Circular RNAs (circRNAs) are more stable than linear RNAs, enabling expanding applications in RNA vaccines and therapeutics. We previously developed an in vitro circRNA preparation method based on end-to-end self-targeting and splicing (STS) using the Tetrahymena group I intron, which generates circRNAs without extraneous sequences. However, self-circularization efficiency declines as gene of interest (GOI) length increases, limiting its application to longer GOIs. Here, we systematically optimized key determinants of STS efficiency, including target site selection and P1 construct engineering. Target site screening revealed that selection of optimal target sites within each GOI markedly improved self-circularization efficiency. Moreover, engineering of the P1 construct, including incorporation of a polyA10 sequence upstream of the internal guide sequence of the intron and an antisense sequence complementary to the target site and its upstream region at the 5' side of polyA10, further enhanced efficiency. Notably, the optimized STS strategy achieved up to two-fold higher self-circularization efficiency than the conventional permuted intron-exon (PIE) method for long GOIs (∼8 K-nt). Collectively, these results establish an improved STS workflow for efficient circRNA production without extraneous sequences across a wide range of GOI lengths, outperforming the PIE method for long GOIs, and broadening biomedical applications.
Overexpression of PDGFR-β (platelet-derived growth factor receptor beta) kinase contributes to diverse human diseases, including cancers, cardiovascular disorders, and fibrosis. G-quadruplexes (G4s) formed in the PDGFR-β promoter act as transcriptional repressors and represent attractive therapeutic targets. We previously reported that the major G4-forming region of the PDGFR-β promoter adopts a unique broken-strand G4, whereas truncation of this sequence generates a vacancy G4 (vG4) that can be filled-in by external guanine analogs or metabolites and further stabilized by small molecules, suggesting a potential regulatory mechanism and opportunity for selective drug targeting. However, the relationship between broken-strand G4s and vG4s remains unclear. Here, we demonstrate that the PDGFR-β promoter sequence forms a dynamic equilibrium between two broken-strand G4 conformations that interconvert on the millisecond timescale, with vG4 serving as an intermediate. We determined the high-resolution NMR structures of these interconverting G4s, which share a conserved vG4 core but differ in their intramolecular guanine "fill-in." Both conformations feature a stabilizing G-G capping base pair unique to the PDGFR-β promoter. These findings elucidate the structural details of broken-strand PDGFR-β promoter G4s and the mechanism of vG4 formation, providing critical insights for selective drug targeting and establishing a framework for rational design of small molecules to modulate PDGFR-β transcription.
Chromatin environment influences all nuclear processes, including DNA repair. Conversely, DNA damage itself triggers chromatin modifications and remodeling, which are essential for efficient DNA repair and its coordination with transcription, replication, and epigenetic regulation to preserve genome function. While chromatin dynamics associated with double-strand break repair and nucleotide excision repair are well understood, those accompanying base excision repair (BER) remain comparatively poorly characterized. Yet, BER is responsible for eliminating a wide spectrum of chemically diverse and non-helix-distorting base modifications, arising from both endogenous and exogenous sources, and is implicated in numerous pathologies. This review examines how BER operates in the context of chromatin, with a focus on its interplay with other repair factors, chromatin modifications, and remodeling. It also explores the diversity of BER substrates, the blurred distinction between base lesions and programmed modifications, and the intricate link between BER, transcriptional regulation, and epigenetic reprogramming. Together, these insights highlight BER's pivotal role in maintaining genome stability, shaping transcriptional programs, and preventing disease.
Natural transformation enables bacteria to internalize extracellular DNA, driving adaptation and the spread of antibiotic resistance. The membrane protein ComEC mediates translocation of single-stranded DNA (ssDNA) across the cytoplasmic membrane while degrading the complementary strand, yet the structural basis of its activity remains incompletely defined. Here, we report a cryo-electron microscopy structure of full-length ComEC from Neomoorella carbonis in a pre-translocation state, revealing a three-domain architecture and a conserved transmembrane channel captured in a closed conformation. Structural analysis indicates that conformational rearrangements of channel-lining helices would be required to accommodate ssDNA. Biochemical assays show that, relative to the isolated β-lactamase-like domain, full-length ComEC degrades DNA more efficiently and exhibits position-dependent cleavage of phosphodiester bonds within the DNA substrate. Importantly, coating of the DNA by the periplasmic DNA receptor ComEA suppresses endonucleolytic cleavage and enhances 5'' terminal cleavage, thereby directing ComEC towards productive processing of transforming DNA during natural transformation.
