Biology subjects

Ignatova, Z.

Publications and source records attributed to Ignatova, Z..

17 recordsLinked to original sources

bioRxiv2025

ADAM-tRNA-seq: An Optimized Approach for Demultiplexing and Enhanced Hierarchal Mapping in Direct tRNA Sequencing

Transfer RNAs (tRNAs) play an essential role in protein synthesis and cellular homeostasis, with their dysregulation associated with various human pathologies. Recent advances in direct RNA sequencing by the Nanopore platform have enabled simultaneous profiling of tRNA abundance, modifications, and aminoacylation status. However, the high sequence similarity among tRNAs and the lack of robust demultiplexing strategies reduce the accuracy and limit the scalability of current approaches. Here, we developed ADAM-tRNA-seq, a framework that addresses two key limitations of the Nanopore-based direct tRNA sequencing. First, we develop an RNA-based barcode demultiplexing method, employing a barcode embedded within the sequencing adapter that is recognized by the Dorado basecaller. Second, we designed a hierarchy-based mapping strategy that mitigates read loss due to multimapping by classifying reads at the isodecoder, isoacceptor, or isotype levels, thereby enhancing quantification accuracy. We validated ADAM-tRNA-seq using both synthetic tRNAs and a complex human tRNA pool, and systematically optimized it to achieve up to 99% classification precision. Together, these developments enable more accurate, scalable, and comprehensive characterization of tRNA pools in diverse sample types.

biochemistry↗

bioRxiv2024

Glycyl-tRNA sequestration is a unifying mechanism underlying GARS1-associated peripheral neuropathy

Dominantly inherited mutations in eight cytosolic aminoacyl-tRNA synthetase genes cause hereditary motor and sensory neuropathy, characterized by degeneration of peripheral motor and sensory axons. We previously identified a pathogenic gain-of-toxic function mechanism underlying peripheral neuropathy (PN) caused by heterozygous mutations in the GARS1 gene, encoding glycyl-tRNA synthetase (GlyRS). Specifically, PN-mutant GlyRS variants sequester tRNAGly, which depletes the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly available to the ribosome and consequently ribosome stalling at glycine codons. Given that GlyRS functions as a homodimer, a subset of PN-GlyRS mutations might alternatively cause peripheral neuropathy through a dominant negative loss-of-function mechanism. To explore this possibility, we here generated three novel PN-GlyRS Drosophila models expressing human PN-GlyRS (hGlyRS) variants that do not alter the overall GlyRS protein charge (S211F and H418R) or the single reported PN-GlyRS variant that renders the GlyRS protein charge more negative (K456Q). High-level expression of hGlyRS-K456Q did not induce peripheral neuropathy and the K456Q variant does not affect aminoacylation activity, suggesting that K456Q is not a pathogenic mutation. Expression of hGlyRS-S211F or hGlyRS-H418R in Drosophila did induce peripheral neuropathy and de novo protein synthesis defects. Genetic and biochemical evidence indicates that these phenotypes were attributable to tRNAGly sequestration rather than a dominant negative mechanism. Our data identify tRNAGly sequestration as a unifying pathogenic mechanism underlying PN-GlyRS. Thus, elevating tRNAGly levels may constitute a therapeutic approach for all PN-GlyRS patients, irrespective of their disease-causing mutation.

neuroscience↗

bioRxiv2024

Automated high-throughput selection of DNA aptamers using a common optical next-generation sequencer

Aptamers are conventionally selected via Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This process is, however, laborious, time-consuming, and has a relatively low efficacy. Here, we developed an automated high-throughput screening platform for the selection of DNA aptamers which consists of an optical next-generation sequencer with a modified software and hardware to automatically perform fluorescence-based binding assays on the displayed DNA sequences subsequent to sequencing. Using this platform, after only three to five SELEX rounds we selected highly affine DNA aptamers for the lectins LecA and LecB of Pseudomonas aeruginosa as well as for the Pseudomonas Exotoxin A. In comparison, twelve rounds of conventional SELEX resulted in three-fold less affine aptamers for LecA and PEA and none for LecB. Our high throughput-approach bears great potential to augment SELEX as it significantly increases time efficiency, enabling the selection of aptamers within only one week.

