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At least 433 records · Page 24Linked to original sources
PubMed2026

Clinically actionable stratification of uncommon MET fusions: a precision oncology framework.

BACKGROUND: MET fusions represent emerging therapeutic targets in solid tumors; however, functional interpretation of non-canonical variants remains poorly understood, posing a major challenge for precision oncology. METHODS: We conducted a multicenter, pan-cancer study analyzing 23,299 clinical samples using DNA-based next-generation sequencing (NGS) to profile MET fusions. Transcriptional validation was performed using RNA-based NGS on available samples. Preliminary clinical outcomes were assessed in four patients with advanced malignancies harboring uncommon MET fusions who received MET tyrosine kinase inhibitor therapy. RESULTS: We identified 116 MET fusions (incidence: 0.5%), with 55.2% (64/116) classified as uncommon fusions. These uncommon fusions were stratified into: Group A (5’-retained, n = 12), Group B (intergenic/exonic breakpoints, n = 19), Group C (rare partners, n = 23), and Group D (dual fusions, n = 10). RNA validation revealed an overall low transcriptional consistency of 43.8% (14/32) for uncommon fusions, versus 100% for canonical fusions (PTPRZ1::MET, CAPZA2::MET). Notably, most 5’-retained fusions were transcriptionally silent, while some intergenic fusions resolved into expressed canonical partners (e.g. PTPRZ1::MET). Therapeutically, all four MET inhibitor-treated patients achieved partial responses, including pediatric diffuse midline gliomas (DMG) (median OS: 11.2 months) and lung adenocarcinoma (median OS: 34 months), demonstrating preliminary clinical activity. CONCLUSIONS: uncommon MET fusions are heterogeneous at genomic and transcriptional levels. DNA-level findings often do not predict functional transcripts, underscoring the necessity of RNA-based confirmation for clinical interpretation. Despite low overall consistency, a subset retains therapeutic potential. We propose a refined diagnostic framework integrating DNA-based stratification and RNA validation to guide the management of MET-altered cancers in precision oncology workflows.

Humans↗
PubMed2002

Gender difference in HIV RNA levels: a meta-analysis of published studies.

Plasma HIV RNA copy number is a strong prognostic marker of progression to AIDS in antiretroviral-naive persons. Recent research suggests women have lower HIV RNA levels than comparable men. Because clinical care recommendations currently include HIV RNA thresholds as a guide to initiating antiretroviral therapy (ART), the authors undertook the present quantitative meta-analysis to explore the relation between gender and plasma HIV RNA levels. A gender difference in HIV RNA levels was observed in the CD4-unadjusted and -adjusted analyses. The summary estimate including only CD4-adjusted results with the lowest heterogeneity indicated that on average, women have 41% lower plasma HIV RNA levels than men (-.23 log; 95% confidence interval [CI], -.16 to -.31 log ). Because numerous studies have found similar HIV disease progression rates in men and women, adjusted for CD4 but not HIV RNA, the present meta-analysis supports the use of lower HIV RNA thresholds in women than in men to guide initiation of ART. Given the patient characteristics in the original studies used in this meta-analysis, the results are most likely to apply to therapy-naive persons with CD4 lymphocyte counts >200 cells/mm, a subgroup for whom HIV RNA levels may strongly influence the decision to initiate therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1996

RNA editing: a mechanism for gRNA-specified uridylate insertion into precursor mRNA.

In the mitochondria of trypanosomatid protozoa the precursors of messenger RNAs (pre-mRNAs) have their coding information remodeled by the site-specific insertion and deletion of uridylate (U) residues. Small trans-acting guide RNAs (gRNAs) supply the genetic information for this RNA editing. An in vitro system was developed to study the mechanism of U insertion into pre-mRNA. U-insertion editing occurs through a series of enzymatic steps that begin with gRNA-directed pre-mRNA cleavage. Inserted U's are derived from free uridine triphosphate and are added to the 3' terminus of a 5' pre-mRNA cleavage product. gRNA specifies edited RNA sequence at the subsequent ligation step by base pairing-mediated juxtaposition of the 3' cleavage product and the processed 5' cleavage product. gRNA/pre-mRNA chimeras, purported intermediates, seem to be abortive end products of the same reaction.

Animals↗
PubMed2004

RNA silencing in viral infections: insights from poliovirus.