Modified nucleotides are essential determinants of RNA function, and identifying the enzymes that install them is fundamental to understanding their cellular roles. Here, we show that the human RNA methyltransferase TRMT11 and its cofactor TRMT112 are imported into mitochondria via N-terminal targeting signals. Using a recently developed N2-methylguanosine (m2G)-sensitive DNAzyme, we demonstrate that TRMT11 catalytic activity and interaction with TRMT112 are required for installation of m2G at position 10 in 13 mitochondrial (mt-)tRNAs. The crosslinking profile of TRMT11 on mt-tRNAs experimentally supports a model of the TRMT11-TRMT112-mt-tRNATrp complex in which the THUMP domain contacts the 3' end of the acceptor stem, and G10 is flipped into the S-adenosylmethionine binding pocket for methylation. Transcriptome-wide mapping reveals that TRMT11 interacts with most nuclear-encoded and mt-tRNAs, but only methylates a subset. In vitro reconstitution of TRMT11-TRM112-mediated methylation defines key structural requirements for m2G10 installation across different mt-tRNAs, and reveals how pathogenic mutations influence this modification. TRMT11-TRMT112 recognizes folded mt-tRNAs, and in the degenerate mt-tRNALys, m1A9 strongly enhances m2G10 methylation efficiency. Loss of m2G10 modifications alters the conformation of numerous mt-tRNAs, perturbs mitochondrial protein synthesis, and impairs oxidative phosphorylation, highlighting an essential role of this modification in maintaining mitochondrial function.
The Structural Maintenance of Chromosome (SMC) protein family plays a central role in higher-order genome organization through ATP-dependent DNA loop extrusion by cohesin and condensin and other processes. Whether these activities fully account for the complexity of chromosome architecture remains unknown. Here, we uncover a conserved ATP-independent mechanism of chromatin condensation by SMC complexes, occurring via biomolecular condensation. Using single-molecule fluorescence imaging, we show that a variety of SMCs form dynamic DNA-bound condensates that exhibit key features of biomolecular condensates, including droplet coalescence, fluorescence recovery after photobleaching, and rapid exchange with free SMC complexes. Atomic force microscopy analysis of human cohesin-DNA assemblies reveals DNA-length-dependent clustering, providing evidence for bridging-driven condensation. Analyses of in vivo super-resolution imaging and high-throughput chromosome conformation capture (Hi-C) data indicate that these condensates form chromatin-associated clusters with multi-loop structures. Together, our results establish that SMC complexes employ ATP-independent phase condensation as well as ATP-dependent activities to shape genome architecture. This work reveals a broadly conserved principle of chromosomal organization across eukaryotes.
The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by suppressing transposable elements in animal germlines. Despite its well-established function in the animal germline, piRNAs and PIWI proteins are expressed in somatic tissues across arthropod species, and their functions outside the gonads remain poorly understood. Aedes albopictus mosquitoes express four PIWI genes, Piwi4, Piwi5, Piwi6, and Ago3, in both gonadal and somatic tissues. Here, we generated Piwi6 knockout (KO) Ae. albopictus cell lines and observed a substantial upregulation of long terminal repeat retrotransposons, including a full-length endogenous retrovirus that we named Aedes albopictus Endogenous Retrovirus-1 (AalERV1). Nascent RNA sequencing and Cleavage Under Targets and Tagmentation (CUT&Tag) analyses revealed that Piwi6 silences AalERV1 transcriptionally by guiding the deposition of the repressive H3K9me3 histone mark. Consistently, Piwi6 localized to both the cytoplasm and nucleus, with sequences in the intrinsically disordered region guiding nuclear translocation. Reintroduction of full-length GFP-Piwi6, but not a mutant GFP-Piwi6 defective in nuclear localization, rescued AalERV1 repression in Piwi6 KO cells. Importantly, Piwi6-mediated control of AalERV1 was recapitulated in vivo as Piwi6 knockdown increased AalERV1 expression in both ovaries and somatic tissues of Ae. albopictus mosquitoes. These results establish Aedes mosquitoes as a model to study nuclear PIWI functions and suggest that somatic piRNA-mediated transposon silencing is evolutionarily conserved across arthropod species.
