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Results for “RNA-binding protein”

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 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

mTOR regulates aerobic glycolysis through NEAT1 and nuclear paraspeckle-mediated mechanism in hepatocellular carcinoma

Hepatocellular Carcinoma (HCC) is a major form of liver cancer and a leading cause of cancer-related death worldwide. New insights into HCC pathobiology and mechanism of drug actions are urgently needed to improve patient outcomes. HCC undergoes metabolic reprogramming of glucose metabolism from respiration to aerobic glycolysis, a phenomenon known as the ‘Warburg Effect’ that supports rapid cancer cell growth, survival, and invasion. mTOR is known to promote Warburg Effect, but the underlying mechanism(s) remains poorly defined. The aim of this study is to understand the mechanism(s) and significance of mTOR regulation of aerobic glycolysis in HCC. We profiled mTORC1-dependent long non-coding RNAs (lncRNAs) by RNA-seq of HCC cells treated with rapamycin. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were used to explore the transcriptional regulation of NEAT1 by mTORC1. [U- 13 C]-glucose labeling and metabolomic analysis, extracellular acidification Rate (ECAR) by Seahorse XF Analyzer, and glucose uptake assay were used to investigate the role of mTOR-NEAT1-NONO signaling in the regulation of aerobic glycolysis. RNA immunoprecipitation (RIP) and NONO-binding motif scanning were performed to identify the regulatory mechanism of pre-mRNA splicing by mTOR-NEAT1. Myristoylated AKT1 (mAKT1)/NRASV 12 -driven HCC model developed by hydrodynamic transfection (HDT) was employed to explore the significance of mTOR-NEAT1 signaling in HCC tumorigenesis and mTOR-targeted therapy. mTOR regulates lncRNA transcriptome in HCC and that NEAT1 is a major mTOR transcriptional target. Interestingly, although both NEAT1_1 and NEAT1_2 are down-regulated in HCC, only NEAT1_2 is significantly correlated with poor overall survival of HCC patients. NEAT1_2 is the organizer of nuclear paraspeckles that sequester the RNA-binding proteins NONO and SFPQ. We show that upon oncogenic activation, mTORC1 suppresses NEAT1_2 expression and paraspeckle biogenesis, liberating NONO/SFPQ, which in turn, binds to U5 within the spliceosome, stimulating mRNA splicing and expression of key glycolytic enzymes. This series of actions lead to enhanced glucose transport, aerobic glycolytic flux, lactate production, and HCC growth both in vitro and in vivo. Furthermore, the paraspeckle-mediated mechanism is important for the anticancer action of US FDA-approved drugs rapamycin/temsirolimus. These findings reveal a molecular mechanism by which mTOR promotes the ‘Warburg Effect’, which is important for the metabolism and development of HCC, and anticancer response of mTOR-targeted therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural basis of tRNA recognition by the widespread OB fold

The widespread oligonucleotide/oligosaccharide-binding (OB)-fold recognizes diverse substrates from sugars to nucleic acids and proteins, and plays key roles in genome maintenance, transcription, translation, and tRNA metabolism. OB-containing bacterial Trbp and yeast Arc1p proteins are thought to recognize the tRNA elbow or anticodon regions. Here we report a 2.6 Å co-crystal structure of Aquifex aeolicus Trbp111 bound to tRNA Ile , which reveals that Trbp recognizes tRNAs solely by capturing their 3' ends. Structural, mutational, and biophysical analyses show that the Trbp/EMAPII-like OB fold precisely recognizes the single-stranded structure, 3' terminal location, and specific sequence of the 3' CA dinucleotide — a universal feature of mature tRNAs. Arc1p supplements its OB – tRNA 3' end interaction with additional contacts that involve an adjacent basic region and the tRNA body. This study uncovers a previously unrecognized mode of tRNA recognition by an ancient protein fold, and provides insights into protein-mediated tRNA aminoacylation, folding, localization, trafficking, and piracy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A compact stem-loop DNA aptamer targets a uracil-binding pocket in the SARS-CoV-2 nucleocapsid RNA-binding domain

