with the aptamer) of each droplet was analysed. Between 1 and 2% green fluorescence droplets were gated for each round of selection. The gated droplets were deflected into a collecting channel by applying a 1 ms AC field  and were collected into a 1.5 mL tube. Collected droplets were recovered by flushing 200 µL of surfactant-free HFE 7500 fluorinated oil (3 M) through the tubing. 1H, 1H, 2H, 2H-perfluoro-1-octanol of 100 µL (Sigma-Aldrich) and 200 µL of 200 µg/mL yeast total RNA solution (Ambion) were then added, the droplets broken by vortexing the mixture and DNA-containing aqueous phase was recovered.(iii) Droplet analysis and sorting: the emulsion was finally re-injected into an analysis and sorting microfluidic device mounted on a Thermo plate (Tokai Hit) holding the temperature at 45 °C as previously describedA PCR mixture supplemented with DNA coding for RNA Mango  was emulsified in 2.5 pL droplets and the DNA amplified as above. The droplets were paired and fused with droplets of IVT mixture containing either a low (70 U of enzyme from New England Biolabs) or a high (20 µg/mL of enzyme purified in the lab) concentration of T7 RNA polymerase and the resulting emulsions were incubated for respectively 30 min or 120 min at 37 °C. After incubation, the RNA-containing phase was recovered using 1H, 1H, 2H, 2H-perfluoro-1-octanol (Sigma-Aldrich) and the transcription was stopped by a phenol extraction followed by an ethanol precipitation in the presence of 300 mM sodium acetate pH 5.5 (Sigma-Aldrich). After centrifugation and a wash in 70% ethanol, the pellets were re-suspended in water. Baseline-Zero™ DNase of 10 U (Epicentre) and the corresponding buffer were added and a second incubation of 60 min at 37 °C was performed. The DNase was removed by phenol extraction and RNA recovered by ethanol precipitation.Recovered RNAs were reverse-transcribed for 60 min at 55 °C, followed by 5 min at 95 °C, in a mixture containing 1 µM of reverse primer, 0.5 mM of each dNTP, 8 U/µL RT Maxima (Thermo Scientific) and the supplied buffer according to recommended concentrations. The complementary DNA (cDNA) was amplified using SsoFast™ Evagreen supermix (Bio-Rad) supplemented with 0.2 µM of each primer (forward and reverse) using a CFX96 Touch™ real-time PCR detection system (Bio-Rad). Finally, the cDNA was quantified using the calibration curve obtained with reactions performed with purified Mango II RNA.2, 40 mM Tris-HCl pH 8.0, 5 mM DTT, 1 mM Spermidine and 70 µg/mL T7 RNA polymerase. After 4 h of incubation at 37 °C, 10 U of Baseline-Zero™ DNase (Epicentre) and the corresponding buffer were added and the mixture was incubated for 60 min at 37 °C. RNAs were recovered by phenol extraction followed by an ethanol precipitation in the presence of 300 mM sodium acetate pH 5.5 (Sigma-Aldrich). After centrifugation and a wash in 70% ethanol, the pellets were dissolved in denaturing loading buffer  and the solution loaded onto a 12% denaturing 8 M urea polyacrylamide gel. The piece of gel containing RNA was identified by ultraviolet (UV) shadowing, sliced from the gel and transferred into dialysis tubing (molecular weight cut-off (MWCO) = 3500, Spectrum Lab) filled with TBE. RNA was electro-eluted by placing the montage in TBE for 60 min at 100 V. Eluted RNA were filtered in centrifuge tube  and ethanol precipitated in the presence of 300 mM sodium acetate pH 5.5. After centrifugation and a wash in 70% ethanol, the pellets were dissolved in DEPC-Treated water and quantified with Nanodrop (Thermo Scientific).The pool molecules contained in 2 µL recovered from the sorted fractions were introduced into 100 µL of PCR mixture containing 0.1 µM of each primer (fwd and rev), 0.2 mM of each dNTP, 0.05 U/µL of DreamTaq™ and its corresponding buffer (Fermentas). The mixture was then subjected to an initial denaturation step of 30 s at 95 °C, followed by 20 cycles of: 5 s at 95 °C and 30 s at 60 °C. 20 µL of PCR products were then in vitro-transcribed in 250 µL of mixture containing 2 mM of each NTP, 25 mM MgCl2 and TO1-B fluorescence (ex. 492 nm/em. 516 nm) was measured. In the case of NMM resistance measurement, the mixture was further supplemented with 3 µM NMM. In the case of TO3-Biotin screenings, 300 nM of RNA and 100 nM of TO1-B were used with or without 110 nM of TO3-Biotin. Both green (ex. 492 nm/em. 516 nm) and red (ex. 635 nm/em. 665 nm) fluorescence were measured.In the case of NMM screenings, 2 µM of RNA were incubated with 100 