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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.

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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles

Cell-free protein synthesis is an important tool for studying gene expression and harnessing it for applications. In cells, gene expression is regulated in part by the spatial organization of transcription and translation. Unfortunately, current cell-free approaches are unable to control the organization of molecular components needed for gene expression, which limits the ability to probe and utilize its effects. In this work, we show, using complementary computational and experimental approaches, that macromolecular crowding can be used to control the spatial organization and translational efficiency of gene expression in cell-sized vesicles. Computer simulations and imaging experiments reveal that, as crowding is increased, DNA plasmids become localized at the inner surface of vesicles. Ribosomes, in contrast, remain uniformly distributed, demonstrating that crowding can be used to differentially organize components of gene expression. We further carried out cell-free protein synthesis reactions in cell-sized vesicles and quantified mRNA and protein abundance. At sufficiently high levels of crowding, we observed localization of mRNA near vesicle surfaces, a decrease in translational efficiency and protein abundance, and anomalous scaling of protein abundance as a function of vesicle size. These results are consistent with high levels of crowding causing altered spatial organization and slower diffusion. Our work demonstrates a straightforward way to control the organization of gene expression in cell-sized vesicles and provides insight into the spatial regulation of gene expression in cells.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Structural basis of RNA cap modification by SARS-CoV-2

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5'-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2'-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2'-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping

N 4 -acetylcytidine (ac 4 C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. In this work, we report ac 4 C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac 4 C at single-nucleotide resolution. In human and yeast mRNAs, ac 4 C sites are not detected but can be induced—at a conserved sequence motif—via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac 4 C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac 4 C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac 4 C and its potential thermoadaptive role. Our studies quantitatively define the ac 4 C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt

The intestinal epithelium is replaced weekly by non-quiescent stem cells with kinetics that rely on a rapid loss of stemness and choice for secretory or absorptive lineage differentiation. To determine how the cellular transcriptome and proteome changes during these transitions, we developed a new cell sorting method to purify stem cells, secretory and absorptive progenitor cells, and mature, differentiated cells. Transcriptome analyses revealed that as stem cells transition to the progenitor stage, alternative mRNA splicing and polyadenylation dominate changes in the transcriptome. In contrast, as progenitors differentiate into mature cell types, alterations in gene expression and mRNA levels drive the changes. RNA processing targets mRNAs encoding regulators of cell cycle, RNA regulators, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling of sorted cell populations detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

High-throughput Single-Cell Proteomics and Transcriptomics from the Same Cells with a Nanoliter-Scale Spin-Transfer Approach

Single-cell multiomic platforms provide a comprehensive snapshot of cellular states and cell types by offering critical insights into the spatiotemporal regulation of biomolecular networks at a systems level, thereby defining the basis of multicellularity. Here, we introduce nanoSPINS, an advanced platform that enables high-throughput profiling and integrative analysis of the transcriptome and proteome from the same single cells using RNA sequencing and isobaric labeling LC-MS-based proteomics, respectively. NanoSPINS can efficiently transfer mRNA-containing droplets across two microarrays via a centrifugation-based approach, while proteins are retained on the initial platform. Benchmarking of nanoSPINS on two cell lines demonstrates its ability to generate global proteomic and transcriptomic profiles that align well with previously established methodologies/platforms. The incorporation of isobaric TMTpro labeling into this single-cell multiomics platform significantly enhances the throughput of single-cell proteomic analyses. Through the high-throughput quantification of the proteome and transcriptome, nanoSPINS not only facilitates the identification of molecular features at both mRNA and protein level but also provides larger sample sizes for improved statistical power in clustering and differential abundance. Given the broad applicability of single-cell multiomics in biological research and clinical settings, we believe nanoSPINS represents a powerful platform for the characterization of heterogeneous cell populations.

multi 'omics↗
DOE OSTI2020

Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu∙GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu∙GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Pseudouridine synthase 7 is an opportunistic enzyme that binds and modifies substrates with diverse sequences and structures

Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, in this study, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UGUAR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structure of saguaro cactus virus 3′ translational enhancer mimics 5′ cap for eIF4E binding

The genomes of several plant viruses contain RNA structures at their 3' ends called cap-independent translation enhancers (CITEs) that bind the host protein factors such as mRNA 5' cap-binding protein eIF4E for promoting cap-independent genome translation. However, the structural basis of such 5' cap-binding protein recognition by the uncapped RNA remains largely unknown. Here, we have determined the crystal structure of a 3' CITE, panicum mosaic virus-like translation enhancer (PTE) from the saguaro cactus virus (SCV), using a Fab crystallization chaperone. The PTE RNA folds into a three-way junction architecture with a pseudoknot between the purine-rich R domain and pyrimidine-rich Y domain, which organizes the overall structure to protrude out a specific guanine nucleotide, G18, from the R domain that comprises a major interaction site for the eIF4E binding. The superimposable crystal structures of the wild-type, G18A, G18C, and G18U mutants suggest that the PTE scaffold is preorganized with the flipped-out G18 ready to dock into the eIF4E 5' cap-binding pocket. The binding studies with wheat and human eIF4Es using gel electrophoresis and isothermal titration calorimetry, and molecular docking computation for the PTE–eIF4E complex demonstrated that the PTE structure essentially mimics the mRNA 5' cap for eIF4E binding. Such 5' cap mimicry by the uncapped and structured viral RNA highlights how viruses can exploit RNA structures to mimic the host protein-binding partners and bypass the canonical mechanisms for their genome translation, providing opportunities for a better understanding of virus-host interactions and non-canonical translation mechanisms found in many pathogenic RNA viruses.

3' cap-independent translation enhancers↗
DOE OSTI2024

Stress-regulated Arabidopsis GAT2 is a low affinity γ-aminobutyric acid transporter

Abstract The four-carbon non-proteinogenic amino acid γ-aminobutyric acid (GABA) accumulates to high levels in plants in response to various abiotic and biotic stress stimuli, and plays a role in C:N balance, signaling, and as a transport regulator. Expression in Xenopus oocytes and voltage-clamping allowed the characterization of Arabidopsis GAT2 (At5g41800) as a low affinity GABA transporter with a K0.5GABA ~8 mM. l-Alanine and butylamine represented additional substrates. GABA-induced currents were strongly dependent on the membrane potential, reaching the highest affinity and highest transport rates at strongly negative membrane potentials. Mutation of Ser17, previously reported to be phosphorylated in planta, did not result in altered affinity. In a short-term stress experiment, AtGAT2 mRNA levels were up-regulated at low water potential and under osmotic stress (polyethylene glycol and mannitol). Furthermore, AtGAT2 promoter activity was detected in vascular tissues, maturating pollen, and the phloem unloading region of young seeds. Even though this suggested a role for AtGAT2 in long-distance transport and loading of sink organs, under the conditions tested neither AtGAT2-overexpressing plants, atgat2 or atgat1 T-DNA insertion lines, nor atgat1 atgat2 doubleknockout mutants differed from wild-type plants in growth on GABA, amino acid levels, or resistance to salt and osmotic stress.

Plant Sciences↗
DOE OSTI2022

CRISPR-RNAa: targeted activation of translation using dCas13 fusions to translation initiation factors

Abstract Tools for synthetically controlling gene expression are a cornerstone of genetic engineering. CRISPRi and CRISPRa technologies have been applied extensively for programmable modulation of gene transcription, but there are few such tools for targeted modulation of protein translation rates. Here, we employ CRISPR-Cas13 as a programmable activator of translation. We develop a novel variant of the catalytically-deactivated Cas13d enzyme dCasRx by fusing it to translation initiation factor IF3. We demonstrate dCasRx-IF3’s ability to enhance expression 21.3-fold above dCasRx when both are targeted to the start of the 5′ untranslated region of mRNA encoding red fluorescent protein in Escherichia coli. Activation of translation is location-dependent, and we show dCasRx-IF3 represses translation when targeted to the ribosomal binding site, rather than enhancing it. We provide evidence that dCasRx-IF3 targeting enhances mRNA stability relative to dCasRx, providing mechanistic insights into how this new tool functions to enhance gene expression. We also demonstrate targeted upregulation of native LacZ 2.6-fold, showing dCasRx-IF3’s ability to enhance expression of endogenous genes. dCasRx-IF3 requires no additional host modification to influence gene expression. This work outlines a novel approach, CRISPR-RNAa, for post-transcriptional control of translation to activate gene expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Adrenal Stress Hormone Regulation of Hepatic Homeostatic Function After an Acute Ozone Exposure in Wistar-Kyoto Male Rats

