Biology subjects

Machowska, M.

Publications and source records attributed to Machowska, M..

3 recordsLinked to original sources

bioRxiv2025

Whole-miRNome sequencing (WMS) - a panel for targeted sequencing of all human miRNA genes

There is a growing interest in the genetic variation of noncoding genomic elements, including miRNAs, and several mutations in miRNA genes implicated in human diseases, including cancer, have already been detected. However, the lack of dedicated analytical tools severely hampers progress in this area. In this study, we developed whole-miRNome sequencing (WMS), which enables targeted sequencing of all human miRNA genes (n[~]2000) and 28 miRNA biogenesis genes. Herein, by sequencing almost 600 samples, including [~]300 tumor/normal pairs of samples from different cancer types, we identified [~]2, 000 mutations, including 1, 435 cancer somatic mutations, with 879 occurring in miRNA genes. These mutations were located in all parts of the genes, including seed or cleavage sites essential for the functioning of miRNA genes. The high reliability of the mutations was confirmed through various approaches, including different sequencing methods. The analysis identified several miRNA genes with functional enrichment of cancer mutations, including MIR3928, specifically mutated in basal cell carcinoma (BCC), indicating its potential role in this cancer. WMS also allowed the identification of multiple copy number alterations, hotspots of which often encompassed miRNA genes. WMS provides highly effective, low-cost sequencing of all miRNA genes in different types of samples, including highly degraded FFPE samples.

genomics↗

bioRxiv2024

Human iPSCs-derived muscle cells as a new model for investigation of EDMD1 pathogenesis.

Emery-Dreifuss muscular dystrophy type 1 (EDMD1) is a rare genetic disease caused by mutations in the EMD gene, which encodes the nuclear envelope protein emerin. Despite understanding the genetic basis of the disease, the molecular mechanism underlying muscle and cardiac pathogenesis remains elusive. Progress is restricted by the limited availability of patient-derived samples, therefore there is an urgent need for human-specific cellular models. In this study, we present the generation and characterization of induced pluripotent stem cell (iPSC) lines derived from EDMD1 patients carrying EMD mutations that lead to truncated or absent emerin, together with iPSCs from healthy donor. The patient-specific iPSCs exhibit stable karyotypes, maintain appropriate morphology, express pluripotency markers and demonstrate the ability to differentiate into three germ layers. To model EDMD1, these iPSCs were differentiated into myogenic progenitors, myoblasts and multinucleated myotubes, which represent all stages of myogenesis. Each developmental stage was validated by the presence of stage-specific markers, ensuring the accuracy of the model. We present the first iPSC-based in vitro platform that captures the complexity of EDMD1 pathogenesis during myogenesis. This model can significantly contribute to understanding disease mechanisms and develop the targeted therapeutic strategies for EDMD1.

molecular biology↗

bioRxiv2024

Heat shock alters the distribution and in vivo interaction of major nuclear structural proteins, lamin and DNA topoisomerase II, with nucleic acids

Lamins and topoisomerases play a critical role in the structural support of cell nuclei, in the regulation of chromatin structure, chromatin distribution, topology of DNA, gene expression, transcription, splicing and transport. Here, we report the role of lamins and Top2 during transition from normal conditions (N), in heat shock (HS) and recovery (R) in Drosophila since the fly genome contains a single gene for B-type lamin (lamin Dm), for A-type lamin (lamin C) and Top2. Heat shock increases transient phosphorylation of lamin Dm on S25, induces changes in solubility of lamin Dm, Top2, HSF, HDAC1 and HP1 proteins, especially in S2 cells and relocates Top2 and chromatin closer to the nuclear lamina with induction of granular staining for Top2 in Kc, S2 and embryonic cells. Lamin Dm interacts with Top2 protein and HS increases the interaction. In vivo photocrosslinking and immunoprecipitation revealed a significant increase in binding to chromatin and nucleic acids upon HS induction for Top2 and lamin Dm. All the detected changes in the properties and location of proteins returned to "normal" after recovery from heat shock. This suggests an important role for lamin Dm, Top2 and their complexes in nuclear functions during HS and recovery. We propose a model in which relocation of Top2 chromatin complexes closer to the nuclear lamina and lamin Dm may help to rearrange the gene expression pattern by tethering HS inactivated genes to nuclear lamina and NPCs which might also help to bind nuclear fraction of non-HS-related transcripts at the nuclear lamina/NPCs area.

cell biology↗