The reaction provides the corresponding azide‐modified RNA in nearly quantitatively yields without affecting the nucleobase amino groups. The obtained azido‐RNA can then be further processed utilizing well‐established bioortho‐gonal reactions, such as azide‐alkyne cycloadditions (Click) or Staudinger ligations. We exemplify the robustness of this approach for the synthesis of peptidyl‐tRNA mimics and for the pull‐down of 3‐(3‐amino‐3‐carboxypropyl)uridine (acp3U)‐ and lysidine (k2C)‐containing tRNAs of an Escherichia coli tRNA pool isolated from cellular extracts. Our approach therefore adds a new dimension to the targeted chemical manipulation of diverse RNA species.A major challenge in the field of RNA chemistry is the identification of selective and quantitative conversion reactions on RNA that can be used for tagging and any other RNA tool development. Here, we introduce metal‐free diazotransfer on native RNA containing an aliphatic primary amino group using the diazotizing reagent fluorosulfuryl azide (FSO 2N3 selectively provides azide‐modified RNA in quantitative yields. The stereochemically controlled reaction opens new avenues for the synthesis of RNA‐peptide conjugates and for the isolation of cellular RNA containing post‐transcriptional nucleotide modifications with an aliphatic primary amino group, such as acp3U.Metal‐free diazotransfer on native RNA using the reagent FSO Ribonucleic acid (RNA) is a fragile molecule that does not survive harsh reaction conditions and thus makes selective modification by synthetic organic chemistry challenging.4, 5 WThe functionalities of RNA are more diverse when one considers naturally occurring RNA with more than 140 modifications known to date, most of them found in tRNA, and also in rRNA, mRNA and non‐coding RNAs.12, 133U),2C),5s2U),In the present work, we have searched for a solution to convert an RNA containing a primary amino group into the corresponding azide‐modified RNA, leaving the nucleobase amines unaffected Figure . Finding2N3), originally designed to provide rapid access to azido libraries of small organic compounds.H‐imidazol‐3‐ium trifluoromethanesulfonate,2N3, two 3′‐aminoacylated oligoribonucleotides , and a 5‐aminomethyl uridine containing RNA (1–3) were chosen and individually treated by an excess of FSO2N3 in a biphasic mixture of methyl tert‐butyl ether (MTBE), DMF, and aqueous NaHCO3 solution, thoroughly mixed for 20 minutes  were analyzed by ion exchange chromatography. Indeed, all three oligoribonucleotides gave a very clean HPLC trace with a major peak at slightly higher retention time, from which the yields were estimated to be higher than 95 %  see Supporting Figure S2 and Supporting Table S1). The integrity of the products was confirmed by mass spectrometry amine (THPTA) as CuI stabilizing ligand, we reacted the crude product of the 5‐azidomethyluridine modified RNA 2 az that was obtained after amine‐to‐azide conversion with a biotin‐alkyne derivative .With the highly efficient RNA amine‐to‐azide conversion in hand, we set out to demonstrate the applicability of the so generated azido‐RNAs for bioconjugation reactions and first utilized the most popular one, the copper(I)‐catalyzed alkyne‐azide cycloaddition (CuAAC).37, 38e Figure . Efficied‐amino acids are brought to the ribosome active site.l‐cysteinyl‐3′‐amino‐RNA.A true benefit of the here introduced amine‐to‐azide conversion on native RNAs arises for the synthesis of peptidyl‐tRNA mimics.48, 49l‐isoleucyl‐RNA 7 az that mimics the 3′‐terminal sequence of tRNAIle from E. coli phenyl ester (for preparation see Supporting Information) to yield 7 az⋅fMet  and the use of wet DMF at 60 °C after 4 h. We confirmed the robustness of the ligation by additional examples using azido‐l‐phenylalanyl‐, azido‐glycyl‐, and azido‐l‐valyl‐RNAs  to give 3 az⋅fMet, 5 az⋅fMet, and 6 az⋅fMet, respectively uridine (acp3U) and lysidine (k2C) are known .[3U is a very common modified nucleotide found in the dihydrouridine (D) and variable (V) loops of tRNAs, however, little is known about the physiological functions.[E. coli, acp3U resides in the V‐loop at position 47 of tRNALys, tRNAMet, tRNAArg, tRNAPhe, tRNAIle1, tRNAIle2, tRNAIle3, tRNAVal1, and tRNAVal2, while k2C is located in the anticodon loop at position 34 of tRNAIle3 only .[To further demonstrate the value of the amine‐to‐azide conversion we turned to RNA from natural sources. Our goal was to enrich and analyze RNAs that contain native modifications which possess an aliphatic primary amino group, from a cellular RNA pool. We decided to focus on  and S7).9, 13 3U47 and k2C34 from the total E.coli tRNA pool is schematically shown in Figure 2N3 under the above optimized conditions to convert all aliphatic primary amino groups into azides. Then, CuAAC with desthiobiotin‐PEG4‐alkyne is applied, based on the conditions optimized for the short az5U oligoribonucleotide shown above. Subsequently, the resulting tRNA reaction mixture is subjected to streptavidin covered magnetic beads (SMBs) to immobilize the tRNA fraction that has been labeled with desthiobiotin. After several washing steps to remove unlabeled tRNAs, the labeled tRNA fraction is eluted with a biotin solution under non‐denaturing conditions at room temperature. In order to identify the individual tRNAs, we carried out northern blotting. Indeed, all tRNA species annotated with the acp3U47 modification and tRNAIle3 annotated also with the k2C34 modification were unequivocally confirmed in the enriched pool  should be addressed to the authors.SupplementaryClick here for additional data file.


Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5′ end of viral RNA via a 5′ to 5′ triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5′ capping of viral RNAs. The formation of the 5′ 7-methylguanosine (m Coronaviruses pose a serious threat to human health as they can cause severe respiratory disease and have pandemic potential. Severe acute respiratory syndrome coronavirus (SARS-CoV) was responsible for an epidemic in 2003 which caused nearly 800 deaths, and SARS-CoV-2 is responsible for the ongoing Coronavirus Disease 2019 (COVID-19) pandemic  cap, based on indirect evidence such as the presence of virally-encoded capping enzymes and some direct biochemical evidence  enzyme to covalently link GTP to the 5′ end of an RNA substrate. This activity has recently been demonstrated for nsp12, the RNA-dependent RNA polymerase (RdRp) component of the RTC, using GTP or UTP nucleotides  were mixed with SARS-CoV-2 England/02/2020 (P3) at MOI 1.0 and seeded in each 35mm well of a 6- well culture dish with 2 ml culture medium. At 3, 6, 9 and 12 h post-infection medium was removed and 1 ml TRI reagent (Sigma) was added to the wells, then total cellular RNA extraction and precipitation was performed according to the manufacturer's instructions. Mock infected samples were also included as a negative control.SARS-CoV-2 England/02/2020 isolate was received from Public Health England Virus Reference Laboratory at P1 and propagated in Vero CCL-81 cells (ATCC) in containment level (CL) 3 to produce P3 virus stock, confirmed identical by sequencing. 8.1 × 1070 ng of extracted RNA (quantified on a NanoDrop 2000 Spectrophotometer) was annealed to 1 μM each reverse primer in 5 μl mixtures, by heating to 95°C and cooling to room temperature. Reverse primer sequences (purchased from Sigma) are as follows: Full-length, 5′-AATTAGTTATTAATTATACTGCGTG-3′; S, 5′-GCAGGGGGTAATTGAGTT-3′; 3a, 5′-GCGCGAACAAAATCTGAA-3′; E, 5′-CGCTATTAACTATTAACGTACCT-3′; M, 5′-GCTCTTCAACGGTAATAGTAC-3′; 6, 5′-CCAATCCTGTAGCGAC-3′; 7a, 5′-AAAAGTACTGTTGTACCTCT-3′; 8, 5′-TGAGTACATGACTGTAAACTAC-3′; N, 5′-GTTCTCCATTCTGGTTACTG-3′; GAPDH mRNA, 5′-CAAAGTTGTCATGGATGACC-3′; 5S rRNA, 5′-TCCCAGGCGGTCTCCCATCC-3′. Reverse transcription was carried out by addition of 50 U SuperScript III (Invitrogen), 1× first-strand buffer (Invitrogen), 10 U RNasin (Promega) and 10 mM dithiothreitol (DTT) to a total reaction volume of 10 μl. Reactions were incubated at 37°C for 15 min, 50°C for 45 min and then 70°C for 10 min. cDNA was stored at −20°C prior to qPCR analysis. To perform qPCR 1 μl of the resulting cDNA was mixed with 1× qRT-PCR Brilliant III SYBR Master Mix (Agilent) and a further 0.75 μM of the reverse primer, as well as 0.75 μM of the corresponding forward primer in a total reaction volume of 10 μl. Forward primer sequences (purchased from Sigma) are as follows: Leader , 5′-ATTAAAGGTTTATACCTTCCCAG-3′; GAPDH mRNA, 5′-CCATGGAGAAGGCTGGGG-3′; 5S rRNA, 5′-GTCTACGGCCATACCACCCTGAACG-3′. qPCR was performed in 384-well skirted reaction plates (Alpha Laboratories) on a Quantstudio 5 RT-PCR machine (Applied Biosystems) with the following cycling conditions: 95°C 10 min; 95°C 15 s, 60°C 30 s, 60 cycles; melt curve analysis. qPCR data analysis was performed using the included Design and Analysis Software v1.5.2 (Applied Biosystems). Two technical replicates of qPCR were performed per sample and averaged. Data presented were normalised to the GAPDH mRNA Ct value7G antibody  or mouse anti-his antibody  in a total volume of 200 μl wash buffer , 0.01% bovine serum albumin, 2 mM EDTA, 1mM DTT and 0.1 U/μl RNasin). 