Biology subjects

Zheludev, I. N.

Publications and source records attributed to Zheludev, I. N..

9 recordsLinked to original sources

bioRxiv2024

Viroid-like colonists of human microbiomes

Here, we describe the "Obelisks," a previously unrecognised class of viroid-like elements that we first identified in human gut metatranscriptomic data. "Obelisks" share several properties: (i) apparently circular RNA [~]1kb genome assemblies, (ii) predicted rod-like secondary structures encompassing the entire genome, and (iii) open reading frames coding for a novel protein superfamily, which we call the "Oblins". We find that Obelisks form their own distinct phylogenetic group with no detectable sequence or structural similarity to known biological agents. Further, Obelisks are prevalent in tested human microbiome metatranscriptomes with representatives detected in [~]7% of analysed stool metatranscriptomes (29/440) and in [~]50% of analysed oral metatranscriptomes (17/32). Obelisk compositions appear to differ between the anatomic sites and are capable of persisting in individuals, with continued presence over >300 days observed in one case. Large scale searches identified 29,959 Obelisks (clustered at 90% nucleotide identity), with examples from all seven continents and in diverse ecological niches. From this search, a subset of Obelisks are identified to code for Obelisk-specific variants of the hammerhead type-III self-cleaving ribozyme. Lastly, we identified one case of a bacterial species (Streptococcus sanguinis) in which a subset of defined laboratory strains harboured a specific Obelisk RNA population. As such, Obelisks comprise a class of diverse RNAs that have colonised, and gone unnoticed in, human, and global microbiomes.

microbiology↗

bioRxiv2023

Tertiary folds of the SL5 RNA from the 5' proximal region of SARS-CoV-2 and related coronaviruses

Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically-determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses. The SL5 domain of betacoronavirus SARS-CoV-2, resolved at 4.7 [A] resolution, exhibits a T-shaped structure, with its UUYYGU hexaloops at opposing ends of a coaxial stack, the Ts "arms." Further analysis of SL5 domains from SARS-CoV-1 and MERS (7.1 and 6.4-6.9 [A] resolution, respectively) indicate that the junction geometry and inter-hexaloop distances are conserved features across the studied human-infecting betacoronaviruses. The MERS SL5 domain displays an additional tertiary interaction, which is also observed in the non-human-infecting betacoronavirus BtCoV-HKU5 (5.9-8.0 [A] resolution). SL5s from human-infecting alphacoronaviruses, HCoV-229E and HCoV-NL63 (6.5 and 8.4-9.0 [A] resolution, respectively), exhibit the same coaxial stacks, including the UUYYGU-capped arms, but with a phylogenetically distinct crossing angle, an X-shape. As such, all SL5 domains studied herein fold into stable tertiary structures with cross-genus similarities, with implications for potential protein-binding modes and therapeutic targets. SignificanceThe three-dimensional structures of viral RNAs are of interest to the study of viral pathogenesis and therapeutic design, but the three-dimensional structures of viral RNAs remain poorly characterized. Here, we provide the first 3D structures of the SL5 domain (124-160 nt, 40.0-51.4 kDa) from the majority of human-infecting coronaviruses. All studied SL5s exhibit a similar 4-way junction, with their crossing angles grouped along phylogenetic boundaries. Further, across all species studied, conserved UUYYGU hexaloop pairs are located at opposing ends of a coaxial stack, suggesting that their three-dimensional arrangement is important for their as-of-yet defined function. These conserved tertiary features support the relevance of SL5 for pan-coronavirus fitness and highlight new routes in understanding its molecular and virological roles and in developing SL5-based antivirals. Classification: Biological Sciences, Biophysics and Computational Biology

biophysics↗

bioRxiv2023

SPLASH: a statistical, reference-free genomic algorithm unifies biological discovery

The authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2022/497555. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: https://doi.org/10.1101/2022.06.24.497555

genomics↗

bioRxiv2022

Restriction Endonuclease-based Modification-Dependent Enrichment (REMoDE) of DNA for Metagenomic Sequencing

Metagenomic sequencing is a swift and powerful tool to ascertain the presence of an organism of interest in a sample. However, sequencing coverage of the organism of interest can be insufficient due to an inundation of reads from irrelevant organisms in the sample. Here, we report a nuclease-based approach to rapidly enrich for DNA from certain organisms, including enterobacteria, based on their differential endogenous modification patterns. We exploit the ability of taxon-specific methylated motifs to resist the action of cognate methylation-sensitive restriction endonucleases that thereby digest unwanted, unmethylated DNA. Subsequently, we use a distributive exonuclease or electrophoretic separation to deplete or exclude the digested fragments, thus, enriching for undigested DNA from the organism of interest. As a proof-of-concept, we apply this method to enrich for the enterobacteria Escherichia coli and Salmonella enterica by 11- to 142-fold from mock metagenomic samples and validate this approach as a versatile means to enrich for genomes of interest in metagenomic samples. ImportancePathogens that contaminate the food supply or spread through other means can cause outbreaks that bring devastating repercussions to the health of a populace. Investigations to trace the source of these outbreaks are initiated rapidly but can be drawn out due to the labored methods of pathogen isolation. Metagenomic sequencing can alleviate this hurdle but is often insufficiently sensitive. The approach and implementations detailed here provide a rapid means to enrich for many pathogens involved in foodborne outbreaks, thereby improving the utility of metagenomic sequencing as a tool in outbreak investigations. Additionally, this approach provides a means to broadly enrich for otherwise minute levels of modified DNA which may escape unnoticed in metagenomic samples.

