Determination of protein localization and RNA kinetics in human cells

In dieser Dissertation haben wir das Verhalten menschlicher Zellen in Raum und Zeit untersucht. Hochwertige Datensätze subzellulärer Regionen in HEK293-Zellen wurden mit Hilfe der BirA* Proximity-Labelling-Aktivität erstellt, wobei die Lokalisierung auf zelluläre Regionen beschränkt wurde, die mit herkömmlichen Methoden nur schwer zu reinigen sind (d. h. die dem Zytosol zugewandten Seiten des ER, Mitochondrien und Plasma-membranen). Wir entwickelten daraufhin einen Ansatz zur Kartierung der Verteilung von Proteinen, die aktiv an RNA binden, und nannten ihn f-XRNAX. Wir stellten hintergrundkorrigierte Proteome für Zellkerne, Zytoplasma und Membranen von HEK293-Zellen her. Überraschenderweise wurden viele nicht-kanonische RBPs in der Membranfraktion identifiziert, und ihre Peptidprofile waren in Regionen mit hoher Dichte an intrinsisch ungeordneten Regionen angereichert, was auf eine möglicherweise schwache, durch diese nicht-strukturellen Motive vermittelte Interaktion mit RNA hinweist. Schließlich konnten wir die unterschiedliche Bindung desselben Proteins an RNA in verschiedenen HEK293-Kompartimenten nachweisen. Im zweiten Teil dieser Arbeit konzentrierten wir uns auf die Bestimmung und Quantifizierung von neu transkribierten RNAs auf Einzelzellebene. Die Kinetik der RNA-Transkription und -Degradation war bis vor kurzem auf Einzelzellebene nicht messbar. Daher haben wir einen neuen Ansatz (SLAM-Drop-seq genannt) entwickelt, indem wir die veröffentlichte SLAM-seq-Methode an Einzelzellen angepasst haben. Wir haben SLAM-Drop-seq verwendet, um die zeitabhängigen RNA-Kinetikraten der Transkription und des Umsatzes für Hunderte von oszillierenden Transkripten während des Zellzyklus von HEK293-Zellen zu schätzen. Wir fanden heraus, dass Gene ihre Expression mit unterschiedlichen Strategien regulieren und spezifische Modi zur Feinabstimmung ihrer kinetischen Raten entlang des Zellzyklus haben.In this PhD dissertation we investigated the behaviour of human cells through space and time. High quality datasets of subcellular regions in HEK293 cells were generated using BirA* proximity labelling activity and restricting its localization at cellular regions difficult to purified with traditional methods (i.e., the cytosol-facing sides of the endoplasmic reticulum, mitochondria, and plasma membranes). We then developed an approach to map the distribution of proteins actively binding to RNA, and named it f-XRNAX. We recovered background-corrected proteomes for nuclei, cytoplasm and membranes of HEK293 cells. Surprisingly, many non-canonical RBPs were identified in the membrane fraction, and their peptide profiles were enriched in regions with high density of intrinsically disordered regions, indicating a possibly weak interaction with RNA mediated by these non-structural motives. Lastly, we provided evidence of the differential binding to RNA of the same protein in different HEK293 compartments. In the second part of this thesis, we focused on the determination and quantification of newly transcribed RNAs at the single-cell level. The kinetics of RNA transcription, processing and degradation were until recently not measurable at the single-cell level. Thus, we have developed a novel approach (called SLAM-Drop-seq ) by adapting the published SLAM-seq method to single cells. We used SLAM Drop-seq to estimate time-dependent RNA kinetics rates of transcription and turnover for hundreds of oscillating transcripts during the cell cycle of HEK293 cells. We found that genes regulate their expression with different strategies and have specific modes to fine-tune their kinetic rates along the cell cycle

Life’s order, complexity, organization, and its thermodynamic–holistic imperatives

