Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “structured RNA”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

The Importance of Solution Studies for the Structural Characterization of the Enterovirus 5’ Cloverleaf

Enteroviruses initiate genomic replication via a highly conserved mechanism that is controlled by an RNA platform, also known as the 5’ cloverleaf (5’CL). Here, we present a biophysical analysis of the 5’CL conformation of three enterovirus serotypes under various ionic conditions, utilizing CD spectroscopy, size-exclusion chromatography, and small-angle X-ray scattering. In general, a tendency toward a smaller monomeric hydrodynamic radius in the presence of salts was observed, but the exact structural signature of each 5’CL varied depending upon the serotype. Rhinovirus B14 (RVB14) exhibited at least two monomeric conformations and a low propensity for dimerization, while poliovirus 1 (PV1) showed a high propensity for dimerization, which was enhanced by the presence of salts. Enterovirus D70 was observed to be somewhat intermediate, with primarily a monomeric structure, but possessing some potential for dimerization. The equilibrium between the two monomeric and the dimeric conformations is also discussed. These results indicate that the 5’CL conformation may be more complex than the current literature suggests, thus underscoring the need for a combined crystal and solution approach for the accurate representation of the 5’CL conformation, and the conformation of other RNA structural elements, under native conditions.

Virology↗
DOE OSTI2025

Phenotypically anchored transcriptomics across diverse agrichemicals reveals conserved pathways and unique gene expression signatures in zebrafish

Agrichemicals such as herbicides, fungicides, insecticides, and biocides are widely used in agriculture, yet some are associated with adverse effects in humans and the environment. While many of these chemicals have been extensively studied in vitro and are included in the EPA’s ToxCast program, comprehensive in vivo comparisons using RNA sequencing across structurally diverse agrichemicals, in a single screening platform, are lacking. In this study, we examined structurally diverse agrichemicals found in the U.S. Environmental Protection Agency’s (EPA) Toxcast Phase I and II library by statically exposing early life stage zebrafish at 6 h post fertilization (hpf) until 120 hpf at concentrations ranging from 0.25 to 100 µM. Morphological outcomes were assessed at 120 hpf across 10 endpoints, including yolk sac edema, craniofacial malformations, and axis abnormalities. Chemicals that produced robust concentration-response relationships were selected for transcriptomic profiling. For transcriptomic analysis, zebrafish were statically exposed to each chemical and sampled at 48 hpf, prior to the onset of morphological effects observed at 120 hpf. Differential expression analysis identified between 0 and 4,538 differentially expressed genes (DEGs) per chemical, with no clear correlation to morphological severity. Both DEG and co-expression network analyses revealed chemical-specific expression patterns that converged on shared biological pathways, including neurodevelopment and cytoskeletal organization. Key regulatory genes such as mylpfa and krt4 were identified within co-expression modules, suggesting their potential role in conserved toxicity mechanisms. Semantic similarity analysis of enriched gene ontology (GO) terms, when compared to existing datasets, highlighted gaps in the annotation of neurodevelopmental processes, indicating that some in vivo effects may not be fully captured by current curated resources. The results provide new insights into the modes of action of diverse agrichemicals and establish a framework for understanding how agrichemical structure relates to biological function in a vertebrate model.

agrichemical↗
DOE OSTI2021

The three-way junction structure of the HIV-1 PBS-segment binds host enzyme important for viral infectivity

HIV-1 reverse transcription initiates at the primer binding site (PBS) in the viral genomic RNA (gRNA). Although the structure of the PBS-segment undergoes substantial rearrangement upon tRNALys3 annealing, the proper folding of the PBS-segment during gRNA packaging is important as it ensures loading of beneficial host factors. DHX9/RNA helicase A (RHA) is recruited to gRNA to enhance the processivity of reverse transcriptase. Because the molecular details of the interactions have yet to be defined, we solved the solution structure of the PBS-segment preferentially bound by RHA. Evidence is provided that PBS-segment adopts a previously undefined adenosine-rich three-way junction structure encompassing the primer activation stem (PAS), tRNA-like element (TLE) and tRNA annealing arm. Disruption of the PBS-segment three-way junction structure diminished reverse transcription products and led to reduced viral infectivity. Because of the existence of the tRNA annealing arm, the TLE and PAS form a bent helical structure that undergoes shape-dependent recognition by RHA double-stranded RNA binding domain 1 (dsRBD1). Mutagenesis and phylogenetic analyses provide evidence for conservation of the PBS-segment three-way junction structure that is preferentially bound by RHA in support of efficient reverse transcription, the hallmark step of HIV-1 replication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular architecture of the Chikungunya virus replication complex

