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At least 523 records · Page 29
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

cGAS-like receptors sense RNA and control 3'2'-cGAMP signalling in Drosophila

Cyclic GMP–AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cG[2'–5']pA[3'–5']p (2'3'-cGAMP) and controls activation of innate immunity in mammalian cells. Animal genomes typically encode multiple proteins with predicted homology to cGAS, but the function of these uncharacterized enzymes is unknown. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'–5']pA[2'–5']p (3'2'-cGAMP). A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3'2'-cGAMP explains selective isomer recognition, and 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Gene expression analysis of whole blood RNA from pigs infected with low and high pathogenic African swine fever viruses

African swine fever virus (ASFV) is a macrophage-tropic virus responsible for ASF, a transboundary disease that threatens swine production world-wide. Since there are no vaccines available to control ASF after an outbreak, obtaining an understanding of the virus-host interaction is important for developing new intervention strategies. In this study, a whole transcriptomic RNA-Seq method was used to characterize differentially expressed genes in pigs infected with a low pathogenic ASFV isolate, OUR T88/3 (OURT), or the highly pathogenic Georgia 2007/1 (GRG). After infection, pigs infected with OURT showed no or few clinical signs; whereas, GRG produced clinical signs consistent with acute ASF. RNA-Seq detected the expression of ASFV genes from the whole blood of the GRG, but not the OURT pigs, consistent with the pathotypes of these strains and the replication of GRG in circulating monocytes. Even though GRG and OURT possess different pathogenic properties, there was significant overlap in the most upregulated host genes. A small number of differentially expressed microRNAs were also detected in GRG and OURT pigs. These data confirm previous studies describing the response of macrophages and lymphocytes to ASFV infection, as well as reveal unique gene pathways upregulated in response to infection with GRG.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Armillaria root rot fungi host single-stranded RNA viruses

Species of Armillaria are distributed globally and include some of the most important pathogens of forest and ornamental trees. Some of them form large long-living clones that are considered as one of the largest organisms on earth and are capable of long-range spore-mediated transfer as well as vegetative spread by drought-resistant hyphal cords called rhizomorphs. However, the virus community infecting these species has remained unknown. In this study we used dsRNA screening and high-throughput sequencing to search for possible virus infections in a collection of Armillaria isolates representing three different species: Armillaria mellea from South Africa, A. borealis from Finland and Russia (Siberia) and A. cepistipes from Finland. Our analysis revealed the presence of both negative-sense RNA viruses and positive-sense RNA viruses, while no dsRNA viruses were detected. The viruses included putative new members of virus families Mymonaviridae, Botourmiaviridae and Virgaviridae and members of a recently discovered virus group tentatively named “ambiviruses” with ambisense bicistronic genomic organization. We demonstrated that Armillaria isolates can be cured of viruses by thermal treatment, which enables the examination of virus effects on host growth and phenotype using isogenic virus-infected and virus-free strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Environmental RNA as a Tool for Marine Community Biodiversity Assessments

Abstract Microscopic organisms are often overlooked in traditional diversity assessments due to the difficulty of identifying them based on morphology. Metabarcoding is a method for rapidly identifying organisms where Environmental DNA (eDNA) is used as a template. However, legacy DNA is problematically detected from organisms no longer in the environment during sampling. Environmental RNA (eRNA), which is only produced by living organisms, can also be collected from environmental samples and used for metabarcoding. The aim of this study was to determine differences in community composition and diversity between eRNA and eDNA templates for metabarcoding. Using mesocosms containing field-collected communities from an estuary, RNA and DNA were co-extracted from sediment, libraries were prepared for two loci (18S and COI), and sequenced using an Illumina MiSeq. Results show a higher number of unique sequences detected from eRNA in both markers and higher α-diversity compared to eDNA. Significant differences between eRNA and eDNA for all β-diversity metrics were also detected. This study is the first to demonstrate community differences detected with eRNA compared to eDNA from an estuarine system and illustrates the broad applications of eRNA as a tool for assessing benthic community diversity, particularly for environmental conservation and management applications.