nM of TO1-B in 40 mM Tris-HCl pH 8.0, 50 mM KCl, and 22 mM MgClGenes contained in the libraries were diluted in a PCR mixture as immediately above and thermocycled 25 times using a final extension step of 10 min at 72 °C. PCR products were inserted in pTZ57R/T vector following manufacturer’s instruction . Ligation products were recovered by phenol/chloroform extraction and ~100 ng of DNA used to transform Electro-10 blue bacteria (Agilent) placed in a 2 mm electroporation . After an hour of recovery at 37 °C under agitation, bacteria were plated on Luria broth (LB)-ampicillin agar plate and incubated overnight at 37 °C. The colonies were picked, used to inoculate liquid LB and grown at 37 °C until saturation. Plasmids DNA were extracted using ‘GeneJet Plasmid Miniprep kit’ (Thermo Scientific), and sequences determined by Sanger approach (GATC Biotech).2, 40 mM Tris-HCl pH 8.0, 50 mM KCl, 5 mM DTT, 1 mM Spermidine, 70 µg/mL T7 RNA polymerase and 100 nM TO1-B. The mix was then split in two and one aliquot was supplemented with 3 µM of NMM. The reaction was incubated in a real-time thermocycler  for 2 h at 37 °C and the green fluorescence (ex. 492 nm/em. 516 nm) measured every minute.Single colonies were introduced in 10 µL of a PCR mixture identical to that used for TA cloning and the DNA amplified as above. Two microlitres of PCR product added to 18 µL of IVT mixture containing 2 mM of each NTP, 25 mM MgCl36. RNA of 50 nM was 3′-end-labelled with 32P pCp and gel-purified. The resulting RNA was incubated in 50 mM HEPES pH 7.5 (volume 50 µL) at room temperature for 30 min. After incubation, 10 µg carrier RNA was added. The sample was then heated to 90 °C for 3 min before the addition of 0.5 µL of 25% DMS (diluted in ethanol) and heated to 80 °C for 1 min, 150 µL ice cold ethanol + 5 µL 3 M NaCl was then immediately added and the sample moved to −20 °C for 30 min. DMS-modified RNA was pelleted by centrifuge at 16,300 RCF at 4 °C for 20 min.DMS probing consists of four main steps: (i) DMS (denaturing): protocol is adapted from Lorsch and Szostak2, 140 mM either KCl or NaCl, with or without 500 nM TO1-B  at room temperature for 30 min. After incubation, 10 µg carrier RNA was added. The sample was then incubated at room temperature for 15 min after the addition of 0.5 µL of 100% DMS. Ice cold ethanol of 150 µL + 5 µL 3 M NaCl was then immediately added and pelleted as for the denaturing DMS protocol.(ii) DMS (native): 50 nM 3′-end-labelled RNA was incubated in 50 mM HEPES pH 7.5, 1 mM MgCl(iii) Reduction: pellets were re-suspended in 10 µL 1 M Tris buffer pH 8 and 10 µL of freshly prepared 0.2 M sodium borohydride was added. Reaction was carried out on ice and in the dark for 30 min. Reactions were stopped by ethanol precipitation as above.2O, 3 parts glacial acetic acid) were added and incubated at 60 °C for 15 min in the dark. Samples were flash-frozen by placing tubes in liquid nitrogen and lyophilized by speed vacuum centrifuge. Once dry, 20 µL ddH2O was added, the sample was refrozen and lyophilized once again. The pellet was re-suspended in a 50% formamide denaturing solution before being loaded on a 15% polyacrylamide gel (19:1 acrylamide:bis).(iv) Aniline cleavage: to the resulting pellet, 20 µL  at 50 °C for 10 min. Samples was flash-frozen in liquid nitrogen for 5 min, heat denatured in denaturing solution at 95 °C for 5 min prior to gel loading. Hydrolysis ladders were generated by incubating in 50 mM NaHCO260, where extinction coefficients were estimated based on an average 11,000 M−1 cm−1 per base.To identify the minimal functional motif of each Mango, truncated constructs were designed as shown in Supplementary Fig. 2, 10 mM NaH2PO4 pH 7.2, 0.05% Tween-20) to determine binding affinities. Fluorescence was measured at the maximum excitation and emission wavelengths of each complex , F0 is the unbound fluorescence and Fmax the maximal complex fluorescence, respectively. When F0 was undetectable, it was set to zero.Fluorescence data were gathered using a Varian Cary Eclipse Spectrophotometer unless otherwise stated. Fluorescent titrations were measured in a buffer mimicking cellular conditions .RNA Mango aptamers were incubated with TO1-B in WB buffer for at least 1 h until equilibrium fluorescence was reached. Formaldehyde was then added such that final concentrations after dilution were 50 nM RNA, 100 nM TO1-B, and 0, 2, 4 or 8% formaldehyde. Fluorescence was measured as a kinetic run at a rate of 2 readings per min using a Varian Cary Eclipse Fluorescence Spectrophotometer, ex/em = 510 ± 2.5/535 ± 5 nm.