The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA.

GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.

Uridine-derived ribose fuels glucose-restricted pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease notoriously resistant to therapy1,2. This is mediated in part by a complex tumour microenvironment3, low vascularity4, and metabolic aberrations5,6. Although altered metabolism drives tumour progression, the spectrum of metabolites used as nutrients by PDA remains largely unknown. Here we identified uridine as a fuel for PDA in glucose-deprived conditions by assessing how more than 175 metabolites impacted metabolic activity in 21 pancreatic cell lines under nutrient restriction. Uridine utilization strongly correlated with the expression of uridine phosphorylase 1 (UPP1), which we demonstrate liberates uridine-derived ribose to fuel central carbon metabolism and thereby support redox balance, survival and proliferation in glucose-restricted PDA cells. In PDA, UPP1 is regulated by KRAS-MAPK signalling and is augmented by nutrient restriction. Consistently, tumours expressed high UPP1 compared with non-tumoural tissues, and UPP1 expression correlated with poor survival in cohorts of patients with PDA. Uridine is available in the tumour microenvironment, and we demonstrated that uridine-derived ribose is actively catabolized in tumours. Finally, UPP1 deletion restricted the ability of PDA cells to use uridine and blunted tumour growth in immunocompetent mouse models. Our data identify uridine utilization as an important compensatory metabolic process in nutrient-deprived PDA cells, suggesting a novel metabolic axis for PDA therapy.

Why U matters: detection and functions of pseudouridine modifications in mRNAs.

The uridine modifications pseudouridine (Ψ), dihydrouridine, and 5-methyluridine are present in eukaryotic mRNAs. Many uridine-modifying enzymes are associated with human disease, underscoring the importance of uncovering the functions of uridine modifications in mRNAs. These modified uridines have chemical properties distinct from those of canonical uridines, which impact RNA structure and RNA-protein interactions. Ψ, the most abundant of these uridine modifications, is present across (pre-)mRNAs. Recent work has shown that many Ψs are present at intermediate to high stoichiometries that are likely conducive to function and at locations that are poised to influence pre-/mRNA processing. Technological innovations and mechanistic investigations are unveiling the functions of uridine modifications in pre-mRNA splicing, translation, and mRNA stability, which are discussed in this review.

Exploring a unique class of flavoenzymes: Identification and biochemical characterization of ribosomal RNA dihydrouridine synthase.

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives.

Selective prebiotic formation of RNA pyrimidine and DNA purine nucleosides.

The nature of the first genetic polymer is the subject of major debate1. Although the 'RNA world' theory suggests that RNA was the first replicable information carrier of the prebiotic era-that is, prior to the dawn of life2,3-other evidence implies that life may have started with a heterogeneous nucleic acid genetic system that included both RNA and DNA4. Such a theory streamlines the eventual 'genetic takeover' of homogeneous DNA from RNA as the principal information-storage molecule, but requires a selective abiotic synthesis of both RNA and DNA building blocks in the same local primordial geochemical scenario. Here we demonstrate a high-yielding, completely stereo-, regio- and furanosyl-selective prebiotic synthesis of the purine deoxyribonucleosides: deoxyadenosine and deoxyinosine. Our synthesis uses key intermediates in the prebiotic synthesis of the canonical pyrimidine ribonucleosides (cytidine and uridine), and we show that, once generated, the pyrimidines persist throughout the synthesis of the purine deoxyribonucleosides, leading to a mixture of deoxyadenosine, deoxyinosine, cytidine and uridine. These results support the notion that purine deoxyribonucleosides and pyrimidine ribonucleosides may have coexisted before the emergence of life5.

Rational design of base, sugar and backbone modifications improves ADAR-mediated RNA editing.

AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.

Combining Nanopore direct RNA sequencing with genetics and mass spectrometry for analysis of T-loop base modifications across 42 yeast tRNA isoacceptors.

