Biology subjects

Labun, K.

Publications and source records attributed to Labun, K..

10 recordsLinked to original sources

bioRxiv2026

Ribosome profiling to study translation in cortical synaptoneurosomes on group 1 mGluR activation

Local protein synthesis in neurons occurs in both axons and dendrites and plays a central role in synaptic function. High-throughput-based sequencing and imaging studies have demonstrated the presence and translation of synaptically localised mRNAs. However, quantification of activity-dependent translation dynamics at synapses at the transcriptome-wide scale remains limited. Here, we apply ribosome profiling to synapse-enriched fractions (synaptoneurosomes) derived from rat cortical tissue following stimulation with the group 1 mGluR agonist DHPG. DHPG stimulation induced translation of mRNAs involved in synaptic processes, including synaptic vesicle exocytosis and axo-dendritic transport. Notably, translation of ribosomal protein mRNAs was upregulated upon mGluR activation, consistent with the expected increase in de novo protein synthesis. Together, these results demonstrate the use of ribosome profiling to capture changes in local mRNA translation from isolated preparations.

neuroscience↗

bioRxiv2025

CHOPOFF: symbolic alignments enable fast and sensitive CRISPR off-target detection

CRISPR/Cas systems offer powerful tools for genome editing, but their therapeutic application is hampered by the risk of unintended off-target mutations. Many molecular methods have been established to detect off-target editing, however, their sensitivity depends on first identifying potential sites using in silico methods. However, these in silico prediction methods are challenged by a trade-off between speed and sensitivity, and can fail to comprehensively detect all edited off-target sites. Here, we demonstrate that ignoring bulges has led to missing editing at off-target sites in previous studies and that continuing this practice can lead to inflated claims of fidelity. As a solution, we introduce the concept of symbolic alignments to efficiently identify off-targets without sacrificing sensitivity. We further present specialized data structures that enable rapid, alignment-free probabilistic ranking of guide RNAs based on their predicted off-target burden. Implemented in the tool CHOPOFF, these innovations accommodate mismatches, bulges (insertions/deletions), and incorporate genomic sequence variants for personalized off-target assessment. Benchmarking demonstrates that CHOPOFF significantly outperforms state-of-the-art tools in both prediction accuracy and computational speed. AvailabilityCHOPOFF command line available at https://github.com/JokingHero/CHOPOFF.jl CHOPOFF web server available at https://crisprtools.org/chopoff

bioinformatics↗

bioRxiv2024

Transcriptome-wide mapping of small ribosomal subunits elucidates scanning mechanisms of translation initiation in the mammalian brain

Protein synthesis in neurons is highly compartmentalised and regulated, with key roles for translation initiation and elongation factors. The most widely used transcriptome-wide method for measuring translation, ribosome profiling, characterises the elongation phase of translation but does not provide insight into the initiation phase with scanning of the small ribosomal subunit (SSU). Here, we adapted and optimised ribosome complex profiling (RCP-seq) for brain tissue, capturing SSUs and analysis of translation initiation dynamics in mouse dentate gyrus and cerebral cortex. In both tissues, SSUs accumulate upstream of the start codon on synaptically localised RNAs and this poised SSU configuration is associated with enhanced translational efficiency. Upstream open reading frames (uORFs) are extensively translated and associated with less SSU poising downstream, suggesting that uORFs may have a buffering effect on poised SSUs. Ribosome occupancy analysis suggests that neuron-specific transcripts recruit more ribosomes and are more translated than glia-specific transcripts. Furthermore, monosome-preferring neuronal mRNAs exhibit reduced scanning and elongation relative to polysome-preferring transcripts implying reduced recruitment of ribosomes. In sum, RCP-seq elucidates translation initiation dynamics in the mammalian brain and uncovers cell-type- and transcript-specific regulation.

