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PubMed1997

The mitochondrion in dividing Leishmania tarentolae cells is symmetric and circular and becomes a single asymmetric tubule in non-dividing cells due to division of the kinetoplast portion.

Kinetoplastid protozoa have a single mitochondrion that extends throughout the cell. The disk-shaped portion of the mitochondrion adjacent to the basal body of the flagellum contains the kinetoplast DNA nucleoid body which consists of thousands of catenated minicircles and a smaller number of catenated maxicircles. The maxicircles contain structural genes and cryptogenes, rRNA genes, and a few guide RNA genes The minicircles contain the majority of the guide RNA genes. The long slender non-dividing stationary phase Leishmania tarentolae cells in culture have an asymmetric mitochondrion that consists of a single tubule extending from one edge of the kinetoplast portion. This presents a problem for cell division, in that one daughter cell will receive significantly less mitochondrial membranes than the other cell. We show in this paper that the solution to this problem is that dividing cells, which are normally shorter and rounder than stationary phase cells, possess a symmetric circular mitochondrion that has mitochondrial tubules extending from both edges of the kinetoplast which are joined in the posterior region of the cell. This implies that growth of the mitochondrion occurs after cell division, either from elongation of the longitudinal tubule towards the anterior of the cell, or from elongation of the kinetoplast portion of the mitochondrion towards the posterior region and fusion of the tubules.

Animals↗

PubMed2025

Unlocking Zeptomolar Single-Molecule Detection by Synergizing Digital Microfluidics and Digital CRISPR.

Accurate diagnosis relies on the highly sensitive and quantitative detection of multiple immune-related biomarkers. However, current detection methods still face significant limitations in sensitivity, specificity, and background signal control. Here, we introduce DDA (Dual-Digital immunoAssay), a fully automated, universal immunoassay platform that synergizes digital microfluidics with digital Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based amplification. This "dual-digital" strategy pushes the detection limit into the zeptomolar (zM) regime, enabling unprecedented sensitivity for single-molecule analysis. The DDA platform is built upon a digital microfluidic microwell array chip, integrating magnetic bead-based immunocapture with RNA-guided CRISPR/Cas13a signal amplification. This system enables a fully automated, "sample-in, answer-out" workflow. By systematically optimizing the entire process, DDA significantly reduces background noise and enhances detection sensitivity, achieving a limit of detection (LOD) down to 100 zM for key protein biomarkers. This represents a >100-fold improvement over leading commercial ultrasensitive assays. With single-molecule resolution and full automation, DDA provides a robust solution for the precise quantification of low-abundance immune biomarkers. As a proof-of-concept, we demonstrate its ability to accurately quantify key heart-failure-associated biomarkers, including NT-proBNP (LOD: 1 aM), IL-6 (LOD: 1.5 aM), and TNF-α (LOD: 2.5 aM), directly in complex serum samples. This platform holds great promise for automated multibiomarker screening and risk assessment, showcasing its powerful potential for the early diagnosis of major diseases such as cardiovascular diseases, cancers, neurodegenerative disorders, and infectious diseases.

Humans↗

PubMed1995

Editing domains of Trypanosoma brucei mitochondrial RNAs identified by secondary structure.

The posttranscriptional insertion and deletion of U residues in trypanosome mitochondrial transcripts called RNA editing initiates at the 3' end of precisely defined editing domains that can be identified independently of the cognate guide RNA. The regions where editing initiates in Trypanosoma brucei cytochrome b and cytochrome oxidase subunit II preedited mRNAs are specifically cleaved by a trypanosome mitochondrial endonuclease that acts like mung bean nuclease and therefore is single strand specific. The regions where editing initiates in virtually all examined preedited mRNAs are predicted to form loop structures, suggesting that editing domains could generally be recognized as prominent single-stranded loops. In contrast to preedited mRNA, edited mRNA can be either resistant or sensitive to cleavage by trypanosome mitochondrial endonuclease, depending on the reaction conditions. This selectivity appears dependent on the availability of extract RNAs, and in model reactions, edited mRNA becomes resistant to cleavage upon base pairing with its guide RNA. Natural partially edited mRNAs are also specifically cleaved with a sensitivity like preedited and unlike edited mRNAs, consistent with their being intermediates in editing. These results suggest that in vivo, the structure of editing domains could initially be recognized by the mitochondrial endonuclease, which could target its associated RNA ligase and terminal U transferase to begin cycles of enzymatic editing modifications.