biochemistry↗

bioRxiv2024

The integrated stress response/eIF2α pathway controls cytokine production in tissue-resident memory CD4+ T cells

Tissue-resident memory T (Trm) cells are a specialized T cell population that resides in tissues and can play both a protective and pathogenic role. The mechanism that enables Trm cells to provide a rapid protective response while restricting their function in homeostasis remains unclear. Here, we show that human and mouse CD4+ Trm cells exist in a poised state, characterized by storage of proinflammatory type-1 and type-3 cytokine mRNAs without protein production. In steady-state conditions, cytokine mRNA translation in Trm cells is suppressed by the integrated stress response (ISR)/eIF2 pathway, whereas Trm-cell activation under inflammatory conditions results in eIF2 dephosphorylation, leading to derepression and rapid translation of the cytokine mRNAs stored in stress granules. Pharmacological inhibition of eIF2 dephosphorylation resulted in reduced cytokine production from Trm cells, and ameliorated autoimmune kidney disease in mice. Consistent with these results, the ISR pathway in Trm cells was downregulated in patients with immune-mediated diseases of the kidney and the intestine. Our results identify ISR/eIF2-mediated control of cytokine mRNA translation as an underlying mechanism that restricts Trm cell activity in homeostasis but also promotes rapid response upon local infection or autoimmune reaction.

immunology↗

bioRxiv2022

Tailored synthetic sRNAs dynamically tune multilayer genetic circuits

Predictable and controllable tuning of genetic circuits to regulate gene expression, including modulation of existing circuits or constructs without the need for redesign or rebuilding, is a persistent challenge in synthetic biology. Here, we propose a rational design of de novo small RNAs (sRNAs) to dynamically modulate gene expression within a broad range, from high, medium to low repression, and implemented them in Escherichia coli. We designed multiple multilayer genetic circuits, in which the variable effector element is a transcription factor (TF) controlling downstream the production of a reporter protein. Our approach harnesses the intrinsic RNA-interference pathway in E. coli to exert dynamic and modular control of the multilayer genetic circuits. The sRNAs were designed to target TFs instead of the reporter gene, which allowed for wide range of expression modulation of the reporter protein, including the most difficult to achieve dynamic switch to an OFF state. Out work provides a frame for achieving independent modulation of gene expression, by only including an independent control circuit expressing synthetic sRNAs, without altering the structure of existing genetic circuits.

synthetic biology↗

bioRxiv2022

Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events

BackgroundThe Ccr4-Not complex is most well known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics have been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts. ResultsIn this work, by analyzing soluble and insoluble mRNA decay intermediates in yeast, we determine that insoluble mRNAs are enriched for ribosomes dwelling at non-optimal codons compared to soluble mRNAs. mRNA decay is higher for soluble RNAs, but the proportion of co-translational degradation relative to the overall mRNA decay is higher for insoluble mRNAs. We show that depletion of Not1 and Not4 inversely impact mRNA solubilities and, for soluble mRNAs, ribosome dwelling according to codon optimality. Depletion of Not4 solubilizes mRNAs with lower non-optimal codon content and higher expression that are rendered insoluble by Not1 depletion. By contrast, depletion of Not1 solubilizes mitochondrial mRNAs, which are rendered insoluble upon Not4 depletion. ConclusionOur results reveal that mRNA solubility defines dynamics of co-translation events and is oppositely regulated by Not1 and Not4, a mechanism that we additionally determine may already be set by Not1 promoter association in the nucleus.

molecular biology↗

bioRxiv2021

Improving the robustness of engineered bacteria to nutrient stress using programmed proteolysis

