The RNA world hypothesis is supported by the discovery of RNA polymerase ribozymes that can perform RNA-catalyzed RNA replication processes on different RNA templates. Recently, RNA-catalyzed rolling circle synthesis (RCS) on small circular RNA (scRNA) templates has been demonstrated. However, the structural and dynamic properties of scRNA replication and its products and intermediates have not been

Retrotransposons, multi-copy sequences that propagate via copy-and-paste mechanisms, occupy large portions of eukaryotic genomes. A great majority of their manifold copies remain silenced in somatic cells; nevertheless, some are transcribed, often in a tissue-specific manner, and a small fraction retains its ability to mobilize. While it is well characterized that retrotransposon sequences may provide

8-Oxoguanine (8-oxoG) is a common DNA oxidative lesion prevalent in telomeric regions. However, the impact of 8-oxoG modification on the Watson–Crick base pairing energy remains controversial, potentially due to the formation of partially folded intermediates. Here, we used single-molecule magnetic tweezers to characterize the mechanical stability and equilibrium folding/unfolding transitions of human

Spontaneous backbone cleavage of ribonucleic acids (RNAs) in basic aqueous solution has a preference for the 5′ side of guanosine. This phenomenon can also be observed in fully desolvated RNA (M − nH)n− ions subjected to vibrational activation by low-energy collisionally activated dissociation. However, the underlying chemical mechanism of the cleavage reaction has so far remained elusive. Using RNA

Long-range tertiary interactions are a widespread structural feature in viral RNAs (vRNAs) and mRNAs. In the orthoflaviviruses, conserved complementary sequences in the 5′ and 3′ terminal regions have an essential role in viral replication. Long-range pairing of these conserved sequences is proposed to facilitate a switch between two alternative vRNA conformations. Yet the detailed nature of these

HiBiT is an engineered luciferase’s 11-amino-acid component that can be introduced as a tag at either terminus of a protein of interest. When the LgBiT component and a substrate are present, HiBiT and LgBiT dimerize forming a functional luciferase. The HiBiT technology has been extensively used for high-throughput protein turnover studies in cells. Here, we have adapted the use of the HiBiT technology

Naïve mouse embryonic stem cells (mESCs) are characterized by a mixed population of cells in an interconvertible pluripotent state and a totipotent 2-cell (2C)-like state. It remains to be understood how the pluripotent state is maintained while the 2C-like state is suppressed. We show that N-acetyltransferase 10 (Nat10) maintains the pluripotent state and suppresses the 2C-like state in mESCs through

DNA cytosine methylation is an important epigenetic mechanism for transposon silencing and gene regulation in fungi, particularly during sexual reproduction. However, its occurrence in vegetative hyphae and role in defense against mycoviruses is unclear. In this study, we demonstrated that genomic-wide cytosine methylation of the tripartite genomovirus FgGMTV1 occurs in the hyphae of Fusarium graminearum

RecQ helicases can unwind a wide spectrum of DNA structures and thereby protect the cells from genome instability. Unwinding mechanisms have been extensively studied for bacterial and human RecQ helicases. DNA-induced winged helix (WH) domain repositioning and allosteric remodeling of the ATPase domain have been shown to be important for unwinding activity of bacterial RecQ helicases. In contrast,

Circular RNAs (circRNAs), an emerging subclass of noncoding RNAs, have been increasingly recognized as critical regulators in diverse biological functions and cellular processes. Despite their functional significance, the epigenetic mechanisms governing circRNA biogenesis remain poorly understood. Our study reveals that H3K27ac-marked super-enhancers (SEs) significantly enhance both circRNA splicing

Bidirectional promoters (BDPs) hold great promise for applications in synthetic biology by enabling co-expression of multiple genes with minimized promoter size. However, the lack of well-characterized BDPs along with an incomplete understanding of their regulatory mechanisms limits broader applications. Here, we conducted genome-wide screening and characterization of 749 BDP candidates containing

Viral DNA-binding proteins, a type of protamine-like protein, pack the viral genome and promote late viral gene expression. In baculovirus Autographa californica multiple nucleopolyhedrovirus, DNA-binding protein P6.9 plays a crucial role in the production of progeny viruses. Earlier studies suggested the importance of P6.9 phosphorylation states in its function, while the detailed mechanism remains