Animals↗

PubMed2002

The expanding snoRNA world.

In eukaryotes, the site-specific formation of the two prevalent types of rRNA modified nucleotides, 2'-O-methylated nucleotides and pseudouridines, is directed by two large families of snoRNAs. These are termed box C/D and H/ACA snoRNAs, respectively, and exert their function through the formation of a canonical guide RNA duplex at the modification site. In each family, one snoRNA acts as a guide for one, or at most two modifications, through a single, or a pair of appropriate antisense elements. The two guide families now appear much larger than anticipated and their role not restricted to ribosome synthesis only. This is reflected by the recent detection of guides that can target other cellular RNAs, including snRNAs, tRNAs and possibly even mRNAs, and by the identification of scores of tissue-specific specimens in mammals. Recent characterization of homologs of eukaryotic modification guide snoRNAs in Archaea reveals the ancient origin of these non-coding RNA families and offers new perspectives as to their range of function.

Animals↗

PubMed2003

Peptide synthesis with a template-like RNA guide and aminoacyl phosphate adaptors.

The genetic code is thought to have developed from an early system of RNA-dependent peptide synthesis. To investigate one kind of template-like peptide synthesis that might emerge from an RNA world, we constructed highly reactive aminoacyl phosphate oligonucleotides as adaptors that bound to RNA guide sequences. The reactive aminoacyl groups mimic a chemistry found in modern protein biosynthesis. Guide sequence interactions with adaptors were borrowed in part from universal contacts seen between tRNAs and rRNA. With these constructions, di- and tripeptides formed in a single guide sequence-dependent reaction. The order of amino acids was not random but directional in a way consistent with substrate reactivities. No ribosomes or ribozymes were required. Thus, aminoacyl phosphate adaptors and RNA guides could, in principle, have been intermediates in the transition from the RNA world to modern template-dependent protein synthesis.

Amino Acid Sequence↗

PubMed2026

Dense RNA motif modifications enable robust in vivo prime editing and enhance efficiencies of diverse editing systems.

Prime editing holds promise for therapeutic applications. However, viral delivery of the prime editor presents challenges for clinical translation due to concerns regarding long-term expression. Meanwhile, systemic delivery using non-viral vectors has been limited by low efficiency, the need for repeated injections and reliance on doses that exceed clinically translatable levels. Here we develop engineered prime editing guide RNAs (pegRNAs) with densely modified RNA motifs and demonstrate their application for efficient in vivo prime editing. By systemically delivering the prime editor in RNA format via a single injection of lipid nanoparticles, we achieved nearly 70% editing efficiency in the bulk mouse liver, indicating successful editing of the majority of hepatocytes. Notably, a single injection at a clinically translatable lipid nanoparticle dose was sufficient to suppress target protein expression in vivo, resulting in a near 80-fold increase in editing efficiency compared with conventional end-modified pegRNAs. Furthermore, incorporating densely modified RNA motifs, including the widely used MS2 motif, proved broadly applicable across various RNA sequences and split RNA-guided genome editing platforms, resulting in up to an 11-fold increase in base editing efficiency. These findings present a generalizable approach for enhancing the therapeutic potential of prime editing and expanding the utility of RNA-based therapeutics.

Journal Article↗

PubMed2026

RNA splicing and cardiovascular disease: a guide for cardiologists.

Alternative splicing (AS) is a fundamental RNA processing mechanism, which generates different RNA transcripts and consequently different protein isoforms from a single gene. This increases the diversity of proteins within an organism and can fine-tune biological processes. This review examines how cardiac-enriched RNA-binding proteins establish heart-specific splicing programs governing aspects of cardiac development, function, and disease. Developmentally, coordinated sarcomeric isoform switches underpin the foetal-to-adult transition and further isoform rewiring in ion channel and kinase genes determine electrophysiology and excitation-contraction coupling. AS contributes to the pathogenesis of several cardiomyopathies and emerging datasets suggest that pathological hypertrophy engages distinct splicing signatures compared with physiological hypertrophy. This review summarizes diagnostic and prognostic opportunities arising from bulk, long-read, and single-cell/nucleus transcriptomics, which resolve cell type-specific isoforms and disease-associated switches. Circulating RNA biomarkers (including splice ratios and circularRNAs) may signify myocardial remodelling and arrhythmic risk. Integrative approaches that link AS with proteomics and genomics improve variant interpretation, reveal previously unannotated protein isoforms, and enable tracking of disease progression and therapy response. Finally, an outline of therapeutic strategies to modulate AS in cardiovascular disease (CVD), including antisense oligonucleotides, small molecules, and genome-editing modalities (CRISPR, base, and prime editing), is provided. The major challenges that remain before splice-targeting therapeutics can be targeted to treat cardiovascular disease are highlighted. Lessons from neuromuscular indications establish clinical feasibility of splicing correction and motivate translation to cardiology. Together, mechanistic insight, biomarker development, and therapeutic innovation position RNA splicing as a tractable axis for precision cardiovascular medicine.

