Biophysical methods are widely used for drug discovery, with surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) being amongst the most used techniques. However, these types of bulk or ensemble methods do not provide single-molecule resolution or structural and mechanistic insights, so that researchers are forced to rely upon a battery of complementary techniques. Here, we used a scaled magnetic force spectroscopy (MFS) instrument system capable of analysing hundreds of single molecules in parallel and obtaining dynamic conformational insights. Taking the S-adenosylmethionine-I (SAM-I) riboswitch as a model system, we demonstrated that MFS can be used to characterize the RNA structure in conjunction with the published crystal structures. Next, we compared the performance of MFS to SPR and ITC for measuring binding affinities of two ligands with highly different affinities, SAM and SAH in both high and low magnesium concentrations. MFS gave similar binding affinities to SPR and ITC but was more closely aligned to ITC. We then demonstrated the power of MFS to study the molecular dynamics of SAM-I and the mechanisms of SAM and S-adenosylhomocysteine (SAH) binding. By subjecting the RNA to constant forces for extended periods, we were able to observe that the conformation dynamics of SAM-I are impacted differently by SAM and SAH binding, pointing to different stabilization mechanisms between the two ligands.
BACKGROUND:Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis have transformed cancer therapy, but their efficacy remains limited in glioblastoma (GBM) and heterogeneous in colorectal cancer (CRC). MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level, including immune checkpoint molecules, yet conserved regulatory miRNA networks across distinct cancers remain poorly defined. RESULTS:Five conserved miRNAs (miR-106a-5p, miR-106b-5p, miR-20a-5p, miR-20b-5p, miR-138-5p) fulfilled the selection criteria and were consistently dysregulated in GBM and CRC. MiR-106a-5p and miR-106b-5p were upregulated in both cancers and showed favourable prognostic associations, with higher expression correlating with improved survival. miR-20a-5p and miR-20b-5p were preferentially expressed in microsatellite-stable (MSS) CRC, and correlated with favourable outcomes in both cancers, whereas miR-138-5p was downregulated in both tumours compared to normal tissue, but showed opposite survival associations, with higher levels linked to worse prognosis. Correlation analysis revealed significant inverse associations between several miRNAs and checkpoint gene expression, including moderate inverse correlations for CD274-miR-106a-5p in GBM, CD274-miR-20a-5p in CRC, and PDCD1LG2-miR-20a-5p in both cancers. Pan-cancer profiling demonstrated broad and heterogeneous dysregulation, with expression absent in ovarian cancer for four of the five miRNAs. Pathway enrichment implicated the TGF-β, Hippo, FoxO, and cell cycle pathways, consistent with their known roles in tumour immune evasion. CONCLUSION:We identified a conserved set of miRNAs that are dysregulated in both GBM and CRC, correlate with survival, and display inverse relationships withPD-1/PD-L1/PD-L2expression. These miRNAs represent candidate regulators of the PD-1/PD-L1/PD-L2 axis and potential biomarkers of tumour biology that may influence immune checkpoint signalling.
T cell exhaustion is a dysfunctional state that arises during chronic infections and cancer, characterized by impaired effector functions and sustained expression of inhibitory receptors. While transcriptional, epigenetic, and metabolic rewiring have been well documented in exhausted T cells, a comprehensive understanding of how translation is regulated in this state remains incomplete. To address this gap, we performed ribosome profiling and RNA sequencing on in vitro chronically activated human CD8+ T cells to globally assess translational control during a model of T cell exhaustion. Our analyses reveal a marked repression of 5’ terminal oligopyrimidine (TOP) mRNAs during chronic activation. Unexpectedly, we demonstrate that this translational repression occurs despite evidence of elevated mTOR activity. These findings uncover a previously unknown layer of translational control in exhausted T cells.
The Mediator complex functions as a central multi-subunit coactivator essential for RNA polymerase II-dependent transcription in eukaryotes, integrating signals from enhancers to promoters across the transcription cycle. This review summarizes its modular composition and structural dynamics revealed by cryo-EM, and details its mechanistic roles in pre-initiation complex assembly, promoter-proximal pausing release, elongation progression, co-transcriptional splicing, termination via 3’-end processing, and chromatin changes. Recent progress elucidates allosteric signal transmission, TF multivalency, cohesin-mediated looping, and phase separation, while dysregulation implicates Mediator in cancer and developmental disorders. Future directions emphasize in vivo kinetics, and therapeutic targeting of kinase modules to decode context-specific gene control.
