The story of rRNA expansion segments: Finding functionality amidst diversity.

Expansion segments (ESs) are multinucleotide insertions present across phyla at specific conserved positions in eukaryotic rRNAs. ESs are generally absent in bacterial rRNAs with some exceptions, while the archaeal rRNAs have microexpansions at regions that coincide with those of eukaryotic ESs. Although there is an increasing prominence of ribosomes, especially the ribosomal proteins, in fine-tuning gene expression through translation regulation, the role of rRNA ESs is relatively underexplored. While rRNAs have been established as the major catalytic hub in ribosome function, the presence of ESs widens their scope as a species-specific regulatory hub of protein synthesis. In this comprehensive review, we have elaborately discussed the current understanding of the functional aspects of rRNA ESs of cytoplasmic eukaryotic ribosomes and discuss their past, present, and future. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems Translation > Ribosome Structure/Function Translation > Regulation.

Endosymbiotic selective pressure at the origin of eukaryotic cell biology.

The dichotomy that separates prokaryotic from eukaryotic cells runs deep. The transition from pro- to eukaryote evolution is poorly understood due to a lack of reliable intermediate forms and definitions regarding the nature of the first host that could no longer be considered a prokaryote, the first eukaryotic common ancestor, FECA. The last eukaryotic common ancestor, LECA, was a complex cell that united all traits characterising eukaryotic biology including a mitochondrion. The role of the endosymbiotic organelle in this radical transition towards complex life forms is, however, sometimes questioned. In particular the discovery of the asgard archaea has stimulated discussions regarding the pre-endosymbiotic complexity of FECA. Here we review differences and similarities among models that view eukaryotic traits as isolated coincidental events in asgard archaeal evolution or, on the contrary, as a result of and in response to endosymbiosis. Inspecting eukaryotic traits from the perspective of the endosymbiont uncovers that eukaryotic cell biology can be explained as having evolved as a solution to housing a semi-autonomous organelle and why the addition of another endosymbiont, the plastid, added no extra compartments. Mitochondria provided the selective pressures for the origin (and continued maintenance) of eukaryotic cell complexity. Moreover, they also provided the energetic benefit throughout eukaryogenesis for evolving thousands of gene families unique to eukaryotes. Hence, a synthesis of the current data lets us conclude that traits such as the Golgi apparatus, the nucleus, autophagosomes, and meiosis and sex evolved as a response to the selective pressures an endosymbiont imposes.

Ancestral State Reconstructions Trace Mitochondria But Not Phagocytosis to the Last Eukaryotic Common Ancestor.

Two main theories have been put forward to explain the origin of mitochondria in eukaryotes: phagotrophic engulfment (undigested food) and microbial symbiosis (physiological interactions). The two theories generate mutually exclusive predictions about the order in which mitochondria and phagocytosis arose. To discriminate the alternatives, we have employed ancestral state reconstructions (ASR) for phagocytosis as a trait, phagotrophy as a feeding habit, the presence of mitochondria, the presence of plastids, and the multinucleated organization across major eukaryotic lineages. To mitigate the bias introduced by assuming a particular eukaryotic phylogeny, we reconstructed the appearance of these traits across 1789 different rooted gene trees, each having species from opisthokonts, mycetozoa, hacrobia, excavate, archeplastida, and Stramenopiles, Alveolates and Rhizaria. The trees reflect conflicting relationships and different positions of the root. We employed a novel phylogenomic test that summarizes ASR across trees which reconstructs a last eukaryotic common ancestor that possessed mitochondria, was multinucleated, lacked plastids, and was non-phagotrophic as well as non-phagocytic. This indicates that both phagocytosis and phagotrophy arose subsequent to the origin of mitochondria, consistent with findings from comparative physiology. Furthermore, our ASRs uncovered multiple origins of phagocytosis and of phagotrophy across eukaryotes, indicating that, like wings in animals, these traits are useful but neither ancestral nor homologous across groups. The data indicate that mitochondria preceded the origin of phagocytosis, such that phagocytosis cannot have been the mechanism by which mitochondria were acquired.

Transcription Regulators and Membraneless Organelles Challenges to Investigate Them.

Eukaryotic cells are composed of different bio-macromolecules that are divided into compartments called organelles providing optimal microenvironments for many cellular processes. A specific type of organelles is membraneless organelles. They are formed via a process called liquid-liquid phase separation that is driven by weak multivalent interactions between particular bio-macromolecules. In this review, we gather crucial information regarding different classes of transcription regulators with the propensity to undergo liquid-liquid phase separation and stress the role of intrinsically disordered regions in this phenomenon. We also discuss recently developed experimental systems for studying formation and properties of membraneless organelles.

Motif-pattern dependence of biomolecular phase separation driven by specific interactions.

Eukaryotic cells partition a wide variety of important materials and processes into biomolecular condensates-phase-separated droplets that lack a membrane. In addition to nonspecific electrostatic or hydrophobic interactions, phase separation also depends on specific binding motifs that link together constituent molecules. Nevertheless, few rules have been established for how these ubiquitous specific, saturating, motif-motif interactions drive phase separation. By integrating Monte Carlo simulations of lattice-polymers with mean-field theory, we show that the sequence of heterotypic binding motifs strongly affects a polymer's ability to phase separate, influencing both phase boundaries and condensate properties (e.g. viscosity and polymer diffusion). We find that sequences with large blocks of single motifs typically form more inter-polymer bonds, which promotes phase separation. Notably, the sequence of binding motifs influences phase separation primarily by determining the conformational entropy of self-bonding by single polymers. This contrasts with systems where the molecular architecture primarily affects the energy of the dense phase, providing a new entropy-based mechanism for the biological control of phase separation.