Ozone-induced lung injury, inflammation, and pulmonary/hypothalamus gene expression changes are diminished in adrenalectomized (AD) rats. Acute ozone exposure induces metabolic alterations concomitant with increases in epinephrine and corticosterone. We hypothesized that adrenal hormones are responsible for observed hepatic ozone effects, and in AD rats, these changes would be diminished. In total, 5–7 days after sham (SH) or AD surgeries, male Wistar-Kyoto rats were exposed to air or 0.8-ppm ozone for 4 h. Serum samples were analyzed for metabolites and liver for transcriptional changes immediately post-exposure. Ozone increased circulating triglycerides, cholesterol, free fatty-acids, and leptin in SH but not AD rats. Ozone-induced inhibition of glucose-mediated insulin release was absent in AD rats. Unlike diminution of ozone-induced hypothalamus and lung mRNA expression changes, AD in air-exposed rats (AD-air/SH-air) caused differential hepatic expression of ~1000 genes. Likewise, ozone in AD rats caused differential expression of ~1000 genes (AD-ozone/AD-air). Ozone-induced hepatic changes in SH rats reflected enrichment for pathways involving metabolic processes, including acetyl-CoA biosynthesis, TCA cycle, and sirtuins. Upstream predictor analysis identified similarity to responses produced by glucocorticoids and pathways involving forskolin. These changes were absent in AD rats exposed to ozone. However, ozone caused unique changes in AD liver mRNA reflecting activation of synaptogenesis, neurovascular coupling, neuroinflammation, and insulin signaling with inhibition of senescence pathways. In these rats, upstream predictor analysis identified numerous microRNAs likely involved in glucocorticoid insufficiency. These data demonstrate the critical role of adrenal stress hormones in ozone-induced hepatic homeostasis and necessitate further research elucidating their role in propagating environmentally driven diseases.

Toxicology↗
DOE OSTI2025

CsrA-mediated regulation of a virulence switch in Acinetobacter baumannii

CsrA is an RNA binding protein that functions as a global regulator in bacteria. We demonstrate that, in Acinetobacter baumannii, CsrA acts as a positive regulator of the switch from virulent (VIR-O) to avirulent (AV-T) subpopulations. This regulation is mediated, in part, by CsrA interfering with Rho-dependent termination in the mRNA leader region of the ABUW_1645 gene, encoding the primary TetR-type transcriptional regulator that drives cells from the VIR-O to the AV-T state. We demonstrate that CsrA directly binds to the ABUW_1645 mRNA leader region and interferes with Rho binding. We identify three small RNAs (sRNAs) designated CsrB, CsrC, and CsrD that bind to CsrA and inhibit its activity. Individual overexpression of each sRNA greatly decreased the rate of VIR-O to AV-T switching. Individual deletions of each sRNA increased the frequency of VIR-O to AV-T switching, with loss of CsrB giving the highest increase at 2.4-fold. The expression of each sRNA was strongly dependent on the GacA response regulator and the expression of each sRNA was higher in VIR-O cells than in AV-T variants. Furthermore, this regulation provides a mechanism for the differential control of CsrA activity between VIR-O and AV-T variants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Intelligent resolution: Integrating Cryo-EM with AI-driven multi-resolution simulations to observe the severe acute respiratory syndrome coronavirus-2 replication-transcription machinery in action