microbiology↗

bioRxiv2022

Extant hybrids of RNA viruses and viroid-like elements

Earths life may have originated as self-replicating RNA. Some of the simplest current RNA replicators are RNA viruses, defined by linear RNA genomes encoding an RNA-dependent RNA polymerase (RdRP), and subviral agents with single-stranded, circular RNA genomes, such as viroids encoding paired self-cleaving ribozymes. Amongst a massive expansion of candidate viroid and viroid-like elements, we report that fungal pathogens, ambiviruses, are viroid-like elements which undergo rolling circle replication and encode their own viral RdRP, thus they are a distinct hybrid infectious agent. These findings point to a deep evolutionary history between modern RNA viruses and sub-viral elements and offer new perspectives on the evolution of primordial infectious agents, and RNA life. One-Sentence SummaryNovel infectious agents resembling self-cleaving viroid-like RNAs whilst encoding a viral RNA-dependent RNA polymerase.

microbiology↗

bioRxiv2020

Exploring the evolutionary history of kinetic stability in the alpha-lytic protease family

In addition to encoding the final tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determines the speed of folding. How these kinetic barriers are encoded by the sequence is not well understood. Here, we use evolutionary sequence variation in the alpha-lytic protease (LP) protein family to probe the relationship between sequence and energy landscape. LP has an unusual energy landscape: the native state of LP is not the most thermodynamically favored conformation and, instead, it remains folded due to a large kinetic barrier preventing unfolding. In order to fold, LP utilizes an N-terminal pro region of similar size to the protease itself that functions as a folding catalyst. Once folded, the pro region is removed, and the native state does not unfold on a biologically relevant timescale. Without the pro region, LP folds on the order of millennia. A phylogenetic search uncovers LP homologs with a wide range of pro-region sizes, including some with no pro region at all. In the resulting phylogenetic tree, these homologs cluster by pro-region size. Homologs naturally lacking pro regions are thermodynamically stable, fold much faster than LP, yet retain the same fold as LP. Key amino acids thought to contribute to LPs extreme kinetic stability are lost in these homologs, further supporting their role in kinetic stability. This study highlights how the entire energy landscape plays an important role in determining the evolutionary pressures on and changes to the protein sequence.

biophysics↗

bioRxiv2020

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome1, 2. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides3-6. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 [A], presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

biochemistry↗

bioRxiv2020

RNA genome conservation and secondary structure in SARS-CoV-2 and SARS-related viruses

As the COVID-19 outbreak spreads, there is a growing need for a compilation of conserved RNA genome regions in the SARS-CoV-2 virus along with their structural propensities to guide development of antivirals and diagnostics. Using sequence alignments spanning a range of betacoronaviruses, we rank genomic regions by RNA sequence conservation, identifying 79 regions of length at least 15 nucleotides as exactly conserved over SARS-related complete genome sequences available near the beginning of the COVID-19 outbreak. We then confirm the conservation of the majority of these genome regions across 739 SARS-CoV-2 sequences reported to date from the current COVID-19 outbreak, and we present a curated list of 30 SARS-related-conserved regions. We find that known RNA structured elements curated as Rfam families and in prior literature are enriched in these conserved genome regions, and we predict additional conserved, stable secondary structures across the viral genome. We provide 106 SARS-CoV-2-conserved-structured regions as potential targets for antivirals that bind to structured RNA. We further provide detailed secondary structure models for the 5 UTR, frame-shifting element, and 3 UTR. Last, we predict regions of the SARS-CoV-2 viral genome have low propensity for RNA secondary structure and are conserved within SARS-CoV-2 strains. These 59 SARS-CoV-2-conserved-unstructured genomic regions may be most easily targeted in primer-based diagnostic and oligonucleotide-based therapeutic strategies.

genetics↗

bioRxiv2019

Ribosolve: Rapid determination of three-dimensional RNA-only structures

The discovery and design of biologically important RNA molecules is dramatically outpacing three-dimensional structural characterization. To address this challenge, we present Ribosolve, a hybrid method integrating moderate-resolution cryo-EM maps, chemical mapping, and Rosetta computational modeling, and demonstrate its application to thirteen previously unknown 119-to 338-nucleotide protein-free RNA-only structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length V. cholerae and F. nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and computer-designed spinach-TTR-3, eterna3D-JR_1, and ATP-TTR-3 with and without AMP. Blind challenges, prospective compensatory mutagenesis, internal controls, and simulation benchmarks validate the Ribosolve models and establish that modeling convergence is quantitatively predictive of model accuracy. These results demonstrate that RNA-only 3D structure determination can be rapid and routine.

biophysics↗