Piwi proteins, a subclass of Argonaute-family proteins, carry approximately 24-30-nt Piwi-interacting RNAs (piRNAs) that mediate gonadal defense against transposable elements (TEs). We analyzed the ...Drosophila ovary somatic sheet (OSS) cell line and found that it expresses miRNAs, endogenous small interfering RNAs (endo-siRNAs), and piRNAs in abundance. In contrast to intact gonads, which contain mixtures of germline and somatic cell types that express different Piwi-class proteins, OSS cells are a homogenous somatic cell population that expresses only PIWI and primary piRNAs. Detailed examination of its TE-derived piRNAs and endo-siRNAs revealed aspects of TE defense that do not rely upon ping-pong amplification. In particular, we provide evidence that a subset of piRNA master clusters, including flamenco, are specifically expressed in OSS and ovarian follicle cells. These data indicate that the restriction of certain TEs in somatic gonadal cells is largely mediated by a primary piRNA pathway.
Abstract
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, mostly, lying in large pools on the surface of sand at low tide. As a member of the Acoela it has been thought as a proxy for ancestral bilaterian animals; however, its phylogenetic position remains still debated. In order to understand the basic structural characteristics of the acoel genome, we sequenced and assembled the genome of aposymbiotic species S. roscoffensis. The size of this genome was measured to be in the range of 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 it. 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 trans-spliced. The completeness of this genome suggests that it can be used extensively to characterize gene families and conduct accurate phylogenomic reconstructions.
Differences in Vertebrate MicroRNA Expression Ason, Brandon; Darnell, Diana K.; Wittbrodt, Beate ...
Proceedings of the National Academy of Sciences - PNAS,
09/2006, Volume:
103, Issue:
39
Journal Article
Peer reviewed
Open access
MicroRNAs (miRNAs) attenuate gene expression by means of translational inhibition and mRNA degradation. They are abundant, highly conserved, and predicted to regulate a large number of transcripts. ...Several hundred miRNA classes are known, and many are associated with cell proliferation and differentiation. Many exhibit tissue-specific expression, which aids in evaluating their functions, and it has been assumed that their high level of sequence conservation implies a high level of expression conservation. A limited amount of data supports this, although discrepancies do exist. By comparing the expression of ≈100 miRNAs in medaka and chicken with existing data for zebrafish and mouse, we conclude that the timing and location of miRNA expression is not strictly conserved. In some instances, differences in expression are associated with changes in miRNA copy number, genomic context, or both between species. Variation in miRNA expression is more pronounced the greater the differences in physiology, and it is enticing to speculate that changes in miRNA expression may play a role in shaping the physiological differences produced during animal development.
Cardiac hypertrophy accompanies many forms of cardiovascular diseases. The mechanisms behind the development and regulation of cardiac hypertrophy in the human setting are poorly understood, which ...can be partially attributed to the lack of a human cardiomyocyte-based preclinical test system recapitulating features of diseased myocardium. The objective of our study is to determine whether human embryonic stem cell-derived cardiomyocytes (hESC-CMs) subjected to mechanical stretch can be used as an adequate in vitro model for studying molecular mechanisms of cardiac hypertrophy. We show that hESC-CMs subjected to cyclic stretch, which mimics mechanical overload, exhibit essential features of a hypertrophic state on structural, functional, and gene expression levels. The presented hESC-CM stretch approach provides insight into molecular mechanisms behind mechanotransduction and cardiac hypertrophy and lays groundwork for the development of pharmacological approaches as well as for discovering potential circulating biomarkers of cardiac dysfunction.
•Cyclic stretch leads to the changes in hESC-CM size, contractility, and elasticity•hESC-CM stretch results in release of cardiac injury mediators•RNA-seq reveals signaling pathways associated with cardiac hypertrophy•Knockdown of FSTL3 and SP6 genes blunted the hypertrophic response
In this article, Berezikov, van der Meer, and colleagues used stem cell-derived cardiomyocytes to model human cardiac hypertrophy. Their approach provides novel insights into molecular mechanisms behind mechanotransduction and cardiac hypertrophy and lays groundwork for the development of new pharmacological approaches as well as for discovering new potential circulating biomarkers of cardiac dysfunction.
MicroRNAs (miRNAs) are a class of non-coding RNAs of ∼22 nucleotides in length, and constitute a novel class of gene regulators by imperfect base-pairing to the 3'UTR of protein encoding messenger ...RNAs. Growing evidence indicates that miRNAs are implicated in several pathological processes in myocardial disease. The past years, we have witnessed several profiling attempts using high-density oligonucleotide array-based approaches to identify the complete miRNA content (miRNOME) in the healthy and diseased mammalian heart. These efforts have demonstrated that the failing heart displays differential expression of several dozens of miRNAs. While the total number of experimentally validated human miRNAs is roughly two thousand, the number of expressed miRNAs in the human myocardium remains elusive. Our objective was to perform an unbiased assay to identify the miRNOME of the human heart, both under physiological and pathophysiological conditions. We used deep sequencing and bioinformatics to annotate and quantify microRNA expression in healthy and diseased human heart (heart failure secondary to hypertrophic or dilated cardiomyopathy). Our results indicate that the human heart expresses >800 miRNAs, the majority of which not being annotated nor described so far and some of which being unique to primate species. Furthermore, >250 miRNAs show differential and etiology-dependent expression in human dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM). The human cardiac miRNOME still possesses a large number of miRNAs that remain virtually unexplored. The current study provides a starting point for a more comprehensive understanding of the role of miRNAs in regulating human heart disease.