® CRL-3216™) were grown in Dulbecco Modified Eagle’s Medium containing 10% foetal bovine serum, 2 mM d-Glucose, 2 mM l-Glutamine, 1 mM sodium pyruvate and 100 U/mL penicillin/streptomycin (Thermo Fisher) and maintained at 37 °C with 5% CO2 in a humidified incubator. Cells used for imaging were cultured in Ibidi glass-bottomed eight-well chamber slides (Ibidi GmbH).HEK293T cells . RNA was purified from an 8 M urea, 12% denaturing polyacrylamide gel using 29:1 acylamide:bis solution (Fisher Scientific). The RNA was excised and eluted in RNA elution buffer  followed by ethanol precipitation. Fluorescence measurements were taken for each of the RNA constructs using a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent) containing 40 nM TO1-B, 200 nM RNA, 10 mM sodium phosphate, 100 mM KCl and 1 mM MgCl2 at pH 7.2. Similar measurements were also taken with a limiting amount of RNA (40 nM) in an excess of TO1-B (200 nM) and the results showed a similar trend. U6-Mango/control RNA was synthesized by the PCR amplification of a 5′ T7 sequence to each construct followed by IVT and purification as described above.DNA encoding the F30 folding scaffold2 was incubated at room temperature with a 1:1 dilution in OptiMEM prior to transfection. The RNA transfected was incubated at 37 °C for 1 h in complete growth medium. FuGene 6 was used to transfect 400 ng of the pSLQ--Mango and control plasmids directly in the eight-well chamber slides following the manufacturer’s instructions. Plasmids were left to express between 12–48 h before fixation as described below.RNA was transfected directly into eight-well chamber slides using the lipofectamine-based CRISPRMAX reagent following the manufacturers guidelines (Invitrogen). A final concentration of 62.5 nM RNA in 10 mM sodium phosphate buffer (pH 7.2), 100 mM KCl, and 1 mM MgCl2) followed by a 10 min incubation in 200 nM TO1-B diluted in PKM buffer before replacing with imaging media . For immunostaining, cells were first blocked (2% BSA in PBS) for 30 min followed by primary antibody (1:50 – 1:500 dilutions) incubation for 120 min in blocking solution.Cells were fixed in PBS containing 4% paraformaldehyde for 10 min on ice followed by permeabilization in 0.2% Triton X-100 for 10 min at room temperature. Cells not requiring immunostaining were washed three times for 5 min each with PKM buffer , anti-ribosomal protein L7 , anti-ATP5B , anti-GW182 , anti-EEA-1 , anti-LSm3 , anti-TIAR . Secondary antibodies used were donkey anti-mouse and donkey anti-rabbit Alexa Fluor 680 (Molecular Probes). Primary antibodies were washed three times for 20 min each in blocking solution followed by incubation with secondary antibody at 1:500 dilution for 60 min, which was subsequently washed as above. After immunostaining the cells were washed and stained in TO1-B and Hoechst 33258 as described previously.Z-stacks and time series experiments containing more than one colour were acquired by alternating between each channel for an individual frame, leading to recovery and minimal loss of the Mango signal throughout the acquisition. To visualize the nuclear boundary in live cells, a plasmid expressing a fluorescently tagged histone protein  was transfected using FuGene 6 (Promega) 24 h prior to RNA transfection. RNA was transfected directly into eight-well chamber slides (Ibidi GmbH) as described above, with an additional pre-incubation step with 125 nM of TO1-B prior to the addition of the CRISPRMAX transfection reagent. Upon addition to the imaging chambers, the final effective concentrations of RNA and fluorophore were 10 and 20 nM, respectively. Following incubation of the RNA transfection, the cells were washed once with PBS and replaced with live-cell imaging media . Live cells were maintained at 37 °C with 5% CO2 in a stage top incubator (Tokai Hit).Live and fixed cell images were taken directly in the eight-well chamber slides using a Zeiss Elyra wide-field microscope by exciting at 405 nm (blue), 488 nm (green), 561 nm (red) and 642 nm (far-red) and detecting emission at 420–480 nm, 495-550 nm, 570–640 nm and >650 nm, respectively. Image acquisition for the Mango signal (488 nm laser) used 5 mW of power and 200 ms exposure time, except in the photobleaching-assisted microscopy experiments where 50 ms was used. Due to the observation that the Mango signal is stabilized under pulsed illumination in