Transfer RNAs (tRNAs) contain dozens of chemical modifications. These modifications are critical for maintaining tRNA tertiary structure and optimizing protein synthesis. Here we advance the use of Nanopore direct RNA-sequencing (DRS) to investigate the synergy between modifications that are known to stabilize tRNA structure. We sequenced the 42 cytosolic tRNA isoacceptors from wild-type yeast and five tRNA-modifying enzyme knockout mutants. These data permitted comprehensive analysis of three neighboring and conserved modifications in T-loops: 5-methyluridine (m5U54), pseudouridine (Ψ55), and 1-methyladenosine (m1A58). Our results were validated using direct measurements of chemical modifications by mass spectrometry. We observed concerted T-loop modification circuits-the potent influence of Ψ55 for subsequent m1A58 modification on more tRNA isoacceptors than previously observed. Growing cells under nutrient depleted conditions also revealed a novel condition-specific increase in m1A58 modification on some tRNAs. A global and isoacceptor-specific classification strategy was developed to predict the status of T-loop modifications from a user-input tRNA DRS dataset, applicable to other conditions and tRNAs in other organisms. These advancements demonstrate how orthogonal technologies combined with genetics enable precise detection of modification landscapes of individual, full-length tRNAs, at transcriptome-scale.

Crosslinking of base-modified RNAs by synthetic DYW-KP base editors implicates an enzymatic lysine as the nitrogen donor for U-to-C RNA editing.

Sequence-specific cytidine to uridine (C-to-U) and adenosine to inosine editing tools can alter RNA and DNA sequences and utilize a hydrolytic deamination mechanism requiring an active site zinc ion and a glutamate residue. In plant organelles, DYW-PG domain containing enzymes catalyze C-to-U edits through the canonical deamination mechanism. Proteins developed from consensus sequences of the related DYW-KP domain family catalyze what initially appeared to be uridine to cytidine (U-to-C) edits leading to this investigation into the U-to-C editing mechanism. The synthetic DYW-KP enzyme KP6 was found sufficient for C-to-U editing activity stimulated by the addition of carboxylic acids in vitro. Despite addition of putative amine/amide donors, U-to-C editing by KP6 could not be observed in vitro. C-to-U editing was found not to be concomitant with U-to-C editing, discounting a pyrimidine transaminase mechanism. RNAs containing base modifications were highly enriched in interphase fractions consistent with covalent crosslinks to KP6, KP2, and KP3 proteins. Mass spectrometry of purified KP2 and KP6 proteins revealed secondary peaks with mass shifts of 319 Da. A U-to-C crosslinking mechanism was projected to explain the link between crosslinking, RNA base changes, and the ∼319 Da mass. In this model, an enzymatic lysine attacks C4 of uridine to form a Schiff base RNA-protein conjugate. Sequenced RT-PCR products from the fern Ceratopteris richardii indicate U-to-C base edits do not preserve proteinaceous crosslinks in planta. Hydrolysis of a protonated Schiff base conjugate releasing cytidine is hypothesized to explain the completed pathway in plants.

Expanded tRNA methyltransferase family member TRMT9B regulates synaptic growth and function.

Nervous system function rests on the formation of functional synapses between neurons. We have identified TRMT9B as a new regulator of synapse formation and function in Drosophila. TRMT9B has been studied for its role as a tumor suppressor and is one of two metazoan homologs of yeast tRNA methyltransferase 9 (Trm9), which methylates tRNA wobble uridines. Whereas Trm9 homolog ALKBH8 is ubiquitously expressed, TRMT9B is enriched in the nervous system. However, in the absence of animal models, TRMT9B's role in the nervous system has remained unstudied. Here, we generate null alleles of TRMT9B and find it acts postsynaptically to regulate synaptogenesis and promote neurotransmission. Through liquid chromatography-mass spectrometry, we find that ALKBH8 catalyzes canonical tRNA wobble uridine methylation, raising the question of whether TRMT9B is a methyltransferase. Structural modeling studies suggest TRMT9B retains methyltransferase function and, in vivo, disruption of key methyltransferase residues blocks TRMT9B's ability to rescue synaptic overgrowth, but not neurotransmitter release. These findings reveal distinct roles for TRMT9B in the nervous system and highlight the significance of tRNA methyltransferase family diversification in metazoans.