Ribonucleotides can serve as a strand discrimination signal in reconstituted in vitro biochemical mismatch repair (MMR) assays, but the influence of ribonucleotides on mismatch correction has not been measured directly in vivo. We have developed a fluorescence-based host cell reactivation assay that reports correction of a mismatch in proximity of a site-specifically incorporated ribonucleotide. A ribonucleotide leads to enhanced mismatch correction. While neither inactivation of a single allele nor knockdown of RNaseH2 is sufficient to suppress ribonucleotide directed MMR, a modest but statistically significant impairment for repair of mismatches in the presence of an embedded ribonucleotide is observed in RNaseH2 knockout cell lines. Reporter plasmids with ribonucleotides located in either the 3' or 5' orientation are robustly repaired in MMR-proficient cells but are weakly repaired in MMR-deficient cells, underscoring their utility as effective MMR reporters. Significant ribonucleotide-enhanced mismatch correction was consistently observed in MMR-deficient cells when the ribonucleotide is in the 3' orientation. The presence of a ribonucleotide led to enhanced MMR even in RNaseH2 knockout cells, suggesting that other enzymes may promote ribonucleotide-directed MMR. Loss of RNaseH2 was not sufficient to confer significant resistance to the alkylating agent, temozolomide, in support of a model in which ribonucleotide-directed repair events make minor contributions to the canonical MMR pathway in mammalian cells. We propose a model in which MMR-independent ribonucleotide enhanced correction of mismatches can proceed by ribonucleotide excision repair when the ribonucleotide is in the 5' direction, and proceeds by an unknown mechanism when the ribonucleotide is in the 3' direction.
Activation-induced deaminase (AID) initiates immunoglobulin class switch recombination (CSR) by deaminating cytosines within transcription-generated single-stranded DNA in switch regions. R-loops formed during switch-region transcription are thought to expose AID substrates; however, how AID engages and organizes these complex nucleic acid structures remain unclear. Here, combining ensemble biochemistry with single-molecule colocalization and fluorescence resonance energy transfer (FRET) analyses, we uncover an unexpected role for AID as a DNA synapsis factor. AID preferentially promotes synapsis between multistranded DNA substrates, including R-loops and tailed D-loops, and stabilizes these higher-order synaptic complexes. Mutational analyses reveal that two distinct nucleic acid-binding pockets cooperate to drive efficient synapsis. Single-molecule FRET further reveals that AID promotes intramolecular synapsis of tailed D-loops that mimic key CSR intermediates. Moreover, three-color single-molecule analyses indicate that DNA binding-associated AID self-assembly, consistent with AID assemblies observed in cells, accompanies with synaptic complex formation. Notably, a catalytically active AID mutant with impaired AID-AID interactions shows severely compromised DNA synapsis, indicating that higher-order AID organization is essential for synaptic complex formation and synapsis is mechanistically separable from cytosine deamination. Together, our findings establish AID as a DNA synapsis factor and support a model in which AID self-assembly and multistranded DNA binding drive higher-order synapsis during CSR.
In the wake of a replication fork, nascent chromatin is composed of a 1:1 mixture of parental histones and newly synthesized histones. The parental histones contain the pattern of post-translational modifications that dictated the chromatin state prior to DNA replication. The parental histones clearly play a critical role in epigenetic inheritance as they serve as the template that directs the maturation of the nascent chromatin into the appropriate mature chromatin state. An essential step that ensures faithful epigenetic inheritance is the transfer of the parental histone modification state to the newly synthesized histones. But what of the newly synthesized histones that make up 50% of nascent chromatin? Are they merely blank slates or do they play an active role in epigenetic inheritance? This review will address this question by exploring the nature of newly synthesized histones and their contribution to the assembly of specific chromatin states.