50 μl washed Sheep Anti-Mouse IgG Dynabeads (Invitrogen) were added and incubated with the mixture at room temperature for 30 min. Beads were then washed 3x in 1 ml wash buffer and RNA was eluted by addition of 1 ml TRI reagent. RNA extraction was then performed according to the manufacturer's instructions, except that 20 μg glycogen (Roche) was added to aid RNA precipitation. Input and eluted RNA was analysed using RT-qPCR as described above. Data presented were normalized to the Ct value of 5S rRNA, and then the input sample was set to 0. Data were analysed using Prism 9 (GraphPad).2 μg total cellular RNA extracted from Vero cells 9 h post-infection with SARS-CoV-2 (see above) was mixed with 2 μg mouse anti-m2 and the NaCl concentration was changed to 300 mM  protease cleavable protein-A tag at the C-terminus . Nsp12 wo 300 mM . After oEscherichia coli BL21 (DE3) cells, then purified on Glutathione Sepharose resin . After overnight cleavage with PreScission protease, the released proteins were further purified on a Superdex 75 Increase 10/300 GL column  using 25 mM HEPES–NaOH, pH 7.5, 300 mM NaCl and 0.1 mM MgCl2. Fractions containing target proteins were pooled, concentrated, and stored at 4°C.The NiRAN construct (nsp12 amino acid residues 1–259) and full-length nsp7 and nsp8 genes from SARS-CoV-2 were cloned into pGEX-6P-1 vector  with an N-terminal GST tag followed by a PreScission protease site. Proteins were expressed in E. coli BL21 (DE3) cells, then 5 ml of overnight culture was used to inoculate 1 l of terrific broth (TB) supplemented with 100 mg/l ampicillin and 10 μM ZnCl2. The culture was grown to an OD600 of 2, then induced with 1mM IPTG and incubated at 18°C for 16 h. Cells were harvested and resuspended in a buffer containing 50 mM Tris pH 8, 500 mM NaCl, 5% (v/v) glycerol, 1 mM TCEP, 1× protease inhibitor (Roche) and 10 mg of lysozyme, then sonicated. Lysate was clarified by centrifugation, applied to Glutathione Sepharose resin and incubated for 3 h before washing 3× in a buffer containing 25 mM HEPES pH 7.5, 500 mM NaCl, 5% (v/v) glycerol and 1 mM DTT. After overnight cleavage with PreScission protease, eluate was concentrated before further purification on a Superdex 200 Increase 10/300 GL column using 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT. Fractions containing nsp14 were pooled, concentrated, and stored at −80°CThe full-length nsp14 gene from SARS-CoV-2 was cloned into a pGEX-6P-1 vector  with an N-terminal GST tag followed by a PreScission protease site. The plasmid was transformed into 2, 4.75 μM ATP and 0.25 μM γ-32P-ATP for the indicated time at 30°C, then reactions were stopped by addition of 80% formamide and 10 mM EDTA, followed by heating to 95°C for 3 min. Inactivated nsp13 was heated to 70°C for 5 min prior to incubation. As a positive control, 0.5 U/μl FastAP Thermosensitive Alkaline Phosphatase (Thermo) was incubated with 4.75 μM ATP and 0.25 μM γ-32P-ATP for 1 h at 37°C. Reaction products were resolved by 20% denaturing PAGE with 7 M urea, and visualized by phosphorimaging on a Fuji FLA-5000 scanner. Data were analysed using ImageJ and Prism 9 (GraphPad).Purified SARS-CoV-2 nsp13 with an N-terminal His6-ZBasic tag in 25 mM HEPES–NaOH, pH 7.5, 300 mM NaCl and 5% glycerol buffer was a kind gift from Yuliana Yosaatmadja and Opher Gileadi. 250 nM nsp13 was incubated with 5 mM MgCl2 for 5 min at 30°C, followed by heat inactivation at 70°C for 5 min. The resulting diphosphorylated RNA stock was used for all GTase reactions.To generate diphosphorylated RNA for GTase reactions 5 μM 20mer model RNA (5′-AAUCUAUAAUAGCAUUAUCC-3′), purchased as a 1:1 mixture of di- and triphosphorylated RNA (Chemgenes), was incubated with 250 nM nsp13 in the presence of 5 mM MgClWhere indicated, 5 μM diphosphorylated 20mer RNA substrate (see above) was pre-treated with 0.3 U/μl FastAP Thermosensitive Alkaline Phosphatase (Thermo) at 37°C for 1 h. Alternatively, 5 μM diphosphorylated 20mer RNA substrate was pre-treated with 0.5 U/μl RNA 5′ Pyrophosphohydrolase (NEB) in 1× NEBuffer 2 (NEB) at 37°C for 1 h. Enzymes were heat inactivated at 75°C for 5 min before the resulting RNA substrates were used in GTase reactions.32P-UTP or α-32P-GTP, 5 mM MgCl2, 10 mM KCl, 1 U/μl RNasin and 1 mM DTT in a 3 μl reaction at 30°C for 240 min unless stated otherwise. Where indicated, nsp7 and/or nsp8 were included at a 5:1 excess over nsp12. GTase reactions involving vaccinia capping enzyme were run for 60 min under the same conditions, using 0.01 U/μl vaccinia capping enzyme (NEB) instead of SARS-CoV-2 protein.To run the GTase reaction, 500 nM SARS-CoV-2 nsp12 or NiRAN domain was incubated with 1 μM diphosphorylated or phosphatase-treated 20mer RNA, 0.05 μM α-Where indicated, completed GTase reactions were treated with 0.3 U/μl FastAP Thermosensitive Alkaline Phosphatase (Thermo) at 37°C for 1 h. Alternatively, reactions were treated with 0.5 U/μl RNA 5′ Pyrophosphohydrolase (NEB) in 1× NEBuffer 2 (NEB) at 37°C for 1 h. All reactions were stopped by addition of 80% formamide and 10 mM EDTA, followed by heating to 95°C for 3 min. Reaction products were resolved by 20% denaturing PAGE with 7 M urea, and visualized by phosphorimaging on a Fuji FLA-5000 or Typhoon FLA-9500 scanner. Data were analysed using ImageJ and Prism 9 (GraphPad).32P-radiolabelled 20mer LS2 (5′-GUCAUUCUCCUAAGAAGCUA-3′) RNAs corresponding to the 3′ end of the SARS-CoV genome (without the polyA tail) were pre-annealed by heating to 70°C for 5 mins followed by cooling to room temperature. 50 nM pre-annealed RNA was incubated for the indicated time at 30°C with 500 nM nsp7/8/12 complex, in a 3 μl reaction containing 5 mM MgCl2, 0.5 mM of each ATP, UTP, GTP and CTP, 10 mM KCl, 1 U/μl RNasin and 1 mM DTT. Remdesivir triphosphate (MedChemExpress) was included where indicated, and for these experiments the ATP, UTP, GTP and CTP concentration was reduced to 1 μM. Reactions were stopped by addition of 80% formamide and 10 mM EDTA, followed by heating to 95°C for 3 min. Reaction products were resolved by 20% denaturing PAGE with 7 M urea, and visualized by phosphorimaging on a Fuji FLA-5000 scanner. Data were analysed using ImageJ and Prism 9 (GraphPad).Nsp7, nsp8 and nsp12 were mixed at a molar ratio of 5:5:1 to form the nsp7/8/12 complex. Activity assays were performed essentially as described for SARS-CoV nsp7/8/12 . Briefly32P-GTP, the diphosphorylated RNA concentration was increased to 10 μM and the reaction volume was increased to 5 μl. Reactions were incubated at 30°C for 240 min then heated to 95°C for 3 min. 500 nM SARS-CoV-2 nsp14 and 0.1 mM S-adenosylmethionine (NEB) were then added and reactions were incubated at 30°C for a further 60 min, then heated to 95°C for 3 min.SARS-CoV-2 nsp12 GTase activity assays were set up as described above, except that 1 mM GTP was used instead of α-7G primary antibody  followed by goat anti-mouse HRP-conjugated secondary antibody  in blocking buffer. Membranes were washed 3× in 1× PBS, 0.1% Tween-20 then HRP activity was detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and exposure on Super RC Fuji Medical X-ray film (Kodak). Data were analysed using ImageJ and Prism 9 (GraphPad).Reaction mixtures were dotted onto nylon membrane and allowed to air dry for 10 min, then heated to 80°C for 10 min under a light vacuum. Membranes were blocked in 1× PBS, 0.1% Tween-20 (Sigma) and 2.5% skimmed milk powder (Marvel), then incubated with mouse anti-m7G capped, though there is little direct evidence to support this (7G capped.Coronavirus genomic RNA is presumed to be mort this . Therefo7G capped RNAs we performed immunoprecipitation (IP) on RNA extracted from SARS-CoV-2-infected cells using an anti-m7G antibody, with an anti-his tag antibody as a negative control -qPCR method to detect full-length genomic and subgenomic viral RNAs, and using this approach could detect all positive-sense viral RNA species in infected cells with the exception of 7b subgenomic RNA Figure , B 28)..28). As l Figure . Full-les Figure . S, 3a a7G capped RNA requires several distinct enzymatic activities, which are well characterised for coronaviruses apart from the viral GTase , and nsp13 is thought to generate the diphosphorylated RNA substrate by acting as a 5′ triphosphatase Figure  9,29). ,29. 9,2932P-GTP, then separated the reaction products by denaturing PAGE ..13). To E Figure . In reacc Figure  (13).in vitro.To confirm that the radiolabelled product resulted from GTase activity we compared the activity of nsp12 with vaccinia capping enzyme, a known GTase . Under tNext, we aimed to identify the GTase active site in nsp12. A recent cryo-EM study showed that the nsp12 NiRAN domain can bind to GDP, so we hypothesised that this nucleotide binding pocket could be important for GTase activity Figure , B 13)..13). We To first examine the effect of these mutations on the RNA polymerase activity of nsp12, we established an assay to measure the extension of a 20 nucleotide (nt) primer (LS2) along a 40nt RNA template (LS1) Figure  21). In. In21). In contrast, when we performed GTase reactions using the mutant nsp12 proteins we found that all NiRAN domain mutations significantly reduced GTase activity Figure . The D21To then investigate whether the isolated NiRAN domain has GTase activity, we expressed and purified the SARS-CoV-2 NiRAN domain and performed GTase reactions alongside full-length nsp12 Figure , B. The 7G capped viral RNA the product of the nsp12 GTase reaction must be subsequently methylated by the nsp14 N7-methyltransferase ..32).50 of 6.56 μM ..50 of 6.50 of 0.43 mM  as a by-product .,40.de not Figure ,41,42. Ht Figure .Canonical GTases use a nucleotidylated enzyme intermediate (Gp-E) to transfer GMP to a diphosphorylated RNA substrate . This in32P-UTP cannot be transferred to a diphosphorylated RNA substrate .,18.32P-Ue Figure ,29,44. Atrans-nucleotidylation of nsp8 and nsp9 with UTP   . The funreaction ,20. Intereaction  19,20).,20.transin vitro assays indicate that remdesivir triphosphate inhibits SARS-CoV-2 RNA polymerase activity with an IC50 of 6.56 μM, higher than the 0.77 μM IC50 reported for SARS-CoV-2 infections in cell culture . Th. Thin vio Figure . This iso Figure . Interesn Figure . While wive site . The GTaIn summary, we have demonstrated that SARS-CoV-2 nsp12 is a GTase enzyme and identified the active site as the nsp12 NiRAN domain. These findings not only improve understanding of coronavirus RNA synthesis, but also highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.Source data as well as plasmids are available upon request.