RNA interference or RNA silencing is a dsRNA guided mechanism that mediates sequence specific degradation of RNA. The recent demonstration that RNA interference can be used to inhibit virus replication has initiated an exciting field of research: first, as a potential novel antiviral therapeutic approach and, second, as a tool for dissecting virus-host interactions. Here we review and discuss the current data and perspectives on the use of RNA interference in the study of poliovirus as a model for positive strand RNA viruses.

Animals↗
PubMed2006

HIV-l and the microRNA-guided silencing pathway: an intricate and multifaceted encounter.

MicroRNAs (miRNAs) are approximately 21-24 nucleotide RNAs that mediate repression of messenger RNA (mRNA) translation through recognition of specific miRNA binding sites usually located in the 3' non-translated region. Designed to simulate miRNAs, small interfering RNAs represent a powerful genetic approach to potently inhibit gene expression by mediating cleavage of the intended mRNA target. This strategy has been applied successfully to suppress replication of several viruses, including human immunodeficiency virus type 1 (HIV-1). However, recent evidences indicate that viral RNAs may themselves be processed, to some extent, by the endogenous miRNA biosynthetic machinery in mammalian cells, extending previous observations in plants. The resulting viral miRNAs may exert regulatory effects towards host and/or viral genes that may influence viral replication and modulate the course of infection. Viral miRNA generation and/or action may be limited by counteraction through inhibitory viral RNAs and/or proteins. This review article will focus on the relationship between HIV-1 and miRNA-guided RNA silencing, and discuss the different aspects of their interaction. As we learn more about the mechanism and importance of small RNA-based antiviral systems, a more intricate picture of the interaction between HIV-1 and a proven antiviral defense mechanism in lower eukaryotes is emerging.

Animals↗
PubMed1997

Insertional and deletional RNA editing in trypanosome mitochondria.

The mitochondrial mRNAs of trypanosomes are often post-transcriptionally modified by an RNA processing event, termed RNA editing, which results in the insertion or deletion of uridylate (U) residues in mRNAs. RNA editing is necessary for the formation of complete coding sequences for several essential mitochondrial proteins. The number and site of U addition and deletion is directed by small guide RNAs (gRNAs). Recent studies indicate that the mechanism of RNA editing in trypanosomes involves a series of enzymatic steps. We show that the initial step in this enzymatic cascade requires the formation of a binary RNA complex between the gRNA and its cognate pre-mRNA. Depletion of specific gRNAs inhibits cleavage of the pre-mRNA by an editing site specific endoribonuclease. Addition of synthetic gRNAs reverses this inhibition. All of the activities needed for RNA editing in vitro are present within a 19S ribonucleo-protein complex (RNP) composed of gRNAs, the editing site specific endonuclease, an RNA ligase, a terminal uridylate transferase (TUTase) and approximately 15 other unidentified proteins. We have recently identified and cloned the gene for a 45kDa protein, the RNA Editing Associated Protein-1 (REAP-1), which is a component of trypanosome editing complexes. REAP-1 co-purifies with RNA ligase and TUTase activities and is part of a > 700 kDa RNP containing gRNAs. Antibodies against REAP-1 inhibit in vitro RNA editing reactions confirming its role in RNA editing.

Animals↗
PubMed2026

Spatially guided in vivo single-cell functional genomics of postnatal heart.

Understanding how spatial organization and cell-cell interactions shape gene regulatory programs is central to decoding tissue development and function. The transition at birth, marked by increased circulatory demands and rapid tissue growth, requires precise spatiotemporal coordination of cardiac maturation. In this study, we generated a high-resolution spatial and temporal atlas of the postnatal mouse heart by integrating single-nucleus RNA sequencing with image-based spatial transcriptomics. This framework revealed dynamic cellular interactions, niche-specific signaling and transcriptional programs guiding cardiomyocyte maturation. To functionally test prioritized regulators in vivo and at scale, we developed PIP-seq (probe-based indel-detectable Perturb-seq), a high-throughput platform that detects single guide RNA identity, infers gene editing and profiles transcription from fixed nuclei. Applying PIP-seq to the developing postnatal heart, we identified 21 previously uncharacterized regulators of cardiomyocyte maturation, including genes essential for sarcomere assembly, metabolic reprogramming and electrophysiological transitions. Together, our findings define how microenvironmental signals and intrinsic gene programs cooperate to guide heart maturation and establish a broadly applicable framework for functional genomics in complex tissues.