The use of short peptide tags in synthetic genetic circuits allows for the tuning of gene expression dynamics and release of amino acid resources through targeted protein degradation. Here, we use elements of the Escherichia coli and Mesoplasma florum transfer-messenger RNA (tmRNA) ribosome rescue systems to compare endogenous and foreign proteolysis systems in E. coli. We characterize the performance and burden of each and show that while both greatly shorten the half-life of a tagged protein, the endogenous system is approximately ten times more efficient. Based on these results, we then demonstrate using mathematical modelling and experiments how proteolysis can improve cellular robustness through targeted degradation of a reporter protein in auxotrophic strains, providing a limited secondary source of essential amino acids that help partially restore growth when nutrients become scarce. These findings provide avenues for controlling the functional lifetime of engineered cells once deployed and increasing their tolerance to fluctuations in nutrient availability.

synthetic biology↗

bioRxiv2020

Structural basis of resistance to lincosamide, streptogramin A, and pleuromutilin antibiotics by ABCF ATPases in Gram-positive pathogens

Target protection proteins bind to antibiotic targets and confer resistance to the host organism. One class of such proteins, termed antibiotic resistance (ARE) ATP binding cassette (ABC) proteins of the F-subtype (ARE ABCFs), are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition by antibiotics that target the large ribosomal subunit. Using single-particle cryo-EM, we have solved the structure of ARE ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a comparative approach to understanding how these proteins mediate antibiotic resistance on the ribosome. We present evidence of mechanistically diverse allosteric relays converging on a few peptidyltransferase center (PTC) nucleotides, and propose a general model of antibiotic resistance mediated by these ARE ABCFs.

microbiology↗

bioRxiv2020

Tautomerization constraints the accuracy of codon-anticodon decoding

G{circ}U(T) mismatch has the highest contribution to the error rate of base pair recognition in replication, as well as in codon-anticodon decoding in translation. Recently, this effect was unambiguously linked to keto-enol tautomerization, which enables the Watson-Crick (WC) geometry of the base pair. Structural studies of the ribosome revealing G{circ}U in the WC geometry in the closed state of the A-site challenge the canonical induced-fit model of decoding and currently lack a physicochemical explanation. Using computational and theoretical methods, we address effects of the ribosomal A-site on the wobble{leftrightarrow}WC tautomerization reaction in G{circ}U (wb-WC reaction), and the consequent implications for the decoding mechanism in translation. The free energy change of the wb-WC reaction in the middle codon-anticodon position was calculated with quantum-mechanical/molecular-mechanical umbrella sampling simulations. The wb-WC reaction was endoergic in the open A-site, but exoergic in the closed state. This effect can be explained in part by the decreased polarity of the closed A-site. We developed a model of initial selection in translation that incorporates the wb-WC reaction parameters in the open and closed states of the A-site. In the new model, the exoergic wb-WC reaction is kinetically restricted by the decoding rates, which explains the observations of the WC geometry at equilibrium conditions. Moreover, the model reveals constraints imposed by the exoergic wb-WC reaction on the decoding accuracy: its equilibration counteracts the favorable contribution from equilibration of the open-closed transition. The similarity of the base-pair recognition mechanism in DNA polymerases allows extending this model to replication as well. Our model can be a step towards a general recognition model for flexible substrates.

biophysics↗

bioRxiv2020

Structural basis for bacterial ribosome quality control

In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality-control (RQC) pathways. RQC begins with splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal tails. How such tailing can occur without the regular suite of translational components is, however, unclear. Using ex vivo single-particle cryo-EM, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with YabO, a protein homologous to, yet distinct from, Hsp15. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.

biochemistry↗

bioRxiv2020

A proteogenomics workflow to uncover the world of small proteins in Staphylococcus aureus