When publishing qualitative research in international journals, researchers studying non‐English‐speaking participants provide quotes in English language. This is an issue of increasing concern given the need to be rigorous to represent a diversity of participants within their context, beyond how language  situates them.To argue for providing English and native language quotes in qualitative research reports.Discussion.This paper is based on the literature on use of quotes and translation in qualitative research and authors’ experiences of publishing qualitative research.Provision of native and English language quotes may allow for greater transparency of findings, thereby reflecting that the researchers adequately captured the socially and culturally dependent experiences of participants.Presentation of findings with eloquent quotes serves as the gateway into the sociocultural experiences of individuals. We argued against the norm of providing translated quotes in qualitative reports and build a case for the provision of native as well as English language quotes to promote cross‐cultural understanding. The tools may include online software, glossaries, internet searches and IT tools  requires authors to provide English and non‐English quotes and encourages adding a second abstract in the authors’ native language to avoid losing the context of findings. Therefore, it seems reasonable to provide both English and native quotes when reporting qualitative studies in the fields of nursing and the health sciences. Finally, the authors of cross‐cultural qualitative studies should explicitly discuss any potential challenges and issues encountered during the translation and analysis of native language quotes in their manuscripts. It is important that readers are aware of the issues that may have affected the transparent reporting of qualitative quotes and their underlying contextual meanings.We are also conscious of the fact that sometimes journals do not have adequate space to publish qualitative studies with a long list of quotes. Therefore, we offer three recommendations to overcome this limitation. First, the translated and original quotes could be presented in a supplementary online file, only if the journal restricts the word count. If the journal space permits, the authors should provide both English and original language quotes under each theme or sub‐theme in the research report. Second, as Bazeley  suggesteDr. Parveen Ali is an Associate Editor for Nursing Open.


Saccharomyces cerevisiae. We present their interaction mode with different chemicals  or proteins (such as the RNase III) and their recent employment into clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas) systems. Particular attention is paid, throughout the whole paper, to their usage and performance into synthetic gene circuits.Among noncoding RNA sequences, riboswitches and ribozymes have attracted the attention of the synthetic biology community as circuit components for translation regulation. When fused to aptamer sequences, ribozymes and riboswitches are enabled to interact with chemicals. Therefore, protein synthesis can be controlled at the mRNA level without the need for transcription factors. Potentially, the use of chemical-responsive ribozymes/riboswitches would drastically simplify the design of genetic circuits. In this review, we describe synthetic RNA structures that have been used so far in the yeast  RNA is a polymeric biomacromolecule that carries out crucial functions in diverse biological systems. Since it was discovered, the messenger RNA (mRNA) has shown its fundamental role in gene expression as the intermediate step from DNA to proteins in the central dogma. Further, noncoding RNA elements are ubiquitous both in prokaryotic and eukaryotic cells. Over the last two decades, with the advent of synthetic biology, noncoding RNAs have been largely used to control protein synthesis within genetic circuits . To thisRiboswitches are regions of the mRNA that contain specific evolutionarily conserved ligand-binding domains (sensors) along with a variable sequence, termed the expression platform, which enables regulation of the downstream coding sequence (CDS) . They weRibozymes—whose existence have been known since the 1980s of the last century —are funcSaccharomyces cerevisiae. We first described aptamers that react to different kinds of commonly used chemicals. Then, we presented how ribozymes and other hairpin structures have been utilized together with clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas) systems. We illustrated switches arising from hairpins that are cleaved by RNase III, and, finally, before the conclusions, we listed some other RNA structures that do not belong to any specific groups.In this paper, we reviewed RNA structures such as aptamers, ribozymes, and riboswitches that have found synthetic biology application in the yeast S. cerevisiae respond to well-tolerated drugs, such as theophylline, tetracycline, and aminoglycosides. They are thoroughly described in the following. Examples of other RNA structures sensing different chemicals are given as well, at the end of this section.Most of the ribozymes and riboswitches that have shown high functionality in Camellia sinensis (tea plant) and Theobroma cacao. In 1994, Jenison et al. [D decreased from 0.6 to 0.1 µM [S. cerevisiae determined a sharp reduction (OFF switch) or increase (ON switch)—about 90% in both cases—in GFP expression in the presence of 1 to 10 mM of theophylline. This work represented the first successful application of theophylline-dependent riboswitches in eukaryotic cells.Theophylline  sensing theophylline (5–10 mM) and/or tetracycline (0.25–0.5 mM) were engineered by means of two modified HHRs or a single one carrying an aptamer at both stem I and stem II or two aptamers in series at stem II. A remarkable ~14-fold gain in fluorescence expression was achieved by the AND gate [S. cerevisiae could be assessed by the fluorescence level triggered by an ON switch sensing the amount of theophylline (up to 140 µM) derived from (up to 1 mM) caffeine transformation. This screening process permitted us to find out mutations that increased, considerably, both the activity and the selectivity of caffeine demethylase [In a later work, Win and Smolke  modifiedAND gate . A singlAND gate . This moethylase .A peculiar implementation of RNA-based Boolean gates and an apoptosis controller was presented by Anzalone et al.  who engiPromoters activated or repressed by tetracycline tc—see a have beD equals 0.8 nM [S. cerevisiae. Green fluorescence was measured after placing the tc-responsive riboswitch in the 5′UTR of the strong ADH1 promoter (pADH1), either close to the fluorescent protein start codon or in a cap-proximal location. The latter position appeared more effective in repression of translation (6-fold in the presence of tetracycline). Interestingly, stabilization of the aptamer structure led to an increased ON-to-OFF ratio but lowered basal fluorescence expression . In another work published in 2003, the tetracycline riboswitch was shown to lead, in the presence of the antibiotic, to a 9-fold downregulation in luciferase synthesis from the constitutive TEF1 promoter [ADH1 promoter and the GFP gene. As already experienced in [GPD promoter, were then adopted to construct universal insertion cassettes that could be integrated, via homologous recombination, into the yeast genome. Five endogenous genes  were completely switched OFF upon induction with at most 500 µM of tetracycline. NOP8 and SEC1 had never been regulated previously. More recently, the tandem configuration of this riboswitch was optimized in silico via machine learning techniques. In vivo, the newly designed 5′UTR sequence guaranteed a 40-fold repression of fluorescence in the presence of 250 µM tetracycline [As we have seen in the previous section, the tc aptamer has been used in several works together with the theophylline aptamer. The tetracycline aptamer consists of three stems (P1–P3), two single-stranded regions , and the loops L2 and L3 see b. Portios 0.8 nM ). In 200s 0.8 nM  reportedpromoter . 5′UTRs promoter . Initialenced in ,45, riboacycline .One or two tetracycline-responsive riboswitches were also shown to be effective in controlling pre-mRNA splicing when placed within an intron nearby the 5′ splice site (SS). The most efficient regulation was obtained by burying the 5′ SS into the P1 stem at the basis of the tetracycline aptamer. Upon insertion of the modified intron along the sequence of GFP, fluorescence was emitted by the cells only in the absence of tetracycline, i.e., when the mRNA was spliced successfully. In contrast, in the presence of tetracycline a drastic reduction in cell fluorescence level was observed .Schistosoma mansoni via a linker region optimized through 11 rounds of in vitro selection (SELEX). Synthetic ribozymes completely self-cleaved in the presence of 1 µM of tetracycline, whereas self-cleavage was inhibited up to 333-fold in the absence of the antibiotic [In a different approach to control gene expression, the tetracycline aptamer was fused to the full-length HHR N79 from tibiotic .Aminoglycosides are positively charged antibiotics characterized by a high affinity towards RNA sequences ,50,51. AS. mansoni HHR. Neomycin-responsive ribozymes, engineered in this way, were inserted in the 3′UTR of the gal4 gene. Hence, gal4 expression was maximal in the absence of neomycin. The Gal4 activator induced the synthesis of lacZ such that the ribozyme performance was assessed, indirectly, via beta-galactosidase assay. This represented a new protocol for the in vivo selection of neomycin-sensing ribozymes. The best one reported in this work showed a ~25-fold reporter downregulation in the presence of 100 µg/mL neomycin. A similar design was followed, in a later work by the same group [lacZ gene expression.Klauser et al. ,61 introme group , for theS. mansoni HHR. The connection between aptamer and stem I was realized through an addressable three-way junction [gfp gene such that its activity was quantified, directly, by measuring cell fluorescence. Libraries of artificial ribozymes, obtained by randomizing, first, the three-way junction and, then, the three-nucleotide bulge, were screened in the presence and absence of, usually, 100 µg/mL of neomycin. Differently from [In a recent work, Sack et al.  presentejunction , whose ftly from , both OF15 RNA sequences via the Capture-SELEX method [D = 20 nM) and allows an 8.5-fold translation downregulation to be registered when exposed to 250 µM of the antibiotic. On it, a working NOR gate was engineered by replacing P1 with the neomycin aptamer. In the presence of 250 µM of both inputs, the logic gate gave a 13.8-fold repression factor.A new synthetic riboswitch responding to paromomycin  was engiX method . With reD = 64.2 nM).As we have seen above, antibiotics such as tetracycline and aminoglycosides have been largely used for the engineering of novel aptamers. Groher et al.  realizedS. cerevisiae cell cycle via a dye-responsive aptamer placed in the 5′UTR of the CLB2 gene. Upon binding tetramethylrosamine , the aptamer underwent a drastic structural change that reduced CLB2 translation in a considerable way. As a consequence, cell growth slowed down and elongated buds appeared during cell division.In 2001, Grate and Wilson  describeMore recently, a novel kind of aptamer, indirectly activated by light, was realized by Lotz et al. . This RNCandida parapsilosis, and are functional in S. cerevisiae. Once placed on an intron inside a reporter protein gene, the TPP riboswitch allowed splicing and, therefore, fluorescence expression only in the absence of thiamine, whereas the pre-mature mRNA was not spliced in the presence of 10 µM thiamine.After describing many synthetic aptamers, we shall mention the thiamine pyrophosphate (TPP) riboswitches, i.e., the only natural eukaryotic riboswitches discovered so far. They were found—usually in introns, as splicing regulators—in the genome of plants , filamenAn overview of the performance of the aptamers described in this section is given in So far, we have described RNA structures that are regulated by chemicals and represent possible interface between synthetic gene circuits and the environment. However, ribozymes and other hairpins that are inert to small molecules can also carry out important functions as components of genetic circuits. For instance, both HH and HDV ribozymes have been recently combined with the CRISPR-Cas9 system.SNR52 promoter and the SUP4 terminator [ADH1 promoter and terminator, can be adopted as in [GFP gene with a dramatic reduction in the cell fluorescence level  has been widely adopted for gene editing and synthetic biology applications in eukaryotic cells . Cas9  and leads the transcription of an RGR cassette that releases a third sgRNA (output). The RGR was designed in silico to assure a correct folding of the ribozymes. Moreover, in order to increase repression efficiency, dCas9 was fused to the Mxi1 repression domain. NOR gates’ shared input–output allowed gates’ assembly into larger circuits such as an XOR gate and a six-step cascade.A similar strategy was followed by Gander et al.  to desigS. cerevisiae. A possible decrease in the affinity between the synthetic sgRNA and Cas9 has been proposed as an explanation