Animals↗
PubMed2001

RNA editing in Trypanosoma brucei: characterization of gRNA U-tail interactions with partially edited mRNA substrates.

Guide RNAs (gRNAs), key components of the RNA editing reaction in Trypanosoma brucei, direct the insertion and deletion of uridylate (U) residues. Analyses of gRNAs reveal three functional elements. The 5'-end of the gRNA contains the anchor, which is responsible for selection and binding to the pre-edited mRNA. The second element (the guiding region) provides the information required for editing. At the 3'-end of the gRNA is a non-encoded U-tail, whose function remains unclear. However, the cleavage-ligation model for editing proposes that the U-tail binds to purine-rich regions upstream of editing sites, thereby strengthening the interaction and holding onto the 5' cleavage product. Our previous studies demonstrated that the U-tail interacts with upstream sequences and may play roles in both stabilization and tethering. These studies also indicated that the U-tail interactions involved mRNA regions that were to be subsequently edited. This raised the question of what happens to the mRNA-U-tail interaction as editing proceeds in the 3'-->5' direction. We examined gCYb-558 and its U-tail interaction with 5'CYbUT and two partially edited 5'CYb substrates. Our results indicate that the 3'-end of the U-tail interacts with the same sequence in all three mRNAs. Predicted secondary structures using crosslinking data suggest that a similar structure is maintained as editing proceeds. These results indicate that the role of the U-tail may also involve maintenance of important secondary structure motifs.

Animals↗
PubMed2006

Post-transcriptional small RNA pathways in plants: mechanisms and regulations.

Small RNAs are riboregulators that have critical roles in most eukaryotes. They repress gene expression by acting either on DNA to guide sequence elimination and chromatin remodeling, or on RNA to guide cleavage and translation repression. This review focuses on the various types of post-transcriptional small RNA-directed pathways in plants, describing their roles and their regulations.

DNA Methylation↗
PubMed2025

Optimization of Structure-Guided Development of Chemical Probes for the Pseudoknot RNA of the Frameshift Element in SARS-CoV-2.

Targeting the RNA genome of SARS-CoV-2 is a viable option for antiviral drug development. We explored three ligand binding sites of the core pseudoknot RNA of the SARS-CoV-2 frameshift element. We iteratively optimized ligands, based on improved affinities, targeting these binding sites and report on structural and dynamic properties of the three identified binding sites. Available experimental 3D structures of the pseudoknot element were compared to SAXS and NMR data to validate its dominant folding state in solution. In order to experimentally map in silico predicted binding sites, NMR assignments of the majority of nucleobases were achieved by segmental labeling of the pseudoknot RNA and isotope-filtered NMR experiments at 1.2 GHz, demonstrating the value of NMR spectroscopy to supplement modelling and docking data. Optimized ligands with enhanced affinity were shown to specifically inhibit frameshifting without affecting 0-frame translation in cell-free translation assays, establishing the frameshift element as target for drug-like ligands of low molecular weight.

SARS-CoV-2↗
PubMed2006

Mono- and dicotyledonous plant-specific RNA editing sites are correctly edited in both in organello systems.

We set out to analyse the phylogenetic distribution of cox2 RNA editing sites. Database searches have revealed the presence of mono- and dicotyledonous-specific RNA editing sites. Therefore, to better understand tRNA editing system in plants, we developed a new dicotyledonous in organello RNA editing system using cauliflower mitochondria and analysed the transcription of the cox2 gene for both maize and Arabidopsis. These results were compared with those obtained from a maize mitochondrial in organello system. Surprisingly, both the mono- and dicotyledonous cox2 transcripts were efficiently edited in the mitochondrial cauliflower and maize in organello systems, respectively, even for RNA editing sites not present in the endogenous cox2 sequences. Taken together, our observations support a self-guiding-transcript model for RNA editing in higher plants.

Arabidopsis↗
PubMed2005

Lentiviral-mediated delivery of combined HIV-1 decoy TAR and Vif siRNA as a single RNA molecule that cleaves to inhibit HIV-1 in transduced cells.