RNA viruses like SARS-CoV-2 have high mutation rates, which contribute to their rapid evolution. Mutation rates depend on mutation type and can vary between sites in a virus’s genome. Understanding this variation can shed light on the mutational processes at play, and is crucial for quantitative modeling of viral evolution. Using millions of SARS-CoV-2 full-genome sequences, we estimate rates of synonymous

During mitosis, the microtubule depolymerase KIF2C, the tumor suppressor BRCA2, and the kinase PLK1 contribute to the control of kinetochore-microtubule attachments. Both KIF2C and BRCA2 are phosphorylated by PLK1, and BRCA2 phosphorylated at T207 (BRCA2-pT207) serves as a docking site for PLK1. Reducing this interaction results in unstable microtubule-kinetochore attachments. Here we identified that

The arms race between phages and bacteria is dynamic and ongoing, with both continuously acquiring new strategies to outcompete each other during co-evolution. Here, we report bacteriophage T4 exonuclease DexA and an uncharacterized Escherichia coli exonuclease as a rare pair of attack and defense duo arising from the same mechanism. DexA, highly conserved among phages, has two well-characterized biological

Manganese (Mn)-sensing riboswitches protect bacteria from Mn toxicity by upregulating expression of Mn exporters. The Mn aptamers share key features but diverge in other important elements, including within the metal-binding core. Although X-ray crystal structures of isolated aptamers exist, these structural snapshots lack crucial details about how the aptamer communicates the presence or absence of

The mammalian non-homologous end joining (NHEJ) is required for class switch and V(D)J recombination as well as repairing DNA double-strand breaks (DSBs). Initiated by the binding of Ku70/Ku80 (Ku) dimer to DNA ends and the recruitment of the DNA-dependent protein kinase catalytic subunit, NHEJ plays a key role in DSB repair. While the overall function of Ku70 in NHEJ is well documented, the specific

RNA editing modifies cytidines to uridines in plant organelle transcripts so that their sequences differ from the ones predicted from the genomic DNA. This process involves a family of RNA-binding proteins that has significantly expanded, the pentatricopeptide repeat (PPR)-containing proteins. In angiosperms, PPR proteins are found in editosomes associated with accessory proteins. The exact function

Mutation, deletion, or silencing of genes encoding cellular metabolism factors occurs frequently in human malignancies. Neomorphic mutations in isocitrate dehydrogenases 1 and 2 (IDH1/2) promoting the production of R-2-hydroxyglutarate (R-2HG) instead of α-ketoglutarate (αKG) are recurrent in human brain cancers and constitute an early event in low-grade gliomagenesis. Due to its structural similarity

The nuclease prime editor (PEn) combines double-strand break (DSB) induction with reverse transcription for editing. Recently, high-activity PEn forms (e.g. uPEn) have been developed via the concomitant application of DNA repair regulator(s). While the standard uPEn introduces edits only downstream of the nuclease-induced DNA break, we seek innovative designs to enable upstream-directed editing by

The CRISPR/Cas12a [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a] system is known for its intrinsic RNA-guided trans-cleavage activity; however, its RNA detection sensitivity is limited, with conventional methods typically achieving detection limits in the nanomolar range. Here, we report the development of a “pseudo hybrid DNA–RNA” (PHD) assay that significantly

Tn7 transposable elements are known for their sophisticated target-site selection mechanisms. For the prototypical Tn7 element, dedicated transposon-encoded proteins direct insertions to either a conserved site in the chromosome or replicating DNA structures in conjugal plasmids, ensuring the vertical and horizontal spread of the element. While the pathway targeting the attTn7 site in the bacterial

Pseudomonas syringae, a highly destructive plant bacterial pathogen causing severe disease and significant yield losses in agriculture globally, has complex regulatory systems involving many transcriptional factors (TFs). Although the LysR-type transcriptional regulator (LTTR) protein family is a well-known group of TFs involved in diverse physiological functions, the roles of LTTRs in P. syringae

Influenza A viruses (IAVs) contain eight negative-sense single-stranded viral RNA (vRNA) molecules, which are transcribed into messenger RNA (mRNA) and replicated via complementary RNA (cRNA). These processes are tightly regulated, but the precise molecular mechanisms governing the switch from transcription to replication remain elusive. Here, we introduce multiple direct RNA-assisted padlock probing

