Strand RNA Research Articles

Barcode demultiplexing of nanopore sequencing raw signals by unsupervised machine learning.

Introduction: Oxford Nanopore Technologies (ONT) is a third generation sequencing approach that allows the analysis of individual, full-length nucleic acids. ONT records the alterations of an ionic current flowing across a nano-scaled pore while a DNA or RNA strand is threading through the pore. Basecalling methods are then leveraged to translate the recorded signal back to the nucleic acid sequence. However, basecall generally introduces errors that hinder the process of barcode demultiplexing, a pivotal task in single-cell RNA sequencing that allows for separating the sequenced transcripts on the basis of their cell of origin. Methods: To solve this issue, we present a novel framework, called UNPLEX, designed to tackle the barcode demultiplexing problem by operating directly on the recorded signals. UNPLEX combines two unsupervised machine learning methods: autoencoders and self-organizing maps (SOM). The autoencoders extract compact, latent representations of the recorded signals that are then clustered by the SOM. Results and Discussion: Our results, obtained on two datasets composed of in silico generated ONT-like signals, show that UNPLEX represents a promising starting point for the development of effective tools to cluster the signals corresponding to the same cell.

Synthesis and properties of a novel modified nucleic acid, 2′-N-methanesulfonyl-2′-amino-locked nucleic acid

2′-Amino-locked nucleic acid has a functionalizable nitrogen atom at the 2′-position of its furanose ring that can provide desired properties to a nucleic acid as a scaffold. In this study, we synthesized a novel nucleic acid, 2′-N-methanesulfonyl-2′-amino-locked nucleic acid (ALNA[Ms]) and conducted comparative studies on the physical and pharmacological properties of the ALNA[Ms] and on conventional nucleic acids, such as 2′-methylamino-LNA (ALNA[Me]), which is a classical 2′-amino-LNA derivative, and also on 2′,4′-BNA/LNA (LNA). ALNA[Ms] oligomers exhibited binding affinities for the complementary RNA strand that are similar to those of conventional nucleic acids. Four types of ALNA[Ms] nucleosides exhibited no genotoxicity in bacterial reverse mutation assays. The knockdown abilities of Malat1 RNA using the Matat1 antisense oligonucleotide (ASO) containing ALNA[Ms] were higher than those of ALNA[Me] and were closer to those of LNA. Furthermore, the ASO containing ALNA[Ms] showed different tissue tropism from that containing LNA. ALNA[Ms] exhibited biological activities that were distinct from conventional constrained nucleic acids, suggesting the possibility that ALNA[Ms] can serve as novel modified nucleic acids in oligonucleotide therapeutics.

Mixed anhydrides at the intersection between peptide and RNA autocatalytic sets: evolution of biological coding.

We present a scenario for the origin of biological coding, a semiotic relationship between chemical information stored in one location that links to chemical information stored in a separate location. Coding originated from cooperation between two, originally separate, collectively autocatalytic sets (CASs), one for nucleic acids and one for peptides. Upon interaction, a series of RNA folding-directed processes led to their joint cooperativity. The aminoacyl adenylate was the first covalent association made by these two CASs and solidified their interdependence, and is a palimpsest of this era, a relic of the original semiotic relationship between RNA and proteins. Coding was driven by selection pressure to eliminate waste in CASs. Eventually a 1 : 1 relationship between single amino acids and short RNA pieces was established, i.e. the 'genetic code'. The two classes of aaRS enzymes are remnants of the complementary information in two RNA strands, as postulated by Rodin and Ohno. Every stage in the evolution of coding was driven by the downward selection on the components of a system to satisfy the Kantian whole. Coding was engendered because there were two chemically distinct classes of polymers needed for open-ended evolution; systems with only one polymer cannot exhibit this characteristic. Coding is thus synonymous with life as we know it.

Knock-down of YME1L1 induces mitochondrial dysfunction during early porcine embryonic development.