Humans↗

PubMed2027

Inferring Gene Regulatory Networks in Stem Cells: Methods and Applications.

Gene regulatory networks (GRNs) represent the complex interplay of transcription factors, regulatory elements, and target genes that orchestrate cellular identity and function, playing a crucial role in the differentiation and maintenance of stem cells. This chapter provides an overview of experimental and computational methodologies for inferring GRNs, with particular emphasis on single-cell approaches. We first review key experimental techniques for detecting transcription factor binding sites, chromatin accessibility, and DNA motifs, alongside essential databases that support GRN reconstruction. We then introduce computational inference methods that can be categorized into four principal frameworks: correlation-based approaches, regression and machine learning models, probabilistic and deep learning methods, and integrative or message-passing frameworks. To illustrate practical application, we present a case study applying the pySCENIC workflow to a peripheral blood mononuclear cell single-cell RNA sequencing dataset from mouse, demonstrating how regulon-based analysis can reveal cell-type-specific regulatory programs. This chapter aims to serve as a practical guide for researchers seeking to understand and implement GRN inference methodologies in stem cell biology and related fields.

Gene Regulatory Networks↗

PubMed2001

Guide DNA technique reveals that the protein component of bacterial ribonuclease P is a modifier for substrate recognition.

We developed a guide DNA technique with which the cleavage efficiency of pre-tRNA substrate raised in the RNase P reaction. The 20-mer guide DNAs hybridizing to the upstream region of the cleaving site enhanced the cleavage reactions of RNA substrates by Escherichia coli RNase P. This guide DNA technique was also applicable to cleavage site selection by choosing the DNA-hybridizing site. Results showed that RNase P accepts DNA/RNA double-stranded 5'-leader region with high catalytic efficiency as well as single-stranded RNA region in pre-tRNAs as substrates, which suggests that the protein component of bacterial RNase P prefers bulky nucleotides. The protein component did not affect the normal 5'-processing reaction of pre-tRNAs, but enhanced the mis-cleaving (hyperprocessing) reactions of tRNA in non-cloverleaf folding. Our results suggested that the protein component of RNase P is a modifier for substrate recognition.

Bacterial Proteins↗

PubMed2004

The RNA-induced silencing complex is a Mg2+-dependent endonuclease.

In the Drosophila and mammalian RNA interference (RNAi) pathways, target RNA destruction is catalyzed by the siRNA-guided, RNA-induced silencing complex (RISC). RISC has been proposed to be an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. Although 5' cleavage products are readily detected for RNAi in vitro, only 3' cleavage products have been observed in vivo. Proof that RISC acts as an endonuclease requires detection of both 5' and 3' cleavage products in a single experimental system. Here, we show that siRNA-programmed RISC generates both 5' and 3' cleavage products in vitro; cleavage requires Mg(2+), but not Ca(2+), and the cleavage product termini suggest a role for Mg(2+) in catalysis. Moreover, a single phosphorothioate in place of the scissile phosphate blocks cleavage; the phosphorothioate effect can be rescued by the thiophilic cation Mn(2+), but not by Ca(2+) or Mg(2+). We propose that during catalysis, a Mg(2+) ion is bound to the RNA substrate through a nonbridging oxygen of the scissile phosphate. The mechanism of endonucleolytic cleavage is not consistent with the mechanisms of the previously identified RISC nuclease, Tudor-SN. Thus, the RISC-component that mediates endonucleolytic cleavage of the target RNA remains to be identified.

Animals↗

PubMed1992

Native mRNA editing complexes from Trypanosoma brucei mitochondria.