Giroux, Marissa S.↗
DOE OSTI2021

Community RNA-Seq: multi-kingdom responses to living versus decaying roots in soil

Abstract Roots are a primary source of organic carbon input in most soils. The consumption of living and detrital root inputs involves multi-trophic processes and multiple kingdoms of microbial life, but typical microbial ecology studies focus on only one or two major lineages. We used Illumina shotgun RNA sequencing to conduct PCR-independent SSU rRNA community analysis (“community RNA-Seq”) and simultaneously assess the bacteria, archaea, fungi, and microfauna surrounding both living and decomposing roots of the annual grass, Avena fatua. Plants were grown in 13CO2-labeled microcosms amended with 15N-root litter to identify the preferences of rhizosphere organisms for root exudates (13C) versus decaying root biomass (15N) using NanoSIMS microarray imaging (Chip-SIP). When litter was available, rhizosphere and bulk soil had significantly more Amoebozoa, which are potentially important yet often overlooked top-down drivers of detritusphere community dynamics and nutrient cycling. Bulk soil containing litter was depleted in Actinobacteria but had significantly more Bacteroidetes and Proteobacteria. While Actinobacteria were abundant in the rhizosphere, Chip-SIP showed Actinobacteria preferentially incorporated litter relative to root exudates, indicating this group’s more prominent role in detritus elemental cycling in the rhizosphere. Our results emphasize that decomposition is a multi-trophic process involving complex interactions, and our methodology can be used to track the trajectory of carbon through multi-kingdom soil food webs.

Nuccio, Erin E. (ORCID:000000030189183X)↗
DOE OSTI2018

Structure of HIV-1 reverse transcriptase cleaving RNA in an RNA/DNA hybrid

HIV-1 reverse transcriptase (RT) contains both DNA polymerase and RNase H activities to convert the viral genomic RNA to dsDNA in infected host cells. Here we report the 2.65-Å resolution structure of HIV-1 RT engaging in cleaving RNA in an RNA/DNA hybrid. A preferred substrate sequence is absolutely required to enable the RNA/DNA hybrid to adopt the distorted conformation needed to interact properly with the RNase H active site in RT. Substituting two nucleotides 4 bp upstream from the cleavage site results in scissile-phosphate displacement by 4 Å. Here, we also have determined the structure of HIV-1 RT complexed with an RNase H-resistant polypurine tract sequence, which adopts a rigid structure and is accommodated outside of the nuclease active site. Based on this newly gained structural information and a virtual drug screen, we have identified an inhibitor specific for the viral RNase H but not for its cellular homologs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Robust reconstruction of single-cell RNA-seq data with iterative gene weight updates

Single-cell RNA-sequencing technologies have greatly enhanced our understanding of heterogeneous cell populations and underlying regulatory processes. However, structural (spatial or temporal) relations between cells are lost during cell dissociation. These relations are crucial for identifying associated biological processes. Many existing tissue-reconstruction algorithms use prior information about subsets of genes that are informative with respect to the structure or process to be reconstructed. When such information is not available, and in the general case when the input genes code for multiple processes, including being susceptible to noise, biological reconstruction is often computationally challenging. We propose an algorithm that iteratively identifies manifold-informative genes using existing reconstruction algorithms for single-cell RNA-seq data as subroutine. We show that our algorithm improves the quality of tissue reconstruction for diverse synthetic and real scRNA-seq data, including data from the mammalian intestinal epithelium and liver lobules.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Viral RNA and Infectious Influenza Virus on Mobile Phones of Patients With Influenza in Hong Kong and the United States

Abstract Mobile phones are among the most highly touched personal objects. As part of a broader study on the contribution of fomites to influenza transmission, between 2017 and 2019, we swabbed mobile phones from 138 patients with influenza in 2 locations. Influenza viral RNA detection rates were 23% (23 of 99 phones) and 36% (14 of 39) in Hong Kong and Maryland, respectively. In Hong Kong, infectious influenza virus was recovered from 3 of 23 mobile phones which had influenza viral RNA detected. Mobile phone influenza contamination was positively associated with upper respiratory tract viral load and negatively associated with age. Cleaning personal objects of patients with influenza should be recommended, and individuals should avoid sharing objects with these patients.

Xiao, Jingyi↗
DOE OSTI2020

Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation

Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3'-OH RNA strands in the context of 3'-OH/5'-PO 4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl–AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn 2+ ) 2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn 2+ . The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mechanism of efficient double-strand break repair by a long non-coding RNA

Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1’s ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA–RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes

Abstract RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection

Abstract Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli

Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.