YME1L1, a mitochondrial metalloproteinase, is an Adenosine triphosphate (ATP)-dependent metalloproteinase and locates in the mitochondrial inner membrane. The protease domain of YME1L1 is oriented towards the mitochondrial intermembrane space, which modulates the mitochondrial GTPase optic atrophy type 1 (OPA1) processing. However, during embryonic development, there is no report yet about the role of YME1L1 on mitochondrial biogenesis and function in pigs. In the current study, the mRNA level of YME1L1 was knocked down by double strand RNA microinjection to the 1-cell stage embryos. The expression patterns of YME1L1 and its related proteins were performed by immunofluorescence and western blotting. To access the biological function of YME1L1, we first counted the preimplantation development rate, diameter, and total cell number of blastocyst on day-7. First, the localization of endogenous YME1L1 was found in the punctate structures of the mitochondria, and the expression level of YME1L1 is highly expressed from the 4-cell stage. Following significant knock-down of YME1L1, blastocyst rate and quality were decreased, and mitochondrial fragmentation was induced. YME1L1 knockdown induced excessive ROS production, lower mitochondrial membrane potential, and lower ATP levels. The OPA1 cleavage induced by YME1L1 knockdown was prevented by double knock-down of YME1L1 and OMA1. Moreover, cytochrome c, a pro-apoptotic signal, was released from the mitochondria after the knock-down of YME1L1. Taken together, these results indicate that YME1L1 is essential for regulating mitochondrial fission, function, and apoptosis during porcine embryo preimplantation development.

Abstract 2732: A self-replicating RNA precision medicine approach to therapeutic protein delivery of narrow therapeutic index biomolecules

Abstract Protein drug replacement using nucleic acid technologies has been a sought-after solution for in situ production of proteins with poor half-lives and challenging manufacturability. Linear mRNA approaches have failed clinically in this technological use case due to low levels of protein expression and poor durability. Self-replicating RNA (srRNA) is a new technology for expression of biotherapeutics combining the advantages of fully synthetic drug products with higher peak levels of protein expression with active expression persisting out to 49 days. RBI-2000 is a novel srRNA encapsulated in a lipid nanoparticle and encoding two distinct proteins on the same strand of RNA. The first protein is a pro-inflammatory multimeric cytokine to promote de novo immune cell generation and infiltration. The second molecule is a single chain high affinity protein antagonist of downstream mediators of the inflammasome to prevent sterile inflammation, aberrant angiogenesis, and tumor invasiveness. We selected a novel alphaviral vector which demonstrated an enhanced peak level of protein expression vs. the prototypical vector that has been used in other RNA products. These encoded proteins elicited a pharmacodynamic response that had no evidence of decay in the first 7 days following a single treatment down to doses of 0.01 mcg. Using an implanted MC38 tumor model, tumor control was obtained at the lowest, single dose tested at 0.1 mcg of RBI-2000. When tested in combination with a checkpoint inhibitor (CPI), a single dose of the CPI and a single dose of RBI-2000 at 10 mcg resulted in 80% tumor control in the mice (8/10 mice). Tumor re-challenge of the mice at a site distal to the original implantation showed 100% protection. Collectively, these data demonstrate the feasibility of novel srRNA vectors as a protein drug replacement approach including for the expression of complex, multimeric cocktails of proteins. Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle P. Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shigeki Miyake-Stoner, Christopher A. Rabiola, Erika J. Crosby, Zachary C. Hartman, Herbert K. Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA precision medicine approach to therapeutic protein delivery of narrow therapeutic index biomolecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2732.

Suppression of Lung Cancer Malignancy by Micellized siRNA through Cell Cycle Arrest (Adv. Healthcare Mater. 11/2023)

Gene Therapy In article 2202358, Minseok Kwak, Peter Chang-Whan Lee, and co-workers show effective tumor suppression using RNAi with just three DNA and RNA strands. This transfection reagent-free technique self-assembles siRNAs and covers DNA micelles to create nanomedicine for lung cancer. Raw data files are used to create a montage background, recognizing the work of graduate students.