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replication transcription complex (RTC) is a multi-domain protein responsible for replicating and transcribing the viral mRNA inside a human cell. Attacking RTC function with pharmaceutical compounds is a pathway to treating COVID-19. Conventional tools, e.g., cryo-electron microscopy and all-atom molecular dynamics (AAMD), do not provide sufficiently high resolution or timescale to capture important dynamics of this molecular machine. Consequently, we develop an innovative workflow that bridges the gap between these resolutions, using mesoscale fluctuating finite element analysis (FFEA) continuum simulations and a hierarchy of AI-methods that continually learn and infer features for maintaining consistency between AAMD and FFEA simulations. We leverage a multi-site distributed workflow manager to orchestrate AI, FFEA, and AAMD jobs, providing optimal resource utilization across HPC centers. Our study provides unprecedented access to study the SARS-CoV-2 RTC machinery, while providing general capability for AI-enabled multi-resolution simulations at scale.

Trifan, Anda↗
DOE OSTI2024

Systemic immunological responses are dependent on sex and ovarian hormone presence following acute inhaled woodsmoke exposure

Background: Rural regions of the western United States have experienced a noticeable surge in both the frequency and severity of acute wildfire events, which brings significant challenges to both public safety and environmental conservation efforts, with impacts felt globally. Identifying factors contributing to immune dysfunction, including endocrinological phenotypes, is essential to understanding how hormones may influence toxicological susceptibility. Methods: This exploratory study utilized male and female C57BL/6 mice as in vivo models to investigate distinct responses to acute woodsmoke (WS) exposure with a focus on sex-based differences. In a second set of investigations, two groups were established within the female mouse cohort. In one group, mice experienced ovariectomy (OVX) to simulate an ovarian hormone-deficient state similar to surgical menopause, while the other group received Sham surgery as controls, to investigate the mechanistic role of ovarian hormone presence in driving immune dysregulation following acute WS exposure. Each experimental cohort followed a consecutive 2-day protocol with daily 4-h exposure intervals under two conditions: control HEPA-filtered air (FA) and acute WS to simulate an acute wildfire episode. Results: Metals analysis of WS particulate matter (PM) revealed significantly increased levels of 63 Cu, 182 W, 208 Pb, and 238 U, compared to filtered air (FA) controls, providing insights into the specific metal components most impacted by the changing dynamics of wildfire occurrences in the region. Male and female mice exhibited diverse patterns in lung mRNA cytokine expression following WS exposure, with males showing downregulation and females displaying upregulation, notably for IL-1β, TNF-α, CXCL-1, CCL-5, TGF-β, and IL-6. After acute WS exposure, there were notable differences in the responses of macrophages, neutrophils, and bronchoalveolar lavage (BAL) cytokines IL-10, IL-6, IL-1β, and TNF-α. Significant diverse alterations were observed in BAL cytokines, specifically IL-1β, IL-10, IL-6, and TNF-α, as well as in the populations of immune cells, such as macrophages and polymorphonuclear leukocytes, in both Sham and OVX mice, following acute WS exposure. These findings elucidated the profound influence of hormonal changes on inflammatory outcomes, delineating substantial sex-related differences in immune activation and revealing altered immune responses in OVX mice due to ovarian hormone deficiency. In addition, the flow cytometry analysis highlighted the complex interaction between OVX surgery, acute WS exposure, and their collective impact on immune cell populations within the hematopoietic bone marrow niche. Conclusions: In summary, both male and female mice, alongside females subjected to OVX and those who had sham surgery, exhibit significant variations in the expression of proinflammatory cytokines, chemokines, lung mRNA gene expression, and related functional networks linked to signaling pathways. These differences potentially act as mediators of sex-specific and hormonal influences in the systemic inflammatory response to acute WS exposure during a wildfire event. Understanding the regulatory roles of genes expressed differentially under environmental stressors holds considerable implications, aiding in identifying sex-specific therapeutic targets for addressing acute lung inflammation and injury.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Odor exposure during imprinting periods increases odorant-specific sensitivity and receptor gene expression in coho salmon ( Oncorhynchus kisutch )