Kammerdiener, Elise K. [Oak Ridge National Laborat↗
DOE OSTI2014

A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2',3'-cyclic phosphate or 3'-phosphate termini to 5'-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3'-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases

In many bacteria and archaea, small RNAs derived from clustered regularly interspaced short palindromic repeats (CRISPRs) associate with CRISPRassociated (Cas) proteins to target foreign DNA for destruction. In Type I and III CRISPR/Cas systems, the Cas6 family of endoribonucleases generates functional CRISPR-derived RNAs by site-specific cleavage of repeat sequences in precursor transcripts. CRISPR repeats differ widely in both sequence and structure, with varying propensity to form hairpin folds immediately preceding the cleavage site. To investigate the evolution of distinct mechanisms for the recognition of diverse CRISPR repeats by Cas6 enzymes, we determined crystal structures of two Thermus thermophilus Cas6 enzymes both alone and bound to substrate and product RNAs. These structures show how the scaffold common to all Cas6 endonucleases has evolved two binding sites with distinct modes of RNA recognition: one specific for a hairpin fold and the other for a single-stranded 5' -terminal segment preceding the hairpin. These findings explain how divergent Cas6 enzymes have emerged to mediate highly selective pre-CRISPR-derived RNA processing across diverse CRISPR systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

A dynamic intron retention program enriched in RNA processing genes regulates gene expression during terminal erythropoiesis

Differentiating erythroblasts execute a dynamic alternative splicing program shown here to include extensive and diverse intron retention (IR) events. Cluster analysis revealed hundreds of developmentallydynamic introns that exhibit increased IR in mature erythroblasts, and are enriched in functions related to RNA processing such as SF3B1 spliceosomal factor. Distinct, developmentally-stable IR clusters are enriched in metal-ion binding functions and include mitoferrin genes SLC25A37 and SLC25A28 that are critical for iron homeostasis. Some IR transcripts are abundant, e.g. comprising ~50% of highly-expressed SLC25A37 and SF3B1 transcripts in late erythroblasts, and thereby limiting functional mRNA levels. IR transcripts tested were predominantly nuclearlocalized. Splice site strength correlated with IR among stable but not dynamic intron clusters, indicating distinct regulation of dynamically-increased IR in late erythroblasts. Retained introns were preferentially associated with alternative exons with premature termination codons (PTCs). High IR was observed in disease-causing genes including SF3B1 and the RNA binding protein FUS. Comparative studies demonstrated that the intron retention program in erythroblasts shares features with other tissues but ultimately is unique to erythropoiesis. Finally, we conclude that IR is a multi-dimensional set of processes that post-transcriptionally regulate diverse gene groups during normal erythropoiesis, misregulation of which could be responsible for human disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Structure of mycobacterial 3'-to-5' RNA:DNA helicase Lhr bound to a ssDNA tracking strand highlights distinctive features of a novel family of bacterial helicases

Mycobacterial Lhr is a DNA damage-inducible superfamily 2 helicase that uses adenosine triphosphate (ATP) hydrolysis to drive unidirectional 3'-to-5' translocation along single-stranded DNA (ssDNA) and to unwind RNA:DNA duplexes en route. ATPase, translocase and helicase activities are encompassed within the N-terminal 856-amino acid segment. The crystal structure of Lhr-(1–856) in complex with AMPPNP•Mg 2+ and ssDNA defines a new helicase family. The enzyme comprises two N-terminal RecA-like modules, a winged helix (WH) domain and a unique C-terminal domain. The 3' ssDNA end binds in a crescent-shaped groove at the interface between the first RecA domain and the WH domain and tracks 5' into a groove between the second RecA and C domains. A kissing interaction between the second RecA and C domains forms an aperture that demarcates a putative junction between the loading strand tail and the duplex, with the first duplex nucleoside bookended by stacking on Trp597. Intercalation of Ile528 between nucleosides of the loading strand creates another bookend. Coupling of ATP hydrolysis to RNA:DNA unwinding is dependent on Trp597 and Ile528, and on Thr145 and Arg279 that contact phosphates of the loading strand. The structural and functional data suggest a ratchet mechanism of translocation and unwinding coupled to ATP-driven domain movements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

RNAcentral: a hub of information for non-coding RNA sequences

RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences, collating information on ncRNA sequences of all types from a broad range of organisms. We have recently added a new genome mapping pipeline that identifies genomic locations for ncRNA sequences in 296 species. We have also added several new types of functional annotations, such as tRNA secondary structures, Gene Ontology annotations, and miRNA-target interactions. A new quality control mechanism based on Rfam family assignments identifies potential contamination, incomplete sequences, and more. The RNAcentral database has become a vital component of many workflows in the RNA community, serving as both the primary source of sequence data for academic and commercial groups, as well as a source of stable accessions for the annotation of genomic and functional features. These examples are facilitated by an improved RNAcentral web interface, which features an updated genome browser, a new sequence feature viewer, and improved text search functionality.

Sweeney, Blake A.↗