Resalis takes aim at noncoding RNA

Since the advent of the Moderna and Pfizer-BioNTech COVID-19 vaccines, RNA-based drug and vaccine candidates have garnered significant cash and attention. RNA is a hot topic. According to the team at Resalis Therapeutics , it’s also a hot target. The Italy-based company launched Feb. 22 with €10 million ($10.7 million) to develop therapies targeting noncoding RNA to treat metabolic diseases. Noncoding RNA is RNA that does not get translated into a protein. One such type, microRNA, gloms onto a strand of messenger RNA (mRNA) and blocks a portion of that strand from being translated. As the name suggests, microRNAs are very small—usually around 20–24 nucleotides—and they can bind to several different mRNAs, says Riccardo Panella, a Resalis cofounder and its chief scientific officer. With its lead candidate, Resalis is targeting microRNA-22 (miR-22). “We say it’s the master regulator of metabolism,” says Sakari Kauppinen, also a cofounder, as well as Resalis’s

Efficient CRISPR-Cas13d-Based Antiviral Strategy to Combat SARS-CoV-2

The current SARS-CoV-2 pandemic forms a major global health burden. Although protective vaccines are available, concerns remain as new virus variants continue to appear. CRISPR-based gene-editing approaches offer an attractive therapeutic strategy as the CRISPR-RNA (crRNA) can be adjusted rapidly to accommodate a new viral genome sequence. This study aimed at using the RNA-targeting CRISPR-Cas13d system to attack highly conserved sequences in the viral RNA genome, thereby preparing for future zoonotic outbreaks of other coronaviruses. We designed 29 crRNAs targeting highly conserved sequences along the complete SARS-CoV-2 genome. Several crRNAs demonstrated efficient silencing of a reporter with the matching viral target sequence and efficient inhibition of a SARS-CoV-2 replicon. The crRNAs that suppress SARS-CoV-2 were also able to suppress SARS-CoV, thus demonstrating the breadth of this antiviral strategy. Strikingly, we observed that only crRNAs directed against the plus-genomic RNA demonstrated antiviral activity in the replicon assay, in contrast to those that bind the minus-genomic RNA, the replication intermediate. These results point to a major difference in the vulnerability and biology of the +RNA versus -RNA strands of the SARS-CoV-2 genome and provide important insights for the design of RNA-targeting antivirals.

A shape-shifting nuclease unravels structured RNA

RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts. Dis3-like 2 (Dis3L2) is a 3′–5′ exoribonuclease that plays a critical role in human development. Dis3L2 independently degrades structured substrates, including coding and noncoding 3′ uridylated RNAs. While the basis for Dis3L2’s substrate recognition has been well characterized, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining cryogenic electron microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for RNA processing. We discovered a dramatic conformational change that is triggered by double-stranded RNA (dsRNA), repositioning two cold shock domains by 70 Å. This movement exposes a trihelix linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not single-stranded RNA, degradation. These findings reveal the conformational plasticity of Dis3L2 and detail a mechanism of structured RNA degradation.

Order and Complexity in the RNA World

The basic idea of the RNA world as an early step towards life relies on a molecular evolution process based on self-replicating RNA strands. It is probably the oldest and most convincing model for efficient prebiotic evolution. Obviously, the functionality of RNA sequences depends on order (i.e., the definition of their sequence) as well as on complexity (i.e., the length of their sequence). Order and complexity seem to be crucial parameters in the course of RNA evolution. In the following, an attempt is made to define these parameters and to identify characteristic mechanisms of their development. Using a general RNA world scenario including the free monomer units, the sequential order is defined based on statistical thermodynamics. The complexity, on the other hand, is determined by the size of a minimal algorithm fully describing the system. Under these conditions, a diagonal line in an order/complexity-diagram represents the progress of molecular evolution. Elementary steps such as repeated random polymerization and selection follow characteristic pathways and finally add up to a state of high system functionality. Furthermore, the model yields a thermodynamic perspective on molecular evolution, as the development of a defined polymer sequence has a distinct influence on the entropy of the overall system.