of the negative result.A recent study showed that the fusion of a chemical-responsive aptamer to the sgRNA could lead, in bacteria, to ligand-activated/deactivated CRISPR-Cas9 systems . HoweverPseudomonas aeruginosa. Csy4 processes the CRISPR array transcript into mature CRISPR-RNA (crRNA) after binding a 28-nt-long hairpin and cleaving the RNA at its base [S. cerevisiae, cleavage by Csy4 led to an effective gene silencing with an over ten-fold decrease in the fluorescent signal.A different engineering approach  took advits base ,86. Qi eits base  insertedS. cerevisiae RNase III (Rnt1protein—Rnt1p) targets RNA hairpins capped, generally, with the tetraloop AGNN [Csy4 is not an isolate case of a protein that induces RNA degradation upon recognition and cleavage of an RNA secondary structure. Another typical example is given by the RNase III endoribonucleases that discern and split double-stranded RNA (dsRNA) sequences. In this way, they induce the decay of various kinds of RNAs  both in prokaryotic and eukaryotic cells . S. cereoop AGNN  and cleaoop AGNN ,90. Moreoop AGNN   [A few years later, Babiskin and Smolke  developeB (8 nt) . InteresB (8 nt) . TherefoB (8 nt)  also buiAmong the RNA structures which are of interest for the yeast synthetic biology community and do not belong to any of the above-described categories, it is worth mentioning the snorbozyme , i.e., aS. cerevisiae and mammalian cells, was also realized as a hybrid construct by combing the MS2-coat-protein-binding aptamer [GFP gene. Fluorescence intensity increased from 20% to 97% of the maximal GFP level by using the ON-gene switch, whereas it decreased from 55% to 13% with the OFF-gene switch. Moreover, only the MS2 concentration was found to be an important parameter for the working of the two switches, i.e., MS2 localization in the nucleus or in the cytoplasm did not play a major role in the activity of the switches.A protein-responsive ribozyme, able to work both in  aptamer  and the  aptamer . The MS2S. cerevisiae [As a final work, we want to mention a recent study focused on the design of novel RNA hairpin modules that allow for fine-tuning in a predictive way and translation in revisiae . The exprevisiae ) of the S. cerevisiae synthetic gene circuits. These RNA structures can be regulated, directly, with chemicals. Hence, they allow for a close control of protein synthesis to be achieved. Several synthetic ribozymes/riboswitches have proved their utility in the assembly of genetic networks, especially Boolean gates where a switch-like behavior is essential for a proper reproduction of a truth table. However, these RNA structures have, basically, never been tested into different kinds of even basic circuits (such as feedback loops and oscillators) and their general applicability to synthetic gene networks, in S. cerevisiae, is still to be confirmed.Ribozymes and riboswitches have been discovered in bacteria and quickly repurposed in eukaryotic cells. In this review, we presented the progress made, mostly over the last two decades, in the constructions of artificial aptamers for applications into If employed into digital circuits, riboswitches could drastically reduce the number of components necessary to mimic complex logic functions made of several literals  and clauses (the Boolean gates), as we have shown in a previous work . Hence, S. cerevisiae by Grilly et al. [Protein decay, moreover, can be achieved indirectly by inducing the degradation of the corresponding mRNA via a ribozyme or a Csy4-bound hairpin placed at the 3′UTR of a CDS. These represent simpler bio-engineering solutions than those so-far adopted, based on tagging proteins with a degron element recognized by the bacterial ClpP-ClpX system—repurposed in y et al. —or couply et al. .S. cerevisiae.Even though the working mechanisms of ribozymes and riboswitches look rather simple, engineering new ribozymes and riboswitches can be challenging. In order to carry out their function, these RNA molecules have to fold properly, once inserted along an mRNA sequence, and show high specificity towards a single chemical. Moreover, so far, not many chemical-aptamer systems have been characterized. Despite the fact that, over the past years, new ribozymes and riboswitches have been engineered in mammalian cells ,104,105,Notwithstanding the non-negligible hurdles that currently hinder a diffuse usage of aptamer-like structures in genetic circuits, we think that the application of ribozymes to the CRISPR-Cas technology for the expression of sgRNAs and the ongoing studies about using hairpins to build chemical-responsive crRNAs are a clear indication that the engineering of novel synthetic ribozymes/riboswitches will play a central role in synthetic biology in the coming years.


S-adenosylmethionine (SAM). SAM and other nucleotide-derived cofactors are viewed as evolutionary leftovers from an RNA world, in which riboswitches have regulated, and ribozymes have catalyzed essential metabolic reactions. Here, we disclose the thus far unrecognized direct link between a present-day riboswitch and its inherent reactivity for site-specific methylation. The key is O6-methyl pre-queuosine (m6preQ1), a potentially prebiotic nucleobase which is recognized by the native aptamer of a preQ1 class I riboswitch. Upon binding, the transfer of the ligand’s methyl group to a specific cytidine occurs, installing 3-methylcytidine (m3C) in the RNA pocket under release of pre-queuosine (preQ1). Our finding suggests that nucleic acid-mediated methylation is an ancient mechanism that has offered an early path for RNA epigenetics prior to the evolution of protein methyltransferases. Furthermore, our findings may pave the way for the development of riboswitch-descending methylation tools based on rational design as a powerful alternative to in vitro selection approaches.Methylation is a prevalent post-transcriptional modification encountered in coding and non-coding RNA. For RNA methylation, cells use methyltransferases and small organic substances as methyl-group donors, such as  In humans, protein methyltransferase is responsible for RNA methylation using S-adenosylmethionine as a methyl group donor. Here the authors report a self-methylation activity of a bacterial riboswitch. One hypothesis suggests that present-day riboswitches for common enzyme cofactors may have evolved from ancient ribozymes that depended on these cofactors7. What has stood the test of time are RNA sensors of cofactors for the regulation of gene expression8 while the reactivity part has been taken over by more efficient protein-based modification machineries12. Riboswitches typically employ a highly conserved aptamer domain to sense the small molecule ligand with high specificity and selectivity, and an adjoining expression platform to regulate the expression of genes associated with ligand biosynthesis and transport14. Thereby, the partially overlapping RNA sequences of the two domains assist in converting the ligand-binding event into a change in gene expression by employing ligand-mediated RNA folding changes. It seems reasonable to assume that ligand-induced structural adaptions may also be exploited to trigger a chemical reaction. A single example exists for a known naturally occurring RNA, namely the glms riboswitch-ribozyme which binds glucosamine-6-phosphate whose protonated amino group then directly participates in reaction catalysis of RNA backbone cleavage15. Moreover, support for an advanced reactivity scope of cofactor-RNA systems has been provided by ribozyme engineering. The first methyltransferase ribozyme that catalyzes the site-specific installation of 1-methyladenosine in a substrate RNA, using a small-molecule cofactor has been created by in vitro evolution very recently16.In recent years, numerous discoveries of metabolite-sensing riboswitches hint at RNA World ribozymes that could have promoted chemical transformations17. The aptamers of these riboswitches bind their dedicated ligands in the nano- to micromolar range. For all of them, different subclasses are found, meaning that the ligand is recognized by distinct RNA architectures with both specific binding pockets as well as overall folds18. For SAM-binding riboswitches, a total of six subclasses is known to date; they accommodate the ligand with its reactive methyl group in conformations that strictly avoid self-methylation20.Numerous natural riboswitches are known to selectively sense nucleotide-derived metabolites connected to methyl-group transfer or one-carbon metabolism, comprising SAM, methylene tetrahydrofolate (THF) and adenosylcobalamin (vitamin B12)11. Besides the above-mentioned ubiquitous enzyme cofactors, other small organic compounds could serve for RNA-catalyzed RNA methylation in modern riboswitches, as implicated by the recently in vitro selected RNA methyltransferase that utilizes O6-methyl guanine16. An obvious link between guanine riboswitches and the near-cognate ligand O6-methyl guanine was tested even prior to the in vitro selected methyltransferase, however, methylation was not observed21. Nevertheless, we speculated that other riboswitches sensing guanine-type ligands may possess the inherent reactivity for methyl group transfer that we were searching for. Here, we report on the identification of a riboswitch motif that uses O6-methyl pre-queuosine (m6preQ1) as a small-molecule methyl-group donor, catalyzing site-specific methylation of a cytosine at the N3-atom, resulting in RNA sequence-specific installation of 3-methylcytidine (m3C)  preQ1 riboswitch which adopts an H-type pseudoknot structure exhibiting low nanomolar affinity for preQ1  mass spectrometry (MS), which can determine the mass-to-charge ratio (m/z) of ions with very high precision by measuring the frequency of their cyclotron motion in a static magnetic field26. A major strength of such a set-up is that an RNA can be directly sequenced along with the identification of modifications and their localization (top-down RNA characterization) as we have demonstrated earlier27.A methylation product of the preQTt preQ1 riboswitch by RNA solid-phase synthesis, synthesized the m6preQ1 ligand, and for initial trials, incubated both in a riboswitch-typical aqueous buffer system . The reaction mixture was left overnight at room temperature, after which ultrafiltration with centrifugal concentrator devices was applied to remove the ligand and salts from the RNA for direct characterization by electrospray ionization (ESI) FT-ICR MS. Encouragingly, the high-resolution FT-ICR mass spectrum indeed showed the anticipated signal with a 14.0157 Da mass increase, consistent with a methyl group attached to the RNA 28 released during the methylation reaction will be bound by the riboswitch/ribozyme, thereby blocking the binding of m6preQ1  and observed the expected m3C-to-m3U conversion product (beside emerging fragments from RNA degradation) which additionally confirms that m3C15 RNA was formed selectively , U, A, or G , methylation occurred almost as efficiently as for wildtype RNA , reaction yields decreased to one-fourth compared to the wild-type RNA , we first determined the dissociation constant of the cognate preQ1 ligand to wildtype preQ1 RNA at pH 6 as reference  RNA is higher than to the unreactive C15U mutant because the imino tautomer imC15 as proposed in Fig. Finally, we set out to estimate the affinity of mnce Fig. . The KdI6preQ1, namely O6-ethyl-preQ1 (e6preQ1), O6-benzyl-preQ1 (bn6preQ1), and O6-methyl-preQ0 (m6preQo)  and O6-(4-aminomethyl)benzylguanine (abn6G) did not result in detectable amounts of m3C15 RNA product 22 and G (at least 25-fold lower)29 compared to the cognate ligand preQ1. Therefore, it is reasonable to assume that m6preQ1 is also significantly stronger bound compared to m6G and m6preQ0. It is the 7-aminomethyl substituent of preQ1 and m6preQ1 that has to be present together with its interaction partner, the G5-C16 base pair to reach high affinities22 and high methylation yields  Fig. . These dQo) Fig. . Of furtuct Fig. . The obs1 riboswitch classes also possess self-methylation properties29. Both, preQ1 class II and III riboswitches have an identical architecture of their preQ1-bound pockets, however, this pocket architecture is different from class I31. Characteristically, class II and III apply a preQ1–C trans base pair instead of a cis Watson–Crick pair (class I), and significantly, the O6 atom of preQ1 is rather solvent-exposed with no obvious RNA functionalities for nucleophilic attack nearby. Nevertheless, we tested the well-studied preQ1 II riboswitch from Lactobacillus rhamnosus for methyl transfer of m6preQ1; as expected, methylation was not observed  and a type 3 preQ1 riboswitch (Shigella dysenteriae). Both revealed no methyltransferase activity  that is found in the wobble position of tRNAs containing the GUN anticodon32. The presence of Q in tRNAs improves their ability to read degenerate codons, while the absence of enzymes participating in Q biosynthesis or salvage results in deleterious phenotypes in many organisms34. Noteworthily, O6-methylated versions of the pre-queuosine heterocycle have been found in nature, in form of 2-amino-5-cyano-4-methoxypyrrolopyrimidine produced by Streptomyces35, and as the corresponding N2 -glyosylated natural products huimycin and dapiramicin36. Their occurrence hints at potential cellular