RNA interference (RNAi) silences gene expression via short interfering 21-23 mer double-stranded RNA (siRNA) segments that guide cognate mRNA degradation in a sequence-specific manner. On the other hand, HIV-1 decoy TAR RNA are known to competitively interact with the HIV-1 Tat protein, to downregulate the enhanced gene expression from the long terminal repeat (LTR) promoters. Here we report that a novel expression construct, encoding both HIV-1 decoy TAR and Vif siRNA, as a single RNA substrate, was expressed under the control of the human U6 promoter, and later the TAR and siRNA were cleaved into their respective separate RNA by the endogenous RNase III-like enzyme. Each of the cleaved HIV-1 anti-genes then synergistically contributed toward enhancing the inhibition efficacy (>80%) of HIV-1 replication in transduced Jurkat cells. These results suggest that targeting HIV-1 mRNA with simultaneously expressed intracellular decoy TAR and Vif-siRNA could lead to an effective gene therapy strategy for the control and management of HIV-AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Trypanosoma brucei RNA editing. A full round of uridylate insertional editing in vitro mediated by endonuclease and RNA ligase.

RNA editing in kinetoplastids is the post-transcriptional insertion and deletion of uridylate residues in mitochondrial transcripts, directed by base pairing with guide RNAs. Models for editing propose transesterification or endonuclease plus RNA ligase reactions and may involve a guide RNA-mRNA chimeric intermediate. We have assessed the feasibility of the enzymatic pathway involving chimeras in vitro. Cytochrome b chimeras generated with mitochondrial extract were first found to have junctions primarily at the major endonuclease cleavage sites, supporting the role of endonuclease in chimera formation. Such cytochrome b chimeras are then specifically cleaved by extract endonuclease within the oligo(U) tract at the editing site, and the mRNA cleavage products are then joined by RNA ligase to generate partially edited mRNAs with uridylate residues transferred to an editing site. These in vitro generated partially edited mRNAs mimic partially edited mRNAs generated in vivo. Specific endonuclease cleavage in the editing region of the partially edited RNA demonstrates the potential for further in vitro editing. Finally, sensitivity to various ATP analogs suggests that all editing-like activities reported thus far utilize a mechanism involving RNA ligase.

Animals↗
PubMed2002

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex.

The genomes of hyperthermophilic Archaea encode dozens of methylation guide, C/D box small RNAs that guide 2'-O-methylation of ribose to specific sites in rRNA and various tRNAs. The genes encoding the Sulfolobus homologues of eukaryotic proteins that are known to be present in C/D box small nucleolar ribonucleoprotein (snoRNP) complexes were cloned, and the proteins (aFIB, aNOP56, and aL7a) were expressed and purified. The purified proteins along with an in vitro transcript of the Sulfolobus sR1 small RNA were reconstituted in vitro, into an RNP complex. The order of assembly of the three proteins onto the RNA was aL7a, aNOP56, and aFIB. The complex was active in targeting S-adenosyl methionine (SAM)-dependent, site-specific 2'-O-methylation of ribose to a short fragment of ribosomal RNA (rRNA) that was complementary to the D box guide region of the sR1 small RNA. The presence of aFIB was essential for methylation; mutant proteins having amino acid replacements in the SAM-binding motif of aFIB were able to assemble into an RNP complex, but the resulting complexes were defective in methylation activity. These experiments define the minimal number of components and the conditions required to achieve in vitro RNA guide-directed 2'-O-methylation of ribose in a target RNA.

Amino Acid Motifs↗
PubMed1995

Hemodynamic regulation of tyrosine hydroxylase messenger RNA in medullary catecholamine neurons: a c-fos-guided hybridization histochemical study.