Effect of temperature and IRF-9 gene-knockout on dynamics of vRNA, cRNA, and mRNA of viral hemorrhagic septicemia virus (VHSV)

The replication of viral hemorrhagic septicemia virus (VHSV) in appropriate host cells depends on environmental factors and the host cell's immunity. The dynamics of each VHSV RNA strand (vRNA, cRNA, and mRNA) in different conditions can provide a clue on the viral replication strategies, which can be a base for the development of efficient control measures. As VHSV is known to be sensitive to temperature and type I interferon (IFN) responses, in this study, we analyzed the effect of temperature difference (15 °C and 20 °C) and IRF-9 gene knockout on the dynamics of the three VHSV RNA strands in Epithelioma papulosum cyprini (EPC) cells using a strand-specific RT-qPCR. The tagged primers designed in this study successfully worked to quantify the three strands of VHSV. In the results of the temperature effect, the higher speed in viral mRNA transcription and the significantly higher (more than 10 times at 12–36 h) copy number of cRNA at 20 °C compared to those at 15 °C suggested the positive effect of high temperature on VHSV replication. In the results of the IRF-9 gene knockout effect, although IRF-9 gene knockout did not bring a dramatic effect on VHSV replication compared to the temperature effect, the increase of mRNA in IRF-9 KO cells was faster than normal EPC cells, which was reflected in the copy numbers of cRNA and vRNA. The IRF-9 gene knockout effect was not dramatic even in the replication of rVHSV-ΔNV-eGFP that harbors eGFP gene ORF instead of NV gene ORF. These results suggest that VHSV may be highly susceptible to pre-activated type I IFN responses but not highly susceptible to post-infection-mediated type I IFN responses or lowered type I IFN before infection. In both experiments of temperature effect and IRF-9 gene knockout effect, the copy number of cRNA never exceeded the copy number of vRNA at all assay times, suggesting that the binding efficiency of the RNP complex to the 3′ end of cRNA might be lower than that to the 3′ end of vRNA. Further research is needed to elucidate the regulatory mechanism that limits the amount of cRNA at an appropriate level during VHSV replication.

Biochemical and mutational studies of an endonuclease V from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A.

Endonuclease V (EndoV), which is widespread in bacteria, eukarya and Archaea, can cleave hypoxanthine (Hx)-containing DNA or RNA strand, and play an essential role in Hx repair. However, our understanding on archaeal EndoV's function remains incomplete. The model archaeon Sulfolobus islandicus REY15A encodes a putative EndoV protein (Sis-EndoV). Herein, we probed the biochemical characteristics of Sis-EndoV and dissected the roles of its seven conserved residues. Our biochemical data demonstrate that Sis-EndoV displays maximum cleavage efficiency at above 60°C and at pH 7.0-9.0, and the enzyme activity is dependent on a divalent metal ion, among which Mg2+ is optimal. Importantly, we first measured the activation energy for cleaving Hx-containing ssDNA by Sis-EndoV to be 9.6 ± 0.8kcal/mol by kinetic analyses, suggesting that chemical catalysis might be a rate-limiting step for catalysis. Mutational analyses show that residue D38 in Sis-EndoV is essential for catalysis, but has no role in DNA binding. Furthermore, we first revealed that residues Y41 and D189 in Sis-EndoV are involved in both DNA cleavage and DNA binding, but residues F77, H103, K156 and F161 are only responsible for DNA binding.

Altered Mechanisms for Acid-Catalyzed RNA Cleavage and Isomerization Reactions Models.