To better understand how positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) to synthesize, process, and transport viral RNA in virus-infected cells, we determined both the high-resolution structure of the core RNA replicase of chikungunya virus and the native RC architecture in its cellular context at subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase, the viral polymerase nsP4, which is in complex with nsP2 helicase-protease, sits in the central pore of the membrane-anchored nsP1 RNA-capping ring. The addition of a large cytoplasmic ring next to the C terminus of nsP1 forms the holo-RNA-RC as observed at the neck of spherules formed in virus-infected cells. These results represent a major conceptual advance in elucidating the molecular mechanisms of RNA virus replication and the principles underlying the molecular architecture of RCs, likely to be shared with many pathogenic (+) RNA viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis for Tpt1-catalyzed 2'-PO 4 transfer from RNA and NADP(H) to NAD +

Tpt1 is an essential agent of fungal and plant tRNA splicing that removes an internal RNA 2'-phosphate generated by tRNA ligase. Tpt1 also removes the 2'-phosphouridine mark installed by Ark1 kinase in the V-loop of archaeal tRNAs. Tpt1 performs a two-step reaction in which the 2'-PO 4 attacks NAD + to form an RNA-2'-phospho-(ADP-ribose) intermediate, and transesterification of the ADP-ribose O2" to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1",2"-cyclic phosphate. Here, we present structures of archaeal Tpt1 enzymes, captured as product complexes with ADP-ribose-1"-PO 4 , ADP-ribose-2"-PO 4 , and 2'-OH RNA, and as substrate complexes with 2',5'-ADP and NAD + , that illuminate 2'-PO 4 junction recognition and catalysis. We show that archaeal Tpt1 enzymes can use the 2'-PO 4 -containing metabolites NADP + and NADPH as substrates for 2'-PO 4 transfer to NAD + . A role in 2'-phospho-NADP(H) dynamics provides a rationale for the prevalence of Tpt1 in taxa that lack a capacity for internal RNA 2'-phosphorylation.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Development of high throughput and in vitro assays for analyzing RNA modifications

Modifications on RNAs play major roles in their stability, translation, and enzymatic activity. Despite its importance, the current techniques are insufficient to study the structure and function of RNA modifications. Indeed, the National Academies of Science, Engineering and Medicine indicate that developing new tools and further study the function of RNA modifications is strategically a high priority for advancing science in the coming years (https://www.nationalacademies.org/our-work/toward-sequencing-and-mapping-of-rna-modifications). RNA modifications occur in all domains of life controlling processes such as RNA turnover, translation regulation, cellular defenses and bioproduction. Our preliminary data indicated that the insulin mRNA might get ADP-ribosylated by the ADP-ribosyltransferase PARP12. RNA ADP-ribosylation has been described in Escherichia coli. Combined to the fact that ADP-ribosyltransferase (PARP) genes are conserved throughout evolution we hypothesize that this modification might play essential roles in cells. Therefore, we proposed to develop sequencing techniques and in vitro enzymatic assays to identify and validate ADP-ribosylation motifs and sites. Here we report the development of RNA-seq and qPCR assays to identify ADP-ribosylated RNAs, in addition to a nicotinamide adenosine dinucleotide (NAD – ADP-ribosylation donor) consumption assay and an enzyme-linked immunosorbent assay (ELISA) to measure ADP-ribosyltransferase activity. Testing these assays with the insulin mRNA confirmed that this transcript is ADP-ribosylated. These assays will not only enable studying the function of ADP-ribosylation but can be easily adapted for studying other RNA modifications. This will open opportunities to study RNA modifications in different model systems from bacteria to viruses to plants, bringing insights into their cellular functions and the possibility of targeting them for biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Naturally ornate RNA-only complexes revealed by cryo-EM

The structures of natural RNAs remain poorly characterized and may hold numerous surprises. Here we report three-dimensional structures of three large ornate bacterial RNAs using cryo-electron microscopy (cryo-EM). GOLLD (Giant, Ornate, Lake- and Lactobacillales-Derived), ROOL (Rumen-Originating, Ornate, Large) and OLE (Ornate Large Extremophilic) RNAs form homo-oligomeric complexes whose stoichiometries are retained at lower concentrations than measured in cells. OLE RNA forms a dimeric complex with long co-axial pipes spanning two monomers. Both GOLLD and ROOL form distinct RNA-only multimeric nanocages with diameters larger than the ribosome, each empty except for a disordered loop. Extensive intramolecular and intermolecular A-minor interactions, kissing loops, an unusual A–A helix and other interactions stabilize the three complexes. Sequence covariation analysis of these large RNAs reveals evolutionary conservation of intermolecular interactions, supporting the biological importance of large, ornate RNA quaternary structures that can assemble without any involvement of proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Epitopes recognition of SARS-CoV-2 nucleocapsid RNA binding domain by human monoclonal antibodies