Ribosomal protein L41 (RPL41 or eL41) is the smallest ribosomal protein and forms the eukaryote-specific bridge, eB14, near the decoding center; however, its role in mammalian translation remains unclear. In this study, we established RPL41-deficient models of human HEK293T cells and mice to define its function. Cryo-electron microscopy revealed that RPL41 constrains intersubunit conformational dynamics without inducing major local static rearrangements. Loss of RPL41 altered A-site dynamics, slowed elongation, modestly increased amino acid misincorporation, and modestly enhanced readthrough of collision-inducing reporter sequences. Quantitative proteomic analysis suggested that these translational defects compromise long-protein homeostasis, as evidenced by increased insolubility and reduced abundance of long proteins. In vivo, Rpl41-/- mice were viable but exhibited growth retardation and decreased abundance of long proteins in tissues. Our findings reveal a conserved role for RPL41 in maintaining ribosome dynamics and translational fidelity, indicating that RPL41 supports ribosome function and long-protein homeostasis in mammals.
R-loops are three-stranded nucleic acid structures consisting of a DNA-RNA hybrid strand and a displaced single-stranded DNA, and represent pervasive chromatin structural features in eukaryotic cells. Although it has long been considered that the RNA substrates of R-loops are linear RNAs, emerging evidence has demonstrated that circular RNAs (circRNAs), a class of covalently closed single-stranded RNAs generated via back-splicing, can also hybridize with genomic DNA to form R-loops. These circRNA-derived R-loops are termed ciR-loops herein. Owing to the exceptional stability of circRNAs relative to their linear counterparts, ciR-loops are more difficult to resolve than those derived from linear RNAs. Consequently, the accumulation of unscheduled or deleterious ciR-loops can more readily interfere with gene transcription and replication, posing greater threats to genomic and transcriptomic integrity. Given that functions and regulatory mechanisms of ciR-loops have gained increasing attention in the field of RNA biology and gene expression, particularly over the past 3 years, a timely and comprehensive review is urgently needed. However, the molecular mechanisms underlying ciR-loop metabolism and hot spots of investigations about ciR-loop-mediated chromatin regulation are yet to be summarized. Here, we provide a systematic review of recent advances in ciR-loop biology, focusing on the diverse mechanisms of their formation and resolution, as well as their molecular and physiological functions.
The nuclear receptor Nur77 plays a crucial, protective role in chronic inflammatory diseases and deficiency of Nur77 in macrophages results in excessive pro-inflammatory cytokine secretion. Previous research suggested that Nur77's regulatory function in inflammation is due to repression of the pro-inflammatory transcription factor NF-kB, but the underlying mechanism remains unclear. To address this, we applied a genome-wide, multi-omics approach in LPS-stimulated RAW264.7 macrophages with inducible Nur77 expression. Key findings were validated in wild-type and Nur77-deficient bone marrow-derived macrophages. We show that Nur77 suppresses the expression of inflammatory genes through a dual mechanism wherein Nur77 acts as a repressor of AP-1 targets at two levels: first, Nur77 occupies regulatory elements proximal to AP-1 target genes through AP-1 motifs and second, Nur77 regulates the expression of AP-1 family members themselves. These repressive activities of Nur77 result in diminished RNA Pol II on AP-1 genes and their targets. The first zinc finger of the Nur77 DNA-binding domain is required to reduce AP-1 activity. In summary, Nur77 represses macrophage inflammation through regulation of both immediate-early AP-1 expression, as well as inhibition of AP-1-driven gene programs.
Accurate cell annotation is a primary challenge in spatial transcriptomics (ST). Current approaches primarily rely on label transfer from single-cell RNA sequencing (scRNA-seq) reference or marker-based clustering. While these methods are widely used, they have critical limitations. Label transfer approaches depend on the availability of a well-matched scRNA-seq reference. Marker-based annotation methods often suffer from accuracy and limited coverage. To address these challenges, we developed Binary-SPA (Binary Self-referenced Projection Annotation), a computational framework for cell-type annotation of high-resolution ST. Binary-SPA performs annotation in two stages. First, a binary classification step identifies high-confidence cells using predefined marker sets. These confidently annotated cells are then used as an internal reference for anchor-based label transfer in the second stage. Binary-SPA consistently outperforms conventional marker-based methods and matches or exceeds label-transfer methods across diverse spatial platforms, tissue types, and preservation protocols-without requiring matched reference data. Unlike label transfer methods that decline without same-tissue references, Binary-SPA eliminates external data dependencies entirely while maintaining high accuracy at high annotation coverage. Binary-SPA demonstrates robust performance in challenging specimens such as bone marrow biopsies, and validation against matched COMET protein expression data confirmed strong concordance between transcriptomic- and protein-based cell identities. Binary-SPA thus provides a robust, reference-free solution for ST annotation with broad applicability to research and clinical specimens.