RNA strand cleavage by 2'-O-transphosphorylation is catalyzed not only by numerous nucleolytic RNA enzymes (ribozymes) but also by hydroxide or hydronium ions. In experiments, both cleavage of the 5'-linked nucleoside and isomerization between 3',5'- and 2',5'-phosphodiesters occur under acidic conditions, while only the cleavage reaction is observed under basic conditions. An ab initio path-integral approach for simulating kinetic isotope effects is used to reveal the reaction mechanisms for RNA cleavage and isomerization reactions under acidic conditions. Moreover, the proposed mechanisms can also be combined through the experimental pH-rate profiles.

D-RNA selects L-amino acids in a newly characterized aminoacyl-RNA loop ligation.

Biological life adheres to molecular asymmetry to an extent that chirality is considered a requisite for the earliest biochemical systems. Particularly, L-amino acids and D-sugars (in nucleic acids) are almost exclusive throughout biology, while purely chemical synthesis pathways for these molecules (which necessarily predate biological synthesis pathways) yield an even split of both enantiomers. Thus, the origins-of-life field has outstanding interest in physical and chemical mechanisms for symmetry breaking. Numerous investigations on the origins of homochirality have yielded explanations including magnetic fields, spin-polarized electrons, crystallization, and the Soai reaction. However, proposed mechanisms tend to target molecules of a single species or those resulting from a particular synthesis pathway. These mechanisms are significant only because chemical reactions with such enantioenriched species are assumed to propagate asymmetry throughout chiral molecules. We experimentally suggest such proliferation of asymmetry through an enantiopreferential and self-amplifying reaction between amino acids and RNA - loop ligation. The loop ligation reaction takes place with two RNA strands in a nicked stem-loop, in which the 3’ hydroxyl of one strand is aminoacylated by an amino acid chemically activated with imidazole, forming an aminoacyl ester. The amino acid ligates to the imidazole-activated 5’ phosphate of a second RNA strand, forming a phosphoramidate linkage. The final product is a closed stem loop with an amino acid-bridged backbone. Our results demonstrate five-fold preference for incorporation of L-amino acids in a backbone of D-RNA. We determined that enantioselectivity occurs most prominently in the loop-closing step based on experiments which isolated individual kinetic steps of the loop ligation process. Because aminoacylation is the cornerstone of RNA-coded protein synthesis, the enantiopreference observed in prebiotically plausible loop ligation offers a chemical axis for the selection of L-amino acids in a D-RNA world.

Rapid live-cell sequential multiplexed imaging with fluorogenic RNAs.

Multiplexed detection of different cellular targets is an important goal in cell biology. However, multiplexed imaging in living cells is still extremely challenging. In terms of fluorescence imaging, significant spectral overlaps limit the number of distinguishable fluorophores that can be detected simultaneously. To solve this problem, in this project, we introduce a sequential imaging strategy termed “sequential Fluorogenic RNA Imaging-Enabled Sensor” (seqFRIES), to separately detect these spectrally overlapped fluorophores over time. Fluorogenic RNA aptamers are RNA strands that recognize and activate the fluorescence of otherwise non-fluorescent dye molecules. Here, we explored a list of reported fluorogenic RNA/dye pairs and identified five orthogonal ones. After genetically encoding all these fluorogenic RNAs together in cells, the corresponding dye molecules are added, imaged, and removed in a sequential manner. A very fast fluorescence activation and deactivation (in several minutes) was observed with these RNA/dye pairs. Taking advantages of such “image-and-wash” cycles, the multiplexing capacity of this imaging system is no longer limited by the excitation/emission spectral overlap of the fluorescent molecules. As a proof of concept, we show that the seqFRIES can perform robustly in both bacterial and mammalian cells, enabling multiplexed imaging of different RNAs, metabolites, and signaling molecules. We envision that this versatile multiplexed reporter system can be potentially adapted for imaging the cellular colocalization and dynamics of various target analytes, again inside living cells.

RNA structure controls assembly of multi-layered paraspeckles.