Chromatin structure during meiosis is different from somatic cells due to the assembly of the synaptonemal complex between homologous chromosome axes. However, genome-wide organizing principles of this meiosis-specific multiprotein complex remain mysterious despite intensive super-resolution imaging analysis. Here, we profiled chromatin occupancy of SYCP3, the key chromatin organizer of synaptonemal

Many proteins that bind specifically to DNA and RNA employ a mechanism known as nucleotide flipping, whereby a nucleotide rotates out of the helical stack and into an active site. This conformational change can be monitored using spectroscopic methods, including fluorescence and nuclear magnetic resonance (NMR). We previously showed that flipping of a 2′-fluoroarabino-substituted nucleotide by thymine

Cas12a trans nuclease activity has been leveraged for nucleic acid detection, often coupled with isothermal amplification to increase sensitivity. However, due to the lack of highly efficient thermostable Cas12a orthologs, use of Cas12a in one-pot combination with high temperature (55-65°C) amplification, such as loop-mediated isothermal amplification (LAMP), has remained challenging. Here, we attempt

Ultraviolet (UV) light causes cyclobutane pyrimidine dimers (CPDs) and other DNA lesions that must be efficiently repaired to prevent cell death and mutagenesis. While mammals utilize the nucleotide excision repair (NER) pathway to repair CPDs, many species primarily utilize photolyase enzymes to repair UV damage. Our understanding of how different genomic and chromatin features impact photolyase repair

Telomere elongation is driven by telomerase, which consists of several proteins and the ncRNA component TLC1 in yeast. While many ncRNAs are terminated via the Nrd1-Nab3-Sen1 (NNS) pathway, we found that TLC1 requires cleavage and polyadenylation factor (CPF)-cleavage factor (CF) mediated 3'end processing and the resulting poly(A) tail to mature into a functional ribozyme. The poly(A) tail is predicted

We describe a DNA-array-based method to infer intramolecular connections in a population of RNA molecules in vitro. First we add DNA oligonucleotide “patches” that perturb the RNA connections, and then we use a microarray containing a complete set of DNA oligonucleotide “probes” to record where perturbations occur. The pattern of perturbations reveals couplings between regions of the RNA sequence,

RecD2 is a superfamily 1B helicase involved in DNA replication and repair, modulating replication restart, fork progression, and RecA recombinase activity. In this work, we have characterized the functions of Bacillus subtilis RecD2 using biochemical and single-molecule approaches. ATPγS binding and low MgCl2 concentrations enhance DNA association, with a preference for forked structures and unstructured

The incorporation of ribonucleotides (rNMPs) into the nuclear genome leads to severe genomic instability, including strand breaks and short 2–5 bp deletions at repetitive sequences. Curiously, the detrimental effects of rNMPs are not observed for the human mitochondrial genome (mtDNA) that typically contains several rNMPs per molecule. Given that the nuclear genome instability phenotype is dependent

Pioneer transcription factors can bind to closed chromatin, initiating its opening and subsequent gene activation. However, the specific features that enable transcription factors to activate particular loci remain largely undefined. Here, we show that the transcription factor p53 unexpectedly initiates epigenetic remodeling at the majority of its binding sites and drives transcription at select loci

Genetic instability is a major hazard threatening the fate of cells and ultimately of organisms. DNA double-strand break (DSB) is a highly toxic lesion, jeopardizing genome stability. Using cytogenetic and differential exome sequencing, we show here that upregulation of the kinase PKB/AKT1 leads to genomic rearrangements and chromosome fusions. By combining various approaches, at the genome scale and

Pioneer transcription factors (PTFs) bind to inaccessible chromatin and recruit collaborating transcription factors to promote chromatin accessibility. However, mechanisms driving PTFs to specify collaborating transcription factor recruitment and chromatin remodeling remain unclear. Here, we utilize inducible expression of a PTF, GATA3, in a basal breast cancer cell line (SUM159PT) to mechanistically

Single-stranded DNA binding (SSB) proteins are essential components of genome metabolism in both bacteria and eukaryotes. Recently demonstrated condensation propensities have placed SSB functions in a new context regarding the organization of nucleic acid-modifying complexes. In this work, we provide functional dissection of the condensation and partner binding properties of Escherichia coli (Ec) SSB

