Search

RNA-catalysed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyses the site-specific synthesis of 1-methyladenosine (m\(^1\)A) in RNA, using O\(^6\)-methylguanine (m\(^6\)G) as methyl group donor. Here we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2'-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution.

An RNA duplex modified with a merocyanine dimer was synthesized and exciton dynamics within this dimer were studied using ultrafast third- and fifth-order transient absorption spectroscopy.

Covalent crosslinking of DNA strands provides a useful tool for medical, biochemical and DNA nanotechnology applications. Here we present a light-induced interstrand DNA crosslinking reaction using the modified nucleoside 5-phenylethynyl-2’-deoxyuridine (\(^{Phe}\)dU). The crosslinking ability of \(^{Phe}\)dU was programmed by base pairing and by metal ion interaction at the Watson-Crick base pairing site. Rotation to intrahelical positions was favored by hydrophobic stacking and enabled an unexpected photochemical alkene-alkyne [2+2] cycloaddition within the DNA duplex, resulting in efficient formation of a \(^{Phe}\)dU-dimer after short irradiation times of a few seconds. A \(^{Phe}\)dU dimer-containing DNA was shown to efficiently bind a helicase complex, but the covalent crosslink completely prevented DNA unwinding, suggesting possible applications in biochemistry or structural biology.

Arene‐fluoroarene interactions offer outstanding possibilities for engineering of supramolecular systems, including nucleic acids. Here, we implement the tolane‐perfluorotolane interaction as base pair replacement in DNA. Tolane (THH) and perfluorotolane (TFF) moieties were connected to acyclic backbone units, comprising glycol nucleic acid (GNA) or butyl nucleic acid (BuNA) building blocks, that were incorporated via phosphoramidite chemistry at opposite positions in a DNA duplex. Thermodynamic analyses by UV thermal melting revealed a compelling stabilization by THH/TFF heteropairs only when connected to the BuNA backbone, but not with the shorter GNA linker. Detailed NMR studies confirmed the preference of the BuNA backbone for enhanced polar π‐stacking. This work defines how orthogonal supramolecular interactions can be tailored by small constitutional changes in the DNA backbone, and it inspires future studies of arene‐fluoroarene‐programmed assembly of DNA.

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-d-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir., Quantitative biochemical assays and high-resolution cryo-EM analysis reveal how the COVID-19 antiviral drug candidate molnupiravir causes lethal viral mutagenesis by the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2.

Fluorogenic RNA aptamers are synthetic RNAs that have been evolved by in vitro selection methods to bind and light up conditionally fluorescent organic ligands. Compared with other probes for RNA detection, they are less invasive than hybridization-based methods (FISH, molecular beacons) and are considerably smaller than fluorescent protein-recruiting systems (MS2, Pumilio variants). Fluorogenic aptamers have therefore found widespread use as genetically encodable tags for RNA detection in live cells and have also been used in combination with riboswitches to construct versatile metabolite sensors for in vitro use. Their success builds on a fundamental understanding of their three-dimensional structure to explain the mechanisms of ligand interaction and to rationally design functional aptamer devices. In this protocol, we describe a supramolecular FRET-based structure probing method for fluorogenic aptamers that exploits distance- and orientation-dependent energy transfer efficiencies between site-specifically incorporated fluorescent nucleoside analogs and non-covalently bound ligands, exemplified by 4-cyanoindol riboside (4CI) and the DMHBI

The pseudopeptide backbone provided by N-(2-aminoethyl)-glycine oligomers with attached nucleobases has been widely utilized in peptide nucleic acids (PNAs) as DNA mimics. Here we demonstrate the suitability of this backbone for the formation of structurally defined dye stacks. Toward this goal a series of peptide merocyanine (PMC) dye oligomers connected to a N-(2-aminoethyl)-glycine backbone were prepared through peptide synthesis. Our concentration-, temperature- and solvent-dependent UV/Vis absorption studies show that under the control of dipole-dipole interactions, smaller-sized oligomers consisting of one, two or three dyes self-assemble into defined duplex structures containing two up to six chromophores. In contrast, upon further extension of the oligomer, the chosen peptide backbone cannot direct the formation of a defined duplex architecture anymore due to intramolecular aggregation between the dyes. For all aggregate species a moderate aggregation-induced emission enhancement is observed.