In memoriam Jeffrey S. Wicken (1942–2002)—the evolutionarily minded biochemist, who in the 1970/80s strived for a synthesis of biological and physical theories to fathom the tentative origins of life. Several integrative concepts are worth remembering from Wicken’s legacy. (i) Connecting life’s origins and complex organization to a preexisting physical world demands a thermodynamically sound transition. (ii) Energetic ‘charging’ of the prebiosphere must precede the emergence of biological organization. (iii) Environmental energy gradients are exploited progressively, approaching maximum interactive structure and minimum dissipation. (iv) Dynamic self-assembly of prebiotic organic matter is driven by hydrophobic tension between water and amphiphilic building blocks, such as aggregating peptides from non-polar amino acids and base stacking in nucleic acids. (v) The dynamics of autocatalytic self-organization are facilitated by a multiplicity of weak interactions, such as hydrogen bonding, within and between macromolecular assemblies. (vi) The coevolution of (initially uncoded) proteins and nucleic acids in energy-coupled and metabolically active so-called ‘microspheres’ is more realistic as a kinetic transition model of primal biogenesis than ‘hypercycle replication’ theories for nucleic acid replicators on their own. All these considerations blend well with the current understanding that sunlight UV-induced photo-electronic excitation of colloidal metal sulfide particles appears most suitable as a prebiotic driver of organic synthesis reactions, in tight cooperation with organic, phase-separated, catalytic ‘microspheres’. On the ‘continuist vs. miraculist’ schism described by Iris Fry for origins-of-life considerations (Table 1), Wicken was a fervent early protagonist of holistic ‘continuist’ views and agenda

An inventory of yeast proteins associated with nucleolar and ribosomal components

BACKGROUND: Although baker's yeast is a primary model organism for research on eukaryotic ribosome assembly and nucleoli, the list of its proteins that are functionally associated with nucleoli or ribosomes is still incomplete. We trained a naïve Bayesian classifier to predict novel proteins that are associated with yeast nucleoli or ribosomes based on parts lists of nucleoli in model organisms and large-scale protein interaction data sets. Phylogenetic profiling and gene expression analysis were carried out to shed light on evolutionary and regulatory aspects of nucleoli and ribosome assembly. RESULTS: We predict that, in addition to 439 known proteins, a further 62 yeast proteins are associated with components of the nucleolus or the ribosome. The complete set comprises a large core of archaeal-type proteins, several bacterial-type proteins, but mostly eukaryote-specific inventions. Expression of nucleolar and ribosomal genes tends to be strongly co-regulated compared to other yeast genes. CONCLUSION: The number of proteins associated with nucleolar or ribosomal components in yeast is at least 14% higher than known before. The nucleolus probably evolved from an archaeal-type ribosome maturation machinery by recruitment of several bacterial-type and mostly eukaryote-specific factors. Not only expression of ribosomal protein genes, but also expression of genes encoding the 90S processosome, are strongly co-regulated and both regulatory programs are distinct from each other

Sea spray model study of an origin of life hypothesis, 1996

According to geological evidence, life arose in the sea approximately one billion years following earth formation. A new sea spray model for the origin of life is developed here to calculate the time of appearance of the first living cell, termed a protocell. The model proposes that life arose from sea spray that concentrated organic materials from a dilute primordial sea, and this spray became suspended in the atmosphere for several days and dehydrated to the extent of forming polymers which returned to the primordial sea. Each dehydrated sea spray droplet then became an experiment or trial of the mixture of biochemical activities (e.g., enzymatic activities) contained in each droplet. If the dehydrated spray contains a certain minimal set of essential biochemical activities, and returns to the ocean intact, it is considered a successful cell in this model termed the sea spray hypothesis. This work comprises theoretical model building and estimation of model parameters. The elaboration of this sea spray hypothesis has been done in probabilistic model terms. A range of values is assigned to all model parameters and median values are used to calculate the time of appearance of the first cell. A critical model parameter is an estimate of the number of biochemical activities possible with the organic material captured within a spray droplet. Parameter studies help to delimit the necessary conditions that may have generated life on earth under this sea spray hypothesis

Meta-análise de metilação de DNA em invertebrados: tendências evolutivas e indicações para avaliação ambiental