availability of the near-cognate ligand m6preQ1 for preQ1 riboswitches. In this context it is also interesting that preQ1 and SAM aptamers have been found in a putative tandem riboswitch architecture which might hint at the regulatory utility of linking m6preQ1 biosynthesis to the concentrations of SAM and preQ136. At this time, one can only speculate about the biological relevance of a riboswitch with potential self-methylation activity. Such a scenario implies that m3C methylation in the riboswitch pocket impairs recognition of the cognate ligand preQ1 and thereby impacts the gene regulatory function of the riboswitch. Thus far, the m3C modification itself is known for tRNAs in position 32 in diverse organisms38, ranging from yeast39, S. Pombe40, parasites such as Trypanosoma brucei41, to mice, rat, and human42. The modification has attracted additional interest when m3C has been identified as dynamic modification also in mRNA, and corresponding writer  and eraser enzymes  have been characterized43. The emerging importance of the modification stimulated the development of novel RNA sequencing approaches, AlkAniline-Seq44 and HAC-seq45, respectively, to reveal m3C sites transcriptome-wide.Our study answers the long-standing question of whether contemporary riboswitches can act as cofactor-dependent ribozymes by a clear yes. Direct evidence became possible after careful analysis of three-dimensional riboswitch architectures in combination with rational ligand design and chemical intuition that finally led to the discovery of methyltransferase activity of the 1 class I riboswitch can serve as starting point for engineering in vitro and in vivo methylation tools. Applications for m3C installation are conceivable not only in cis, but also in trans. First experimental attempts towards this aim by splitting the aptamer into two halves have been successful and led to remarkable 38% of methylation for the preQ1 stem-loop by simply adding the 3′-terminal preQ1 RNA fragment in the presence of cofactor  on controlled-pore glass solid supports53. RNA sequences are given in Supplementary Table 55. RNA oligonucleotides were deprotected with ammonia and butylamine instead of ammonia and methylamine to avoid any possible methylated RNA from deprotection, followed by 1 M tetrabutylammonium fluoride in THF, desalted (Sephadex G25), and purified by denaturing anion exchange chromatography  and 20 mM NaClO4 in 20% aqueous acetonitrile; solvent B was 25 mM Tris-HCl (pH 8.0) and 0.6 M NaClO4 in 20% aqueous acetonitrile; the gradient was: linear, 25–40% (25–45 for longer sequences) with slope of 5 % solvent B per column volume). Customized deprotection conditions were used for m3C RNA (3:1 (v/v) 28–30% aqueous NH3 and EtOH, at 40 °C for 5 h), m4C RNA . The quality of RNAs (purity and identity) was analyzed by anion-exchange HPLC , and HR–ESI–MS  or FT ICR MS (see below). Measured and calculated masses are listed in Supplementary Table RNA oligonucleotides were prepared by solid-phase synthesis using phosphoramidite chemistry .Analytical data (NMR spectra) of m2, 100 mM KCl, 50 mM 2-(N-morpholino) ethanesulfonic acid (MES), pH 6.0). After heating the mixture to 90 °C for 2 min, it was incubated for 48 h at 37 °C. After desalting using a Sep-Pak C18 cartridge, the methylated RNA product was directly analyzed in the mixture or isolated by AE HPLC, desalted again (Sep-Pak C18), and subjected to FT ICR-ESI-MS . For experiments at pH 4 and 5, sodium acetate buffer was used instead of MES; for pH 7, 7.5, and 8, MOPS was used. Several of the experiments were additionally analyzed using 60 and 120 equiv of the cofactor.A typical methylation reaction was carried out in a volume of 200 μl containing 0.5 nmol RNA (2.5 µM) and 75 μM ligand (30 equiv) in buffer solution (2 mM MgCl3C to m3U RNA was examined in a volume of 280 μl with 7 nmol RNA (25 µM) in 25 mM Na2CO3 buffer (pH 10) with 1 mM EDTA at 65 °C for 1.5 h. After desalting (Sep-Pak C18) the m3U RNA was isolated by AE HPLC and analyzed by FT ICR–ESI–MS (see below).The hydrolysis of mK ≤ 5 mg/kg), piperidine (≥99.5%), and imidazole  were from Sigma-Aldrich, and H2O was purified to 18 MΩ·cm at room temperature using a Milli-Q system (Millipore). Experiments were performed on a 7T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Bruker APEX ultra) equipped with an ESI source for (M − nH)n− ion generation and a collision cell through which a flow of Ar gas was maintained for CAD. The mass resolving power of this instrument is routinely  ∼220,000, ∼120,000, and ∼80 000 at m/z 500, 1000, and 1500, respectively, and the mass accuracy is ∼1 ppm with internal calibration and ∼20 ppm with external calibration (Supplementary Table 2O/CH3OH vol/vol with piperidine (2–10 mM) and imidazole (0–10 mM) as additives. Prior to dissociation by CAD, the (M − nH)n− ions under study were isolated in a linear quadrupole; for a more detailed description of the experimental setup for CAD see56. For statistical reasons, between 25 and 500 scans were added for each spectrum , and data reduction utilized the SNAP2 algorithm (Bruker). For desalting, 400 µl of an ammonium salt solution (100 mM ammonium acetate in H2O) was added to 100 µl RNA solution (about 1 nmol in H2O) and concentrated to 100 µl using Vivaspin 500 centrifugal concentrators . The process was repeated five to seven times, followed by six to seven cycles of concentration and dilution with H2O. RNA concentration was determined by UV absorption at 260 nm using a NanoPhotometer (Implen).Methanol was HPLC grade (Acros), ammonium acetate , in which k = kobs and Y = final yield, using OriginPro (2020). All kinetic assays were carried out as three independent replicates.The methylation reactions were carried out as described above  and 50-μl aliquots were taken at desired time points and quenched immediately by adding 50 μl of stop solution (12 M urea). The samples were analyzed by AE HPLC and UV-detection. The peak areas were quantified. The yield versus time data were fit to (fraction unreacted) = 200 instrument at 20 °C in 50 mM MES, 100 mM KCl and 2 mM MgCl2 (pH 6.0). For preQ1, the concentration of Tte preQ1 class I RNA in the measuring cell was typically 0.012 mM; preQ1 ligand was in the syringe at a concentration that was 10-fold higher than the RNA. The c-value was typically around 300. For m6preQ1, the concentration of the Tte preQ1 class I RNA in the measuring cell was typically 0.19 mM; m6preQ1 cofactor was in the syringe at a concentration that was 15-fold higher than the RNA. The c-value was typically around 3. Both Kd determinations were carried out as three independent replicates.ITC measurements were performed on a MicroCal iTCFurther information on research design is available in the Supplementary InformationReporting Summary


Multicomponent transporters are used by bacteria to transport a wide range of nutrients. These systems use a substrate-binding protein to bind the nutrient with high affinity and then deliver it to a membrane-bound transporter for uptake. Nutrient uptake pathways are linked to the colonisation potential and pathogenicity of bacteria in humans and may be candidates for antimicrobial targeting. Here we review current research into bacterial multicomponent transport systems, with an emphasis on the interaction at the membrane, as well as new perspectives on the role of lipids and higher oligomers in these complex systems. All bacteria must scavenge and take up nutrients from their environment to survive. The cellular repertoire of transporter proteins is responsible for both the uptake of essential nutrients such as carbohydrates, amino acids, and metals into the cell, as well as the efflux of toxins and antimicrobial agents out of the cell . Transpol-glutamate  to deliver nutrients to the membrane component. These types of transport systems are very substrate specific, which has been shown to provide a competitive advantage to pathogenic bacteria during colonisation and infection; for example, the uptake of carbohydrates such as sialic acid and fucose , amino alutamate , and metlutamate  and ironlutamate . Multicolutamate .Two important families of multicomponent active transporters are of particular interest: the widely studied ATP-binding cassette (ABC) transporters and the less understood tripartite ATP-independent periplasmic (TRAP) transporters. ABC transporters are found across all kingdoms of life, with many eukaryotic ABC transporters implicated in disease states . The rolvia a secondary active transport mechanism, coupling target molecule transport to the movement of cations down an electrochemical gradient  shows that out of the 2,722 prokaryotic genomes analysed in the database, 1,252 (46%) have at least one TRAP system in the genome. Some species have only one TRAP system (E. coli 042), others have over 20   . MoreoveSM 3043) . It seemBuried in the lipid membrane, transporter proteins are intrinsically hydrophobic and traditionally, they are difficult to isolate and characterise. Nonetheless, there have been significant improvements in membrane protein purification methodologies, particularly with the development of new detergents and membrane mimetics. Importantly, these improvements have led to an expansion in the number of membrane protein structures, although they are still under-represented in the PDB and notably, no TRAP transporter membrane protein structure has been experimentally determined.ABC transporter structure: As of writing, 85 experimentally determined structures of ABC transporters have been deposited in the PDB, revealing wide structural diversity. Common to all ABC transporters is a dimeric membrane-bound component (homo- or hetero-) that brings together two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs), otherwise known as ATP-binding cassettes , with five to eight TMH per subunit, or Type II (large), with ten TMH per subunit. A coupling helix on the cytoplasmic surface interacts tightly with the NBD. Each NBD consists of two sub-domains: a larger RecA-like domain and a smaller α-helical domain that is unique to ABC transporters. Several highly conserved motifs help identify NBDs, including the Walker A and B motifs, and the signature ABC motif (The TMD monomer consists of anywhere between four and ten transmembrane helices (TMH), plus a small number of connecting and coupling BC motif .α/β domains linked by a hinge region about which the SBP can open and close around a ligand, which is likened to a “Venus flytrap” mechanism  down an electrochemical gradient, and also utilise a high-affinity SBP. SBPs were previously thought to be unique to ABC transporters, until the discovery of the first TRAP transporter system, dctPQM, from Rhodobacter capsulatus  Haemophilus influenzae SiaPQM systems. These TRAPs transport sialic acids, a family of nine-carbon amino sugars, the most common of which is N-acetylneuraminic acid  shows both SBP lobes interacting with the TMDs , there is no substrate binding pocket, and the substrate is released into a hydrophobic pocket with no measurable affinity—likened to a “Teflon” cavity. The interactions within the BtuCD-F system, as well as the Type I ModBC-A transporter, have been studied using surface plasmon resonance and single-molecule fluorescence resonance energy transfer (FRET), and more recently by native mass spectrometry . Strikingly, a comparison of these two structures shows the remarkable accuracy of these predictions , nanodiscs, and liposomes . Each mi2 . O’Mara via cryo-EM) than that observed when crystallised with detergent , MNG is predicted to pack more efficiently around the transmembrane region , cardiolipin (CL), phosphatidylethanolamine (PE) as well as methylated derivatives of PE such as phosphatidylcholine (PC) . Furtherin vivo studies, X-ray crystallography, and cryo-EM, which are challenging, time-consuming, and often low resolution, not allowing for proper tail and head identification  has been shown to form dimers, although transport via an elevator-type mechanism does not appear to be coupled between protomers  as the structure is undetermined, although dimeric TRAP SBPs have been structurally characterised by crystallography . These dOur current examples of multicomponent transporters are functional without forming larger assemblies, but there are examples of SBP oligomerisation. This area has not expanded much over the last decade, potentially due to the difficulty of studying these systems. Examples could appear with future work in lipid systems such as nanodiscs.Both the TRAP and ABC transporter systems enable bacteria to selectively import nutrients and are therefore important for colonisation and persistence. While much is known about ABC transporters, from how they bind and interact with substrate-binding proteins, to the conformational transitions of the membrane and nucleotide-binding domains, relatively little is known about TRAP transporters. It is clear that in ABC transporter systems there are a number of different mechanisms of transport, and it is not a case of one-size-fits-all. The current model of the TRAP transport cycle needs further experimental testing, with the key knowledge gap being that there are no experimentally determined structures of the membrane domains. Further research into how these transporter systems function in the membrane environment is also required.