Aminoacyl-tRNA synthetases are critical for accurate genetic translation, attaching amino acids to their corresponding transfer RNA molecules. Alanyl-tRNA synthetase (AlaRS) often misactivates Ser or Gly instead of Ala, which is detrimental unless corrected by its editing functions. The paradox of misactivating larger Ser by AlaRS was considered inevitable due to its inherent design, sharing an essential

Intracellular pH (pHi) dynamics regulates diverse cell processes such as proliferation, dysplasia, and differentiation, often mediated by the protonation state of a functionally critical histidine residue in endogenous pH sensing proteins. How pHi dynamics can directly regulate gene expression or whether transcription factors can function as pH sensors has received limited attention. We tested the

The Schlafen (SLFN) family of proteins are a group of DNA/RNA processing enzymes with emerging importance in human health and disease, where their functions are implicated in a variety of immunological and anti-tumor processes. Here, we present the cryo-electron microscopy structure of full-length human SLFN14, a member with antiviral activity and linked to an inherited bleeding disorder. SLFN14 is

Misuse and overuse of antibiotics have led to the rapid emergence of antibiotic-resistant superbugs. In addition, evidence is emerging that antibiotic exposure could impose substantial influence on bacterial virulence, but the underlying mechanisms remain poorly understood. Here, we discovered a highly conserved aminoglycoside-responsive regulator, AmgR, that inversely modulates the production of destructive

Differential digests, also known as test or diagnostic digests, are a standard method in molecular cloning to verify whether a picked clone is indeed the target plasmid or not. However, finding the optimal restriction enzyme for a differential digest by hand may be challenging and time-consuming. To address this problem, we created diffdigester.uni-jena.de (https://diffdigester.uni-jena.de), a free

The formation of condensed heterochromatin is critical for establishing cell-specific transcriptional programs. To reveal structural transitions underlying heterochromatin formation in maturing mouse rod photoreceptors, we apply cryo-electron microscopy (cryo-EM) tomography, AI-assisted denoising, and molecular modeling. We find that chromatin isolated from immature retina cells contains many closely

N 4-Acetylcytidine (ac4C) is an emerging epitranscriptomic mark involved in regulating RNA stability, translation, and gene expression. Despite its emerging role in gene regulation and disease, current methods for in situ detection of ac4C-modified RNA lack sensitivity and specificity. To overcome these challenges, we developed fluorine metabolic labeling mediated proximity ligation assay (FMPLA),

Coronaviruses (CoVs) encode 16 nonstructural proteins (nsps), most of which form the replication–transcription complex (RTC). The RTC contains a core composed of one nsp12 RNA-dependent RNA polymerase (RdRp), two nsp8s, and one nsp7. The core RTC recruits other nsps to synthesize all viral RNAs within the infected cell. While essential for viral replication, the mechanism by which the core RTC assembles

Tryptophanyl-tRNA synthetase (TrpRS) catalyzes the attachment of tryptophan (l-Trp) to tRNATrp, thereby providing the ribosome with a crucial substrate for the decoding of the UGG codon during protein translation. Both bacterial and eukaryotic TrpRSs are unable to efficiently cross-aminoacylate their respective tRNATrp substrates, indicating the evolution of lineage-specific mechanisms for tRNATrp

Ro60 is a conserved RNA-binding protein essential for RNA quality control and implicated in autoimmune responses. In this study, we present the structural and functional characterization of φRo60, an Ro60 homolog from Thermus phage phiLo, with its crystal structure determined at 1.99 Å. Despite limited sequence identity with bacterial and amphibian homologs, φRo60 maintains the canonical doughnut-shaped

Efficient DNA repair is essential for maintaining genome integrity and ensuring cell survival. In Escherichia coli, RecBCD plays a crucial role in processing DNA ends, following a DNA double-strand break (DSB), to initiate repair. While RecBCD has been extensively studied in vitro, less is known about how it contributes to rapid and efficient repair in living bacteria. Here, we use single-molecule

Recently long-read sequencing technologies and bioinformatics have enabled the construction of haplotype-resolved genome assemblies. Here, we present the complete and accurate de novo characterization of two challenging genomic regions, the major histocompatibility complex (MHC) and Killer-cell immunoglobulin-like receptors (KIRs), in phased haplotypic form, using the Oxford Nanopore Technology (ONT)