Identification of a novel 5-aminomethyl-2-thiouridine methyltransferase in tRNA modification.

The uridine at the 34th position of tRNA, which is able to base pair with the 3'-end codon on mRNA, is usually modified to influence many aspects of decoding properties during translation. Derivatives of 5-methyluridine (xm5U), which include methylaminomethyl (mnm-) or carboxymethylaminomethyl (cmnm-) groups at C5 of uracil base, are widely conserved at the 34th position of many prokaryotic tRNAs. In Gram-negative bacteria such as Escherichia coli, a bifunctional MnmC is involved in the last two reactions of the biosynthesis of mnm5(s2)U, in which the enzyme first converts cmnm5(s2)U to 5-aminomethyl-(2-thio)uridine (nm5(s2)U) and subsequently installs the methyl group to complete the formation of mnm5(s2)U. Although mnm5s2U has been identified in tRNAs of Gram-positive bacteria and plants as well, their genomes do not contain an mnmC ortholog and the gene(s) responsible for this modification is unknown. We discovered that MnmM, previously known as YtqB, is the methyltransferase that converts nm5s2U to mnm5s2U in Bacillus subtilis through comparative genomics, gene complementation experiments, and in vitro assays. Furthermore, we determined X-ray crystal structures of MnmM complexed with anticodon stem loop of tRNAGln. The structures provide the molecular basis underlying the importance of U33-nm5s2U34-U35 as the key determinant for the specificity of MnmM.

Online Nucleotide Mapping of mRNAs.

Messenger RNA (mRNA) can be sequenced via indirect approaches such as Sanger sequencing and next generation sequencing (NGS), or direct approaches like bottom-up mass spectrometry (MS). Direct sequencing allows the confirmation of RNA modifications. However, the conventional bottom-up MS approach involves time-consuming in-solution digestions that require a large amount of sample, and can lead to the RNase contamination of the LC-MS system and column. Here, we describe a platform that enables online nucleotide mapping of mRNAs via the use of immobilized RNase cartridges and 2D-LC-MS instrumentation. The online approach was compared to conventional offline digestion protocols adapted from two published studies. For this purpose, five model mRNAs of varying lengths (996-4521 nucleotides) and chemistries (unmodified uridine vs 5-methoxyuridine (5moU)) were analyzed. The profiles and sequence coverages obtained after RNase T1 digestion were discussed. The online nucleotide mapping achieved comparable or slightly greater sequence coverage for the 5 mRNAs (5.8-51.5%) in comparison to offline approaches (3.7-50.4%). The sequence coverage was increased to 65.6-85.6 and 69.7-85.0% when accounting for the presence of nonunique digestion products generated by the RNase T1 and A, respectively. The online nucleotide mapping significantly reduced the digestion time (from 15 to <5 min), increased the signal intensity by more than 10-fold in comparison to offline approaches.

Synthesis and Photophysical Characterization of a pH-Sensitive Quadracyclic Uridine (qU) Analogue.

Fluorescent base analogues (FBAs) have become useful tools for applications in biophysical chemistry, chemical biology, live-cell imaging, and RNA therapeutics. Herein, two synthetic routes towards a novel FBA of uracil named qU (quadracyclic uracil/uridine) are described. The qU nucleobase bears a tetracyclic fused ring system and is designed to allow for specific Watson-Crick base pairing with adenine. We find that qU absorbs light in the visible region of the spectrum and emits brightly with a quantum yield of 27 % and a dual-band character in a wide pH range. With evidence, among other things, from fluorescence lifetime measurements we suggest that this dual emission feature results from an excited-state proton transfer (ESPT) process. Furthermore, we find that both absorption and emission of qU are highly sensitive to pH. The high brightness in combination with excitation in the visible and pH responsiveness makes qU an interesting native-like nucleic acid label in spectroscopy and microscopy applications in, for example, the field of mRNA and antisense oligonucleotide (ASO) therapeutics.