The anthropogenic impact in a variety of ecosystems has been increasing significantly in recent decades. This anthropogenic pressure translates into globally spreading phenomena like habitat fragmentation, mass extinction or biodiversity decline, that threaten ecosystem services. The mitigation of such detrimental ecological impacts is largely dependent on our ability to properly assess effects of the pressures on biological systems, i.e., with sufficient resolution levels that allows establishing reliable cause-effect relationships. High-resolution tools to assess these effects have recently been emerging, e.g., tools based in gene expression and the epigenome. The latter is strongly influenced by environmental conditions, with consequences in gene expression. Thus, the epigenome constitutes a new opportunity for the mechanistic understanding of toxic effects of environmental contaminants and for the development of biomarkers of exposure and effect. In this context, the specific objectives of the present dissertation were as follows: (i) to collect reliable genomic data of various invertebrate species representing different environmental compartments; (ii) quantify the distribution of the potential for gene body methylation based on the prevalence of CpG dinucleotides in the genome of each species; (iii) elaborate on the differential ability of invertebrate species to respond or potentially adapt to challenging and/or highly fluctuating environments based on phenotypic plasticity mediated by DNA methylation and/or related mutations; (iv) conclude on the most favourable models to address gene body methylation within an ecotoxicological context. Among the set of 27 species with reliable genomic data publicly available selected for meta-analysis, 11 recorded an observed-to-expected CpG ratio distribution (CpG O/E) that suggest the existence of gene groups with high susceptibility to methylation, and 16 have distributions suggesting that the entire genome is little or very little susceptible to methylation. Patterns of reduced susceptibility to DNA methylation have been identified among phylogenetically closely related species, which supports the theory that DNA methylation may have played a relevant role in species adaptation to new ecological niches, e.g., during the colonization of terrestrial or freshwater environments from primitive marine environments. The concentration of CG dinucleotides in the analysed genomes also allowed to discuss the suitability of different model organisms used in ecotoxicology for the development of biomarkers of exposure or effect based on DNA methylation. In this context, the following species are noteworthy as those presenting higher relative levels of CG dinucleotides in their genome: N. vectensis and T. californicus in the marine environment; D. magna and D. polymorpha in freshwater ecosystems; I. scapularis and H. saltator in the soil compartment; A. albimanus, A. albopictus and N. vitripennis representing aerial species.O impacto humano no ambiente e nos ecossistemas tem aumentado, especialmente nas décadas mais recentes. Esta pressão antropogénica é traduzida em fenómenos que são globais, como fragmentação de habitat, extinção em massa ou declínio da biodiversidade, que ameaçam os serviços dos ecossistemas. A mitigação destes impactos negativos em diferentes ecossistemas é largamente dependente da nossa capacidade para avaliar os efeitos das pressões nos sistemas biológicos com resolução suficiente, permitindo estabelecer relações fidedignas de causa-efeito. Ferramentas de elevada resolução para avaliar estes efeitos têm emergido recentemente, por exemplo, as baseadas na expressão génica e no epigenoma. Este é fortemente influenciado pelas condições ambientais, com consequências na expressão génica. Assim, o epigenoma constitui uma nova oportunidade para a compreensão mecanicista dos efeitos tóxicos dos contaminantes ambientais e para o desenvolvimento de biomarcadores de exposição e efeito. Neste contexto, os objetivos específicos da presente Dissertação foram: (i) recolher dados genómicos relativos a várias espécies de invertebrados representantes de diferentes compartimentos ambientais; (ii) quantificar a distribuição do potencial para metilação de regiões genéticas codificantes nos genomas destes organismos, com base na prevalência de dinucleótidos citosina-guanina (CpG); (iii) elaborar sobre a capacidade diferencial das espécies de invertebrados para responderem ou potencialmente se adaptarem a pressões ou flutuações ambientais, com base na plasticidade fenotípica mediada pela metilação do DNA e/ou mutações relacionadas; (iv) concluir sobre as espécies-modelo mais favoráveis para abordar a metilação de regiões genéticas codificantes num contexto ecotoxicológico. De entre o conjunto de 27 espécies selecionadas para meta-análise com dados genómicos suficientes disponíveis, 11 apresentaram distribuições do rácio entre frequência de CpG observada e esperada (CpG O/E) que sugerem a existência de grupos de genes muito suscetíveis a metilação, e 16 apresentaram distribuições que sugerem que todo o genoma é pouco ou muito pouco suscetível a metilação. Padrões de redução de suscetibilidade à metilação do ADN foram identificados entre espécies filogeneticamente próximas, que apoiam a teoria de que a metilação do DNA poderá ter tido um papel relevante na adaptação das espécies a novos nichos ecológicos, p.ex. durante a colonização de ambientes terrestres ou dulçaquícolas a partir de ambientes primitivos marinhos. A concentração de dinucleótidos CG nos genomas analisados permitiu ainda discutir sobre a adequação de diferentes organismos-modelo usados em ecotoxicologia para o desenvolvimento de biomarcadores baseados na metilação do ADN. Neste contexto, destacam-se as seguintes espécies, como modelos com uma maior quantidade relativa de dinucleótidos CG: N. vectensis e T. californicus no ambiente marinho; D. magna e D. polymorpha em ecossistemas dulçaquícolas; I. scapularis e H. saltator no compartimento solo; A. albimanus, A. albopictus e N. vitripennis para representar espécies aéreas. Complementarmente a esta evidência, a escolha de organismos-modelo neste contexto deve ser guiada pela informação existente sobre as suas respostas ecotoxicológicas ao nível fenotípico.Mestrado em Biologia Aplicad

