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At least 19 recordsLinked to original sources
PubMed2026

The cellular protein TIAR mediates rapid initiation of West Nile virus genome RNA synthesis.

During the intracellular replication cycle of West Nile virus (WNV), genome RNA synthesis is initially inefficient but increases exponentially as viral replication complexes are sequestered in invaginations in the endoplasmic reticulum. In this study, we investigated the functional role of the cellular protein TIAR (T-cell intracellular antigen-related protein) in the transcription of WNV genome RNA. Close colocalization of cytoplasmic TIAR with viral double-stranded RNA was detected by a proximity ligation assay in WNV-infected cells. TIAR binds specifically to the WNV 3'(-) SL but not to the complementary WNV 5'(+) SL in in vitro RNA binding assays. Only the 3' end of the WNV minus-strand RNA was enriched by immunoprecipitation of infected cell lysates with anti-TIAR antibody. Stable overexpression of TIAR in clonal A549 cells increased the ratio of intracellular viral plus-strand to minus-strand RNA in a dose-dependent manner. TIAR contains three RNA recognition motifs (RRMs). Biophysical data indicated that only RRM2 directly contacts RNA and that up to three TIAR molecules can bind cooperatively to the WNV 3'(-) SL RNA. These data provide additional evidence that TIAR functions as a proviral host factor facilitating exponential amplification of WNV genome production in infected cells.IMPORTANCEWest Nile virus (WNV) is a mosquito-borne orthoflavivirus associated with increasing global human disease incidence. The molecular mechanisms underlying viral replication are not fully understood. In early stages of infection, viral genome transcription is inefficient; however, in late stages, viral genome transcription increases exponentially. T-cell intracellular antigen-related (TIAR) protein is a cellular protein that has been shown to interact with the 3' end of the WNV negative-sense antigenomic RNA. We obtained data showing colocalization of cellular TIAR with viral replication complexes in infected cells and an increased ratio of intracellular genomic to antigenomic viral RNA in TIAR-overexpressing cells, and confirmed preferential binding of TIAR to the 3' end of the WNV antigenome both in vitro and in infected cell extracts. We also demonstrated that multiple TIAR proteins can bind cooperatively to the WNV 3'(-) stem-loop RNA. These data provide supporting evidence for a model of TIAR-mediated rapid initiation of nascent genome RNA synthesis in infected cells.

TIAR↗
PubMed2025

Ebola virus VP35 NNLNS motif modulates viral RNA synthesis and MIB2-mediated signaling.

Ebola virus (EBOV) is a nonsegmented, negative-sense virus (NNSV) with a single-stranded RNA genome. EBOV encodes for a limited number of proteins and thus depends on host factors to facilitate viral replication and pathogenesis. Of the virus-encoded proteins, multifunctional EBOV VP35 (eVP35) is necessary for host immune evasion and viral RNA synthesis. Previous proteomics studies identified an interaction between eVP35 and the host E3 ubiquitin ligase Mindbomb 2 (MIB2). Here, we show how an NNLNS (Asn-Asn-Leu-Asn-Ser) motif (residues 201 to 205) within eVP35 serves as a binding site for MIB2. This motif is critical for eVP35-dependent inhibition of MIB2-mediated interferon induction. It is also important for EBOV RNA synthesis as MIB2 binding to eVP35 inhibited EBOV minigenome activity. Altogether, these findings highlight the importance of the eVP35 protein and the role of host factors in EBOV infection.

Ebolavirus↗
PubMed2025

Defining the networks that connect RNase III and RNase J-mediated regulation of primary and specialized metabolism in Streptomyces venezuelae.