Arabidopsis thaliana, with about 1.5 million strand-specific reads mapped to the chloroplast genome, we could recapitulate most of the complexity of the plastid transcriptome  using virtual Northern blots. Even if the transcripts longer than about 2500 nucleotides were missing, the study of the co-occurrence of editing and splicing events identified 42 pairs of events that were not occurring independently. This study also highlighted a preferential chronology of maturation events with splicing happening after most sites were edited.Plastid gene expression involves many post-transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short-read RNA-Seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full-length transcripts. This information is crucial, however, when it comes to understanding the interplay between the various steps of plastid gene expression. Here, we describe a protocol to study the plastid transcriptome using nanopore sequencing. In the leaf of  Plastids are derived from the endosymbiosis between photosynthetic organisms and an ancestral Eukaryote. Although most of the initial symbiont genes have been transferred to the nucleus during the course of evolution, plastids of land plants and other photosynthetic Eukaryotes still maintain a small but essential genome. It mainly encodes subunits of each of the photosynthetic complexes  and some of the plastid gene expression (PGE) machinery . Most ofA striking feature of PGE is the importance and complexity of the post-transcriptional maturation steps. In addition to the intron removal by RNA splicing  and the ndhA second exon [atpF splicing is severely reduced in the aef1 mutant in which the editing of atpF_12707 is abolished [Arabidopsis thaliana chloroplast RNA editing is affected in a mutant deficient for the exoribonuclease PNPase [A. thaliana, editing of mitochondrial ccmB_17869 by MEF19 depends on the editing of ccmB_17884 by MEF37 [Physcomitrium patens, editing of the mitochondrial ccmFc-C103 by PpPPR_65 controls editing of ccmFc-C122 by PpPPR_71 [Various investigations, both in vitro and in organellar gene expression mutant plants, have indeed revealed situations where the different maturation events can influence each other. For example, intron removal is a prerequisite for editing in the ond exon  and atpFbolished . Arabidoe PNPase . Editingby MEF37 . SimilarPpPPR_71 ,16.These dependencies are usually explained according to two models. First, one maturation event can modify the RNA secondary structure necessary for the second maturation. Second, the proteins responsible for the maturation can interact with each other or, more directly, target several maturation events. Most studies, however, only focused on a limited set of transcripts or RNA maturation events precluding any general conclusions. This illustrates the urgent need for the development of global approaches capable of simultaneously studying all the RNA maturation processes, at the transcriptomic level. This issue has recently been tackled by the increasing use of Illumina-based RNA-Seq strategies to study PGE from transcription to translation ,21,22,23Although this has considerably increased the power and sensitivity of PGE analyses, it is ill-suited to study the potential coordination between maturation steps. The short reads used by Illumina technology (the maximum insert size of Illumina TruSeq RNA libraries reaches around 350 base-pairs) make it impossible to monitor the co-occurrence of these events on single RNA transcripts that can be several kilobases long. An alternative would be to take advantage of other sequencing technologies such as PacBio or Oxford Nanopore. They theoretically allow the sequencing of full-length cDNAs or RNA and should therefore overcome the current technical limitations . A majorA. thaliana plastid transcriptome by sequencing full-length non-polyadenylated and polyadenylated cDNAs using the Oxford Nanopore technology (ONT). This analysis identified all known post-transcriptional maturation events and provided an overview of their coordination in normal growth conditions.In this work, we describe the analysis of the A. thaliana plants grown in long-day conditions at 20 °C, we mapped between 1.55 million and 2.69 million stranded reads (mapping rate between 98.5% and 99.8%) to the A. thaliana genome including between 10% and 40% to the plastid genome and between 0.3% and 0.8% to the mitochondrial genome. The median error rate was between 4% and 4.4%. The rRNA depletion was very efficient with less than 0.1% of reads mapping to rRNA loci. More than 99.5% of the reads mapped to the annotated nuclear genes corresponding to the sense orientation, a proportion similar to Illumina stranded RNA-Seq. Most of the reads (99%) were between 195 and 2141 nucleotides (nt) long with a median size of 852 nt and a maximum size of 4805 nt. In A. thaliana, 7261 genes are producing transcripts longer than 2141 nt and more than 390 genes (including the plastid ycf2 gene) are producing transcripts longer than 4800 nt. Based on the whole transcriptome, the 3′ to 5′ transcript coverage was better with our protocol than for similar samples analyzed using Illumina sequencing for transcripts below 1500 nt  technology developed to synthesize full-length cDNAs . BecausepsbB to petD genomic region  and 1800 nt but also two minor isoforms around 370 nt and 2600 nt. This profile is also compatible with the regular Northern blot. However, Felder et al. [petB showed four major isoforms at 750, 1100, 1450, and 1800 nt. A faint isoform may be present at 2250 nt. These isoforms were also detected by Felder et al. [petD, we found two major isoforms around 1450 and 1800 nt and minor isoforms around 990 and 2225 nt. We missed the larger isoforms detected by Felder et al. [petD transcript which seemed to be replaced by our 990 nt isoform. The detection of sharp “bands” of the expected size in our virtual Northern blots confirms that the majority of the nanopore reads correspond to full-length cDNAs but this result also confirms that transcripts longer than 2–2.5 kb are under-represented in our sequencing libraries.Reads mapping to r et al.  also detr et al.  who founr et al.  but alsopetB and petD Northern blots to a particular isoform.In these complex loci, it is sometimes difficult to identify all the bands on a regular Northern blot. For example, Felder et al. did not associate the 2200 nt transcript of their petB with a spliced petD . For psaA, we observed a strong 5′ to 3′ bias. It may illustrate the pattern of transcript degradation of the 5300 nt long psaA-psaB-rps14 transcript [In anscript  which isp-value < 2.2 × 10−16) and Ruwe et al. [p-value < 2.2 × 10−16).Finally, post-transcriptional maturation events can be quantitatively analyzed. Known editing sites could be detected with rates comparable to leaf datasets  previouse et al.  , it is easy to statistically analyze the possible coordination between these events.clpP introns, petD and petB introns), editing and splicing events  and between editing events . This partial dependency also occurred between different genes  belonging to polycistronic transcripts. Some sites of coordinated events like ndhD_116290 and ndhD_116281, ndhB_95650 and ndhB_95644 or ndhB_95419 and the ndhB intron could be very close but the others were separated by more than 100 nt.Although 1596 co-occurring events could theoretically be expected with 14 splicing and 43 editing events analyzed, only 138 co-occurrences were detected at least once. This is, however, expected as all events are not found on a single transcript . Out of A more detailed analysis shows that maturation intermediates  and FT  columns of psbE_64109 generally occurred before RNA editing at psbF_63985, and that the splicing of petD preferentially occurred before the splicing of petB. The maturation of clpP generally started with RNA editing at clpP_69942 followed by the splicing of the second intron and finished by the splicing of the first intron. For ndhD, the maturation preferentially started with RNA editing at ndhD_116785 followed by ndhD_116494 then both ndhD_116290 and ndhD_116281 to finish with ndhD_117166, the editing site creating the start codon of ndhD. For the ndhB transcript, the chronology of the maturation seemed more convoluted as three sites  were edited independently of the other maturation events. RNA editing at ndhB_97016 seemed to occur first followed by editing at the four sites 96,579, 95,650, 95,608 and 94,999. The maturation of ndhB ended with RNA editing at sites 96,698, 96,419, 95,644 and, probably slightly later, its splicing. To confirm the order deduced from the co-occurrence analysis for transcripts requiring more than three maturation events , we identified the reads covering all the maturation events and counted the frequency of the various intermediates  but 35 (57%) were compatible with the proposed chronology. The reads corresponding to alternative maturation chronologies are probably the result of sequencing errors. Given the nanopore sequencing error rate at around 4% and the fact that this analysis considered five positions in ndhD and 12 in ndhB, only 81.6% (0.965) of the ndhD reads and 62% (0.9612) of the ndhB reads are expected to be error-free at these positions.This analysis suggests that RNA editing at mediates . Out of ndhB (12 events) and ndhD transcripts  strongly suggest that it is associated with the initial RNA-RNA ligation at the 3′ end of transcripts. It has indeed been described that the ligation step was sensitive to secondary structures at the 3′ end . Maybe tFollowing transcription, plastid transcripts undergo a complex array of modifications and maturation and the recent massive use of RNA-Seq based strategies has led to an unprecedented knowledge about its different steps. What is sorely lacking, however, is a global understanding of the interplay between RNA editing, splicing and processing.Initially thought to be mainly independent ,37, therOur results show that co-maturation of several sites tends to occur even when located far apart on their cognate transcript. This implies that all of the actors of these different processing events are grouped or co-localized, likely in the nucleoid .atpF intron and RNA editing at the atpF_12707 site are clearly not independent  maturation of the transcripts before initiating their translation. Although there is currently no known underlying mechanism to support this hypothesis, it could at least explain why partially edited RNA editing sites are generally more edited in ribosome-associated RNAs than on the steady-state pool of transcripts [Editing and splicing of organellar transcripts are required to get mRNA translated into functional proteins as editing often restores conserved amino acids  and splichondria  or maizechondria  showed ter genes . This susplicing . In addinscripts ,50. In anscripts ,52.Despite the modest size of the dataset and its rather simple analysis, the results presented in this study highlight the potential of long-read RNA-Seq for the analysis of plastid and mitochondrial transcriptomes. Even if the molecular protocol still needs improvements to capture the longest transcripts, it provides access to the full complexity of this transcriptome and already showed numerous links between splicing and editing. For analytical reasons, we did not include the analysis of processing in this study but nanopore RNA-Seq is suited for this type of analysis  and we aCol-0 plants were grown in soil in growth chambers with 16 h of light per day at 20 °C for 5 weeks. Fifteen minutes before the onset of lights, 2 adult leaves were flash-frozen in liquid nitrogen. Total RNA was extracted using Nucleozol  followed by a purification with AMPure RNA XP beads . Three independent experiments were performed to get three biological replicates.https://forgemia.inra.fr/guillem.rigaill/nanopore_chloro (accessed on 18 October 2021). Briefly, 10 fmoles of the RNA oligo /5Phos/rNrNrNrNrUrGrArArUrGrCrArArCrArCrUrUrCrUrGrUrArC/3InvdT/  was ligated to the 3′ end of 100 ng of total RNA using 10 U of T4 RNA ligase 1 . Ligated RNA was depleted of rRNA using the QIAseq FastSelect -rRNA Plant Kit  before a full-length cDNA synthesis using the SMARTScribe™ Reverse Transcriptase  and the oligos AAGCAGTGGTATCAACGCAGAGTACrGrG + G and AAGCAGTGGTATCAACGCAGAGTACGTACAGAAGTGTTGCATTC . Full-length cDNAs were amplified with the SeqAmp DNA Polymerase  using the AAGCAGTGGTATCAACGCAGAGTAC primer and purified with AMPure XP beads . 