Abstract SARS-CoV-2 nucleocapsid (N) protein is a structural component of the virus with essential roles in the replication and packaging of the viral RNA genome. The N protein is also an important target of COVID-19 antigen tests and a promising vaccine candidate along with the spike protein. Here, we report a compact stem-loop DNA aptamer that binds tightly to the N-terminal RNA-binding domain of SARS-CoV-2 N protein. Crystallographic analysis shows that a hexanucleotide DNA motif (5′-TCGGAT-3′) of the aptamer fits into a positively charged concave surface of N-NTD and engages essential RNA-binding residues including Tyr109, which mediates a sequence-specific interaction in a uracil-binding pocket. Avid binding of the DNA aptamer allows isolation and sensitive detection of full-length N protein from crude cell lysates, demonstrating its selectivity and utility in biochemical applications. We further designed a chemically modified DNA aptamer and used it as a probe to examine the interaction of N-NTD with various RNA motifs, which revealed a strong preference for uridine-rich sequences. Our studies provide a high-affinity chemical probe for the SARS-CoV-2 N protein RNA-binding domain, which may be useful for diagnostic applications and investigating novel antiviral agents.

Biochemistry & Molecular Biology↗
DOE OSTI2020

CryoEM structure of the low-complexity domain of hnRNPA2 and its conversion to pathogenic amyloid

hnRNPA2 is a human ribonucleoprotein (RNP) involved in RNA metabolism. It forms fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and missense mutations in this domain are found in the disease multisystem proteinopathy (MSP). However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of the hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into β-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment (D290V) of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to a pathogenic form.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A Genome-Scale Atlas Reveals Complex Interplay of Transcription and Translation in an Archaeon

The scale of post-transcriptional regulation and the implications of its interplay with other forms of regulation in environmental acclimation are underexplored for organisms of the domain Archaea. Here, we have investigated the scale of post-transcriptional regulation in the extremely halophilic archaeon Halobacterium salinarum NRC-1 by integrating the transcriptome-wide locations of transcript processing sites (TPSs) and SmAP1 binding, the genome-wide locations of antisense RNAs (asRNAs), and the consequences of RNase_2099C knockout on the differential expression of all genes. This integrated analysis has discovered that 54% of all protein-coding genes in the genome of this haloarchaeon are likely targeted by multiple mechanisms for putative post-transcriptional processing and regulation, with about 20% of genes likely being regulated by combinatorial schemes involving SmAP1, asRNAs, and RNase_2099C. Comparative analysis of mRNA levels (transcriptome sequencing [RNA-Seq]) and protein levels (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry [SWATH-MS]) for 2,579 genes over four phases of batch culture growth in complex medium generated additional evidence for the conditional post-transcriptional regulation of 7% of all protein-coding genes. We demonstrate that post-transcriptional regulation may act to fine-tune specialized and rapid acclimation to stressful environments, e.g., as a switch to turn on gas vesicle biogenesis to promote vertical relocation under anoxic conditions and modulate the frequency of transposition by insertion sequence (IS) elements of the IS200/IS605, IS4, and ISH3 families. Findings from this study are provided as an atlas in a public Web resource (https://halodata.systemsbiology.net).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural basis of template strand deoxyuridine promoter recognition by a viral RNA polymerase

Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the -10 promoter element, is located in the non-template strand of the double-stranded DNA molecule ~10 nucleotides upstream of the transcription start site. Here, we explain the mechanism by which the phage AR9 non-virion RNAP (nvRNAP), a bacterial RNAP homolog, recognizes the -10 element of its deoxyuridine-containing promoter in the template strand. The AR9 sigma-like subunit, the nvRNAP enzyme core, and the template strand together form two nucleotide base-accepting pockets whose shapes dictate the requirement for the conserved deoxyuridines. A single amino acid substitution in the AR9 sigma-like subunit allows one of these pockets to accept a thymine thus expanding the promoter consensus. Our work demonstrates the extent to which viruses can evolve host-derived multisubunit enzymes to make transcription of their own genes independent of the host.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

NASA Discipline Plant Biology↗
DOE OSTI2023

Intra- and inter-molecular regulation by intrinsically-disordered regions governs PUF protein RNA binding