Small proteins play diverse and essential roles in bacterial physiology and virulence. Despite their importance, automated genome annotation algorithms still cannot accurately annotate all respective small open reading frames (sORFs), as they usually provide insufficient sequence information for domain and homology searches, tend to be species specific and only a few experimentally validated examples are covered in standard proteomics studies. The accuracy and reliability of genome annotations, particularly for sORFs, can be significantly improved by integrating protein evidence from experimental approaches that enrich for small proteins. Here we present a highly optimized and flexible workflow for bacterial proteogenomics, which covers all steps from (i) creation of protein databases, (ii) database searches, (iii) peptide-to-genome mapping to (iv) result interpretation and whose automated execution is supported by two open source tools (SALT & Pepper). We used the workflow to identify high quality peptide spectrum matches (PSMs) for both annotated and unannotated small proteins ([≤] 100 aa; SP100) in Staphylococcus aureus Newman. Proteins isolated from cells at the exponential and stationary growth phase were digested with different endopeptidases (trypsin, Lys-C, AspN), the resulting peptides fractionated by gel-based and gel-free methods and measured with highly sensitive mass spectrometers. PSMs or sORF predictions from sORFfinder were stringently filtered allowing us to detect 185 soluble SP100, 69 of which were missing in the used genome annotation. Most interestingly, almost half of the identified SP100 were basic, suggesting a role in binding to more acidic molecules such as nucleic acids or phospholipids. In addition, phage-related functions were proposed for 30 SP100, based on the localization of their coding sequences in the genome.

genomics↗

bioRxiv2020

smORFer: a modular algorithm to detect small ORFs in prokaryotes

Emerging evidence places small proteins ([≤] 50 amino acids) more centrally in physiological processes. Yet, the identification of functional small proteins and the systematic genome annotation of their cognate small open reading frames (smORFs) remains challenging both experimentally and computationally. Ribosome profiling or Ribo-Seq (that is a deep sequencing of ribosome-protected fragments) enables detecting of actively translated open-reading frames (ORFs) and empirical annotation of coding sequences (CDSs) using the in-register translation pattern that is characteristic for genuinely translating ribosomes. Multiple identifiers of ORFs that use 3-nt periodicity in Ribo-Seq data sets have been successful in eukaryotic smORF annotation. Yet, they have difficulties evaluating prokaryotic genomes due to the unique architecture of prokaryotic genomes (e.g. polycistronic messages, overlapping ORFs, leaderless translation, non-canonical initiation etc.). Here, we present our new algorithm, smORFer, which performs with high accuracy in prokaryotic organisms in detecting smORFs. The unique feature of smORFer is that it uses integrated approach and considers structural features of the genetic sequence along with in-register translation and uses Fourier transform to convert these parameters into a measurable score to faithfully select smORFs. The algorithm is executed in a modular way and dependent on the data available for a particular organism allows using different modules for smORF search.

bioinformatics↗

bioRxiv2020

Genome-wide effects of the antimicrobial peptide apidaecin on translation termination

Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here, genome-wide analysis revealed that Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate development of new medicines and research tools for genome exploration.

biochemistry↗

bioRxiv2019

Switch from translation initiation to elongation needs Not4 and Not5 collaboration

Not4 and Not5 are crucial components of the Ccr4-Not complex with pivotal functions in mRNA metabolism. Both associate with ribosomes but mechanistic insights on their function remain elusive. Here we determine that Not5 and Not4 synchronously impact translation initiation and Not5 alone alters translation elongation. Deletion of Not5 causes elongation defects in a codon-dependent fashion, increasing and decreasing the ribosome dwelling occupancy at minor and major codons, respectively. This larger difference in codons translation velocities alters translation globally and enables kinetically unfavorable processes such as nascent chain deubiquitination to take place. In turn, this leads to abortive translation and favors protein aggregation. These findings highlight the global impact of Not4 and Not5 in controlling the speed of mRNA translation and transition from initiation to elongation. SummaryNot4 and Not5 regulate translation synchronously but distinguishably, facilitating smooth transition from initiation to elongation

molecular biology↗

bioRxiv2018

Structure of a hibernating 100S ribosome reveals an inactive conformation of the ribosomal protein S1