both fixed and live cells, 20. Live-cell photobleaching was conducted with a 200 ms frame rate and 5% wide-field laser illumination at 488 nm for 150 frames. To determine the half-life of fluorescence, each photobleaching curve was fit to an exponential decay function. In order to obtain the appropriate signal to noise ratio and time resolution for the analysis of single-step photobleaching, 5S-F30-Mango IV foci were imaged in fixed cells with a 50 ms frame rate, 5% wide-field laser illumination at 488 nm for 800 frames. Maximum likelihood estimation was used to determine each of the photobleaching steps within a trace as previously described27. The step sizes were subsequently binned and the histogram was fit to a double Gaussian equation.To image the photobleaching of 5S-F30-Mango IV foci as compared with dBroccoli in live cells, 5S-F30-Mango I and IV were directly transfected as described above, whereas 5S-F30-dBroccoli was expressed from pAV5S as previously describedy axis) and the AlexaFluor 680 channel (x axis) was plotted. To append a Z-axis density of pixels, Igor Pro was used to carry out a bivariate histogram upon corresponding pixels for both channels and displayed as a heatmap.Images were processed using FIJI and spot detection analysis was performed on each maximum projection by the spot detector plugin in the ICY image analysis software, which detects significant foci with a pixel area ≥3 × 3 pixels and intensity ≥300 a.u. A lower threshold of ≥150 a.u. was used to create a population of the apparent background intensities in the control RNA transfections Fig. . To crea2, and varying pH  for 1 h at room temperature. Mango fluorescence was measured with excitation and emission at 485 nm and 535 nm, respectively.Mango of 50 nM was incubated with 100 nM TO1-B in the presence of 140 mM KCl, 1 mM MgCl2, 20 mM NaH2PO4 pH 7.5) supplemented with 3.6 µM of fluorogenic dye . The mixture was incubated for an hour at room temperature prior to being loaded into a length of PTFE tubing (Thermo Fisher) and infused into a droplet generator microfluidic device where it was dispersed into 100 pL droplets carried by HFE 7500 fluorinated oil (3 M) supplemented with 3% of a fluorosufactant as described previously37. The resulting emulsion was then loaded into 5 µL capillary (Corning) and the montage was imaged on an epifluorescence microscope . Depending on the dye, the emulsion was exposed to a constant illumination wavelength of 470 nm (DFHBI-1T) or 508 nm (TO1-B) at the maximum intensity of the light source , and the emitted fluorescence  was collected by an Orca-Flash IV camera for 200 ms every 100 ms with ×20 objective  0.45). Fluorescence intensity of each picture was then extracted using NiS software (Nikon) and the data were fit to an exponential decay equation to compute the fluorescence half-life.5S-F30-Mango I, II, III and IV as well as 5S-F30-Broccoli template regions were placed under the control of T7 RNA polymerase promoter. Genes were PCR-amplified, in vitro-transcribed, purified and quantified as before (see Enrichment measurement section). Then 1 volume of 3 µM RNA solution was added to 1 volume of 2-times concentrated buffer (280 mM KCl, 2 mM MgClThe data that support the findings of this study are available from the corresponding authors upon reasonable request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


N6-methylation of internal adenosine nucleotides has also been shown to impact flavivirus infection. Here, I summarize recent progress made in understanding roles for methylation in the flavivirus life-cycle and discuss relevant emerging hypotheses.Mosquito-borne flaviviruses are important human pathogens that represent global threats to human health. The genomes of these positive-strand RNA viruses have been shown to be substrates of both viral and cellular methyltransferases. N The genomes of viruses within this family share a similar organization: each contains a single open reading frame flanked by untranslated regions (UTRs) of various sequence, length and structure. The viral UTRs contain functional RNA elements that control viral translation and RNA synthesis , West Nile virus (WNV), Zika virus (ZIKV), Japanese encephalitis virus (JEV) and the four serotypes of dengue viruses (DENV) which are the most prevalent, causing nearly 100 million symptomatic infections world-wide . These vynthesis . Unique ynthesis .Flaviviridae use a fundamental infection strategy: (i) virus particles