To successfully transfer, conjugative plasmids must overcome a recipient cell's defence mechanisms. The leading region of many plasmids-the first DNA to enter-is known to be enriched in anti-defence genes. Here, we investigate whether selective pressure from defence systems has also affected its sequence composition. We consider two possibilities: greater target depletion (to avoid triggering defence systems) or greater codon adaptation (to rapidly express anti-defence genes). We analyse 1751 conjugative plasmids in 13 plasmid taxonomic units in Enterobacterales. First, we test GC content and depletion of short palindromic motifs (4-8 bp), a potential signature of selective pressure from Type II restriction-modification systems. The leading region is GC-rich but not consistently depleted in these motifs. Then, we use the codon adaptation index (CAI) to assess codon usage. Leading regions in plasmids with MOB-F and MOB-P relaxases are more adapted to optimal host codon usage, consistent with higher translational efficiency, with anti-restriction genes showing particular high CAI. We demonstrate the potential of codon adaptation to uncover new plasmid biology by identifying a single-stranded promoter in the F plasmid, consistent with independent experimental findings. Our findings emphasize the intensity of the selective pressure that plasmids face from defence systems.
Protein-nucleic acid interactions (PNIs) are central to fundamental biological processes, and mutations can disrupt these interactions by altering local structural features and binding free energy. Here, we present DeepPNI, a deep learning regression model that integrates sequence- and structure-based features to estimate mutation-induced changes in binding free energy in protein-nucleic acid complexes. The model was developed using a comprehensive dataset of 1754 mutations spanning protein-DNA and protein-RNA complexes, representing one of the largest curated datasets for PNI binding free energy prediction. Structural features were encoded using an edge-aware relational graph convolutional network, while sequence features were represented using the Evolutionary Scale Modeling 2 protein language model. Despite the increased dataset size and heterogeneity, DeepPNI achieved an overall Pearson correlation coefficient of 0.76 in five-fold cross-validation. Consistent performance was observed across protein-DNA and protein-RNA subsets, datasets grouped by experimental temperature, and external blind test datasets, suggesting robustness against dataset heterogeneity. DeepPNI is freely available as a web server at https://research.iitbhilai.ac.in/molinfo/deeppni.
MicroRNAs (miRNAs) are short noncoding RNAs that post-transcriptionally regulate gene expression. Their canonical function depends on a "seed" sequence at the miRNA 5' end that pairs with conserved sites in the 3' untranslated regions of target messenger RNAs to suppress gene expression. Although seed-mediated targeting is well characterized, the role of the miRNA 3' end remains poorly understood and is challenging to study. Here, we identify a noncanonical mechanism by which miR-106a, a member of the miR-17 family, competes with let-7a for binding to let-7 target sites despite their distinct seed sequences. This binding bypasses miR-106a seed pairing and instead relies on extensive complementarity at its 3' end, which shares sequence identity with the let-7a seed. Such binding of miR-106a does not itself silence these targets but shields them from let-7-mediated suppression, resulting in de-repression of let-7 targets. By contrast, miR-106b, which differs from miR-106a and the other miR-17 family members at its 3' end, lacks this de-repressive activity. These findings show that variation in miRNA 3' sequences can generate divergent functions within a miRNA family and underscore the need to account for 3'-end contributions when interpreting miRNA-target networks and considering the development of miRNA-based therapeutics.
Ribosomal RNA (rRNA) modifications cluster around the peptidyl transferase centre (PTC), the catalytic centre of the ribosome, yet their collective functional roles remain unclear. Here we analyse Escherichia coli ribosomes lacking 11 or 12 modifications near the PTC. Using kinetic assays, we show these hypo-modified ribosomes catalyse peptide bond formation at rates twofold to threefold lower than wild-type and exhibit reduced thermal stability. Cryo-electron microscopy of hypo-modified ribosomes reveals multiple alternative conformations of the PTC and exit tunnel regions, disrupting native stacking and hydrogen bonding critical for positioning of transfer RNA substrates. These findings indicate that rRNA modifications stabilize the native PTC structure, preventing formation of alternative, nonfunctional conformations and thereby enhancing catalytic efficiency. Our study provides insight into how rRNA modifications fine-tune ribosome function by maintaining structural integrity essential for efficient translation.
