Biology subjects

Franek, M.

Publications and source records attributed to Franek, M..

4 recordsLinked to original sources

bioRxiv2023

In-section Click-iT detection and super-resolution CLEM: Shedding light on nucleolar ultrastructure and S-phase progression in plants.

Correlative light and electron microscopy (CLEM) is an essential tool that allows for localisation of a particular target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometer scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of serial sections by both, single-molecule localization microscopy and transmission electron microscopy for correlative analysis. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows a dramatic improvement in fluorophore blinking behavior relative to the whole-mount approaches. Here we used commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as FASCIATA1 and NUCLEOLIN1 mutants and applied our on-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

plant biology↗

bioRxiv2022

Hymenoptera (Insecta) telomerase RNAs switched to plant/ciliate-like biogenesis

In contrast to the catalytic subunit of telomerase, its RNA subunit (TR) is highly divergent in size, sequence and biogenesis pathways across eukaryotes. Current views on TR evolution assume a common origin of TRs transcribed with RNA polymerase II in Opisthokonta (the supergroup including Animalia and Fungi) and Trypanosomida on one hand, and TRs transcribed with RNA polymerase III under the control of type 3 promoter, found in TSAR and Archaeplastida supergroups (including e.g., ciliates and Viridiplantae taxa, respectively). Here we focus on unknown TRs in one of the largest Animalia order - Hymenoptera (Arthropoda) with more than 300 available representative genomes. Using a combination of bioinformatic and experimental approaches, we identify their TRs. In contrast to the presumed type of TRs (H/ACA box snoRNAs transcribed with RNA Polymerase II) corresponding to their phylogenetic position, we find here short TRs of the snRNA type, likely transcribed with RNA polymerase III under the control of the type 3 promoter. The newly described insect TRs thus question the hitherto assumed monophyletic origin of TRs across Animalia and point to an evolutionary switch in TR type and biogenesis that was associated with the divergence of Arthropods. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/512496v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@7a691borg.highwire.dtl.DTLVardef@8a8aa2org.highwire.dtl.DTLVardef@193960forg.highwire.dtl.DTLVardef@60d364_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2020

Multiple epigenetic layers accompany the spatial distribution of ribosomal genes in Arabidopsis

45S ribosomal genes in A. thaliana (rDNA) are located in tandem arrays on termini of chromosomes 2 and 4 (NOR2 and NOR4) and encode rRNA, crucial structural elements of the ribosome. Inactive rDNA genes accumulate in the condensed chromocenters in the nucleus and at the nucleolar periphery, while nucleolus delimits the active genes. We show that a subset of nucleolar rDNA assembles into condensed foci marked by H3.1 and H3.3 histones and that progressive rDNA condensation is connected with rDNA transcriptional activity, cell ploidy and rDNA copy number. Interestingly, some nucleolar foci are reminiscent of perinucleolar chromocenters, containing the NOR4 region. We further demonstrate that rDNA promoter is a key regulatory region of the rDNA repeat and describe large involvement of repressive epigenetic mark H3K9me2 and H2A.W histone variant in rDNA activity regulation. In addition, we found euchromatic H3.3 histone enrichment at the rDNA transcription start site in actively dividing tissues, despite its accumulation in nucleolar foci containing condensed rDNA repeats.

cell biology↗

bioRxiv2020

Low ribosomal RNA genes copy number provoke genomic instability and chromosomal segment duplication events that modify global gene expression and plant-pathogen response

Among the hundreds of ribosomal RNA (rRNA) gene copies organized as tandem repeats in the nucleolus organizer regions (NORs), only a portion is usually actively expressed in the nucleolus and participate in the ribosome biogenesis process. The role of these extra-copies remains elusive, but previous studies suggested their importance in genome stability and global gene expression. Because the nucleolus is also a platform for nuclear organization, we tested the impact of a decreased amount of rRNA gene copies on the Arabidopsis thaliana 3D genome organization and stability, using an A. thaliana line only containing 20% of rRNA gene copies (20rDNA line). Compared to the wild-type Col-0, the 20rDNA line shows several signs of genomic instability, such as variations in 3D genome organization, spontaneous double-strand breaks accumulation, transcriptomic changes, and higher DNA methylation level. Strikingly, using genomic and microscopic approaches, we identified seven large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. As a consequence, more than 600 genes were duplicated, often associated with an increase in their expression level. Among them, we found several upregulated genes involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and we discuss their potential implications on plant genome evolution.

plant biology↗