neuroscience↗

bioRxiv2024

T cell correction pipeline for Inborn Errors of Immunity

CRISPR/Cas9 gene editing technology is a promising tool for correcting pathogenic variants for autologous cell therapies for Inborn Errors of Immunity (IEI). The present IEI correction strategies mainly focus on the knock-in of therapeutic cDNAs, or knockout of the disease-causing gene when feasible. These strategies address many single-gene defects but may disrupt gene expression and require significant optimization for each newly discovered IEI-causing gene, highlighting the need for complementary platforms that can precisely correct diverse pathogenic variants. Here, we present a safe and efficient T cell single nucleotide variant (SNV) correction pipeline based on homology-directed repair (HDR), suitable for diverse monogenic mutations. By using founder mutations of Deficiency of ADA2 (DADA2), Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and Cartilage Hair Hypoplasia (CHH) as IEI models, we show that our pipeline can achieve up to 80% bi-allelic editing, with resultant functional correction of the disease phenotype in patient T cells. We do not find detectable pre-malignant off-target effects or karyotypic, transcriptomic or proteomic aberrations upon profiling patient T cells with GUIDE-seq, single cell RNA sequencing, PacBio based long-read whole genome sequencing, and high-throughput proteomics. This study demonstrates that HDR-based SNV editing is a safe and effective option for IEI T cell correction and that it could be developed to an autologous T cell therapy, as the presented protocol is scalable for a GMP-compatible workflow. This study is a step towards the development of gene correction platform that targets a broad number of monogenic mutations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/610811v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f1eb47org.highwire.dtl.DTLVardef@18dbcc2org.highwire.dtl.DTLVardef@63862dorg.highwire.dtl.DTLVardef@1fe1561_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

bioengineering↗

bioRxiv2021

Rapid genome editing by CRISPR-Cas9-POLD3 fusion

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the particular cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

molecular biology↗

bioRxiv2021

ORFik: a comprehensive R toolkit for the analysis of translation

* BackgroundWith the rapid growth in the use of high-throughput methods for characterizing translation and the continued expansion of multi-omics, there is a need for back-end functions and streamlined tools for processing, analyzing, and characterizing data produced by these assays. * ResultsHere, we introduce ORFik, a user-friendly R/Bioconductor toolbox for studying translation and its regulation. It extends GenomicRanges from the genome to the transcriptome and implements a framework that integrates data from several sources. ORFik streamlines the steps to process, analyze, and visualize the different steps of translation with a particular focus on initiation and elongation. It accepts high-throughput sequencing data from ribosome profiling to quantify ribosome elongation or RCP-seq/TCP-seq to also quantify ribosome scanning. In addition, ORFik can use CAGE data to accurately determine 5UTRs and RNA-seq for determining translation relative to RNA abundance. ORFik supports and calculates over 30 different translation-related features and metrics from the literature and can annotate translated regions such as proteins or upstream open reading frames. As a use-case, we demonstrate using ORFik to rapidly annotate the dynamics of 5 UTRs across different tissues, detect their uORFs, and characterize their scanning and translation in the downstream protein-coding regions. * Availabilityhttp://bioconductor.org/packages/ORFik

bioinformatics↗

bioRxiv2020

Deep conservation of ribosome stall sites across RNA processing genes

The rate of translation can vary considerably depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the required time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pause and stall sites, but due to library-specific biases, these predictions are often unreliable. Here, we address this by taking advantage of the deep conservation of the protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important positions of ribosome slowdown - here collectively called stall sites. We analyze multiple ribosome profiling datasets from a phylogenetically diverse group of eukaryotes: yeast, fruit fly, zebrafish, mouse, and human and identify conserved stall sites. We find thousands of stall sites across multiple species, with proline, glycine, and negatively charged amino acids being the main facilitators of stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved regulatory effect on RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation.

genomics↗

bioRxiv2020

Single molecule structure sequencing reveals RNA structural dependencies, breathing and ensembles

RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features.

molecular biology↗

bioRxiv2019

tailfindr: Alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

Polyadenylation at the 3-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability and translation, among others. Only recently, strategies have emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3-ends. Concurrently Oxford Nanopore Technologies (ONT) established full-length isoform RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through basecalling has so far not been possible due the inability to resolve long homopolymeric stretches in ONT sequencing.\n\nHere we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, basecalled data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindrs performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.

bioinformatics↗

bioRxiv2018

Accurate analysis of genuine CRISPR editing events with ampliCan

We present ampliCan, an analysis tool for genome editing that unites highly precise quantification and visualization of genuine genome editing events. ampliCan features nuclease-optimized alignments, filtering of experimental artifacts, event-specific normalization, off-target read detection and quantifies insertions, deletions, HDR repair as well as targeted base editing. It is scalable to thousands of amplicon sequencing-based experiments from any genome editing experiment, including CRISPR. It enables automated integration of controls and accounts for biases at every step of the analysis. We benchmarked ampliCan on both real and simulated datasets against other leading tools, demonstrating that it outperformed all in the face of common confounding factors.

bioinformatics↗