The free-living marine flatworm
is a powerful model organism for use in studying mechanisms of regeneration and stem cell regulation due to its 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, many aspects of this animal's biology remain unknown. One such question is the influence of culture temperature on
biology.
We systematically investigated how different culture temperatures affect development time, reproduction rate, regeneration, heat shock response, and gene knockdown efficiency by RNA interference (RNAi) in
We used marker transgenic lines to accurately measure the regeneration endpoint, and to establish the stress response threshold for temperature shock. We found that compared to the culture temperature of 20 °C commonly used for
, temperatures of 25 °C-30 °C substantially increase the speed of development and regeneration, lead to faster manifestation of RNAi phenotypes, and increase reproduction rate without detectable negative consequences for the animal, while temperatures above 30 °C elicit a heat shock response.
We show that altering temperature conditions can be used to reduce the time required to establish
cultures, perform RNAi experiments, store important lines, and optimize microinjection procedures for transgenesis. These findings will help to optimize the design of experiments in
and thus facilitate future research using this model organism.
is a free-living flatworm that is emerging as an attractive experimental animal for research on a broad range of biological questions. One feature setting it apart from other flatworms is the ...successful establishment of transgenesis methods, facilitated by a steady supply of eggs in the form of single-cell zygotes that can be readily manipulated. This, in combination with the transparency of the animal and its small size, creates practical advantages for imaging and fluorescence-activated cell sorting in studies related to stem cell biology and regeneration.
can regenerate most of its body parts, including the germline, thanks to the neoblasts, which represent the flatworm stem cell system. Interestingly, neoblasts seem to have a high capacity of cellular maintenance, as
can survive up to 210 Gy of γ-irradiation, and partially offset the negative consequence of ageing. As a non-self-fertilizing simultaneous hermaphrodite that reproduces in a sexual manner,
is also used to study sexual selection and other evolutionary aspects of sexual reproduction. Work over the past several years has led to the development of molecular resources and tools, including high-quality genome and transcriptome assemblies, transcriptional profiling of the germline and somatic neoblasts, gene knockdown, and in situ hybridization. The increasingly detailed characterization of this animal has also resulted in novel research questions, such as bio-adhesion based on its adhesion-release glands and genome evolution due to its recent whole-genome duplication.
In response to heart failure (HF), the heart reacts by repressing adult genes and expressing fetal genes, thereby returning to a more fetal-like gene profile. To identify genes involved in this ...process, we carried out transcriptional analysis on murine hearts at different stages of development and on hearts from adult mice with HF. Our screen identified
, encoding for 5-oxoprolinase, a member of the γ-glutamyl cycle that functions by scavenging 5-oxoproline. OPLAH depletion occurred as a result of cardiac injury, leading to elevated 5-oxoproline and oxidative stress, whereas OPLAH overexpression improved cardiac function after ischemic injury. In HF patients, we observed elevated plasma 5-oxoproline, which was associated with a worse clinical outcome. Understanding and modulating fetal-like genes in the failing heart may lead to potential diagnostic, prognostic, and therapeutic options in HF.
Members of the fetal-gene-program may act as regulatory components to impede deleterious events occurring with cardiac remodeling, and constitute potential novel therapeutic heart failure (HF) ...targets. Mitochondrial energy derangements occur both during early fetal development and in patients with HF. Here we aim to elucidate the role of DIO2, a member of the fetal-gene-program, in pluripotent stem cell (PSC)-derived human cardiomyocytes and on mitochondrial dynamics and energetics, specifically. RNA sequencing and pathway enrichment analysis was performed on mouse cardiac tissue at different time points during development, adult age, and ischemia-induced HF. To determine the function of DIO2 in cardiomyocytes, a stable human hPSC-line with a DIO2 knockdown was made using a short harpin sequence. Firstly, we showed the selenoprotein, type II deiodinase (DIO2): the enzyme responsible for the tissue-specific conversion of inactive (T4) into active thyroid hormone (T3), to be a member of the fetal-gene-program. Secondly, silencing DIO2 resulted in an increased reactive oxygen species, impaired activation of the mitochondrial unfolded protein response, severely impaired mitochondrial respiration and reduced cellular viability. Microscopical 3D reconstruction of the mitochondrial network displayed substantial mitochondrial fragmentation. Summarizing, we identified DIO2 to be a member of the fetal-gene-program and as a key regulator of mitochondrial performance in human cardiomyocytes. Our results suggest a key position of human DIO2 as a regulator of mitochondrial function in human cardiomyocytes.
MicroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently > 400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 ...and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA candidates using two approaches: testing of computationally predicted microRNAs by a modified microarray-based detection system, and cloning and sequencing of large numbers of small RNAs from different human and mouse tissues. Together these efforts experimentally identified 348 novel mouse and 81 novel human microRNA candidate genes. Most novel microRNAs candidates are not conserved beyond mammals, and ~10% are taxon-specific. Our analyses indicate that the entire microRNA repertoire is not remotely exhausted.
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