attach to various cellular receptors and are internalized via endocytosis, (ii) endosome acidification causes fusion between the viral envelope and endosomal membrane allowing for escape of the viral nucleocapsid into the cytoplasm, (iii) the viral RNA dissociates from capsid and engages the translational machinery to synthesize viral proteins at the cytosolic face of the endoplasmic reticulum (ER), (iv) viral proteins engage the positive-strand genome to synthesize a negative strand intermediate, (v) the negative-strand asymmetrically templates the synthesis of many genomes, (vi) some of which associate with viral structural proteins and bud into the ER to form immature viral particles that (vii) transit through the golgi apparatus where they are modified by host enzymes, and finally, (viii) mature virions are secreted into the extracellular space. Note that multiple phases of the life-cycle, including translation, RNA synthesis and virus assembly, occur concurrently on separate genomes once infection is established although completion of each process is a prerequisite for the following to occur.At the level of the individual cell all 7GpppN) cap structure attached to the first base of the transcript via a 5′-5′ triphosphate linker  and to a lesser extent at the following nucleotide (type 2 cap) (O-methylation is a mark that signifies an mRNA as a “self” versus foreign molecule.Cellular mRNAs are modified in the nucleus with a 7-methylguanosine (mA pol II . The RNAtructure . Importae 2 cap) . The 5′ e 2 cap) . In cont7G-capping machinery and instead have evolved enzymatic activities to carry out all the necessary steps to generate capped genomes. The NS5 protein, in addition to its essential RNA-dependent RNA-polymerase function, harbors guanylyltransferase and MTase enzymes (7mGpppA (cap-0) and then (ii) m7GpppAm (cap-1)  and is enhanced by sequence within the first 20 residues of the genome. Notably, m7GpppA-RNA is strongly preferred over GpppA-RNA as a substrate for 2′-O-methylation, explaining the sequential order of 5′ end methylation reactions (Flaviviruses do not have access to the nuclear m enzymes . NS3 is  enzymes . The lat (cap-1) . The cisral RNAs . Cap metral RNAs . 2′-O-meeactions .O-methylation? Mutational analyses of the NS5 MTase have identified residues that specifically ablate 2′-O-methylation, cap-methylation, or both. Interestingly, loss of 2′-O-methylation can be tolerated whereas cap methylation is essential for infection (7GpppA and m7GpppAm caps on translation and RNA synthesis using the WNV replicon system which encodes the Renilla luciferase (RLuc) reporter. Compared to uncapped (pppA) WNV replicon RNA, the addition of GpppA strongly (∼25-fold) enhanced the accumulation of (RLuc) at 2 h post-transfection. Surprisingly, m7GpppA enhanced replicon RNA translation by only twofold compared to unmethylated GpppA cap, and m7GpppAm did not further increase RLuc expression. Unexpectedly, no differences in replicon RNA levels were detected for the differently capped replicons. By 72 h post-transfection, each of the capped replicon RNAs produced similar RLuc, indicating that methylation is not required for initial negative-strand synthesis as this is a prerequisite for synthesis of downstream positive-strand synthesis and consequent production of RLuc.What are the functional consequences of cap and 2′-nfection . In cons7-methylation  that sense unmethylated cap structures as invading nucleic acid. No such factor has yet been identified but there are many well-defined “pattern recognition receptors” whose tasks are to detect invading non-self nucleic acids and directly or indirectly, through innate immune pathways, antagonize infection . A pertiO-methylation at the penultimate nucleotide? As noted above, loss of this methylation event does not cause virus lethality in contrast to cap methylation. Key insights into this question were made by Diamond and colleagues who observed that a 2′-O-methylation-deficient NS5 mutant (E218A) WNV lacking m7GpppAm was attenuated in immunocompetent mice and primary cells, whereas animals and cells lacking the type I interferon (IFN) receptor (IFNAR-/-) were fully susceptible to infection  and also at the penultimate nucleotide of transcripts that initiate with A . The latter is referred to as m6Am as it is also methylated at the 2′-hydroxyl .It has been recognized for several decades that a prominent modification to cellular mRNA across many diverse organisms is