Biomolecular condensates mediate key cellular functions from gene expression to signaling. Condensates assemble via multivalent interactions amongst proteins and nucleic acids and often behave as liquid-like droplets. An essential requirement for condensate function is the establishment of a distinct compositional identity. How cells maintain coexisting populations of different condensates, despite the propensity of liquids to fuse, is poorly understood. Multi-layered condensates, comprising layers of distinct biomolecular composition, are useful for understanding the establishment of molecular identity and the opposition to mixing. We set out to uncover molecular cues that encode multi-layered condensates by studying nuclear paraspeckles, composed of a core and shell. Paraspeckles are scaffolded by a long noncoding RNA called NEAT1 that bridges the core and shell and recruits different proteins to each layer. The molecular features of NEAT1 that encode for each layer are unknown. We found that isolated core or shell fragments of NEAT1 can assemble condensates with the paraspeckle core-associated protein FUS. When both RNAs are present, condensates do not fuse but instead form a core and shell reminiscent of paraspeckles. Quantitative imaging showed that condensates formed with shell RNA display a higher protein/RNA ratio compared to core RNA. Probing RNA structure uncovered that shell RNA was more well-structured compared to core RNA, suggesting that RNA structure contributes to differential protein recruitment to paraspeckle layers. Consistent with this idea, disrupting the native structures of each RNA via melting and rapid cooling drove dramatic changes in protein and RNA recruitment. Thus, the structural features along an RNA strand likely control the spatial arrangement of the RNA within paraspeckles and tune protein recruitment to each layer. More broadly, our findings suggest that RNA structure is a powerful control knob that determines condensate identity and function in diverse contexts.

Broken up but still living together: how ARGONAUTE's retention of cleaved fragments explains its role during chromatin modification.

Throughout the eukaryotic kingdoms, small RNAs direct chromatin modification. ARGONAUTE proteins sit at the nexus of this process, linking the small RNA information to the programming of chromatin. ARGONAUTE proteins physically incorporate the small RNAs as guides to target specific regions of the genome. In this issue of Genes & Development, Wang and colleagues (pp. 103-118) add substantial new detail to the processes of ARGONAUTE RNA loading, preference, cleavage, and retention, which together accomplish RNA-directed chromatin modification. They show that after catalytic cleavage by the plant ARGONAUTE protein AGO4, the cleaved fragment remains bound. This happens during two distinct RNA cleavage reactions performed by AGO4: first for a passenger RNA strand of the siRNA duplex, and second for a nascent transcript at the target DNA locus. Cleaved fragment retention of the nascent transcript explains how the protein complex accumulates to high levels at the target locus, amplifying chromatin modification.

Towards direct RNA sequencing with electro-optical waveguides.

Long-read sequencing technologies are revolutionizing transcriptome characterization such as decoding full-length transcripts, isoform quantification, splice variants, and RNA containing different modification or mutation sites. Currently, the most widely adopted long-read RNA sequencing technologies are PacBio cDNA isoform sequencing (Iso-Seq) and nanopore-based direct RNA sequencing, both methods having common limitations: First, neither one is capable of sequencing sub-ng levels of DNA/RNA with any practical throughput. Input sample requirements for sequencing (>3 µg/1 Gb genome) are still orders of magnitude higher than the amounts of DNA (∼6 pg) and RNA (10-30 pg) in a human cell. Therefore, typical library preparation from low-input samples involves amplification/conversion steps, which remove any base modifications present in the native strand and biases the sample toward high copy numbers. Second, both technologies suffer from read length bias since they rely on the capture of biomolecules into a nanoscale aperture. Third, PacBio iso-seq detects cDNA products synthesized by a reverse transcriptase rather than the native RNA strand limiting its ability to explore epigenetic modifications. To overcome these issues, we developed electro-optical ZMWs (eZMWs), a novel design of electrically actuatable ZMWs to load biomolecules electrophoretically in order to minimize the length-bias and the input requirements of long-read technologies. Our new devices facilitate high-efficiency length-independent DNA loading into ZMWs and enable rapid identification of DNA molecules. The final goal is to develop a smRNASeq, a single-molecule RNA sequencing system based on electro-optical waveguides that read individual transcripts during reverse transcription in real time using multi-color single-fluorophore detection of labeled nucleotides. Currently, we are in the process of integrating an engineered MarathonRT-streptavidin fusion into eZMWs and developing fluorescently labeled dNTPs compatible with the enzyme to demonstrate MarathonRT binding and smRNA sequencing in eZMWs.

