Biology subjects

Ustyantsev, K.

Publications and source records attributed to Ustyantsev, K..

8 recordsLinked to original sources

bioRxiv2024

Heat stress drives transcription of LTR retrotransposons in the regenerative flatworm Macrostomum lignano

The evolutionary arms race between transposable elements (TEs) and their hosts contributes to genomic complexity. As TEs mobilization is deleterious for individual cells and organisms, their activity is restricted. During stress, TEs can be reactivated; however, the exact mechanisms vary. We discovered that in the flatworm Macrostomum lignano, LTR retrotransposons hijack the heat shock response pathway to boost their transcription at elevated temperatures. While it has been well-described in cruciferous plants, this is the first report of this mechanism in animal LTR retrotransposons. Our results suggest a convergent evolution of the heat stress response in LTR retrotransposons from animals and plants.

genomics↗

bioRxiv2024

One probe fits all: a highly customizable modular RNA in situ hybridization platform expanding the application of SABER DNA probes

In situ hybridization (ISH) of RNA is a key method to visualize gene expression patterns in complex biological samples. The technique is indispensable for biological research related to e.g. development, disease, gene function, and the validation of novel cell types identified by single-cell sequencing methods. Especially in non-mammalian models lacking accessibility to a broad spectrum of antibodies, ISH remains a major research tool. Diverse available ISH protocols require different custom hybridization probe types, design, and/or proprietary signal detection chemistry. This makes it hard to navigate for a beginner and increases the research costs when multiple methods need to be applied. Here, we describe OneSABER - a unified open platform connecting commonly used canonical and recently developed single- and multiplex, colorimetric, and fluorescent ISH approaches. This platform uses a single type of ISH DNA probes adapted from the signal amplification by exchange reaction (SABER) method. We demonstrate applications of the proposed ISH framework in whole-mount samples of the regenerative flatworm Macrostomum lignano, advancing this animal as a powerful model for stem cell and regeneration research.

developmental biology↗

bioRxiv2023

Optimized protocols for RNA interference in Macrostomum lignano

Macrostomum lignano, a marine free-living flatworm, has emerged as a potent invertebrate model in developmental biology for studying stem cells, germline, and regeneration processes. In recent years, many tools have been developed to manipulate this worm and to facilitate genetic modification. RNA interference is currently the most accessible and direct technique to investigate gene functions. It is obtained by soaking worms in artificial seawater containing dsRNA targeting the gene of interest. Although easy to perform, the original protocol calls for daily exchange of dsRNA solutions, usually until phenotypes are observed, which is both time- and cost-consuming. In this work, we have evaluated alternative dsRNA delivery techniques, such as electroporation and osmotic shock, to facilitate the experiments with improved time and cost efficiency. During our investigation to optimize RNAi, we demonstrated that, in the absence of diatoms, regular single soaking in artificial seawater containing dsRNA directly produced in bacteria or synthesized in vitro is, in most cases, sufficient to induce a potent gene knockdown for several days with a single soaking step. Therefore, this new and highly simplified method allows a very significant reduction of dsRNA consumption and lab work. In addition, it enables performing experiments on a larger number of worms at minimal cost.

developmental biology↗

bioRxiv2022

Genome assembly of the acoel flatworm Symsagittifera roscoffensis, a model for research on photosymbiosis

Symsagittifera roscoffensis is a well-known member of the order Acoela that lives in symbiosis with the algae Tetraselmis convolutae during its adult stage. Its natural habitat is the eastern coast of the Atlantic, where at specific locations thousands of individuals can be found lying in large pools on the surface of sand at low tide and in the sandy interstitial web at high tide. As a member of the Acoela it has been used as a proxy for early bilaterian animals; however, its phylogenetic position remains debated. In order to understand the basic structural characteristics of the acoel genome, we sequenced and assembled the genome of aposymbiotic S. roscoffensis. The size of S. roscoffensis genome was measured to be in range 910 - 940 Mb. Sequencing of the genome was performed using PacBio Hi-Fi technology. Hi-C and RNA-seq data were also generated to scaffold and annotate the genome. The resulting assembly is 1.1 Gb large (covering 118% of the estimated genome size) and highly continuous, with N50 scaffold size of 1.04 Mb. The repetitive fraction of the genome is 61%, of which 85% (half of the genome) are LTR retrotransposons. Genome-guided transcriptome assembly identified 34,493 genes, of which 29,351 are protein coding (BUSCO score 97.6%), and 30.2% of genes are spliced leader (SL) trans-spliced. The completeness of this genome suggests that it can be used extensively to characterize gene families and conduct accurate phylogenomic reconstructions. SignificanceSymsagittifera is a representative of the phylum Acoela, the first offshoot of bilaterian animals. This key phylogenetic position adds an extra value to the knowledge of its genome, since it will inform us on how the genome of a bilaterian ancestor might have looked like. Moreover, Symsagittifera roscoffensis is a model organism used in symbiogenesis research. Host and algae can be cultured independently and, after mixing, the symbiosis can be followed. Symbiogenesis was established early on during the evolution of Metazoa. In spite of its biological relevance, very little is known on the molecular mechanisms that control it. Here the genome of the acoel host should provide us with insights on the first adaptations to symbiogenesis occurring in bilateral animals.