Studies of neurological diseases caused by the expansion of nucleotide repeats led to the discovery that RNA can undergo translation by ribosomes in the absence of canonical AUG start signals, a process termed repeat-associated non-ATG translation (RAN). This discovery suggested that RNA transcribed from mammalian telomeres, termed TERRA, could generate RAN products. Indeed, two dipeptide repeat proteins can be produced: repeating arginine-valine (VR) and repeating glycine-leucine (GL). Both VR and GL form amyloid aggregates, and VR was observed to be expressed in cells with elevated TERRA, including a human osteosarcoma line. VR undergoes a change in aggregation state during mitosis, where it becomes dispersed, binds ribosomes, and can depress translation, possibly playing a regulatory role in the cell cycle. The discovery that RAN translation can occur on telomeric RNAs has opened new connections between telomeres, ageing, and the generation of RAN proteins with important biological activities.
MicroRNAs (miRNAs) are essential post-transcriptional regulators implicated in diverse physiological and pathological processes. Recently, miR-181a-5p emerged as a critical modulator of placental function. This review synthesizes literature on miR-181a-5p at the maternal-foetal interface in normal pregnancies and complications, such as preeclampsia, gestational hypertension, gestational diabetes, and Rh-negative haemolytic disease of the foetus and newborn (Rh-HDFN). From a PubMed search, 12 articles were qualitatively synthesized. Evidence shows that early miR-181a-5p suppression is crucial for normal trophoblast proliferation via the renin-angiotensin system. Conversely, its aberrant expression characterizes gestational pathologies. In preeclampsia, miR-181a-5p upregulation suppresses trophoblast viability, migration, and invasion, increasing apoptosis. In gestational hypertension, its downregulation perturbs glycolytic metabolism. Furthermore, elevated miR-181a-5p disrupts endothelial and placental barriers in gestational diabetes and Rh-HDFN, respectively. Ultimately, miR-181a- as a pleiotropic regulator in the placenta. Decoding its molecular networks underscores its clinical potential as a biomarker and therapeutic target.
Accurate RNA polymerase II (RNAPII)-dependent gene expression requires dynamic phosphorylation of the carboxy-terminal domain (CTD) of its largest subunit, Rpb1, whose heptapeptide repeats form a regulatory platform known as the CTD code. Transcription-associated cyclin-dependent kinases (tCDKs) and CTD phosphatases coordinate the phosphorylation - dephosphorylation cycle of RNAPII throughout transcription, coupling RNA synthesis to co-transcriptional processing and chromatin regulation. By controlling stage-specific modification of the CTD, these enzymes integrate RNAPII activity into broader regulatory networks. Disruption of the delicate kinase - phosphatase balance impairs transcriptional fidelity, RNA maturation, and genome stability, either directly through altered CTD phosphorylation or indirectly through associated pathways. Such alterations are increasingly associated with developmental disorders, neurodegeneration, and cancer. Here, we synthesize current knowledge of RNAPII phosphorylation dynamics, highlighting key mechanistic principles, links to human disease, and emerging therapeutic strategies targeting this central phosphorylation-dependent regulatory system.
Replacing maternal colostrum and milk with those from other species is common practice in neonatal ruminant management, but the effects on small non-coding RNA (sncRNA) transmission remain poorly understood. This study investigated whether colostrum and milk replacement alters circulating sncRNA profiles in neonatal goats using a twin-pair model. Six vaccinated goat dams (Mycobacterium avium ssp. paratuberculosis) and their twelve kids were included. One goat twin kid received maternal colostrum and milk, while the other was fed a replacement diet with bovine colostrum and milk replacer. Small RNA sequencing was performed on serum samples from kids, as well as on goat colostrum and milk and on bovine replacers. All colostrum and milk samples contained diverse sncRNAs, including miRNAs, and tRNA- and rRNA-derived fragments. Processing and storage influenced sncRNA abundance and stability. In neonatal serum, sncRNA profiles changed during development and indicated two response waves: after colostrum replacement and after three weeks of continued milk replacement. These changes involved differential abundance of miRNAs and rRNA-derived fragments, including molecules linked to immune regulation, metabolism, and developmental pathways. Although sequence similarity between bovine and caprine sncRNAs limited direct inference of dietary transfer, the observed differences suggest that milk origin and composition may influence circulating sncRNA profiles. Several milk-associated miRNAs were consistently detected in serum, suggesting potential maternal transfer. Overall, these findings indicate that interspecies milk replacement may modify neonatal circulating sncRNAs, highlighting a potential role for milk-derived sncRNAs in shaping early-life immune and developmental programming.