Male Reproductive Anatomy

The male reproductive system, which is made up of the testes, scrotum, epididymis, vas deferens, seminal vesicles, prostate gland, bulbourethral gland, ejaculatory duct, urethra, and penis, functions mainly in the production, nourishment, and temporary storage of spermatozoa. Epigenetic modifications are essential to regulate normal gonadal development and spermatogenesis. The sperm epigenome is highly susceptible influence by a wide spectrum of environmental stimuli. This book focuses on the male reproductive system, discussing topics ranging from aspects of anatomy and risk factors for male infertility to clinical techniques and management of male reproductive health

Ovarian response to vascular and toxic insults in early life : focus on ovarian reserve and fertility preservation

The ovarian reserve is defined as the number of eggs embodied in primordial follicles. Every woman is born with a fixed number of primordial follicles, which are formed during fetal development. Pathological events can affect the ovarian reserve both preand postnatally and cause a premature ovarian insufficiency (POI), i.e. a precocious exhaustion of the endocrine and reproductive potential of the ovaries. In prenatal life, intrauterine growth restriction (IUGR) caused by poor maternal nutrition or insufficient placental flow to the fetus, has been associated with alterations in pubertal development and follicle number in animals and humans, with inconclusive evidence. In postnatal life, major iatrogenic causes of POI are chemo- and radiotherapeutic treatments administered to cancer patients. Fertility preservation strategies, such as ovarian tissue cryopreservation (OTC), are of fundamental importance to preserve fertility in these patients. These procedures are still experimental and in continuous evolution as more data are acquired. In this thesis, we aimed at investigate the impact of IUGR on ovarian follicles and gene expression in neonatal, juvenile and peripubertal rats, using a model of surgicallyinduced placental insufficiency. Secondly, we focused on the effects of chemotherapy exposure on ovarian follicles and stroma in ovarian tissues from cancer patients who underwent OTC for fertility preservation purposes. Additionally, we analyzed the efficacy of fertility preservation criteria in selecting the patients at high risk of infertility, by analysis of ovarian function during follow-up, the fruition rate and the timing of OTC in a large pediatric oncology unit in Finland. Timing of OTC was analyzed in relation to exposure to chemotherapy. A reduced primordial and total follicle count was found in the ovaries of neonate and juvenile rats. Follicle count normalized at peripubertal ages, alongside with the recovery of body weight. The expression of 24 genes was modified in the rat ovaries in response to the placental insufficiency. We interpreted the gene reprogramming as compensatory, possibly explaining the recovery of follicle number (by a downregulation of the physiological rates of follicle activation and/or depletion). The gene modifications could also have unknown long-term consequences on ovarian function, whose definition was beyond the aims of this work. An increased number of atretic follicles and a reduced size and number of residual intact follicles were evidenced in the ovaries of patients exposed to chemotherapy before OTC. Exposure to chemotherapy also reduced the secretion of steroid hormones in culture and damaged the ovarian stroma, by increasing collagen deposition and apoptosis. Finally, analysis of OTC data from Helsinki Children’s Hospital revealed that patients at high risk of infertility were correctly identified by the selection criteria, despite fruition rate was not optimal. We observed an increased risk of ovarian failure in older patients, which is in line with the age-related decline of the ovarian reserve. We also reported longer time frames between indication to OTC and execution of the procedure in patients with malignant diseases, which was associated to increased exposure to alkylating agents. Delays in the execution of OTC should be limited in order to avoid further damage to the ovarian tissue as a consequence of chemotherapy exposure, especially in older patients

The Origin and Early Evolution of Life

What is life? How, where, and when did life arise? These questions have remained most fascinating over the last hundred years. Systems chemistry is the way to go to better understand this problem and to try and answer the unsolved question regarding the origin of Life. Self-organization, thanks to the role of lipid boundaries, made possible the rise of protocells. The role of these boundaries is to separate and co-locate micro-environments, and make them spatially distinct; to protect and keep them at defined concentrations; and to enable a multitude of often competing and interfering biochemical reactions to occur simultaneously. The aim of this Special Issue is to summarize the latest discoveries in the field of the prebiotic chemistry of biomolecules, self-organization, protocells and the origin of life. In recent years, thousands of excellent reviews and articles have appeared in the literature and some breakthroughs have already been achieved. However, a great deal of work remains to be carried out. Beyond the borders of the traditional domains of scientific activity, the multidisciplinary character of the present Special Issue leaves space for anyone to creatively contribute to any aspect of these and related relevant topics. We hope that the presented works will be stimulating for a new generation of scientists that are taking their first steps in this fascinating field