To survive under conditions of stress, such as nutrient deprivation, bacterial 70S ribosomes dimerize to form hibernating 100S particles1. In {gamma}-proteobacteria, such as Escherichia coli, 100S formation requires the ribosome modulation factor (RMF) and the hibernation promoting factor (HPF)2-4. Although structures of E. coli 100S particles have been reported5,6, the low resolution (18-38 [A]) prevented the mechanism of ribosome inactivation and dimerization to be fully elucidated. Here we present single particle cryo-electron microscopy structures of hibernating 70S and 100S particles isolated from stationary phase E. coli cells at 3.0-7.9 [A] resolution, respectively. Preferred orientation bias for the complete 100S particle was overcome using tilting during data collection. The structures reveal the binding sites for HPF and RMF as well as the unexpected presence of deacylated E-site tRNA and ribosomal protein S1 in the 100S particle. HPF interacts with the anticodon-stem-loop of the E-tRNA and occludes the binding site for the mRNA as well as A- and P-site tRNAs. RMF stabilizes a compact conformation of S1, which together sequester the anti-Shine-Dalgarno (SD) sequence of the 16S ribosomal RNA (rRNA), thereby inhibiting translation initiation. At the dimerization interface, S1 and S2 form intersubunit bridges with S3 and S4 and the C-terminus of S2 probes the mRNA entrance channel of the symmetry related particle, thus suggesting that only translationally inactive ribosomes are prone to dimerization. The back-to-back 100S dimerization mediated by HPF and RMF is distinct from that observed previously in Gram-positive bacteria7-10 and reveals a unique function for S1 in ribosome dimerization and inactivation, rather than its canonical role in facilitating translation initiation.

molecular biology↗

bioRxiv2018

Dynamic m6A methylation facilitates mRNA triaging to stress granules

Summary blurbm6A-modification in the 5 vicinity of the coding sequence of transcripts provides a selective mechanism for triaging mRNAs to stress granules and is mediated by YTHDF3 reader protein.\n\nAbstractReversible post-transcriptional modifications on messenger RNA emerge as prevalent phenomena in RNA metabolism. The most abundant among them is N6-methyladenosine (m6A) which is pivotal for RNA metabolism and function, its role in stress response remains elusive. We have discovered that in response to oxidative stress, transcripts are additionally m6A-modified in their 5 vicinity. Distinct from that of the translationally-active mRNAs, this methylation pattern provides a selective mechanism for triaging mRNAs from the translatable pool to stress-induced stress granules. These stress-induced newly methylated sites are selectively recognized by the YTH domain family 3 (YTHDF3) reader protein, thereby revealing a new role for YTHDF3 in shaping the selectivity of stress response. Our findings describe a previously unappreciated function for RNA m6A modification in the oxidative-stress response and expand the breadth of physiological roles of m6A.

molecular biology↗

bioRxiv2018

Absolute quantification of translational regulation and burden using combined sequencing approaches

Translation of mRNAs into proteins is a key cellular process. Ribosome binding sites and stop codons provide signals to initiate and terminate translation, while stable secondary mRNA structures can induce translational recoding events. Fluorescent proteins are commonly used to characterize such elements but require the modification of a parts natural context and allow only a few parameters to be monitored concurrently. Here, we develop an approach that combines ribosome profiling (Ribo-seq) with quantitative RNA sequencing (RNA-seq) to enable the high-throughput characterization of genetic parts controlling translation in absolute units. We simultaneously measure 743 translation initiation rates and 746 termination efficiencies across the Escherichia coli transcriptome, in addition to translational frameshifting induced at a stable RNA pseudoknot structure. By analyzing the transcriptional and translational response, we discover that sequestered ribosomes at the pseudoknot contribute to a {sigma}32-mediated stress response, codon-specific pausing, and a drop in translation initiation rates across the cell. Our work demonstrates the power of integrating global approaches towards a comprehensive and quantitative understanding of gene regulation and burden in living cells.

synthetic biology↗