the methylation of adenosine at the Nposition . This ochydroxyl . In the s in RNA . These folecules . This ha6A mapping and some functional analyses have been performed on multiple viruses including influenza A virus  suppressed viral titers, co-immunoprecipitated HCV RNA and localized to lipid droplets which are known sites of HCV assembly , indicating that m6A acts as a moderate restriction factor for infection in vitro. Nevertheless, the relatively small effects could reflect a viral strategy that is able to counter, in part, otherwise potent restriction by m6A and trans-acting reader proteins. One hypothesis is that the subgenomic flaviviral RNA (sfRNA), a highly stable RNA fragment produced from decay of virus genomes  is essential for infection, at least in part due to its role in stimulating virus translation. Methylation at the 2′-hydroxyl of the penultimate adenosine (m7GpppAm) is inessential for viability but allows the virus to escape the inhibitory actions of IFIT proteins and likely other factor(s)  . In cont viruses . Thus, uThe author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In cancer cells, telomerase reactivates due to the expressionof the reverse transcriptase gene. Telomerase RNA expresses constitutively inthe majority of human cells. This fact suggests that there are alternativefunctions to telomerase RNA that are unknown at the moment. In this manuscript,we review the biogenesis of yeasts and human telomerase RNAs thanks tobreakthroughs achieved in research on telomerase RNA processing by differentyeasts species and humans in the last several years.Telomerase is one of the major components of the telomeres ––linear eukaryotic chromosome ends – maintenance system. Linearchromosomes are shortened during each cell division due to the removal of theprimer used for DNA replication. Special repeated telomere sequences at thevery ends of linear chromosomes prevent the deletion of genome informationcaused by primer removal. Telomeres are shortened at each replication rounduntil it becomes critically short and is no longer able to protect thechromosome in somatic cells. At this stage, a cell undergoes a crisis andusually dies. Rare cases result in telomerase activation, and the cell gainsunlimited proliferative capacity. Special types of cells, such as stem, germ,embryonic cells and cells from tissues with a high proliferative potential,maintain their telomerase activity indefinitely. The telomerase is inactive inthe majority of somatic cells. Telomerase activity  Telomerase is a ribonucleoprotein complex comprising a reverse transcriptase(TERT) – a protein subunit enabling polymerase activity, and telomeraseRNA (TER) , 2. TeloFig. 1).Despite the high degree of variation in term of their sizes and nucleotidesequences, telomerase RNAs in yeast and mammals share four conserved structuralelements necessary for the formation and functioning of the enzyme-11.The Processing and localization of telomerase RNA of Saccharomyces cerevisiae(TLC1)S. cerevisiae yeast is known to beregulated by a strong promoter , which directs the expression ofprotein-encoding genes and leads to the accumulation of the polyadenylatedform, but it does not affect the content of the non-polyadenylated one, whereasdisruption of the polyadenylation system prevents the formation of thepolyadenylated form and greatly reduces the content of “mature”TLC1 in a cell [In the process of RNA transcription, polymerase II synthesizes two forms oftelomerase RNA: a long polyadenylated one and a short non-polyadenylated one.The fate of the long polyadenylated form is poorly understood at the moment. Itis known that this form accounts for 10% of the total telomerase RNA in a cell,but it is not associated with the active telomerase.It is an a cell , 18.TheS. cerevisiae telomerase RNA form independently. Disruption ofpolyadenylation signaling leads to the disappearance of the long polyadenylatedform of TLC1, but it does not affect the formation of the non-polyadenylatedmature form [TLC1 gene contains binding sites for thetermination factors Nab3 and Nrd1, whose deletion results in the accumulationof the polyadenylated primary transcript[Fig. 2).In yeast cells, different transcriptional complexes associated with RNApolymerase II participate in the formation of the two forms of the telomeraseRNA primary transcript. At the transcription initiation step, RNA polymerase IIforms a complex with termination and processing factors, i.e. the promoterdetermines the mechanism of termination. It has been demonstrated that thepolyadenylated and non-polyadenylated forms of the primary transcript ofure form .TLC1 isure form . The 3’-anscript, 19.Theanscript.TRAMP ianscript, 22.TRFanscript,25, 26.anscriptCellular localization and assembly of the active telomerase complex ofS.cerevisiaeFig. 