ABSTRACT Pacific salmon are well known for their homing migrations; juvenile salmon learn odors associated with their natal streams prior to seaward migration, and then use these retained odor memories to guide them back from oceanic feeding grounds to their river of origin to spawn several years later. This memory formation, termed olfactory imprinting, involves (at least in part) sensitization of the peripheral olfactory epithelium to specific odorants. We hypothesized that this change in peripheral sensitivity is due to exposure-dependent increases in the expression of odorant receptor (OR) proteins that are activated by specific odorants experienced during imprinting. To test this hypothesis, we exposed juvenile coho salmon, Oncorhynchus kisutch, to the basic amino acid odorant l-arginine during the parr–smolt transformation (PST), when imprinting occurs, and assessed sensitivity of the olfactory epithelium to this and other odorants. We then identified the coho salmon ortholog of a basic amino acid odorant receptor (BAAR) and determined the mRNA expression levels of this receptor and other transcripts representing different classes of OR families. Exposure to l-arginine during the PST resulted in increased sensitivity to that odorant and a specific increase in BAAR mRNA expression in the olfactory epithelium relative to other ORs. These results suggest that specific increases in ORs activated during imprinting may be an important component of home stream memory formation and this phenomenon may ultimately be useful as a marker of successful imprinting to assess management strategies and hatchery practices that may influence straying in salmon.

Dittman, Andrew H. (ORCID:000000016482359X)↗
DOE OSTI2020

Molecular basis for the distinct cellular functions of the Lsm1–7 and Lsm2–8 complexes

Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1–7 complex initiates mRNA decay, while the nuclear Lsm2–8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2–8 bound to RNA identify the unique 2',3' cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1–7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1–7 loads onto RNA from the 3' end and removal of the Lsm1 carboxy-terminal region allows Lsm1–7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for A Fluorescence-Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants

Scripts for the manuscript "A fluorescence-based transient expression assay for the analysis of upstream open reading frames in plant" by Haas et al. Upstream open reading frames (uORFs) are regulatory elements present in the 5′ leaders of mRNA that can significantly impact downstream gene expression in eukaryotes. In crop engineering, editing of uORFs can provide an avenue to upregulate expression of native genes without the need to add persistent transgenic copies. Even with genome- wide methods to identify translated uORFs such as ribosome profiling, their functional characterization depends on validation through reporter gene assays and mutagenesis studies. Current screening methods for plants use luciferases or protoplasts to measure differential gene expression between wild- type and mutated transcript leaders, which requires tissue processing and/or substrate addition. Here, we present a time- and cost- efficient alternative to investigate transcript leaders by co- expression of two fluorescent proteins in Nicotiana benthamiana leaf tissue and test our assay on genes involved in photoprotection, editing of which could provide a pathway to increase CO2 assimilation during sun–shade transitions.

Gene Editing↗
DOE OSTI2026

Data for FUN-PROSE: A Deep Learning Approach to Predict Condition-Specific Gene Expression in Fungi

mRNA levels of all genes in a genome is a critical piece of information defining the overall state of the cell in a given environmental condition. Being able to reconstruct such condition-specific expression in fungal genomes is particularly important to metabolically engineer these organisms to produce desired chemicals in industrially scalable conditions. Most previous deep learning approaches focused on predicting the average expression levels of a gene based on its promoter sequence, ignoring its variation across different conditions. Here we present FUN-PROSE—a deep learning model trained to predict differential expression of individual genes across various conditions using their promoter sequences and expression levels of all transcription factors. We train and test our model on three fungal species and get the correlation between predicted and observed condition-specific gene expression as high as 0.85. We then interpret our model to extract promoter sequence motifs responsible for variable expression of individual genes. We also carried out input feature importance analysis to connect individual transcription factors to their gene targets. A sizeable fraction of both sequence motifs and TF-gene interactions learned by our model agree with previously known biological information, while the rest corresponds to either novel biological facts or indirect correlations.

Genomics↗