4'-C-Acetamidomethyl-2'-O-methoxyethyl Nucleic Acid Modifications Improve Thermal Stability, Nuclease Resistance, Potency, and hAgo2 Binding of Small Interfering RNAs.

In this study, we designed the 4'-C-acetamidomethyl-2'-O-methoxyethyl (4'-C-ACM-2'-O-MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'-C-ACM-2'-O-MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4'-C-ACM-2'-O-MOE thymidine modification at the penultimate position of the 3'-end of dT20 oligonucleotides in the presence of 3'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2'-O-Me, 2'-O-MOE, and 2'-F. In gene silencing studies, we found that the 4'-C-ACM-2'-O-MOE uridine or thymidine modifications at the 3'-overhang in the passenger strand in combination with two 2'-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3'-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4'-C-ACM-2'-O-MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4'-C-ACM-2'-O-MOE modification at the 3'-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4'-C-ACM-2'-O-MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.

Equipping Uridine with Three Dipeptide Motifs for the Inhibition of Radical-Induced DNA Oxidation.

We report the discovery and characterization of antioxidative effects of uridine linked with three dipeptide motifs against DNA oxidation induced by peroxyl radicals. First, the dipeptide motifs are constructed by using the Ugi four-component reaction (Ugi 4CR), in which caffeic, ferulic, sinapic, and syringic acids are used as the carboxylic acid resources, vanillin, benzaldehyde, and p-hydroxybenzaldehyde are used as the aldehyde resources, tyramine- and dopamine-related isocyanides as well as ethyl isocyanoacetate are used as the isocyanide resources, and 2-(p-aminophenyl)ethanol is used as the amine component. We found that the antioxidative effects of the Ugi 4CR products are 1.3-2.8 times higher than those of caffeic, ferulic, sinapic, and syringic acids in the protection of DNA against peroxyl radical-induced oxidation. Moreover, when three Ugi 4CR products are linked with three hydroxyl groups of uridine by using three succinic anhydrides as the linkage, the inhibitory effects of the afforded uridine-dipeptide hybrids against the DNA oxidation increase 4.4-8.9 times (>3 times) compared to that of the Ugi 4CR product. This is due to the hybrid structure consisting of uridine and three motifs of the Ugi 4CR product enabling binding with the DNA strand more efficiently and quenching free radicals more rapidly. Therefore, the hybrid structure constructed by the nucleoside with antioxidative dipeptides offers an additional advantage for protecting DNA against radical-induced oxidation.

Cell-resistant wavelength-shifting molecular beacons made of L-DNA and a clickable L-configured uridine.

Wavelength-shifting molecular beacons were prepared from L-DNA. The clickable anchor for the two dyes, Cy3 and Cy5, was 2'-O-propargyl-L-uridine and was synthesized from L-ribose. Four clickable molecular beacons were prepared and double-modified with the azide dyes by a combination of click chemistry on a solid support for Cy3 during DNA synthesis and postsynthetic click chemistry for Cy5 in solution. Cy3 and Cy5 successfully formed a FRET pair in the beacons, and the closed form (red fluorescence) and the open form (green fluorescence) can be distinguished by the two-color fluorescence readout. Two molecular beacons were identified to show the greatest fluorescence contrast in temperature-dependent fluorescence measurements. The stability of the L-configured molecular beacons was demonstrated after several heating and cooling cycles as well as in the cell lysate. In comparison, D-configured molecular beacons showed a rapid decrease of fluorescence contrast in the cell lysate, which is caused by the opening of the beacons, probably due to degradation. This was confirmed in cell experiments using confocal microscopy. The L-configured molecular beacons are potential intracellular thermometers for future applications.