RNA modifications have emerged as central regulators of cancer translational control. Unlike transcriptional reprogramming, which unfolds over hours, modification-dependent translational rewiring enables rapid proteomic adaptation to the nutrient-deprived, hypoxic, and immunologically hostile tumour microenvironment. Yet most existing reviews organize epitranscriptomic mechanisms by modification type or cancer hallmark, obscuring the mechanistic logic by which chemical marks collectively reshape the translational apparatus. This review adopts a translation-centric framework, examining how the most abundant modifications on mRNAs, tRNAs, and rRNAs regulate each stage of protein synthesis in malignant cells. We survey the epitranscriptomic toolkit, including modification chemistries, enzymatic writers, readers, and erasers, and detection technologies including nanopore direct RNA sequencing. We then trace how modifications control initiation (m6A-driven mRNA circularization, cap-independent translation via eIF3 and eIF4G2, rRNA 2'-O-methylation-directed cap-to-IRES switching), elongation (m6A-induced ribosome stalling coupled to mRNA decay, tRNA mcm5s2U-mediated codon-biased translation, YTHDF1-dependent elongation factor recruitment), and termination (pseudouridine-mediated stop codon readthrough, NMD evasion). Crucially, we show that mRNA, tRNA, and rRNA modifications do not act in isolation but form integrated networks. For example, mRNA m6A and tRNA mcm5s2U operate on opposing arms of the same regulatory axis, with direct implications for therapeutic design. We assess the expanding drug pipeline, from the METTL3 inhibitor STC-15 now in Phase 1b/2 trials and METTL3-targeting PROTACs to FTO and ADAR1 inhibitors, and argue that biology-informed combination strategies targeting multiple modification axes will be essential for durable clinical responses.
CAG repeat tracts occur in both non-coding and translated RNAs, have tended to lengthen throughout evolution, and are thought to enhance neuronal function. We identified over 600 human RNAs (including mRNAs, lncRNAs, and circRNAs) with at least 10 CAG repeats, originating from 58 genomic loci, which vary, e.g. in the rate of CAG length polymorphism. Several circRNAs originate from the ATXN7 locus, where CAG expansion causes spinocerebellar ataxia type 7 (SCA7). For selected circATXN7(3,4).1 (circ1), we demonstrated its cytoplasmic localization, as well as its presence in 40S, monosome and polysome fractions. We showed that circ1 is expressed in human fibroblasts, blood and cerebellum, and, importantly, we identified a mutation-containing circRNA with potential implications in SCA7.
Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder caused by dystrophin deficiency. Antisense oligonucleotide (ASO)-mediated exon skipping has emerged as a cornerstone of DMD therapy to restore dystrophin expression. This review provides a comprehensive overview of the four FDA-approved ASO therapies - eteplirsen, golodirsen, viltolarsen, and casimersen - tracing their journey from pivotal clinical trials to post-marketing updates. While the development and clinical evaluation of these agents have established a pioneering framework for rare genetic diseases, they have also highlighted critical challenges. These include complexities in clinical trial design, discrepancies between preclinical efficacy and clinical outcomes, real-world burdens, and limited patient eligibility. Furthermore, the FDA's accelerated approval of these therapies based on limited clinical data remains a subject of ongoing debate. Confirmatory trials to verify clinical efficacy and long-term follow-up studies are actively underway. Concurrently, intensive research is focused on developing next-generation ASOs to achieve enhanced therapeutic efficacy and definitive clinical outcomes. Elucidating the trajectory of research and development in this field offers profound insights for shaping future therapeutic strategies in rare diseases.
Since their discovery, Z-nucleic acids (Z-NAs), which adopt a left-handed double helical conformation, have puzzled researchers regarding their physiological functions. These unusual nucleic acids are recognized by proteins containing Zα domains, particularly Z-DNA binding protein 1 (ZBP1) and adenosine deaminase acting on RNA 1 (ADAR1). Utilizing mouse genetics with knockout models and site-specific Zα domain mutations, scientists have revealed that Z-NAs serve as critical regulators of programmed cell death, inflammation, antiviral immunity, and anti-tumour responses. This review systematically examines mechanistic insights from Zbp1- and Adar1-mutant models, illuminating how Z-NAs play a dual role as essential triggers of host defence and as potential drivers of autoinflammatory diseases.
Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic inflammatory disorder of the gastrointestinal tract with a complex aetiology involving genetic, environmental (such as diet/lifestyle), and microbiota factors. The exact pathogenesis of IBD remains unclear, and current therapies show limited effectiveness. Circular RNAs (circRNAs) are a novel class of noncoding RNAs that have been implicated in the regulation of various biological processes. Many circRNAs show specific expression profiles in IBD patients and play important roles in IBD pathogenesis through different signalling pathways, such as the Janus kinase/signal transducer and activator of transcription (JAK/STAT) and nuclear factor-κB (NF-κB) pathways. Although research on circRNAs in IBD is still in its early stages, many circRNAs have emerged as potential diagnostic, prognostic biomarkers, and therapeutic targets for IBD. Here, we summarize the molecular functions and underlying mechanisms of circRNAs in IBD and discuss current challenges and future perspectives for clinical applications.
The RNA exosome is a conserved multi-subunit ribonuclease complex with pivotal roles in RNA biogenesis, surveillance, and processing. It comprises a nine-subunit scaffold that associates with distinct ribonucleases in a cell compartment-specific manner, contributing to the processing and turnover of a broad spectrum of nuclear and cytoplasmic transcripts, including pervasively transcribed and short-lived RNAs, precursors, and abortive and aberrant transcripts. In this review, we examine how the RNA exosome engages a wide spectrum of RNAs via differential adaptor usage and intrinsic substrate features, such as transcript length and 3’ end structure. This also modulates the entry routes of the recruited transcripts. We highlight conserved principles and major differences between yeast and metazoans. We assimilate emerging evidence that suggests that the RNA exosome localization and activity are dynamically regulated in response to cellular context and external stimuli. Finally, drawing on findings from studies in S. cerevisiae, Drosophila, zebrafish, and mice, we discuss how perturbations in RNA surveillance can result in abnormalities in organismal development and homoeostasis. Together, these studies not only enhance our knowledge of the broader relevance of RNA quality control and metabolism but also provide mechanistic insights into pathomechanisms, particularly the tissue-specific vulnerabilities noted in RNA exosome-linked diseases.
B-cell lymphoma 6 (BCL6) functions in various cancers, but its role and regulation in gastric cancer (GC) remain unclear. N-6-methyladenosine (m(6)A) modification is critical for tumorigenesis, and the demethylase FTO (highly expressed in GC) may regulate target gene stability via m(6)A. This study explored FTO's regulation of BCL6 m(6)A modification and its impact on GC progression through ferroptosis. qPCR assessed BCL6 expression in GC cells. BCL6 overexpression models were used to evaluate cell viability, apoptosis, and ferroptosis (via ROS, Fe2+, MDA, LDH, and ferroptosis-related proteins). RIP-PCR, MeRIP-qPCR, and mRNA stability assays examined FTO-BCL6 mRNA interaction and m(6)A effects. YTHDF2 knockdown validated its role, and co-silencing FTO/BCL6 clarified the axis's function. BCL6 was downregulated in GC cells. Its overexpression inhibited cell viability, promoted apoptosis, and induced ferroptosis. FTO bound BCL6 mRNA, removed m(6)A, and reduced its stability. YTHDF2 mediated FTO's negative regulation of BCL6. FTO knockdown enhanced ferroptosis and impaired GC cell function, partially reversed by BCL6 silencing. FTO suppresses BCL6 via m(6)A demethylation, inhibiting ferroptosis to promote GC progression. The FTO/BCL6 axis is a potential therapeutic target for GC.
Cells produce numerous types of RNAs. Among these, transcripts produced by RNA polymerase II include protein-coding mRNAs as well as a variety of long noncoding RNAs. In this latter group, enhancer (e) RNAs constitute a class of RNAs transcribed from enhancer sites. Although eRNAs are typically unstable and degraded rapidly, multiple roles related to enhancer function have been suggested. But eRNAs also share similarities with mRNAs, such as in a limited number the presence of translated open reading frames. Indeed, other “noncoding” RNAs have also been found to contain coding sequences, and together these transcripts blur the line between coding and noncoding. Here, we review current models of eRNA function, the discoveries that led to them, and additional functions, specifically the potential for translation. We also review the characteristics of proteins encoded by such “noncoding” transcripts, and their possible implications regarding the function and evolution of both eRNAs and mRNAs.