The aim of this study was to identify multicomponent complexes involved in kinetoplastid mitochondrial mRNA editing. Mitochondrial extracts from Trypanosoma brucei were fractionated on 10-30% glycerol gradients and assayed for RNAs and activities potentially involved in editing, including pre-edited mRNA, guide RNA (gRNA), endonuclease, terminal uridylyltransferase (TUTase), RNA ligase and gRNA-mRNA chimera-forming activities. These experiments suggest that two distinct editing complexes exist. Complex I (19S) consists of gRNA, TUTase, RNA ligase and chimera-forming activity. Complex II (35-40S) is composed of gRNA, preedited mRNA, RNA ligase and chimera-forming activity. These studies provide the first evidence that editing occurs in a multicomponent complex. The possible roles of complex I, complex II and RNA ligase in editing are discussed.

Animals↗

PubMed1997

Nonhomologous RNA recombination in a cell-free system: evidence for a transesterification mechanism guided by secondary structure.

Extensive nonhomologous recombinations occur between the 5' and 3' fragments of a replicable RNA in a cell-free system composed of pure Qbeta phage replicase and ribonucleoside triphosphates, providing direct evidence for the ability of RNAs to recombine without DNA intermediates and in the absence of host cell proteins. The recombination events are revealed by the molecular colony technique that allows single RNA molecules to be cloned in vitro. The observed nonhomologous recombinations are entirely dependent on the 3' hydroxyl group of the 5' fragment, and are due to a splicing-like reaction in which RNA secondary structure guides the attack of this 3' hydroxyl on phosphoester bonds within the 3' fragment.

Base Sequence↗

PubMed2006

Crystal structure of an H/ACA box ribonucleoprotein particle.

H/ACA ribonucleoprotein particles (RNPs) are a family of RNA pseudouridine synthases that specify modification sites through guide RNAs. They also participate in eukaryotic ribosomal RNA processing and are a component of vertebrate telomerases. Here we report the crystal structure, at 2.3 A resolution, of an entire archaeal H/ACA RNP consisting of proteins Cbf5, Nop10, Gar1 and L7ae, and a single-hairpin H/ACA RNA, revealing a modular organization of the complex. The RNA upper stem is bound to a composite surface formed by L7ae, Nop10 and Cbf5, and the RNA lower stem and ACA signature motif are bound to the PUA domain of Cbf5, thereby positioning middle guide sequences so that they are primed to pair with substrate RNA. Furthermore, Gar1 may regulate substrate loading and release. The structure rationalizes the consensus structure of H/ACA RNAs, suggests a functional role of each protein, and provides a framework for understanding the mechanism of RNA-guided pseudouridylation, as well as various cellular functions of H/ACA RNP.

Archaeal Proteins↗

PubMed1997

The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria.

Recent advances in in vitrosystems and identification of putative enzymatic activities have led to the acceptance of a modified 'enzyme cascade' model for U insertion/deletion RNA editing in kinetoplastid mitochondria. Models involving the transfer of uridines (Us) from the 3'-end of gRNA to the editing site appear to be untenable. Two types of in vitrosystems have been reported: (i) a gRNA-independent U insertion activity that is dependent on the secondary structure of the mRNA; (ii) a gRNA-dependent U insertion activity that requires addition of a gRNA that can form an anchor duplex with the pre-edited mRNA and which contains guiding A and G nucleotides to base pair with the added Us. In the case of the gRNA-mediated reaction, the precise site of cleavage is at the end of the gRNA-mRNA anchor duplex, as predicted by the original model. The model has been modified to include the addition of multiple Us to the 3'-end of the 5'-cleavage fragment, followed by the formation of base pairs with the guiding nucleotides and trimming back of the single-stranded oligo(U) 3'-overhang. The two fragments, which are held together by the gRNA 'splint', are then ligated. Circumstantial in vitroevidence for involvement of an RNA ligase and an endoribonuclease, which are components of a 20S complex, was obtained. Efforts are underway in several laboratories to isolate and characterize specific components of the editing machinery.

Animals↗

PubMed2005

Identification of novel argonaute-associated proteins.

RNA silencing processes are guided by small RNAs known as siRNAs and microRNAs (miRNAs) . They reside in ribonucleoprotein complexes, which guide the cleavage of complementary mRNAs or affect stability and translation of partial complementary mRNAs . Argonaute (Ago) proteins are at the heart of silencing effector complexes and bind the single-stranded siRNA and miRNA . Our biochemical analysis revealed that Ago2 is present in a pre-miRNA processing complex that is able to transfer the miRNA into a target-mRNA cleaving complex. To gain insight into the function and composition of RNA silencing complexes, we purified Ago1- and Ago2-containing complexes from human cells. Several known Ago1- and/or Ago2-associated proteins including Dicer were identified, but also two novel factors, the putative RNA helicase MOV10, and the RNA recognition motif (RRM)-containing protein TNRC6B/KIAA1093. The new proteins localize, similar to Ago proteins, to mRNA-degrading cytoplasmic P bodies, and they are functionally required to mediate miRNA-guided mRNA cleavage.