Nearly all classes of coding and non-coding RNA undergo post-transcriptional modification, including RNA methylation. Methylated nucleotides are among the evolutionarily most-conserved features of transfer (t)RNA and ribosomal (r)RNA1,2. Many contemporary methyltransferases use the universal cofactor S-adenosylmethionine (SAM) as a methyl-group donor. SAM and other nucleotide-derived cofactors are considered to be evolutionary leftovers from an RNA world, in which ribozymes may have catalysed essential metabolic reactions beyond self-replication3. Chemically diverse ribozymes seem to have been lost in nature, but may be reconstructed in the laboratory by in vitro selection. Here we report a methyltransferase ribozyme that catalyses the site-specific installation of 1-methyladenosine in a substrate RNA, using O6-methylguanine as a small-molecule cofactor. The ribozyme shows a broad RNA-sequence scope, as exemplified by site-specific adenosine methylation in various RNAs. This finding provides fundamental insights into the catalytic abilities of RNA, serves a synthetic tool to install 1-methyladenosine in RNA and may pave the way to in vitro evolution of other methyltransferase and demethylase ribozymes. A methyltransferase ribozyme, along with the small-molecule cofactor O6-methylguanine, is shown to catalyse the site-specific installation of 1-methyladenosine in various RNAs, providing insights into the catalytic abilities of RNA.

RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence‐based strategies reveal information on structure and dynamics of RNA aptamers. Herein, we report the incorporation of the universal emissive nucleobase analog 4‐cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to the bound ligands DMHBI+ or DMHBO+. The photophysical properties of the new nucleobase–ligand‐FRET pair revealed structural restraints for the overall RNA aptamer organization and identified nucleotide positions suitable for FRET‐based readout of ligand binding. This strategy is generally suitable for binding‐site mapping and may also be applied for responsive aptamer devices., A spy in the RNA: The fluorescent nucleobase analogue 4‐cyanoindole was site‐specifically incorporated into the fluorogenic Chili RNA aptamer as a reporter for binding of several ligand classes. This first application of FRET between an isomorphic nucleobase donor and an intrinsically fluorogenic ligand revealed position‐dependent quantum yields and FRET efficiencies for mapping of the ligand binding site.

In vitro selected ribozymes are promising tools for site‐specific labeling of RNA. Previously known nucleic acid catalysts attached fluorescently labeled adenosine or guanosine derivatives through 2′,5′‐branched phosphodiester bonds to the RNA of interest. Herein, we report new ribozymes that use orthogonal substrates, derived from the antiviral drug tenofovir, and attach bioorthogonal functional groups, as well as affinity handles and fluorescent reporter units through a hydrolytically more stable phosphonate ester linkage. The tenofovir transferase ribozymes were identified by in vitro selection and are orthogonal to nucleotide transferase ribozymes. As genetically encodable functional RNAs, these ribozymes may be developed for potential cellular applications. The orthogonal ribozymes addressed desired target sites in large RNAs in vitro, as shown by fluorescent labeling of E. coli 16S and 23S rRNAs in total cellular RNA., A strategy for illuminating RNA with fluorescent derivatives of the antiviral acyclic nucleoside phosphonate Tenofovir is presented. Tenofovir transferase ribozymes were identified by in vitro selection and are shown to be orthogonal to nucleotidyl transferase ribozymes, thus enabling site‐specific dual labeling of RNA.

RNA-cleaving deoxyribozymes have found broad application as useful tools for RNA biochemistry. However, tedious in vitro selection procedures combined with laborious characterization of individual candidate catalysts hinder the discovery of novel catalytic motifs. Here, we present a new high-throughput sequencing method, DZ-seq, which directly measures activity and localizes cleavage sites of thousands of deoxyribozymes. DZ-seq exploits A-tailing followed by reverse transcription with an oligo-dT primer to capture the cleavage status and sequences of both deoxyribozyme and RNA substrate. We validated DZ-seq by conventional analytical methods and demonstrated its utility by discovery of novel deoxyribozymes that allow for cleaving challenging RNA targets or the analysis of RNA modification states.

Deoxyribozymes are artificially evolved DNA molecules with catalytic abilities. RNA-cleaving deoxyribozymes have been recognized as an efficient tool for detection of modifications in target RNAs and provide an alternative to traditional and modern methods for detection of ribose or nucleobase methylation. However, there are only few examples of DNA enzymes that specifically reveal the presence of a certain type of modification, including N6-methyladenosine, and the knowledge about how DNA enzymes recognize modified RNAs is still extremely limited. Therefore, DNA enzymes cannot be easily engineered for the analysis of desired RNA modifications, but are instead identified by in vitro selection from random DNA libraries using synthetic modified RNA substrates. This protocol describes a general in vitro selection stagtegy to evolve new RNA-cleaving DNA enzymes that can efficiently differentiate modified RNA substrates from their unmodified counterpart.

Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio‐macromolecules using 19F nuclear spins and nitroxide radicals in combination with high‐frequency (94 GHz/3.4 T) electron–nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the 19F label enables to access distances up to about 1.5 nm with an accuracy of 0.1–1 Å. The experiment is not limited by the size of the bio‐macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin‐labelled RNA duplexes. The results demonstrate that our simple but strategic spin‐labelling procedure combined with state‐of‐the‐art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state., What did the NO say to the 19 F? Dipolar couplings between nitroxide spin labels and 19F nuclear spins report inter‐spin distances in the range of ≲15 Å at sub‐angstrom accuracy. These couplings are measured with sub‐nanomole spin sensitivity by high frequency (94 GHz) electron–nuclear double resonance (ENDOR) and can be employed to obtain detailed structural insights into biomolecules.

Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m3C32 in the human mitochondrial (mt-)tRNAThr and mt-tRNASer(UCN). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U34G35 and t6A37/(ms2)i6A37, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C32. Several lines of evidence demonstrate the influence of U34, G35, and the m3C32 and t6A37/(ms2)i6A37 modifications in mt-tRNAThr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNAThr/Ser(UCN) lacking METTL8-mediated m3C32 are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m3C32 within mt-tRNAs., RNA modifications are key regulators of RNA functions. Here, the authors identify METTL8 as the enzyme installing m3C32 in mitochondrial tRNAThr/Ser(UCN). Lack of these modifications affects tRNA structure and impairs mitochondrial translation.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited., The recognition of the mRNA codon by the tRNA anticodon is crucial for protein synthesis. Here the authors introduce non-standard nucleotides in bacterial and eukaryotic mRNA to reveal the minimal hydrogen bond requirement of codon-anticodon interaction for efficient and accurate translation.

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication., Remdesivir is a nucleoside analog that inhibits the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and is used as a drug to treat COVID19 patients. Here, the authors provide insights into the mechanism of remdesivir-induced RdRp stalling by determining the cryo-EM structures of SARS-CoV-2 RdRp with bound RNA molecules that contain remdesivir at defined positions and observe that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation.

Coronaviruses use an RNA-dependent RNA polymerase to replicate and transcribe their RNA genome. The structure of the SARS-CoV-2 polymerase was determined by cryo-electron microscopy within a short time in spring 2020. The structure explains how the viral enzyme synthesizes RNA and how it replicates the exceptionally large genome in a processive manner. The most recent structure-function studies further reveal the mechanism of polymerase inhibition by remdesivir, an approved drug for the treatment of COVID-19.

Deoxyribozymes are emerging as modification‐specific endonucleases for the analysis of epigenetic RNA modifications. Here, we report RNA‐cleaving deoxyribozymes that differentially respond to the presence of natural methylated cytidines, 3‐methylcytidine (m3C), N 4‐methylcytidine (m4C), and 5‐methylcytidine (m5C), respectively. Using in vitro selection, we found several DNA catalysts, which are selectively activated by only one of the three cytidine isomers, and display 10‐ to 30‐fold accelerated cleavage of their target m3C‐, m4C‐ or m5C‐modified RNA. An additional deoxyribozyme is strongly inhibited by any of the three methylcytidines, but effectively cleaves unmodified RNA. The mXC‐detecting deoxyribozymes are programmable for the interrogation of natural RNAs of interest, as demonstrated for human mitochondrial tRNAs containing known m3C and m5C sites. The results underline the potential of synthetic functional DNA to shape highly selective active sites., In vitro selected deoxyribozymes respond to the presence of methylcytidines m3C, m4C and m5C in RNA with either enhanced or strongly diminished cleavage rates, and thereby directly reveal the presence of the modified cytidines in target RNA.