Using single-molecule fluorescence techniques to understand the enzymatic cycle of sex-biased RNA helicases DDX3X and DDX3Y.

Sex differences are pervasive in human health and disease with the most striking differences lying in the sex chromosomes, which encode a group of sex-specific protein homologs. DDX3X and DDX3Y are a pair of sexually dimorphic non-processive ATP-dependent RNA helicases. We previously showed that DDX3Y forms larger, less dynamic phase separated condensates compared to DDX3X, possibly due to the lower ATPase activity of DDX3Y and different dynamic interactions with RNA. We used single-molecule FRET of immobilized, fluorescently labeled RNA duplexes and reported that DDX3X and DDX3Y show different binding dynamics and partial unwinding activities (Shen and Yanas et al., Molecular Cell 2022). To further investigate the enzymatic activities of DDX3, we used multiparameter confocal spectroscopy to obtain unwinding, time-resolved anisotropy, diffusion coefficients, and structural changes by FRET of freely diffusing RNA duplexes upon addition of DDX3, with and without ATP. Duplex RNA with a 5’ overhang was labeled with a Cy3 and Alexa647 FRET pair at the chain termini. The proportion of duplex RNA was quantified and used as a readout for helicase activity, and DDX3X shows higher helicase activity than DDX3Y. With ns time-resolved fluorescence anisotropy, we found that the shorter, non-overhanging strand RNA is released with helicase activity, whereas protein remains bound to the longer RNA strand. Diffusion constants from fluorescence correlation spectroscopy revealed formation of enzymatically active protein clusters at low DDX concentrations. Removal of N- and C-terminal intrinsically disordered regions suppressed formation of clusters and reduced enzymatic activity. We hypothesize that these clusters act as “helicase hubs,” possibly tiny nuclei of liquid granules, which may be translational centers in the cell, leading to efficient translation initiation.

Using Nanocompore to Identify RNA Modifications from Direct RNA Nanopore Sequencing Data

RNA modifications can alter the behavior of RNA molecules depending on where they are located on the strands. Traditionally, RNA modifications have been detected and characterized by biophysical assays, mass spectrometry, or specific next-generation sequencing techniques, but are limited to specific modifications or are low throughput. Nanopore is a platform capable of sequencing RNA strands directly, which permits transcriptome-wide detection of RNA modifications. RNA modifications alter the nanopore raw signal relative to the canonical form of the nucleotide, and several software tools detect these signal alterations. One such tool is Nanocompore, which compares the ionic current features between two different experimental conditions (i.e., with and without RNA modifications) to detect RNA modifications. Nanocompore is not limited to a single type of RNA modification, has a high specificity for detecting RNA modifications, and does not require model training. To use Nanocompore, the following steps are needed: (i) the data must be basecalled and aligned to the reference transcriptome, then the raw ionic current signals are aligned to the sequences and transformed into a Nanocompore-compatible format; (ii) finally, the statistical testing is conducted on the transformed data and produces a table of p-value predictions for the positions of the RNA modifications. These steps can be executed with several different methods, and thus we have also included two alternative protocols for running Nanocompore. Once the positions of RNA modifications are determined by Nanocompore, users can investigate their function in various metabolic pathways. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: RNA modification detection by Nanocompore Alternate Protocol 1: RNA modification detection by Nanocompore with f5c Alternate Protocol 2: RNA modification detection by Nanocompore using Nextflow.