UNLABELLED: RNA metabolism involves coordinating RNA synthesis with RNA processing and degradation. Ribonucleases play fundamental roles within the cell, contributing to the cleavage, modification, and degradation of RNA molecules, with these actions ensuring appropriate gene regulation and cellular homeostasis. Here, we employed RNA sequencing to explore the impact of RNase III and RNase J on the transcriptome of Streptomyces venezuelae. Differential expression analysis comparing wild-type and RNase mutant strains at distinct developmental stages revealed significant changes in transcript abundance, particularly in pathways related to multicellular development, nutrient acquisition, and specialized metabolism. Both RNase mutants exhibited dysregulation of the BldD regulon, including altered expression of many cyclic-di-GMP-associated enzymes. We also observed precocious chloramphenicol production in these RNase mutants and found that in the RNase III mutant, this was associated with PhoP-mediated regulation. We further found that RNase III directly targeted members of the PhoP regulon, suggesting a link between RNA metabolism and a regulator that bridges primary and specialized metabolism. We connected RNase J function with translation through the observation that RNase J directly targets multiple ribosomal protein transcripts for degradation. These findings establish distinct but complementary roles for RNase III and RNase J in coordinating the gene expression dynamics critical for S. venezuelae development and specialized metabolism. IMPORTANCE: RNA processing and metabolism are mediated by ribonucleases and are fundamental processes in all cells. In the morphologically complex and metabolically sophisticated Streptomyces bacteria, RNase III and RNase J influence both development and metabolism through poorly understood mechanisms. Here, we show that both ribonucleases are required for the proper expression of the BldD developmental pathway and contribute to the control of chloramphenicol production, with an interesting connection to phosphate regulation for RNase III. Additionally, we show that both RNases have the potential to impact translation through distinct mechanisms and can function cooperatively in degrading specific transcripts. This study advances our understanding of RNases in Streptomyces biology by providing insight into distinct contributions made by these enzymes and the intriguing interplay between them.

Streptomyces↗
PubMed2025

Structure of coxsackievirus cloverleaf RNA and 3Cpro dimer establishes the RNA-binding mechanism of enterovirus protease 3Cpro.

In positive-strand RNA viruses, the genome serves as a template for both protein translation and negative-strand RNA synthesis. Enteroviruses use the cloverleaf RNA structure at the 5' end of the genome to balance these two processes. Cloverleaf acts as a promoter for RNA synthesis and forms a complex with viral 3CD protein, the precursor to 3Cpro protease, and 3Dpol polymerase. The interaction between cloverleaf and 3CD is mediated by the 3Cpro domain, yet how 3Cpro promotes specific RNA-binding is not clear. We report the structure of coxsackievirus cloverleaf RNA-3Cpro complex, wherein two 3Cpro molecules interact with cloverleaf stem-loop D. 3Cpro dimer mainly recognizes the shape of the dsRNA helix through symmetric interactions, suggesting that 3Cpro is a previously undiscovered type of RNA binding protein. We show that 3CD protein also dimerizes on cloverleaf RNA and binds the RNA with higher affinity than 3Cpro. The structure provides insight into the RNA-binding mechanism of 3Cpro or 3CD with other cis-acting replication elements.

RNA, Viral↗
PubMed2026

Spatial mapping of RNA turnover kinetics in the mouse brain.

Gene regulation requires coordinated control of RNA synthesis and degradation, yet measuring RNA turnover across intact tissues remains challenging. Here we present spatial NT-seq, a method that combines transgenesis-free metabolic RNA labeling with in situ chemical recoding on spatial transcriptomics platforms to co-map newly synthesized and pre-existing RNAs. Applying spatial NT-seq to the mouse brain reveals pronounced regional heterogeneity in RNA turnover and identifies the dentate gyrus as a spatial hotspot marked by coordinated upregulation of basal RNA synthesis and decay. Moreover, spatial NT-seq uncovers rapid, brain region-specific transcriptional and post-transcriptional responses to electroconvulsive stimulation, a clinically relevant treatment for refractory depression. Finally, we leverage computational modeling to identify sequence features and post-transcriptional regulators that shape transcriptome-wide mRNA stability across spatial and cellular contexts in the mouse brain. Together, this integrated 'in vivo timescope' framework provides a spatially resolved view of RNA turnover kinetics and reveals the regulatory architecture of RNA stability in vivo.

Journal Article↗
PubMed2005

Factors required for the Uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro.

The 5' terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D(pol). To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D(pol) in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV cre has been identified previously to be within the 5' untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

Enhancer Elements, Genetic↗
PubMed2025

Purification and Ultramicroscopic Observation of the Influenza A Virus Ribonucleoprotein Complex.