2).As has been stated previously, the primarytranscript of telomerase RNA in both yeast and human cells is subjected toco-transcription processing, followed by maturation or degradation by exosomes.In S. cerevisiae, properly matured telomerase RNA localizes inthe nucleolus, where its cap is hypermethylated by the Tgs1 enzyme[One of the important stages in the biogenesis of telomerase RNA and telomeraseitself is the proper intracellular localization of their components[Fig. 3).The primary transcript of telomerase RNAin the cells of these organisms contains two exons, an intron, and a3’-terminus poly(A) sequence. The processing of the primary transcript iscarried out by a spliceosome. The first step in the splicing (cut in5’-splicing site) produces a mature form of telomerase RNA. Processing bya spliceosome was first discovered in the cells of Schizosaccharomycespombe yeast [S. pombe, the distance between thebranching point and 3’-terminal splicing site of telomerase RNA is 22nucleotides [S. pombe telomerase RNA [Telomerase RNA has undergone significant changes over the course of evolution,which have affected both its structure and processing mechanism. At the moment,there is no doubt that telomerase RNA is synthesized in all organisms as a longprecursor whose correct processing results in mature catalytically activetelomerase RNA. Telomerase RNA is involved in the fine regulation of the stateof a cell; therefore, the content of telomerase RNA must be maintained at thephysiological level for its proper functioning in a cell. In fission yeast(Schizosaccharomycetes) ,Hansenuetaceae), and othetaceae), a precube yeast . The splbe yeast . It has be yeast , 36.In leotides , which ileotides . Shortenleotides . Furtherleotides , 35.It leotides , high derase RNA .The mutS.cerevisiae yeast [S. pombe cells, a spliceosome cutsTER1 at a distance of one nucleotide from the Sm-proteins binding site andtherefore, may decrease the stability of the complex. It has been discoveredthat Sm-proteins interact with the polyadenylated TER1 precursor and facilitateits sectioning by the spliceosome [Fig. 3),which protect telomerase RNA against degradation by exosome[Sm-proteins are known to be associated with the telomerase RNA of ae yeast ,and theae yeast , 41.Theae yeast .In S. piceosome .Smd2 at exosome.S. cryophilius and S.octoporus, the5’-terminal splicing site comprises a cytosine residue in the thirdposition, which stabilizes the interaction with U6 of snRNA in the first stageand slow transition to the second [Aspergillus sp. and Neurospora crassa, thefirst nucleotide of the 5’-terminal splicing site, adenine, is importantfor the release of the processed product after the first step of splicing[Pezizomycotina andTaphrinomycotina, which are the closest to the commonancestor, prevents the formation of a proper three-dimensional structure andstops the splicing after the first transesterification reaction and subsequentdissociation of the frozen spliceosome[Later, it was shown that other types of fission yeast and fungi maintaintelomerase RNA processing by splicing its precursor with a spliceosome. Ine second .In Aspesplicing, 42.Thesplicing,whereasiceosome, 36, 43.The dramatic differences in the mechanism of telomerase RNA processing inevolutionarily related organisms do not affect the strict control of thequantity and quality of the telomerase RNA in their cells. In yeast cells,exosome degrades improperly processed telomerase RNA as well as RNA which hadnot form complexes with the proteins that regulate its localization andactivity.Processing and localization of human telomerase RNA hTR gene is carried out by RNA polymeraseII [hTRgene is well mapped, but the terminator region is poorly understood[Fig. 1).H/ACA hairpins are associated with a set of four proteins: dyskerin, NHP2, NOP10,and GAR1 [Yeast and human telomerase RNA differ considerably in length and structure, butthey share the main conservative components important for the formation andfunctioning of the telomerase complex. Mature human telomerase RNA (hTR)consists of 451 nucleotides [eraseII . The proderstood. The lenderstood. The 3’-derstood. This