Biology subjects

Kratka, M.

Publications and source records attributed to Kratka, M..

6 recordsLinked to original sources

bioRxiv2026

Decoding pseudoexon activation by deep intronic variants using a high-throughput minigene assay in the NF1 gene

Deep intronic variants can cause aberrant splicing, often manifested as pseudoexon inclusion, which represents a significant challenge in clinical diagnostics. Pseudoexon activations have been repeatedly described in the NF1 gene, making it a promising genomic context for studying underlying mechanisms. Here, we developed a specialized minigene assay by combining high-throughput mutagenesis and next-generation sequencing to evaluate the splicing effect of 501 deep intronic variants within NF1 intron 3. We identified and validated eleven pseudoexon-activating variants, including seven novel ones, which clustered into four activation-prone hotspots. Additionally, we detected a rare, out-of-frame wild-type pseudoexon whose inclusion was significantly enhanced by three specific mutations. Using this experimentally validated dataset, we benchmarked SpliceAI performance and ESRseq scores to refine variant prioritization. This integrated high-throughput framework offers a robust starting point for the systematic functional prioritization of deep intronic variation, providing a scalable model for variant annotation in other disease-associated genes.

molecular biology↗

bioRxiv2026

Nowhere to Hide: The Effect of Centromere Architecture on LTR Retrotransposon Dynamics

Centromere architecture can determine where transposable elements persist, yet its effects on retrotransposon turnover remain poorly understood. Here we test a chromosome-level "nowhere-to-hide" model, in which holocentric chromosomes, owing to distributed centromere activity and reduced chromatin compartmentalization, provide fewer stable repeat-rich refugia than monocentric chromosomes. We combined genome-wide LTR retrotransposon annotation, spatial modelling and FISH across 40 holocentric plant species and 31 closely related monocentric relatives from Poales, Cuscuta, and Melanthiaceae. Overall LTR retrotransposon abundance and Ty1-copia/Ty3-gypsy composition were explained mainly by lineage history and chromosome size, rather than by holocentricity itself. By contrast, element persistence and removal showed dependence on centromere architecture. Intact LTR retrotransposons were younger in holocentric genomes, and holocentric chromosomes lacked the chromosome-size-dependent spatial clustering of element age observed in monocentrics. Solo-LTR profiles further revealed weaker spatial clustering of removal signatures in holocentric chromosomes, consistent with a more homogeneous chromosome-wide landscape of ectopic recombination. Epigenomic analyses of a matched Luzula-Juncus pair indicated that young elements can occur in centromeric chromatin, whereas solo LTRs are associated with more euchromatic contexts. These results support the nowhere-to-hide model, showing that centromere architecture does not shape LTR retrotransposons accumulation, but their persistence and removal efficiency.

evolutionary biology↗

bioRxiv2025

Complete genomes of a multi-generational pedigree to expand studies of genetic and epigenetic inheritance

Pedigree analysis remains the gold standard for rare disease diagnostics, yet whole genome sequencing studies typically omit critical regions like centromeres, telomeres, and acrocentric chromosome p-arms. Here, we present telomere-to-telomere (T2T) reference genomes for four self-identified African American individuals of admixed ancestry spanning three generations. Our parent-of-origin assigned, chromosome-level assemblies revealed precise meiotic recombination breakpoints in previously inaccessible regions, including recombination events across acrocentric and subtelomeric sequences. Centromeric regions were highly stable, with multi-megabase arrays inherited intact across three generations, while the position of kinetochore assembly sites remained consistent and predominantly associated with the p-arm proximal region. The relative lengths of telomeres on individual chromosomes were maintained across generations. Using a targeted rDNA assembly approach, we reconstructed a complete megabase-scale ribosomal DNA (rDNA) array corresponding to the paternal chromosome 14. This openly available pedigree provides a benchmark dataset for studying recombination and genetic and epigenetic variation across the complete genome.

genomics↗

bioRxiv2025

Epigenetic reprogramming guides sexual dimorphism during floral development in Silene latifolia