Influenza A virus (IAV) has an eight-segmented, single-stranded, negative-sense viral genomic RNA (vRNA). Each vRNA strand associates with nucleoproteins and an RNA-dependent RNA polymerase complex to form a viral ribonucleoprotein (vRNP) complex. IAV vRNPs adopt a flexible double-helical configuration that varies in length. Although the transcription and replication of vRNA take place in the context of vRNPs, the precise structural conformation of vRNPs during RNA synthesis remains partially elucidated. To unravel the intricate ultrastructure of the vRNP, it is necessary to purify it while preserving its native functionality. Herein, we introduce a comprehensive protocol for the purification of IAV vRNPs using glycerol gradient ultracentrifugation. Furthermore, we provide a method for the high-speed atomic force microscopy observation of vRNPs during viral RNA synthesis.

Ribonucleoproteins↗
PubMed2026

Structure and dynamics of the Nipah virus RNA-dependent RNA polymerase.

Nipah virus (NiV) is a highly pathogenic, nonsegmented, negative-sense RNA virus (nsNSV) from the Mononegavirales order that causes frequent outbreaks, with no approved treatment available. Replication and transcription of its genome are carried out by a viral RNA-dependent RNA polymerase (RdRp) complex composed of the large catalytic protein (L) and the tetrameric phosphoprotein (P). Recently, structural insights into the NiV RdRp complex have emerged at an unprecedented pace. In particular, snapshots of the complex in precatalytic, early-elongation, and inhibitor-bound states have been reported. In this article, we review how these data shed light on the molecular mechanisms of RNA synthesis and inhibition in NiV and explore how these insights expand our understanding of nsNSV RdRps in general.

Nipah Virus↗
PubMed2004

A bunyamwera virus minireplicon system in mosquito cells.

Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses. Bunyamwera virus (BUN; genus Orthobunyavirus, family Bunyaviridae) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells. To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells. An Aedes albopictus C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established. Viral proteins and artificial minigenomes (containing Renilla luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids. Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells. However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs. We also report that promoter strength is different for vertebrate versus invertebrate cells. The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.

Aedes↗
PubMed2024

Alpha- and betacoronavirus cis-acting RNA elements.

Coronaviruses have exceptionally large RNA genomes and employ multiprotein replication/transcription complexes to orchestrate specific steps of viral RNA genome replication and expression. Most of these processes involve viral cis-acting RNA elements that are engaged in vital RNA-RNA and/or RNA-protein interactions. Over the past years, a large number of studies provided interesting new insight into the structures and, to a lesser extent, functions of specific RNA elements for representative coronaviruses, and there is evidence to suggest that (a majority of) these RNA elements are conserved across genetically divergent coronavirus genera. It is becoming increasingly clear that at least some of these elements do not function in isolation but operate through complex and highly dynamic RNA-RNA interactions. This article reviews structural and functional aspects of cis-acting RNA elements conserved in alpha- and betacoronavirus 5'- and 3'-terminal genome regions, focusing on their critical roles in viral RNA synthesis and gene expression.

RNA, Viral↗
PubMed2026

A novel dimerization site in non-structural protein 5A of hepatitis C virus regulates viral replication fitness.

We previously found that high genome replication fitness of the hepatitis C virus (HCV) was associated with severe disease in immunocompromised patients. Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReED's mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity/resistance. Our data overall suggest that the ReED in NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and/or polymerase, thereby likely facilitating initiation of RNA synthesis.

Viral Nonstructural Proteins↗
PubMed2024

NusG-Spt5 Transcription Factors: Universal, Dynamic Modulators of Gene Expression.