stand GAR1 . H/ACA hA combination of deep sequencing methods and determination of the3’-terminus of RNA (3’-RACE) has revealed the heterogeneity of the3’-terminus of human telomerase RNA .It has It is known that snRNAs containing H/ACA-motifs are processed by exosomes. In mammPARN1 gene encoding poly(A)ribonuclease 1 were recently discovered in patients with severe manifestationsof dyskeratosis, a disease associated with short telomeres[PARN1 gene leads to a decrease in thetotal amount of telomerase RNA in cells with a simultaneous increase in theproportion of non-processed oligoadenylated RNA[Inactivating mutations in the elomeres.It was ated RNA,58, 59.ated RNA, whereasated RNA, inhibitated RNA. Knockdoated RNA.The TRFated RNA.Interesated RNA.The oliated RNA.The proteins interacting with the H/ACA domain play a major role in theprocessing of human telomerase RNA. Dyskerin, NOP10, NHP2, NAF1, and GAR1 areRNA chaperones, and their interaction with telomerase RNA during processingstabilizes it, preventing degradation by exosomes. Dyskerin protects telomeraseRNA from degradation by nuclear 3’-5’-exosomes. Dyskerin knockdownand mutations that disrupt telomerase RNA binding to the protein result in areduced level of mature telomerase RNA in cells, whereas double knockdown ofdyskerin and PARN1 cause the accumulation of telomerase RNA in cytoplasm bodiescalled cyTER (cytoplasmic TER). Degradation of telomerase RNA from the5’-terminus by the decapping protein DCP2 and5’-3’-exonuclease XRN1 also indFig. 4).After primary transcript synthesis by RNA-polymeraseII and co-transcriptionally capping, it interacts with dyskerin, NOP10, NHP2,and NAF1, which stabilize and protect RNA from degradation[Summarizing the data on the processing of human telomerase RNA, it is possibleto suggest the following general scheme for its synthesis and maturation(radation. Part ofradation. This prradation, 60.Theradation, 59, 60.radation.In a HeLa tumor cell line, telomerase RNA accumulates in Cajal bodies. Aso-called CAB box is identified in the structure of telomerase RNA, which isresponsible for its localization in the Cajal bodies, where telomerase andtelomere interaction takes place .MutatioRecent research on the processing and localization of telomerase RNA in yeastand humans demonstrates that the amount of telomerase RNA in a cell is tightlycontrolled. Telomerase RNA processing and degradation are believed to becompeting processes whose balance regulates the amount of telomerase RNA in acell. The detection of telomerase RNA in the cytoplasm raises new questions. Itis unclear whether this step is necessary for the processing or assembly oftelomerase or whether human telomerase RNA performs alternative cell functions,some of which have been previously described.hTERT gene which encodes the secondcomponent of telomerase is finely regulated, and the enzyme activation dependson the appearance of the hTERT protein in a cell. The mechanism of synthesisand processing of telomerase RNA has attracted the attention of scientists formore than 10 years, and recently there was a breakthrough in the study of thisimportant stage of telomerase biogenesis. One of the most important features oftelomerase RNA processing is the fine regulation of the content of thismolecule in the cell. Both in yeast and human cells, telomerase RNA processinginvolves an exosome that rapidly degrades RNA that is not protected byRNA-chaperones. It has been established that most of the telomerase RNA genetranscription product is degraded in the process of biogenesis. Disruptions inthe processing result in a degradation of the telomerase RNA that causes anumber of diseases classified as telomeropathies.Telomerase maintains the proliferative potential of cells, which makes it oneof the most important objects in studies of aging and cell transformation.Disruption of telomerase functioning leads to the development of tumors andtelomeropathies. One of the essential components of telomerase is telomeraseRNA, whose gene is expressed in most cell types throughout their lifetime. Theexpression of the Despite recent progress in understanding the mechanisms of telomerase RNAprocessing, there are questions to which we still have no answers. A full anddetailed understanding of the mechanisms of both the functioning and biogenesisof telomerase will allow us to develop new approaches to the treatment ofdiseases whose development is associated with an impaired telomere maintenancesystem.