Dioecy, the condition in which male and female individuals exist as separate plants, represents a fascinating and relatively rare reproductive strategy, offering unique opportunities to study the genetic and epigenetic regulation of sexual dimorphism. While sex determining genes underlying dioecy have already been described for several plant species, the role of epigenetic modifications in meristematic cell populations remains poorly understood. In this study we describe the spatio-temporal deposition of three epigenetic markers during early stages of floral development in model dioecious species Silene latifolia. We selected H3K4me1, H3K9me2 and active Ser2 phosphorylated form of RNA Polymerase II (Pol-IIS2ph), to assess levels of chromatin condensation and transcriptional activity of meristematic cells during key developmental stages. Utilizing the novel approach of an AI-assisted nuclei segmentation and high-content imaging we created a single-cell resolution atlas for male and female floral meristems. Our results show a relationship between transcription activity and sex determination during early meristem development. Moreover, our results suggest that H3K9me2 deposition in the developing meristem is linked to sex-specific chromatin reprogramming events, such as pollen mother cell formation during anther maturation. Overall, these results offer new insights into the role of chromatin regulation during floral meristem development and improves our understanding of sexual dimorphism in dioecious species.

plant biology↗

bioRxiv2024

Detection and classification of long terminal repeat sequences in plant LTR-retrotransposons and their analysis using explainable machine learning.

BackgroundLong terminal repeats (LTRs) represent important parts of LTR retrotransposons and retroviruses found in high copy numbers in a majority of eukaryotic genomes. LTRs contain regulatory sequences essential for the life cycle of the retrotransposon. Previous experimental and sequence studies have provided only limited information about LTR structure and composition, mostly from model systems. To enhance our understanding of these key compounds, we focused on the contrasts between LTRs of various retrotransposon families and other genomic regions. Furthermore, this approach can be utilized for the classification and prediction of LTRs. ResultsWe used machine learning methods suitable for DNA sequence classification and applied them to a large dataset of plant LTR retrotransposon sequences. We trained three machine learning models using (i) traditional model ensembles (Gradient Boosting - GBC), (ii) hybrid CNN-LSTM models, and (iii) a pre-trained transformer-based model (DNABERT) using k-mer sequence representation. All three approaches were successful in classifying and isolating LTRs in this data, as well as providing valuable insights into LTR sequence composition. The best classification (expressed as F1 score) achieved for LTR detection was 0.85 using the CNN-LSTM hybrid network model. The most accurate classification task was superfamily classification (F1=0.89) while the least accurate was family classification (F1=0.74). The trained models were subjected to explainability analysis. SHAP positional analysis identified a mixture of interesting features, many of which had a preferred absolute position within the LTR and/or were biologically relevant, such as a centrally positioned TATA-box, and TG..CA patterns around both LTR edges. ConclusionsOur results show that the models used here recognized biologically relevant motifs, such as core promoter elements in the LTR detection task, and a development and stress-related subclass of transcription factor binding sites in the family classification task. Explainability analysis also highlighted the importance of 5- and 3-edges in LTR identity and revealed need to analyze more than just dinucleotides at these ends. Our work shows the applicability of machine learning models to regulatory sequence analysis and classification, and demonstrates the important role of the identified motifs in LTR detection.

bioinformatics↗

bioRxiv2024

Repeat-based holocentromeres of the woodrush Luzula sylvatica reveal new insights into the evolutionary transition from mono- to holocentricity

Although the centromere is restricted to a single region of the chromosome in most studied eukaryotes, members of the rush family (Juncaceae) harbor either monocentric (Juncus) or holocentric (Luzula) chromosomes. This provides an opportunity to study the evolutionary mechanisms involved in the transition to holocentricity. Here by combining chromosome-scale genome assembly, epigenetic analyses, immuno-FISH, and super-resolution microscopy, we report the occurrence of repeat-based holocentromeres in L. sylvatica. We found an irregular distribution of genes, centromeric units, and most repeats along the chromosomes. We determined the centromere function predominantly associated with two satellite DNA repeats, Lusy1 and Lusy2 of 124- and 174-bp monomer length, respectively, while CENH3 also binds satellite-free gene-poor regions. Comparative repeat analysis revealed that Lusy1 is present in most Luzula species, suggesting a conserved centromere role of this repeat. Synteny between L. sylvatica (n = 6) and J. effusus (n = 21) genomes further evidenced a chromosome number reduction in Luzula derived from multiple chromosome fusions of ancestral J. effusus-like chromosomes. We propose that the transition to holocentricity in Luzula involves: (i) fusion of small chromosomes resembling Juncus-like centromeres; (ii) expansion of atypical centromeric units; and (iii) colonization of satellite DNA for centromere stabilization.

genomics↗