Design principles of cell-free replicators

Der heilige Gral der synthetischen Biologie ist die Erschaffung einer minimalen Zelle, welche sowohl zu autonomer Selbstreplikation als auch zu natürlicher Evolution befähigt ist. Bereits heute ist es möglich das zentrale Dogma der Molekularbiologie, also die Implementierung des genetischen Codes mittels Transkription-Translation, in vitro zu rekonstruieren. Doch die Kopplung dieses Prozesses mit einem vollständigen DNA-Selbstreplikationssystem war bisher nur auf ein paar Kilobasen (kbp) beschränkt, weit entfernt von den vorgeschlagenen 113 kbp die für eine minimale Zelle nötig wären. In dieser Arbeit wird die Entwicklung einer Plattform für die transkriptions-translations-gekoppelte DNA-Replikation vorgestellt, genannt PURErep, welche in der Lage ist Genome mit der vorhergesagten Größe einer Minimalzelle zu replizieren. Als wichtiger Schritt in Richtung natürlicher Evolution kann sich der hier beschriebene Selbstreplikator pREP über mehrere Generationen fortpflanzen, sowohl in vitro als auch in vivo. PURErep ist modular aufgebaut und frei verfügbar, sodass es mit beliebigen Funktionen erweitert werden kann. Neben der DNA gibt es weitere Komponenten, die zum Selbsterhalt einer Zelle vermehrt werden müssen. Es konnte gezeigt werden, dass PURErep die simultane Co-Expression mehrerer seiner Proteinkomponenten ermöglicht. Diese Faktoren waren in der Lage sich aktiv an der Selbst-Regeneration des Systems beteiligen, was einen wichtigen Schritt in Richtung biochemischer Autonomie darstellt. Weiterhin wurden Möglichkeiten zur Selbstreplikation des komplexen Ribosoms erforscht, einem wesentlichen Bestandteil des Translationsapparates. Die de novo Synthese und Assemblierung solcher Ribosomen wird eine entscheidende Rolle für zukünftige Entwicklungen spielen. Ein weiteres Merkmal von Zellen stellt ihre Hülle dar, die Zellmembran. Eine von Grund auf neu geschaffene Minimalzelle müsste in der Lage sein, eine ähnliche Hülle selbst zu produzieren. Es wurde ein effizientes Konzept zur Selbst-Verkapselung des pREP Replikators entwickelt, welches vollkommen ohne zusätzlichen Energiebedarf auskommt. Es konnte gezeigt werden, dass diese sogenannten DNA-Nanoflowers Kernstrukturen bildeten und sich über Generation hinweg vermehren können. Insgesamt dienen die in dieser Arbeit dargelegten Entwürfe der Weiterentwicklung unabhängiger Selbstreplikatoren, welche vielleicht in der Lage sein werden eines Tages natürliche Zellen zu imitieren.The holy grail of bottom-up synthetic biology is the creation of a minimal cell capable of autonomous self-replication and open-ended Darwinian evolution. Reconstituting molecular biology’s central dogma, the implementation of genetic information via transcription-translation, is already feasible in vitro. Yet coupling this process to a DNA self-replication system has so far been limited to only a few kilobases (kbp), a far cry from the proposed 113 kbp proposed for a minimal cell. This work presents the development of a transcription-translation coupled DNA replication platform, called PURErep, which is capable of replicating DNA genomes approaching the proposed size of a minimal cell. As an important step towards Darwinian evolution, the herein described self-replicator pREP can propagate over several generations, both in vitro and in vivo. PURErep is modular and freely available, so that it can be extended with further functions as desired. In addition to DNA, there are other components that need to be replicated for the self-preservation of a cell. It could be shown that PURErep enables the simultaneous co-expression for several of its protein components. These factors were able to actively participate in the self-regeneration of the system, representing an important hallmark of biochemical autonomy. Furthermore, the self-reproduction of the complex ribosome was investigated, an essential component of the translational apparatus. The de novo synthesis and assembly of such ribosomes will be a crucial step towards future developments. Another feature of cells is their envelope, the cell membrane. A minimal cell created from scratch should be able to produce a similar compartment by itself. An efficient concept for the self-compartmentalization of the pREP replicator has been developed, which requires no additional energy and is entirely based on self-organization. It could be shown that these so-called DNA nanoflowers formed nuclear structures and could reproduce over generations. Overall, the designs laid out in this work serve to further develop independent self-replicators, which may one day be able to mimic a natural cell