N3-Methyluridine and 2'-O-Alkyl/2'-Fluoro-N3-methyluridine functionalized nucleic acids improve nuclease resistance while maintaining duplex geometry.

Herein, we report the synthesis of 2'-O-alkyl/2'-fluoro-N3-methyluridine (2'-O-alkyl/2'-F-m3U) phosphoramidites and their incorporation in DNA and RNA oligonucleotides. The duplex binding affinity and base discrimination studies showed that all 2'-O-alkyl/2'-F-m3U modifications significantly decreased the thermal stability and base-pairing discrimination ability. Serum stability study of dT20 with 2'-O-alkyl-m3U modification exhibited excellent nuclease resistance when incubated with 3'-exonucleases (SVPD) or 5'-exonucleases (PDE-II) as compared to m3U, 2'-F, 2'-OMe modified oligonucleotides. MD simulation studies with RNA tetradecamer duplexes illustrated that the m3U and 2'-O-methyl-m3U modifications reduce the duplex stabilities by disrupting the Watson-Crick hydrogen bonding and base-stacking interactions. Further molecular modelling investigations demonstrated that the 2'-O-propyl-m3U modification exhibits steric interactions with amino acid residues in the active site of 3'- and 5'-exonuclease, leading to enhanced stability. These combined data indicate that the 2'-modified-m3U nucleotides can be used as a promising tool to enhance the stability, silencing efficiency, and drug-like properties of antisense/siRNA-based therapeutics.

A path from synthesis to emergency use authorization of molnupiravir as a COVID-19 therapy.

Coronaviruses are a group of enveloped viruses with non-segmented, single-stranded, and positive-sense RNA genomes. It belongs to the 'Coronaviridae family', responsible for various diseases, including the common cold, SARS, and MERS. The COVID-19 pandemic, which began in March 2020, has affected 209 countries, infected over a million people, and claimed over 50,000 lives. Significant efforts have been made by repurposing several approved drugs including antiviral, to combat the COVID-19 pandemic. Molnupiravir is found to be the first orally acting efficacious drug to treat COVID-19 cases. It was approved for medical use in the UK in November 2021 and other countries, including USFDA, which granted approval an emergency use authorization (EUA) for treating adults with mild to moderate COVID-19 patients. Considering the importance of molnupiravir, the present review deals with its various synthetic strategies, pharmacokinetics, bio-efficacy, toxicity, and safety profiles. The comprehensive information along with critical analysis will be very handy for a wide range of audience including medicinal chemists in the arena of antiviral drug discovery especially anti-viral drugs against any variant of COVID-19.

Codon-specific translation reprogramming promotes resistance to targeted therapy.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

Structure-Function Insights into the Dual Role in Nucleobase and Nicotinamide Metabolism and a Possible Use in Cancer Gene Therapy of the URH1p Riboside Hydrolase.

The URH1p enzyme from the yeast Saccharomyces cerevisiae has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD+ synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.

Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms.

Eukaryotic genes often generate a variety of RNA isoforms that can lead to functionally distinct protein variants. The synthesis and stability of RNA isoforms is poorly characterized because current methods to quantify RNA metabolism use short-read sequencing and cannot detect RNA isoforms. Here we present nanopore sequencing-based isoform dynamics (nano-ID), a method that detects newly synthesized RNA isoforms and monitors isoform metabolism. Nano-ID combines metabolic RNA labeling, long-read nanopore sequencing of native RNA molecules, and machine learning. Nano-ID derives RNA stability estimates and evaluates stability determining factors such as RNA sequence, poly(A)-tail length, secondary structure, translation efficiency, and RNA-binding proteins. Application of nano-ID to the heat shock response in human cells reveals that many RNA isoforms change their stability. Nano-ID also shows that the metabolism of individual RNA isoforms differs strongly from that estimated for the combined RNA signal at a specific gene locus. Nano-ID enables studies of RNA metabolism at the level of single RNA molecules and isoforms in different cell states and conditions.