35 fmoles of amplified cDNAs were converted to a nanopore sequencing library with the PCR barcoding kit  and then sequenced on an R10.3 MinIon flow-cell .The step-by-step protocol for the construction of the sequencing library is available online at https://forgemia.inra.fr/guillem.rigaill/nanopore_chloro (accessed on 18 October 2021). They were mapped on the col-0 genomic sequence with Minimap2 v2.1 [The raw data were base-called and demultiplexed with Guppy v5.0.7 (Oxford Nanopore Technologies) using the dna_r10.3_450 bps_hac model. Reads were then oriented using the in-house script “fastq_processing.sh” which uses LAST v1179  and CUTAap2 v2.1 . Transcrap2 v2.1 . The Illap2 v2.1 . The plap-values were adjusted with an FDR [p-value < 0.1 in at least 2 of the 3 replicates and an adjusted p-value < 0.005 on the pool of the 3 replicates were considered significant. Commented R scripts to annotate reads, create contingency table, perform Fisher’s exact tests and generate the result table are available online at https://forgemia.inra.fr/guillem.rigaill/nanopore_chloro (accessed on 18 October 2021).The maturation events analyzed in this study are listed in h an FDR . Only papetB), from position 77200 to position 77500 on the Watson strand (petD), from position 74487 to position 74706 on the Watson strand (psbH) or from position 74254 to position 74378 on the Crick strand (psbN) using samtools [https://forgemia.inra.fr/guillem.rigaill/nanopore_chloro (accessed on 18 October 2021).The splicing and editing rates were measured from pooled reads of the 3 replicates. Virtual Northern blots were generated by extracting the length of the reads mapping from position 75700 to position 76000 on the Watson strand (samtools  and bedtsamtools . The siz


Here, we compare the technical features of different ‘in cell’ hybridization approaches with a focus on their advantages, limitations, and current and potential future applications.RNA-binding proteins are crucial to the function of coding and non-coding RNAs. The disruption of RNA–protein interactions is involved in many different pathological states. Several computational and experimental strategies have been developed to identify protein binders of selected RNA molecules. Amongst these, ‘ RNA-binding proteins (RBPs) are of primary importance to the structure and function of different RNA species. Messenger RNAs are specifically associated with proteins in dynamic mRNA–protein complexes (RNPs) during their entire cell life cycle . RNA-binDisruption of ‘physiological’ RNA–protein interactions can cause aberrant mRNA splicing events and/or alteration of ncRNA function, affecting genetic, autoimmune, neurological, and tumoral diseases as well as cancer drug resistance ,9,10,11.Both computational and experimental methods have been developed to study RNA–protein interactions ,18,19. C‘in cell’ because these methods are applied to cell culture samples that do not adequately represent the complex environment of a living organism. Conversely, vIPR (in vivo Interactions by Pulldown of RNA) can be considered properly an in vivo method because proteins binding to a target RNA are identified from a crosslinked and lysed living system; in this case, C. elegans [Both protein-centric and RNA-centric experimental approaches are commonly defined as ‘in vivo’ if they reveal the RNA–protein interactions as present in the cell or as ‘in vitro’ if they show an interplay outside the physiological cell context. In this review, we substitute the term ‘in vivo’ with  elegans .In cell methods identifying the global protein interactomes of selected RNA molecules (RNA-centric) can be divided into the primary categories of tag-mediated/CRISPR-based approaches and hybridization capture approaches. In the tag-mediated approach, the target tagged RNA construct is transfected and expressed in a specific cell system and the RNA–protein complexes formed are purified through the associated tag. Examples of tag-mediated methods are: tandem RNA affinity purification (TRAP) [n (TRAP) , RNA affn (TRAP) , RNA–pron (TRAP) , RBP purn (TRAP) ,27, MS2 n (TRAP) , tobramyn (TRAP)  and simin (TRAP) ,31,32. Rn (TRAP) . Despitein cell hybridization methods have been developed: ChIRP-MS (comprehensive identification of RNA binding proteins by mass spectrometry), CHART-MS , RAP-MS (RNA antisense purification and mass spectrometry), HyPR-MS (hybridization purification of RNA–protein complexes followed by mass spectrometry), and PAIR (peptide nucleic acid (PNA)-assisted identification of RBPs). Other similar approaches have been applied to specific biological questions [Hybridization approaches are able to identify RBPs associated with specific transcripts reflecting the biological cell environment because they are based on purification of the endogenous target RNA that has been cross-linked with the associated proteins ,34. Seveuestions ,36. in cell RNA-centric hybridization method should be easy to set up and to perform in terms of cost and time, should guarantee a high efficiency and specificity, and should be versatile enough to function in different biological environments. Here, based on these parameters, we compare five recently developed hybridization approaches in terms of their user-friendliness, purification, and post-capture phases. We also discuss possible future directions of these methods.The ideal Capture Oligonucleotide (CO) design represents the first critical step that impacts on the success of the entire experiment. The ideal COs should hybridize to the target RNA with high specificity and efficiency. CO number, length, composition, modification, and the choice of the target RNA region are tightly interconnected with the different capture strategies and hybridization conditions , consist of unmodified DNA or RNA sequences, and are biotinylated in order to permit the isolation of the hybridized RNA–protein complexes with streptavidin-coated magnetic beads . DiffereFocusing heavily on the fundamental aspect of probe design, CHART utilizes an RNAse H assay to identify the accessible single-stranded regions, under the same crosslinking conditions as in the capture experiment. This approach is highly accurate but expensive and time-consuming due to the high number of probes and qPCR assays needed to perform the RNAse H assays . The bes8 cells. On the other hand, the discovery of the protein interactors of c-Myc (60 copies per cell) [Ankh mRNAs [4–1 × 108 cells), shows the efficacy of in cell hybridization methods for less abundant targets. Increasing the cell number represents the most readily accessible experimental parameter to isolate an adequate number of capture-eluted transcripts and consequently of the associated proteins in order to be above the limit of detection of the mass spectrometer. In reality, selecting a cell system expressing high levels of the studied transcript while also obtaining high capture efficiency and mass spectrometry sensitivity is not always straightforward. On the other hand, growing a large number of cells can increase costs and potentially increase non-specific background. Therefore, it is important to choose a system that results in a ‘sweet spot’ for the number of cells needed. By considering mass spectrometry sensitivity for peptides in complex biological samples  [Cell number and cell type choices should take into account both technical and biological aspects. Ideally, the RNA to be studied should be analyzed in a cell system where it is expressed at high levels. At the same time, a cell line should be chosen where the selective RNA–protein interactome identification to be investigated supports the biological motivation for the study. Theoretically, the lower the RNA abundance in the chosen cell type, the greater the number of cells that will need to be employed. er cell)  and Ankhkh mRNAs , of Norakh mRNAs , SAMMSONkh mRNAs , and SPRkh mRNAs , still ulecules)  and measlecules) ,45,46.Finally, the use of a ‘comparative’ strategy, either using different cell types or conditions that serve to highlight differences, may help in identification of RBPs with specific biological roles. For example, an impressive ChIRP study  used thrin cell hybridization protocols  of hybridization. Usually, hybridization temperature (Thy) should be set to 18–24 °C below the calculated CO Tm (perfect hybrids) to achieve a greater hybridization rate ,59. Denain cell hybridization methods can be divided into full tiling and single/multiple probe strategies. RAP-MS and ChIRP-MS use COs covering the target RNA along its full length (full tiling strategies), avoiding the potential loss of fragments created by crosslinking and/or solubilization steps and permitting a homogeneous and robust capture  transcript levels (using RT-qPCR), (b) translated protein levels , and (c) transcript function of non-coding RNAs or biological processes accomplished by the product of the target coding transcript . Examples of this approach include specific Xist interactors identified in RAP-MS and ChIRP-MS that affect lncRNA mediated silencing ,40, or oin cell hybridization methods have shown accuracy in identifying protein interactomes of selected RNA molecules  and, consequently, time and economic requirements. The improvement of capture efficiency and/or mass spectrometry (MS) sensitivity becomes primary for this issue. In this context, the further expansion of multiplex capabilities of HyPR-MS, past the three-fold multiplex capability, and MS based proteomics technological advances, like single-cell proteomics, could allow for a scaling down of material. The main challenge for the widespread adoption of in vivo hybridization methods is the study of low copy number RNAs (<50 copies per cell), which represent the most abundant transcripts , decreasFurthermore, although most of RNA species, like mRNA, lncRNA, small nuclear RNA (snRNA), viral RNA, and rRNA, have been studied using in vivo hybridization methods, it is yet to be defined if tRNA or miRNA are accessible for hybridization purification.New biological approaches should be considered to improve the current state of the art. For example, cell cycle synchronization and transcript activation or silencing may be performed to have a more controlled experiment with reduced potential noise arising from the cell cycle and functional state heterogeneity. In ChIRP-MS, this issue has been partially addressed by engineering a cell system with a doxycycline-inducible Xist cDNA .Another issue that should be further explored is related to the comparison of the RNA–protein interactomes. Several conditions can be compared  and each one can be useful for a specific purpose. Currently, the information obtained by a ‘comparative’ strategy is focused on the protein interactors differing between the two tested conditions. For a more in-depth analysis, quantitative and post-translational modification (PTM) differences of the shared proteins should be considered. In fact, it is possible that the same RNA protein interactor found in both conditions could exhibit a distinct biological function if present at a different level or with a different PTM pattern.in cell hybridization methods to tissue samples (in tissue hybridization methods) to obtain information more pertinent to the reality of complex living organisms. Finally, another challenge could be the application of