Brain tumor (Brat) is a Drosophila TRIM-NHL protein required for embryogenesis and neural stem cell differentiation. Although structural and biochemical studies established that the Brat NHL domain specifically binds RNA, the in vivo requirement for this activity has not been directly tested. Here, we used structure-guided mutagenesis and genome engineering to determine whether RNA recognition is essential for Brat function during development. The direct interaction between Brat's NHL domain and RNA containing Brat Binding Sites (BBS) can be abolished by alanine substitution of three separate residues on the NHL surface. We introduced these point mutations into the endogenous brat locus by CRISPR-mediated Scarless Gene Editing to generate three independent RNA-binding defective mutant (RBDmt) alleles. Complementation tests demonstrated that each allele behaves as a strong loss-of-function mutation: homozygotes and hemizygotes are inviable, and RBDmt alleles fail to complement classical brat null and hypomorphic alleles. Lethal phase analysis revealed death predominantly during late larval and pupal stages, consistent with known brat alleles. Consistent with the namesake brat phenotype, RBDmt larval brains exhibited widespread expression of neuroblast markers and a marked reduction of neuronal differentiation. In embryos, these alleles failed to complement female sterile brat alleles and recapitulated characteristic abdominal segmentation defects. Finally, RT-qPCR showed increased expression of endogenous Brat target mRNAs in mutant larvae, consistent with loss of Brat-mediated repression. Together, these results demonstrate that direct RNA binding is an essential molecular activity of Brat and that post-transcriptional regulation of Brat target mRNAs underlies its critical roles across development.
Preeclampsia (PE), a gestational disorder diagnosed with hypertension and proteinuria, is recognized as a significant cause of both maternal and perinatal morbidity and mortality around the world. Oxidative stress (OS) is found to act as a major positive role in PE progression, which is regulated by the function derived from multiple microRNAs (miRNAs), a group of cellular small endogenous RNAs that modulate gene expression at post-transcriptional level. This review summarizes the role of different miRNAs in the PE development, focusing on their involvement in OS regulation. In addition, the paper also discusses the current knowledge of the molecular mechanisms regarding PE and highlights the possibly therapeutic implications of targeting miRNAs in the management of this condition.
Plants possess an expansive suite of epigenetic control mechanisms to keep their large, repetitive, and unruly genomes in check. Small non-coding RNAs constitute one such mechanism by directing transcriptional and post-transcriptional gene silencing. Small RNAs come in several forms that are produced by distinct pathways, including microRNAs and small interfering RNAs, such as tasiRNAs, phasiRNAs, and RNA Polymerase IV (Pol IV)-dependent small RNAs. Compared to other classes of small RNAs, Pol IV-dependent small RNAs and phasiRNAs appear to have the greatest phenotypic impact during the reproductive phase of the life cycle, from meiosis through seed development. Here, we focus on the function of 21-24 nucleotide small RNAs during reproduction, with a primary focus on Pol IV-dependent small RNAs. We critically assess the evidence surrounding their mode of action, specifically whether they act in cis or in trans, cell autonomously or non-autonomously, and within or between generations.
Pigmentation is an orchestrated process involved in cuticle melanization and sclerotization in insects, and plays a critical role in maintaining the structural integrity and functional completeness of the insect cuticle. Although the cascade reactions underlying pigmentation have been extensively studied, our understanding of the involvement of miRNA in this process remains limited. Here, we investigate the role of conserved microRNA-34 (miR-34) in regulating cuticular colouration in the silkworm, bombyx mori. Overexpression of miR-34 led to pronounced melanization in the larval abdomen. Mechanistically, LC/MS analysis revealed that miR-34 overexpression alters the epidermal amino acid composition, with a particularly notable increase in tyrosine and dopamine content. Enzyme activity assays confirmed the activation of phenoloxidase (PO). Through experimental validation, we identified two key target genes of miR-34, including Bm-iAANAT (a critical gene in melanin synthesis) and Bmserpin3 (a regulator of the serine protease cascade). This study uncovers a previously unreported miRNA mediated regulatory mechanism in insects and systematically elucidates how miR-34 coordinately modulates pigmentation through distinct molecular pathways, providing novel theoretical insights into this field.