Medullary catecholamine cell groups are involved in multiple modes of cardiovascular regulation and display indices of functional activation, including widespread c-fos expression, in response to hypotensive hemorrhage. Assessments of the impact of such challenges on transmitter-related gene expression are complicated by the biochemical and connectional heterogeneity that characterize these cell groups. Quantitative hybridization histochemical methods were used to follow the effects of 15% hemorrhage on levels of messenger RNA encoding tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, in medullary aminergic neurons; concurrent staining for nuclear Fos-immunoreactivity permitted comparisons between cells that ostensibly were and were not targeted by the challenge. Increased levels of tyrosine hydroxylase messenger RNA were detected in Fos-immunoreactive neurons in all cell groups examined. Mean maximal increases ranged between 133 and 192% of control values, and were attained within 0.5-1 h post-hemorrhage in noradrenergic (A1 and A2) cell groups, and at 2 h in adrenergic ones (C1, C2, and C2d or dorsal strip). By 4 h after the challenge, tyrosine hydroxylase messenger RNA levels in Fos-immunoreactive neurons in all cell groups had returned to control values. By contrast, tyrosine hydroxylase messenger RNA in non-Fos-immunoreactive cells either did not change significantly over the course of the experiment (C2 and C2d), or showed a rapid and transient increase, whose magnitude tended to be less than that seen in Fos-immunoreactive cells. c-fos messenger RNA was prominently induced in catecholaminergic neurons in each of the medullary cell groups examined at 0.5 h after hemorrhage, suggesting that the early tyrosine hydroxylase messenger RNA response to hemorrhage in non-Fos-immunoreactive cells preceded the capacity of responsive neurons to manifest detectable Fos protein expression. These findings indicate that hemorrhage up-regulates tyrosine hydroxylase messenger RNA levels in medullary catecholaminergic cell groups which have access to adaptive neuroendocrine and/or autonomic control systems. The approach employed here should prove of general utility in assessing the impact of environmental events on messenger RNA expression in connectionally heterogeneous cell groups that share a common biochemical phenotype.

Adrenergic Fibers↗
PubMed2025

Transcript-guided targeted cell enrichment for scalable single-nucleus RNA sequencing.

Large-scale single-cell atlases have revealed many aging- and disease-associated cell types, yet these populations are often underrepresented in heterogeneous tissues, limiting detailed molecular analyses. To address this, we developed EnrichSci-a scalable, microfluidics-free platform that combines hybridization chain reaction RNA fluorescence in situ hybridization (FISH) with combinatorial indexing to profile single-nucleus transcriptomes of target cell types with full gene-body coverage. Applied to oligodendrocytes in the aging mouse brain, EnrichSci uncovered aging-associated molecular dynamics across distinct oligodendrocyte subtypes, revealing both shared and subtype-specific gene expression changes. Additionally, we identified aging-associated exon-level signatures missed by conventional gene-level analyses, highlighting post-transcriptional regulation as a critical dimension of cell-state dynamics in aging. By coupling transcript-guided enrichment with a scalable sequencing workflow, EnrichSci provides a versatile approach to decode dynamic regulatory landscapes in diverse cell types from complex tissues.

Animals↗
PubMed2005

A human, ATP-independent, RISC assembly machine fueled by pre-miRNA.

RNA interference (RNAi) is mediated by RNA-induced silencing complexes (RISCs), which are guided by microRNAs (miRNAs) or short interfering RNAs (siRNAs) to cognate RNA targets. In humans, the catalytic engine of RISC is a ribonucleoprotein formed by the Argonaute2 (Ago2) protein and either miRNA (miRNP) or siRNA (siRNP). The Dicer nuclease produces mature miRNAs and siRNAs from pre-miRNAs and double-stranded RNA (dsRNA), respectively, and associates with Ago2. Here, we studied the assembly of human RISC by presenting pre-miRNA to immunopurified complexes that contain Ago2, Dicer, and TRBP. Mature miRNAs were produced in an ATP-independent manner and guided specific cleavage of cognate RNA targets in a pattern that is typical of RISC. This de novo formed RISC activity dissociated from Dicer. The asymmetry of the RISC loading process was fully recapitulated in this system, which, however, could not efficiently assemble RISC from siRNA duplexes. Our findings demonstrate that, in humans, a miRNA loading complex (miRLC) is formed by Ago2 and Dicer prior to their encounter with pre-miRNA. We suggest that the miRLC couples the processing of the pre-miRNA substrate to the unwinding of the product and that after loading of the mature miRNA to Ago2, the miRLC disassembles and the miRNP is released.

Animals↗
PubMed2006

The silent treatment: RNAi as a defense against virus infection in mammals.

RNA interference (RNAi) is a mechanism for sequence-specific gene silencing guided by double-stranded RNA. In plants and insects it is well established that RNAi is instrumental in the response to viral infections; whether RNAi has a similar function in mammals is under intense investigation. Recent studies to address this question have identified some unanticipated interactions between the RNAi machinery and mammalian viruses. Furthermore, introduction of virus-specific small interfering RNAs (siRNAs) into cells, thus programming the RNAi machinery to target viruses, is an effective therapeutic approach to inhibit virus replication in vitro and in animal models. Although several issues remain to be addressed, such as delivery and viral escape, these findings hold tremendous potential for the development of RNAi-based antiviral therapeutics.

Animals↗