Argonaute Proteins↗

PubMed2005

Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism.

Yeast U2 small nuclear RNA (snRNA) contains three pseudouridines (Psi35, Psi42, and Psi44). Pus7p and Pus1p catalyze the formation of Psi35 and Psi44, respectively, but the mechanism of Psi42 formation remains unclear. Using a U2 substrate containing a single (32)P radiolabel at position 42, we screened a GST-ORF library for pseudouridylase activity. Surprisingly, we found a Psi42-specific pseudouridylase activity that coincided with Nhp2p, a protein component of a Box H/ACA sno/scaRNP (small nucleolar/Cajal body-specific ribonucleoprotein). When isolated by tandem affinity purification (TAP), the other protein components of the H/ACA sno/scaRNP also copurified with the pseudouridylase activity. Micrococcal nuclease-treated TAP preparations were devoid of pseudouridylase activity; however, activity was restored upon addition of RNAs from TAP preparations. Pseudouridylation reconstitution using RNAs from a Box H/ACA RNA library identified snR81, a snoRNA known to guide rRNA pseudouridylation, as the Psi42-specific guide RNA. Using the snR81-deletion strain, Nhp2p- or Cbf5p-conditional depletion strain, and a cbf5 mutation strain, we further demonstrated that the pseudouridylase activity is dependent on snR81 snoRNP in vivo. Our data indicate that snRNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms.

Catalysis↗

PubMed2026

Unveiling the power of TIIC: A prognostic tool for esophageal adenocarcinoma.

BACKGROUND: Esophageal adenocarcinoma (EAC) remains a lethal malignancy with limited prognostic tools for guiding immunotherapy. Tumor-infiltrating immune cells (TIICs) play a critical role in EAC prognosis and treatment response. METHODS: We integrated single-cell RNA sequencing and bulk transcriptome data from TCGA and GEO databases. TIIC-specific RNAs were identified via tissue specificity index calculation combined with machine learning feature selection. Twenty machine learning algorithms were benchmarked to construct an optimal TIIC signature score (TIIC-Score) based on the comprehensive C-index. Immunotherapy response, genomic mutation, and copy number variation were analyzed. Summary-data-based Mendelian randomization (SMR) and two-sample Mendelian randomization (MR) were performed to explore genetic associations. Core prognostic TIIC-related genes were functionally validated in esophageal cancer cell lines through loss-of-function assays. RESULTS: The TIIC-Score demonstrated robust prognostic value for 1-, 2-, and 3-year overall survival across multiple cohorts, outperforming 22 published models. High TIIC-Score was associated with poor survival and increased chromosomal instability. Mutation profiling revealed high frequencies of TP53 (78.2%), TTN (48.7%), and SYNE1 (30.8%). MR analysis identified a significant association between gastro-oesophageal reflux and EAC risk at SNP rs8130507. Functionally, CCNI was upregulated in esophageal cancer cells, and its knockdown suppressed malignant phenotypes while promoting apoptosis, supporting its pro-tumorigenic role. CONCLUSION: The TIIC-Score provides a novel prognostic framework for EAC that effectively stratifies patient risk and may help identify individuals most likely to benefit from immunotherapy.

Esophageal adenocarcinoma↗

PubMed2026

A critical appraisal of base-resolution m6A profiling techniques.

N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic mRNA, influencing RNA fate and gene regulation. Early antibody-based approaches enabled transcriptome-wide profiling but lacked resolution and quantitative accuracy. Newer approaches now achieve base-resolution m6A detection using improved crosslinking, chemical or enzymatic conversion, and single-molecule sequencing. Antibody-free methods provide quantitative stoichiometry from minimal input, while nanopore direct RNA sequencing offers real-time, single-molecule readouts across entire transcriptomes. Collectively, these methods form a versatile toolkit that integrates global mapping with precise site-level analysis, advancing knowledge of context-dependent m6A regulation in physiology and disease. This review compares their principles, strengths, and limitations to guide method selection and highlight how next-generation epitranscriptomic tools are paving the way for clinical and therapeutic applications.

Humans↗