The accurate and efficient biogenesis of RNA by cellular RNA polymerase (RNAP) requires accessory factors that regulate the initiation, elongation, and termination of transcription. Of the many discovered to date, the elongation regulator NusG-Spt5 is the only universally conserved transcription factor. With orthologs and paralogs found in all three domains of life, this ubiquity underscores their ancient and essential regulatory functions. NusG-Spt5 proteins evolved to maintain a similar binding interface to RNAP through contacts of the NusG N-terminal domain (NGN) that bridge the main DNA-binding cleft. We propose that varying strength of these contacts, modulated by tethering interactions, either decrease transcriptional pausing by smoothing the rugged thermodynamic landscape of transcript elongation or enhance pausing, depending on which conformation of RNAP is stabilized by NGN contacts. NusG-Spt5 contains one (in bacteria and archaea) or more (in eukaryotes) C-terminal domains that use a KOW fold to contact diverse targets, tether the NGN, and control RNA biogenesis. Recent work highlights these diverse functions in different organisms. Some bacteria contain multiple specialized NusG paralogs that regulate subsets of operons via sequence-specific targeting, controlling production of antibiotics, toxins, or capsule proteins. Despite their common origin, NusG orthologs can differ in their target selection, interacting partners, and effects on RNA synthesis. We describe the current understanding of NusG-Spt5 structure, interactions with RNAP and other regulators, and cellular functions including significant recent progress from genome-wide analyses, single-molecule visualization, and cryo-EM. The recent findings highlight the remarkable diversity of function among these structurally conserved proteins.

Archaea↗
PubMed2005

Attenuation of bunyavirus replication by rearrangement of viral coding and noncoding sequences.

Bunyamwera virus (BUN) is the prototype virus of the family Bunyaviridae. BUN has a tripartite negative-sense RNA genome comprising small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments, while the internal regions are unique. The UTRs direct replication and transcription of viral RNA and are sufficient to allow encapsidation of viral RNA into ribonucleoprotein complexes. To investigate the segment-specific functions of the UTRs, we have used reverse genetics to recover a recombinant virus (called BUN MLM) in which the L segment open reading frame (ORF) is flanked by the M segment UTRs. Compared to wild-type virus, BUN MLM virus shows growth attenuation in cultured mammalian cells and a slower disease progression in mice, produces small plaques, expresses reduced levels of L mRNA and L (RNA polymerase) protein, synthesizes less L genomic and antigenomic RNA, and has an increased particle-to-PFU ratio. Our data suggest that the packaging of BUN RNAs is not segment specific. In addition, the phenotype of BUN MLM virus supports the finding that BUN UTRs differ in their regulation of RNA synthesis but suggests that the interplay between each segment UTR and its cognate ORF may contribute to that regulation. Since BUN MLM virus is attenuated due to an essentially irreversible mutation, the rearrangement of UTRs is a feasible strategy for vaccine design for the more pathogenic members of the Bunyaviridae.

Animals↗
PubMed2025

Approaches to Study Proteins Encoded by Essential Genes.

Although the phenotypes and functions of nonessential proteins can be studied by deletion of their coding sequences (both gene copies in diploid organisms), essential genes cannot be deleted unless loss of the encoded protein can be bypassed. Bypass is often achieved by supplementation with the product of the enzyme. However, supplementation cannot bypass loss of essential genes such as those encoding enzymes of DNA or RNA synthesis. To study proteins encoded by essential genes that cannot be bypassed, the mutations must be conditional in nature. The mutant cells must be able to grow under a permissive condition, but fail to grow under a different condition, the nonpermissive condition. Several methods have been developed to obtain conditional mutations in essential genes. Mutations that result in proteins abnormally sensitive to high temperatures are called temperature-sensitive (Ts) mutants and are a widely used type of conditional mutation. An alternative to Ts mutants is the "degron" system to target proteins for destruction by cellular proteases. Approaches to conditionally control the functions of proteins encoded by essential genes, plus the advantages and disadvantages of these and other approaches, will be considered.

Genes, Essential↗
PubMed2023

Emerging roles of nucleotide metabolism in cancer.

Nucleotides are substrates for multiple anabolic pathways, most notably DNA and RNA synthesis. Since nucleotide synthesis inhibitors began to be used for cancer therapy in the 1950s, our understanding of how nucleotides function in tumor cells has evolved, prompting a resurgence of interest in targeting nucleotide metabolism for cancer therapy. In this review, we discuss recent advances that challenge the idea that nucleotides are mere building blocks for the genome and transcriptome and highlight ways that these metabolites support oncogenic signaling, stress resistance, and energy homeostasis in tumor cells. These findings point to a rich network of processes sustained by aberrant nucleotide metabolism in cancer and reveal new therapeutic opportunities.