PUF proteins are characterized by globular RNA-binding domains. They also interact with partner proteins that modulate their RNA-binding activities. Caenorhabditis elegans PUF protein fem-3 binding factor-2 (FBF-2) partners with intrinsically disordered Lateral Signaling Target-1 (LST-1) to regulate target mRNAs in germline stem cells. Here, we report that an intrinsically disordered region (IDR) at the C-terminus of FBF-2 autoinhibits its RNA-binding affinity by increasing the off rate for RNA binding. Moreover, the FBF-2 C-terminal region interacts with its globular RNA-binding domain at the same site where LST-1 binds. This intramolecular interaction restrains an electronegative cluster of amino acid residues near the 5' end of the bound RNA to inhibit RNA binding. LST-1 binding in place of the FBF-2 C-terminus therefore releases autoinhibition and increases RNA-binding affinity. This regulatory mechanism, driven by IDRs, provides a biochemical and biophysical explanation for the interdependence of FBF-2 and LST-1 in germline stem cell self-renewal.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region

Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.

NASA Discipline Regulatory Physiology↗
NASA NTRS2000

A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

NASA Discipline Plant Biology↗
NASA NTRS1992

The origin and early evolution of nucleic acid polymerases

The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA-polymerase beta-prime subunit and its homologues is discussed. It is shown that, in the DNA-dependent RNA polymerases from three cellular lineages, a very conserved sequence of eight amino acids, also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein, is present. The optimal conditions for the replicase activity of the avian-myeloblastosis-virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is discussed.

Lazcano, A.↗
DOE OSTI2025

Structure of coxsackievirus cloverleaf RNA and 3C pro dimer establishes the RNA-binding mechanism of enterovirus protease 3C pro

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 3C pro protease, and 3D pol polymerase. The interaction between cloverleaf and 3CD is mediated by the 3C pro domain, yet how 3C pro promotes specific RNA-binding is not clear. We report the structure of coxsackievirus cloverleaf RNA-3C pro complex, wherein two 3C pro molecules interact with cloverleaf stem-loop D. 3C pro dimer mainly recognizes the shape of the dsRNA helix through symmetric interactions, suggesting that 3C pro 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 3C pro . The structure provides insight into the RNA-binding mechanism of 3C pro or 3CD with other cis-acting replication elements.

Science & Technology - Other Topics↗
DOE OSTI2023

The Helix-Loop-Helix motif of human EIF3A regulates translation of proliferative cellular mRNAs

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro . Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC . We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

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 OSTI2026

Structural Analysis of 23S rRNA Methylating Enzyme Cfr Reveals RNA-Binding Determinants for Methylation Regiospecificity and Antibiotic Resistance

The 23S rRNA methylating enzyme Cfr, found in pathogens including Staphylococcus aureus, Clostridium difficile, Escherichia coli, and Klebsiella pneumoniae, confers resistance to phenicols, lincosamides, oxazolidinones (including linezolid), pleuromutilins, and streptogramins A (the PhLOPS A phenotype). Cfr catalyzes methylation of the C8 position of the A2503 base in 23S rRNA, the recognition site of the above antibiotic classes. Along with the RlmN housekeeping enzyme, Cfr can also promote methylation of the C2 position of the same base. The molecular and structural basis of Cfr’s dual substrate specificity is not known, which hinders our ability to design Cfr-targeting inhibitors necessary to curb PhLOPS A resistance. Here, we present the first crystal structure of Cfr and a detailed analysis of its possible interactions with rRNA. Using structure-guided mutagenesis, mass spectrometry analysis of in cellulo 23S rRNA methylated species, and in cellulo resistance studies, we identify the key amino acids essential for Cfr methylation and multidrug resistance activity. In particular, we found that Cfr’s Q329 residue is important for C8- specific methylation. These data provide a framework for further studies of the biochemistry, structure, and inhibition of this important resistance determinant.

AlphaFold↗
DOE OSTI2025

CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d

Bacteriophages constitute one of the largest reservoirs of genes of unknown function in the biosphere. Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because phages subvert many modern functional genomics tools. Here we leverage RNA-targeting dCas13d to selectively interfere with protein translation and to measure phage gene fitness at a transcriptome-wide scale. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective across phage phylogeny, from model ssRNA, ssDNA and dsDNA phages to nucleus-forming jumbo phages. Using CRISPRi-ART, we determine a conserved role of diverse rII homologues in subverting phage Lambda RexAB-mediated immunity to superinfection and identify genes critical for phage fitness. CRISPRi-ART establishes a broad-spectrum phage functional genomics platform, revealing more than 90 previously unknown genes important for phage fitness.

59 BASIC BIOLOGICAL SCIENCES↗