This review aims at juxtaposing common versus distinct structural and functional strategies that are applied by aptamers, riboswitches, and ribozymes/DNAzymes. Focusing on recently discovered systems, we begin our analysis with small-molecule binding aptamers, with emphasis on in vitro-selected fluorogenic RNA aptamers and their different modes of ligand binding and fluorescence activation. Fundamental insights are much needed to advance RNA imaging probes for detection of exo- and endogenous RNA and for RNA process tracking. Secondly, we discuss the latest gene expression–regulating mRNA riboswitches that respond to the alarmone ppGpp, to PRPP, to NAD+, to adenosine and cytidine diphosphates, and to precursors of thiamine biosynthesis (HMP-PP), and we outline new subclasses of SAM and tetrahydrofolate-binding RNA regulators. Many riboswitches bind protein enzyme cofactors that, in principle, can catalyse a chemical reaction. For RNA, however, only one system (glmS ribozyme) has been identified in Nature thus far that utilizes a small molecule – glucosamine-6-phosphate – to participate directly in reaction catalysis (phosphodiester cleavage). We wonder why that is the case and what is to be done to reveal such likely existing cellular activities that could be more diverse than currently imagined. Thirdly, this brings us to the four latest small nucleolytic ribozymes termed twister, twister-sister, pistol, and hatchet as well as to in vitro selected DNA and RNA enzymes that promote new chemistry, mainly by exploiting their ability for RNA labelling and nucleoside modification recognition. Enormous progress in understanding the strategies of nucleic acids catalysts has been made by providing thorough structural fundaments (e.g. first structure of a DNAzyme, structures of ribozyme transition state mimics) in combination with functional assays and atomic mutagenesis.

Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.

Deoxyribozymes (DNAzymes) are small, synthetic, single-stranded DNAs capable of catalyzing chemical reactions, including RNA ligation. Herein, we report a novel class of RNA ligase deoxyribozymes that utilize 5&prime, adenylated RNA (5&prime, AppRNA) as the donor substrate, mimicking the activated intermediates of protein-catalyzed RNA ligation. Four new DNAzymes were identified by in vitro selection from an N40 random DNA library and were shown to catalyze the intermolecular linear RNA-RNA ligation via the formation of a native 3&prime, 5&prime, phosphodiester linkage. The catalytic activity is distinct from previously described RNA-ligating deoxyribozymes. Kinetic analyses revealed the optimal incubation conditions for high ligation yields and demonstrated a broad RNA substrate scope. Together with the smooth synthetic accessibility of 5&prime, adenylated RNAs, the new DNA enzymes are promising tools for the protein-free synthesis of long RNAs, for example containing precious modified nucleotides or fluorescent labels for biochemical and biophysical investigations.

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.

RNA‐cleaving deoxyribozymes can serve as selective sensors and catalysts to examine the modification state of RNA. However, site‐specific endonuclease deoxyribozymes that selectively cleave post‐transcriptionally modified RNA are extremely rare and their specificity over unmodified RNA is low. We report that the native tRNA modification N6‐isopentenyladenosine (i6A) strongly enhances the specificity and has the power to reconfigure the active site of an RNA‐cleaving deoxyribozyme. Using in vitro selection, we identified a DNA enzyme that cleaves i6A‐modified RNA at least 2500‐fold faster than unmodified RNA. Another deoxyribozyme shows unique and unprecedented behaviour by shifting its cleavage site in the presence of the i6A RNA modification. Together with deoxyribozymes that are strongly inhibited by i6A, these results highlight that post‐transcriptional RNA modifications modulate the catalytic activity of DNA in various intricate ways., The natural tRNA modification i6A carries an isopentenyl (prenyl) group at the N 6 position of adenine. This modified nucleotide finetunes the active site of RNA‐cleaving deoxyribozymes, resulting in a distinct shift of the cleavage site. DNA catalysts are reported to exclusively cut post‐transcriptionally modified RNAs.

General and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direct

The Chili RNA aptamer is a 52 nt long fluorogen-activating RNA aptamer (FLAP) that confers fluorescence to structurally diverse derivatives of fluorescent protein chromophores. A key feature of Chili is the formation of highly stable complexes with different ligands, which exhibit bright, highly Stokes-shifted fluorescence emission. In this work, we have analyzed the interactions between the Chili RNA and a family of conditionally fluorescent ligands using a variety of spectroscopic, calorimetric and biochemical techniques to reveal key structure–fluorescence activation relationships (SFARs). The ligands under investigation form two categories with emission maxima of ∼540 or ∼590 nm, respectively, and bind with affinities in the nanomolar to low-micromolar range. Isothermal titration calorimetry was used to elucidate the enthalpic and entropic contributions to binding affinity for a cationic ligand that is unique to the Chili aptamer. In addition to fluorescence activation, ligand binding was also observed by NMR spectroscopy, revealing characteristic signals for the formation of a G-quadruplex only upon ligand binding. These data shed light on the molecular features required and responsible for the large Stokes shift and the strong fluorescence enhancement of red and green emitting RNA–chromophore complexes.