Humans↗
PubMed2026

Cold-adapted RNA polymerase from Pseudomonas phage Njord improves synthesis of therapeutic mRNA.

An RNA polymerase identified in the genome of Pseudomonas phage Njord offers a promising tool for the synthesis of mRNA and other therapeutic nucleic acids. Originating from a marine microbial ecosystem, Njord RNAP transcribes RNA at high yield even under low temperature conditions. Key properties of the enzyme relevant to mRNA synthesis are presented including transcriptional fidelity, promoter specificity, incorporation of modified nucleotides, and the impurity profile of the RNA. Specific attention is given to the formation of contaminating double-stranded RNA (dsRNA) species. Analysis of transcription reactions shows that DNA-templated promoter-independent transcription is a major source of detectable dsRNA impurities and that Njord RNAP displays a minimal level of this activity. Consistent with the known inflammatory role of dsRNA in synthetic mRNA, transcriptomic analysis of cell culture and a live animal study demonstrates that mRNA synthesized with Njord RNAP elicits only a minimal immune response. This natural enzyme enables efficient mRNA synthesis at ambient temperature and produces transcripts essentially free of dsRNA, offering significant potential to streamline mRNA manufacturing processes.

DNA-Directed RNA Polymerases↗
PubMed2026

Retrotransposon-based mechanisms for transgene addition to the human genome.

When human disease arises from a loss of function caused by diverse mutant alleles of the same gene, the patient population could be best served by a clinical therapy that achieves genome safe-harbor supplementation with a functional transgene. Until recently, transgene delivery strategies have shared the disadvantages of induced immune responses and/or genome mutagenesis from untargeted DNA insertion. As a different strategy, several groups recently described the use of retrotransposon proteins to accomplish transgene insertion by RNA-templated cDNA synthesis directly into the genome. In some strategies, gene insertion relies on the retrotransposon protein to bring a transgene-encoding template RNA to the target site. Retrotransposon protein positioning of template RNA for cDNA synthesis minimizes the requirement for RNA base-pairing to target-site DNA. This review presents an overview of RNA-templated DNA synthesis in cells as backdrop for describing recent uses of retrotransposon reverse transcriptases to supplement the human genome.

Journal Article↗
PubMed2026

Dynamic Alterations in the Blood Transcriptome Characterize Drug Use Behavior and Co-Morbidities in Cocaine Use Disorder: A Preliminary Study.

Individuals with cocaine use disorder (CUD) who attempt abstinence experience craving and relapse that can benefit from multimodal treatment monitoring. Longitudinal studies linking behavioral manifestations in CUD to the blood transcriptome are not only limited but also computationally complex. Therefore, we developed an analytical pipeline to investigate the connection between drug use behaviors during abstinence and change in the blood transcriptome. We conducted a longitudinal study with CUD (n = 12 subjects) and collected behavioral metrics and blood RNA-seq at baseline, 3, 6, and 9 months. Our analytical pipeline of the high-dimensional data encompasses hierarchical k-means clustering to classify subjects to responder groups based on behavioral scores and abstinence duration, in silico cell deconvolution, differential analysis with correlated multivariate testing over time, gene set enrichment analysis, and gene co-expression with time splines and RNA-seq data. The pipeline captured dynamic changes in behavioral scores and abstinence duration in responder groups. Genes showing differential transcript-level expression were enriched in substance use and cardiovascular disease-associated genetic risk loci in responder groups. Lastly, time-dependent gene co-expression revealed dynamic changes related to immune processes, cell cycle, RNA-protein synthesis, and second messenger signaling for days of abstinence. This is a preliminary investigation, providing an innovative and scalable pipeline for blood-based longitudinal RNA-seq studies in CUD, potentially applicable to other substance use disorders. It outlines a data-driven approach for analyzing composite longitudinal drug use behavioral phenotypes with blood-based transcriptomics. We also demonstrate changes in drug use behaviors and the blood transcriptome during drug abstinence.

Humans↗