The emergence of numerous variants of SARS-CoV-2 still presents the major challenges in the fight against this disease by reducing the efficacy of vaccines and drugs. RNA G-quadruplexes (G4s) in the SARS-CoV-2 genome are highly conserved and have thus been spotlighted as a promising therapeutic target to combat a wider range of variants. However, very few RNA G4 specific compounds have been reported

Porphyromonas gingivalis, an anaerobic pathogen in chronic periodontitis, belongs to the Bacteroidota phylum and is associated with various virulence factors. Its antibiotic-resistant strains and its propensity to form biofilms pose a challenge to effective treatment. To explore therapeutic avenues, we studied the high-resolution cryogenic electron microscope structures of ribosomes from the wild-type

DNA methylation plays a fundamental role in regulating transcription during development and differentiation. However, its functional role in the regulation of endothelial cell (EC) transcription during state transition, meaning the switch from an angiogenic to a quiescent cell state, has not been systematically studied. Here, we report the longitudinal changes of the DNA methylome over the lifetime

Telomeres ensure genome stability and the levels of telomeric RNA reflect the integrity of telomeric chromatin. The highly conserved RNA-binding protein Ars2 (Arsenite-resistance protein 2) plays an essential role in the RNA nuclear metabolism and negatively regulates the expression of telomeric transcripts in human cells and in Drosophila. We found that germline knockdown of Drosophila Ars2 does not

The A-loop of the 23S ribosomal RNA is a critical region of the ribosome involved in stabilizing the CCA-end of A-site-bound transfer RNA. Within this loop, nucleotide U2552 is frequently 2′-O-methylated (Um2552) in various organisms belonging to the three domains of life. Until now, two enzymatic systems are known to modify this position, relying on either a Rossmann fold-like methyltransferase (RFM)

Bacteria take up environmental DNA using dynamic appendages called type IV pili (T4P) to elicit horizontal gene transfer in a process called natural transformation. Natural transformation is widespread amongst bacteria yet the parameters that enhance or limit this process across species are poorly understood. We show that the most naturally transformable species known, Acinetobacter baylyi, owes this

We characterized the role and regulation mechanism of a pre-mRNA splicing factor, SRSF10, in the development of oligodendrocyte lineage cells (OLCs) and the myelination process during mouse central nervous system (CNS) development. We found that depletion of SRSF10 specifically in OLCs induces hypomyelination and a decrease in OLCs in the developing mouse CNS, whereas depletion of SRSF10 only in differentiated

Short DNA catenanes [circular double-stranded DNA (dsDNA)] have attracted considerable interest for constructing nanostructures and nanomachines, as well as understanding DNA topology. The study of topoisomers of a circular dsDNA with a definite linking number (Lk) is essential but very difficult for simplifying the complex problems about DNA topology. The topoisomers are difficult to prepare, especially

Apart from the iconic Watson–Crick duplex, DNA can fold into different noncanonical structures, of which the most studied are G-quadruplexes (G4s). Despite mounting structural and biophysical evidence, their existence in cells was controversial until their detection using G4-specific antibodies. However, it remains unknown how antibodies recognize G4s at the molecular level and why G4-specific antibodies

Platinum (Pt) drugs are widely utilized in cancer chemotherapy. Although cytotoxic and resistance mechanisms of Pt drugs have been thoroughly explored, it remains elusive what factors affect the receptiveness of DNA to drug-induced damage in nuclei. Here, we demonstrate that nuclear locations of chromatin play a key role in Pt drug-induced DNA damage susceptibility in vivo. By integrating data from

Eukaryotic ribosomes exhibit higher mRNA translation fidelity than prokaryotic ribosomes, partly due to eukaryote-specific ribosomal RNA (rRNA) insertions. Among these, expansion segment 27L (ES27L) on the 60S subunit enhances fidelity by anchoring methionine aminopeptidase (MetAP) at the nascent protein exit tunnel, accelerating co-translational N-terminal initiator methionine (iMet) processing. However

Deciphering cell identity genes is pivotal to understanding cell differentiation, development, and cell identity dysregulation involving diseases. Here, we introduce SCIG, a machine-learning method to uncover cell identity genes in single cells. In alignment with recent reports that cell identity genes (CIGs) are regulated with unique epigenetic signatures, we found CIGs exhibit distinctive genetic