Hepatitis E virus (HEV) is the major cause of acute viral hepatitis in several countries in Europe. HEV is acquired mainly by consumption of contaminated pork but can also be transmitted through blood transfusion. HEV infection is usually self-limited but can become persistent in immunocompromised persons. During the first 30 months of HEV RNA universal screening of blood donations in Catalonia, Spain, we identified 151 HEV RNA–positive donations . Most infected donors reported consumption of pates and sausages, and 58% were negative for HEV IgM and IgG. All HEV isolates belonged to genotype 3. All infected donors spontaneously resolved the infection, and no neurologic symptoms and reinfections were observed after 1 year of follow-up. Since the implementation of HEV RNA universal screening, no new cases of transfusion-transmitted HEV infection were reported. Our data indicate HEV screening of blood donations provides safer blood for all recipients, especially for immunosuppressed persons. Orthohepevirus)  in pools of 16 samples . We analyzed individually all samples in reactive pools by using the same assay  and selected HEV RNA‒containing blood components.During November 1, 2017‒April 30, 2020, we tested 655,523 blood donations, plasma samples, and platelet apheresis samples in Catalonia for HEV RNA by using the Procleix HEV assay . We performed HEV serologic testing by using Mikrogen IgM and IgG Detection Assays (https://www.mikrogen.de) and biochemical analysis  for all HEV RNA‒positive donations.We assessed HEV RNA confirmation and viral load by using an in-house quantitative reverse transcription PCR (RT-PCR) for the open reading frame 3 region adapted from Slot et al. (Some donors might have had a previous (archived) blood donation within 6 months before HEV RNA identification. We retested these archived plasma samples by using the HEV RNA Procleix HEV assay for individual samples.We collected age, sex, postal code, and country of birth for all HEV RNA‒positive donors. All donors received a notification to follow-up on their HEV infection at the blood bank in 1 month. HEV RNA tested individually, HEV IgG and IgM, and biochemical parameters were reanalyzed for the follow-up sample. At the time of follow-up, donors completed a questionnaire evaluating symptoms of acute hepatitis, meat consumption, type of meat products , type of dwelling , wastewater system , and travel history outside Catalonia 2 months before blood donation.In addition, donors who were still HEV RNA reactive at follow-up received the opportunity to visit a clinician at the liver unit of Vall d’Hebron Hospital between 2 and 6 months after HEV RNA detection in the blood bank. The clinician evaluated the hepatic and extrahepatic symptoms related to acute HEV infection, and another serum sample was obtained to determine the presence of HEV RNA, HEV IgG and IgM, and the biochemical parameters. If otherwise qualified, donors were reinstated for donation 6 months after the last HEV RNA‒positive result.https://www.thermofisher.com) and 0.35 nmol/L of each primer (forward: 5′-CCGACAGAATTGATTTCGTCGGC-3′ and reverse: 5′-ACTCCCGRGTYTTACCYACCTT-3′). We performed a nested PCR by using Platinum Taq DNA Polymerase (Invitrogen), 0.32 nmol/L of each primer (forward: 5′-TCGTCTCAGCCAATGGCGAGCC-3′ and reverse: 5′-CASARAANGTCTTNGARTACTGCT-3′), and 30 cycles of standard PCR conditions. We loaded amplicons onto a 2% agarose gel, subjected the amplicons to electrophoresis, and purified specific bands by using the QIAquick Gel Extraction Kit . We sequenced purified material by using the Sanger method and a 3130xL Genetic Analyzer System .We amplified a major fragment of reading frame 2 (nt positions 5145‒7127) by using a nested reverse transcription nested RT-PCR. We performed reverse transcription and the first PCR by using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase  signed an additional research informed consent. The Clinical Research Institutional Review Board of Vall d’Hebron University Hospital in Barcelona approved the study (PR[BST]351/2017).2 test. p values <0.05 were considered significant.Prevalence was expressed as percentages and 95% CIs. Quantitative variables were expressed as means, medians, and SDs. We compared percentages by using the χDuring November 1, 2017‒April 30, 2020, we screened 655,523 blood donations HEV RNA in pools of 16 samples. All reactive pools were resolved to an individual reactive donation, which was confirmed by quantitative RT-PCR. During the 30-month period, we selected 151 HEV RNA‒positive donations representing an overall prevalence of 1 case/4,341 donations . The prevalence of HEV RNA‒positive donations according to month of detection during the 2.5 years of the study fluctuated, and no seasonality of HEV cases was observed , panel AMost (62.9%) donors with acute HEV infection were men; mean age ± SD was 41.5 ± 12.9 years, and 92.7% were born in Spain . A signiHEV-infected blood donors were evenly distributed by province: 107 donors in Barcelona, 18 in Tarragona, 14 in Girona, and 12 in Lleida , panel AThe median viral load was 503 IU/mL  . All HEVAt the time of donation, 63 (42%) of HEV RNA–positive donors were positive for HEV IgM and 45 (30%) were positive for HEV IgG/IgM . IncreasA total of 111 (73.5%) HEV RNA‒positive donors completed the questionnaire with information about clinical symptoms, diet, type of housing, and travel history to determine possible routes of HEV acquisition. Only 2 donors reported symptoms of acute hepatitis, such as pain/aches or abdominal pain. However, 24.3% of HEV RNA donors reported general unrest and 15.3% reported dark urine or light stools during 1 or 2 weeks before or after donation .One HEV RNA‒positive donor was vegetarian, and no risk factor could be associated with HEV infection. However, most HEV-infected donors consumed undercooked meat, especially pork (42.3%); liver pate or sausages (65.6%); or shellfish, especially mollusks (63%), 2 months before blood donation. Moreover, 50% of them reported consuming nonbottled water. In addition, 2 HEV RNA‒positive donors consumed game meat 1 month before blood donation.Regarding travel history, 26 HEV-infected donors traveled outside Spain in the 2-month period before blood donation. Moreover, 11 persons visited farms 2 months before blood donation, and 20 persons lived on a farm or in a rural area.During the first month after RNA detection, 127 HEV RNA‒positive donors attended follow-up appointments at a blood bank . At thisA physician at the Liver Unit of Vall d’Hebron Hospital reevaluated 29 persons who had detectable HEV RNA or altered bilirubin/transaminase levels at the 1-month follow-up. At this point, all persons had shown seroconversion to HEV IgM , 24.1% were still HEV RNA positive, only 1 person had an increased bilirubin level, and all persons had standard levels of transaminases. Despite 2 patients who had mild weakness when raising an arm, an electromyography ruled out proximal myopathy. The remaining persons did not have signs of acute hepatitis E or any extrahepatic manifestation.In this cohort of HEV RNA‒positive blood donors, the maximum duration of detection of RNA was 71 days after blood donation. Long-term follow-up (6 months to >1 year) showed that none of the HEV RNA‒positive donors showed development of chronic HEV infection, and 79% continued to be HEV IgM positive 6 months after the HEV RNA‒positive donation. All these donors remained HEV IgG positive, and none were reinfected with HEV.Thirty archived samples obtained during the 6 months before the HEV RNA‒positive donor identification were analyzed by HEV RNA in individual sample (95% limit of detection 11 IU/mL). Twenty-nine samples were HEV RNA negative in individual sample testing, but 1 archived sample was positive. This sample corresponded to a platelet apheresis that had been collected, tested negative in minipools of 16 samples, and transfused. The viral load was ≈40 IU/mL. In this instance, the hospital that had received the HEV RNA‒contaminated platelet apheresis was informed of potential risks and advised to take appropriate action. The physician informed the blood bank center that no signs of acute hepatitis E were observed, and 3 months after the transfusion, the recipient was negative for HEV RNA and HEV IgG/IgM.The Catalonia blood bank provides all blood components  to all hospitals in Catalonia. Half of blood component recipients are immunocompromised patients. Because we implemented the HEV RNA universal screening in minipools of 16 samples during November 2017, no new cases of transfusion-transmitted hepatitis E have been reported in Catalonia.In Spain, universal screening of blood donations for HEV is not mandatory. During 2013, the prevalence of HEV RNA in blood donations in Catalonia was 1 case/3,333 donations, tested individually, whereas HEV IgG seroprevalence was 20% , in contrast to what was previously reported in England and Ireland, where the highest incidence was seen in young donors (18‒24 years of age) . Genotype 3 virus causes zoonotic infections in pigs, wild boars, and deer. It is clear that most HEV infections are acquired in Spain because only 33% of donors reported travel abroad in the 2-month period before donation (We identified consumption of undercooked meat or pork-derived foods (liver pate or sausages) in 42% and 66% of HEV-infected donors, respectively. A study analyzed the presence of HEV RNA in different organs and tissues of 45 apparently healthy pigs from 9 slaughterhouses in Spain  and did not cause a transfusion-transmitted HEV infection in the recipient. A previous study estimated that the infectious dose is ≈20,000 IU of HEV RNA , and no cases of HEV-transfusion-transmitted infection have been reported in our region. Moreover, most HEV-infected donors were asymptomatic, none showed development of chronic HEV infection, and no HEV reinfection was observed. Our data support the suggestion that HEV RNA screening of blood donations provides safer blood for all recipients, especially for immunosuppressed patients.