Coronavirus nucleocapsid protein (NP) of SARS-CoV-2 plays a central role in many functions important for virus proliferation including packaging and protecting genomic RNA. The protein shares sequence, structure, and architecture with nucleocapsid proteins from betacoronaviruses. The N-terminal domain (NP RBD ) binds RNA and the C-terminal domain is responsible for dimerization. After infection, NP is highly expressed and triggers robust host immune response. The anti-NP antibodies are not protective and not neutralizing but can effectively detect viral proliferation soon after infection. Two structures of SARS-CoV-2 NP RBD were determined providing a continuous model from residue 48 to 173, including RNA binding region and key epitopes. Five structures of NP RBD complexes with human mAbs were isolated using an antigen-bait sorting. Complexes revealed a distinct complement-determining regions and unique sets of epitope recognition. This may assist in the early detection of pathogens and designing peptide-based vaccines. Mutations that significantly increase viral load were mapped on developed, full length NP model, likely impacting interactions with host proteins and viral RNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A minimal complex of KHNYN and zinc-finger antiviral protein binds and degrades single-stranded RNA

Detecting viral infection is a key role of the innate immune system. The genomes of some RNA viruses have a high CpG dinucleotide content relative to most vertebrate cell RNAs, making CpGs a molecular marker of infection. The human zinc-finger antiviral protein (ZAP) recognizes CpG, mediates clearance of the foreign CpG-rich RNA, and causes attenuation of CpG-rich RNA viruses. While ZAP binds RNA, it lacks enzymatic activity that might be responsible for RNA degradation and thus requires interacting cofactors for its function. One of these cofactors, KHNYN, has a predicted nuclease domain. Using biochemical approaches, we found that the KHNYN NYN domain is a single-stranded RNA ribonuclease that does not have sequence specificity and digests RNA with or without CpG dinucleotides equivalently in vitro. We show that unlike most KH domains, the KHNYN KH domain does not bind RNA. Indeed, a crystal structure of the KH region revealed a double-KH domain with a negatively charged surface that accounts for the lack of RNA binding. Rather, the KHNYN C-terminal domain (CTD) interacts with the ZAP RNA-binding domain (RBD) to provide target RNA specificity. We define a minimal complex composed of the ZAP RBD and the KHNYN NYN-CTD and use a fluorescence polarization assay to propose a model for how this complex interacts with a CpG dinucleotide-containing RNA. In the context of the cell, this module would represent the minimum ZAP and KHNYN domains required for CpG-recognition and ribonuclease activity essential for attenuation of viruses with clusters of CpG dinucleotides.

Yeoh, Zoe C. (ORCID:0000000226949068)↗
DOE OSTI2021

The structural plasticity of nucleic acid duplexes revealed by WAXS and MD

Double-stranded DNA (dsDNA) and RNA (dsRNA) helices display an unusual structural diversity. Some structural variations are linked to sequence and may serve as signaling units for protein-binding partners. Therefore, elucidating the mechanisms and factors that modulate these variations is of fundamental importance. While the structural diversity of dsDNA has been extensively studied, similar studies have not been performed for dsRNA. Because of the increasing awareness of RNA’s diverse biological roles, such studies are timely and increasingly important. We integrate solution x-ray scattering at wide angles (WAXS) with all-atom molecular dynamics simulations to explore the conformational ensemble of duplex topologies for different sequences and salt conditions. These tightly coordinated studies identify robust correlations between features in the WAXS profiles and duplex geometry and enable atomic-level insights into the structural diversity of DNA and RNA duplexes. Notably, dsRNA displays a marked sensitivity to the valence and identity of its associated cations.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2020

Structure-function analysis of microRNA 3'-end trimming by Nibbler

Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr’s N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed α/β-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr’s EXO domain exhibits Mn2+-dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'-O-methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The structural basis of mRNA recognition and binding by yeast pseudouridine synthase PUS1

The chemical modification of RNA bases represents a ubiquitous activity that spans all domains of life. Pseudouridylation is the most common RNA modification and is observed within tRNA, rRNA, ncRNA and mRNAs. Pseudouridine synthase or ‘PUS’ enzymes include those that rely on guide RNA molecules and others that function as ‘stand-alone’ enzymes. Among the latter, several have been shown to modify mRNA transcripts. Although recent studies have defined the structural requirements for RNA to act as a PUS target, the mechanisms by which PUS1 recognizes these target sequences in mRNA are not well understood. Here we describe the crystal structure of yeast PUS1 bound to an RNA target that we identified as being a hot spot for PUS1-interaction within a model mRNA at 2.4 Å resolution. The enzyme recognizes and binds both strands in a helical RNA duplex, and thus guides the RNA containing the target uridine to the active site for subsequent modification of the transcript. The study also allows us to show the divergence of related PUS1 enzymes and their corresponding RNA target specificities, and to speculate on the basis by which PUS1 binds and modifies mRNA or tRNA substrates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Binding of Campylobacter jejuni FliW Adjacent to the CsrA RNA-Binding Pockets Modulates CsrA Regulatory Activity

Campylobacter jejuni CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to E. coli CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), C. jejuni CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of C. jejuni CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori

Abstract Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3′-5′ exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The ‘prototypical’ Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, which include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.

Tejada-Arranz, Alejandro↗