genomics↗

bioRxiv2021

DARTS: an Algorithm for Domain-Associated RetroTransposon Search in Genome Assemblies

Retrotransposons comprise a substantial fraction of eukaryotic genomes reaching the highest proportions in plants. Therefore, identification and annotation of retrotransposons is an important task in studying regulation and evolution of plant genomes. A majority of computational tools for mining transposable elements (TEs) are designed for subsequent genome repeat masking, often leaving aside the element lineage classification and its protein domain composition. Additionally, studies focused on diversity and evolution of a particular group of retrotransposons often require substantial customization efforts from researchers to adapt existing software to their needs. Here, we developed a computational pipeline to mine sequences of protein-coding retrotransposons based on the sequences of their conserved protein domains - DARTS. Using the most abundant group of TEs in plants - long terminal repeat (LTR) retrotransposons (LTR-RTs), we show that DARTS has radically higher sensitivity of LTR-RTs identification compared to a widely accepted LTRharvest tool. DARTS can be easily customized for specific user needs. As a result, DARTS returns a set of structurally annotated nucleotide and amino acid sequences which can be readily used in subsequent comparative and phylogenetic analyses. DARTS should facilitate researchers interested in discovery and in-detail analysis of diversity and evolution of retrotransposons, LTR-RTs, and other protein-coding TEs.

bioinformatics↗

bioRxiv2020

Tim29 is required for stem cell activity during regeneration in the flatworm Macrostomum lignano

Domain of Unknown Function (DUF) genes are broadly conserved in metazoa but have an unknown biological function. Previously, we established transcriptional profiles of the germline and stem cells of the flatworm Macrostomum lignano (Grudniewska et al., 2016). Here, we used this information to probe the function of DUF genes in M. lignano. Recently, Kang et al. (2016) found that DUF2366 is a mitochondrial inner membrane protein that interacts with the protein import complex TIM22. DUF2366 was renamed TIM29, and shown to stabilize the TIM22 complex, but its biological role remained largely unknown. We now demonstrate that DUF2366/TIM29 is dispensable in the homeostatic condition in M. lignano, but is essential to adapt to a highly proliferative state required for regeneration. Our results show that de-DUFing the function of DUF genes might require special biological conditions, such as regeneration, and establishes M. lignano as an informative platform to study DUF genes.

cell biology↗

bioRxiv2018

Influence of temperature on the development, reproduction and regeneration in the flatworm model organism Macrostomum lignano

The free-living marine flatworm Macrostomum lignano is a powerful model organism to study mechanisms of regeneration and stem cell regulation due to its convenient combination of biological and experimental properties, including the availability of transgenesis methods, which is unique among flatworm models. However, due to its relatively recent introduction in research, there are still many biological aspects of the animal that are not known. One of such questions is the influence of the culturing temperature on Macrostomum biology. Here we systematically investigated how different culturing temperatures affect the development time, reproduction rate, regeneration, heat shock response, and gene knockdown efficiency by RNA interference in M. lignano. We used marker transgenic lines of the flatworm to accurately measure the regeneration endpoint and to establish the stress response threshold for temperature shock. We found that compared to the culturing temperature of 20{degrees}C commonly used for M. lignano, elevated temperatures of 25{degrees}C-30{degrees}C substantially speed-up the development and regeneration time and increase reproduction rate without detectable negative consequences for the animal, while temperatures above 30{degrees}C elicit a heat shock response.\n\nWe show that altering the temperature conditions can be used to shorten the time required to establish M. lignano cultures, store important lines and optimize the microinjection procedures for transgenesis. Our findings will help to optimize the design of experiments in M. lignano and thus facilitate future research in this model organism.

zoology↗

bioRxiv2017

A platform for efficient transgenesis in Macrostomum lignano, a flatworm model organism for stem cell research

Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration and tissue patterning. However, the lack of transgenesis methods significantly hampers their wider use. Here we report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and showed its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.

bioengineering↗