N6-methyladenosine (m6A) modifications in RNAs play important roles in regulating many different aspects of gene expression. While m6As can have direct effects on the structure, maturation, or translation of mRNAs, such modifications can also influence the fate of RNAs via proteins termed “readers” that specifically recognize and bind modified nucleotides. Several YTH domain-containing proteins have been identified as m6A readers that regulate the splicing, translation, or stability of specific mRNAs. In contrast to the other YTH domain-containing proteins, YTHDC2 has several defined domains and here, we have analyzed the contribution of these domains to the RNA and protein interactions of YTHDC2. The YTH domain of YTHDC2 preferentially binds m6A-containing RNAs via a conserved hydrophobic pocket, whereas the ankyrin repeats mediate an RNA-independent interaction with the 5′–3′ exoribonuclease XRN1. We show that the YTH and R3H domains contribute to the binding of YTHDC2 to cellular RNAs, and using crosslinking and analysis of cDNA (CRAC), we reveal that YTHDC2 interacts with the small ribosomal subunit in close proximity to the mRNA entry/exit sites. YTHDC2 was recently found to promote a “fast-track” expression program for specific mRNAs, and our data suggest that YTHDC2 accomplishes this by recruitment of the RNA degradation machinery to regulate the stability of m6A-containing mRNAs and by utilizing its distinct RNA-binding domains to bridge interactions between m6A-containing mRNAs and the ribosomes to facilitate their efficient translation.

Nitroxide labels are combined with nucleic acid structures and studied using electron paramagnetic resonance experiments (EPR). As X-ray/NMR structures are unavailable with the nitroxide labels, detailed residue level information, down to atomic resolution, about the effect of these nitroxide labels on local RNA structures is currently lacking. This information is critical to evaluate the choice of spin label. In this study, we compare and contrast the effect of TEMPO-based (NT) and rigid spin (Ç) labels (in both 2’-O methylated and not-methylated forms) on RNA duplexes. We also investigate sequence-dependent effects of NT label on RNA duplex along with the more complex G-quadruplex RNA. Distances measured from molecular dynamics simulations between the two spin labels are in agreement with the EPR experimental data. To understand the effect of labeled oligonucleotides on the structure, we studied the local base pair geometries and global structure in comparison with the unlabeled structures. Based on the structural analysis, we can conclude that TEMPO-based and Ç labels do not significantly perturb the base pair arrangements of the native oligonucleotide. When experimental structures for the spin labelled DNA/RNA molecules are not available, general framework offered by the current study can be used to provide information critical to the choice of spin labels to facilitate future EPR studies.Graphical abstract

General and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direct in vitro selection, characterization, and application of versatile trans-acting 2'-5' adenylyl transferase ribozymes for covalent and site-specific RNA labeling. The design of our partially structured RNA pool allowed for in vitro evolution of ribozymes that modify a predetermined nucleotide in cis (i.e., intramolecular reaction) and can then be easily engineered for applications in trans (i.e., in an intermolecular setup). The resulting ribozymes are readily designed for specific target sites in small and large RNAs and accept a wide variety of N6-modified ATP analogues as small-molecule substrates. The most efficient new ribozyme (FH14) shows excellent specificity toward its target sequence also in the context of total cellular RNA.

Mitochondrial gene expression uses a non‐universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt‐)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt‐tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt‐tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m5C34 mt‐tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt‐tRNAMet function. Together, our data reveal how modifications in mt‐tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine. ![][1] RNA methyltransferase NSUN3 acts specifically on mitochondrial tRNAMet, allowing different codons to be recognised by this single tRNA and offering insight on the consequence of reported disease mutations. [1]: /embed/graphic-1.gif

The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA-protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as sub-complexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing.

Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes)1 or synthetic genetic polymers2. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage3. DNA-catalysed reactions include RNA and DNA ligation in various topologies4,5, hydrolytic cleavage6,7 and photorepair of DNA8, as well as reactions of peptides9,10 and small molecules11,12. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold13. Here we report the crystal structure of the RNAligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms. peerReviewed

5-Formylcytosin (5fC) und 5-Formyluracil (5fU) sind naturliche modifizierte Nucleobasen, die durch Oxidation aus 5-Methylcytosin und Thymin (oder 5-Methyluracil) entstehen. Hier beschreiben wir die chemoselektive Markierung von 5-Formylpyrimidinnucleotiden in DNA und RNA mithilfe fluorogener Aldolkondensationsreaktionen mit 2,3,3-Trimethylindolderivaten. Unter milden und spezifischen Reaktionsbedingungen entstehen aus 5fU und 5fC Hemicyanin-artige Chromophore mit charakteristischen photophysikalischen Eigenschaften. Die Reaktionsbedingungen wurden an DNA-Oligonucleotiden optimiert und konnten direkt auf RNA ubertragen werden. Der positionsspezifische Nachweis gelang durch Fluoreszenzmessung und Primerverlangerungsexperimente. Diese Methode der direkten Markierung von 5-Formylpyrimidinen wird zur Untersuchung von Vorkommen, enzymatischen Transformationen und biologischen Funktionen von epigenetischen/epitranskriptomischen Modifikationen von Nucleobasen in DNA und RNA beitragen.

Catalytic DNAs, also known as deoxyribozymes, are of practical value for the synthesis of structurally or topologically complex RNAs, but little is known about the molecular details of DNA catalysis. We have investigated a deoxyribozyme that catalyzes the formation of a specific intramolecular 2′,5′-phosphodiester bond to produce lariat RNA, which is an important biological intermediate in eukaryotic mRNA splicing. The results of combinatorial mutation interference analysis (CoMA) allowed us to shrink the catalytic core to 70 % of its original length and revealed that the essential part of the deoxyribozyme sequence contained more than 50 % guanosines. Nucleotide analogue interference mapping (dNAIM) and dimethyl sulfate interference (DMSi) experiments provided atomic details of individual guanosine functional groups. Additional spectroscopic experiments and structural probing data identified conformational changes upon metal-ion binding and catalysis. Overall, this comprehensive analysis of the DNA-catalyzed reaction has provided specific insights into the synthesis of 2′,5′-branched RNA, and suggested the general features of deoxyribozymes that catalyze nucleic acid ligation reactions.

Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Herein, we report new RNA-cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N

Many evolutionarily conserved microRNAs (miRNAs) in plants regulate transcription factors with key functions in development. Hence, mutations in the core components of the miRNA biogenesis machinery cause strong growth defects. An essential aspect of miRNA biogenesis is the precise excision of the small RNA from its precursor. In plants, miRNA precursors are largely variable in size and shape and can be processed by different modes. Here, we optimized an approach to detect processing intermediates during miRNA biogenesis. We characterized a miRNA whose processing is triggered by a terminal branched loop. Plant miRNA processing can be initiated by internal bubbles, small terminal loops or branched loops followed by dsRNA segments of 15-17 bp. Interestingly, precision and efficiency vary with the processing modes. Despite the various potential structural determinants present in a single a miRNA precursor, DCL1 is mostly guided by a predominant structural region in each precursor in wildtype plants. However, our studies in fiery1, hyl1 and se mutants revealed the existence of cleavage signatures consistent with the recognition of alternative processing determinants. The results provide a general view of the mechanisms underlying the specificity of miRNA biogenesis in plants. Fil: Moro, Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Chorostecki, Uciel Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Arikit, Siwaret. Kasetsart University; Tailandia Fil: Suarez, Irina Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Hobartner, Claudia. Universität Würzburg; Alemania Fil: Rasia, Rodolfo Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Meyers, Blake C.. Donald Danforth Plant Science Center; Estados Unidos. University of Missouri; Estados Unidos Fil: Palatnik, Javier Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

DNA methylation has a profound impact on the regulation of gene expression in normal cell development, and aberrant methylation has been recognized as a key factor in the pathogenesis of human diseases such as cancer. The discovery of modified nucleobases arising from 5-methylcytosine (5mC) through consecutive oxidation to give 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) has stimulated intense research efforts regarding the biological functions of these epigenetic marks. This Review focuses on the sensitive detection and quantitation of 5fC in DNA and RNA by chemoselective labeling, which aims at discriminating between 5fC and its thymine counterpart 5-formyluracil (5fU), and summarizes single-base resolution sequencing methods for locus-specific mapping of 5mC and its oxidized derivatives.