The Emerging Role of Decellularized Plant-Based Scaffolds as a New Biomaterial.

The decellularization of plant-based biomaterials to generate tissue-engineered substitutes or in vitro cellular models has significantly increased in recent years. These vegetal tissues can be sourced from plant leaves and stems or fruits and vegetables, making them a low-cost, accessible, and sustainable resource from which to generate three-dimensional scaffolds. Each construct is distinct, representing a wide range of architectural and mechanical properties as well as innate vasculature networks. Based on the rapid rise in interest, this review aims to detail the current state of the art and presents the future challenges and perspectives of these unique biomaterials. First, we consider the different existing decellularization techniques, including chemical, detergent-free, enzymatic, and supercritical fluid approaches that are used to generate such scaffolds and examine how these protocols can be selected based on plant cellularity. We next examine strategies for cell seeding onto the plant-derived constructs and the importance of the different functionalization methods used to assist in cell adhesion and promote cell viability. Finally, we discuss how their structural features, such as inherent vasculature, porosity, morphology, and mechanical properties (i.e., stiffness, elasticity, etc.) position plant-based scaffolds as a unique biomaterial and drive their use for specific downstream applications. The main challenges in the field are presented throughout the discussion, and future directions are proposed to help improve the development and use of vegetal constructs in biomedical research.

Regulating Expression of Mistranslating tRNAs by Readthrough RNA Polymerase II Transcription.

Transfer RNA (tRNA) variants that alter the genetic code increase protein diversity and have many applications in synthetic biology. Since the tRNA variants can cause a loss of proteostasis, regulating their expression is necessary to achieve high levels of novel protein. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II)-transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4- or TetR-VP16-activated promoters downstream of a mistranslating tRNASer variant that misincorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using this inducible tRNA system, we explore the proteotoxic effects of mistranslation on yeast cells. High levels of mistranslation cause cells to arrest in the G1 phase. These cells are impermeable to propidium iodide, yet growth is not restored upon repressing tRNA expression. High levels of mistranslation increase cell size and alter cell morphology. This regulatable tRNA expression system can be applied to study how native tRNAs and tRNA variants affect the proteome and other biological processes. Variations of this inducible tRNA system should be applicable to other eukaryotic cell types.

Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs.

Viruses exploit the translation machinery of an infected cell to synthesize their proteins. Therefore, viral mRNAs have to compete for ribosomes and translation factors with cellular mRNAs. To succeed, eukaryotic viruses adopt multiple strategies. One is to circumvent the need for m7G-cap through alternative instruments for ribosome recruitment. These include internal ribosome entry sites (IRESs), which make translation independent of the free 5' end, or cap-independent translational enhancers (CITEs), which promote initiation at the uncapped 5' end, even if located in 3' untranslated regions (3' UTRs). Even if a virus uses the canonical cap-dependent ribosome recruitment, it can still perturb conventional ribosomal scanning and start codon selection. The pressure for genome compression often gives rise to internal and overlapping open reading frames. Their translation is initiated through specific mechanisms, such as leaky scanning, 43S sliding, shunting, or coupled termination-reinitiation. Deviations from the canonical initiation reduce the dependence of viral mRNAs on translation initiation factors, thereby providing resistance to antiviral mechanisms and cellular stress responses. Moreover, viruses can gain advantage in a competition for the translational machinery by inactivating individual translational factors and/or replacing them with viral counterparts. Certain viruses even create specialized intracellular "translation factories", which spatially isolate the sites of their protein synthesis from cellular antiviral systems, and increase availability of translational components. However, these virus-specific mechanisms may become the Achilles' heel of a viral life cycle. Thus, better understanding of the unconventional mechanisms of viral mRNA translation initiation provides valuable insight for developing new approaches to antiviral therapy.

A squalene-hopene cyclase in Schizosaccharomyces japonicus represents a eukaryotic adaptation to sterol-limited anaerobic environments.

Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report [C. J. E. A. Bulder, Antonie Van Leeuwenhoek, 37, 353-358 (1971)] that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop-17(21)-ene, hop-21(22)-ene, and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and stimulated anaerobic growth in sterol-free media, thus indicating that one or more of the hopanoids produced by SjShc1 could at least partially replace sterols. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast anaerobic growth of Sch. japonicus in sterol-free media is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.

Liquid-Liquid Phase Separation in Biology: Specific Stoichiometric Molecular Interactions vs Promiscuous Interactions Mediated by Disordered Sequences.

Extensive studies in the past few years have shown that nonmembrane bound organelles are likely assembled via liquid-liquid phase separation (LLPS), a process that is driven by multivalent protein-protein and/or protein-nucleic acid interactions. Both stoichiometric molecular interactions and intrinsically disordered region (IDR)-driven interactions can promote the assembly of membraneless organelles, and the field is currently dominated by IDR-driven biological condensate formation. Here we discuss recent studies that demonstrate the importance of specific biomolecular interactions for functions of diverse physiological condensates. We suggest that phase separation based on combinations of specific interactions and promiscuous IDR-driven interactions is likely a general feature of biological condensation under physiological conditions.

Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.

The heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation sites make mapping the location and timing of replication initiation in human cells difficult. To address this challenge, we have developed optical replication mapping (ORM), a high-throughput single-molecule approach, and used it to map early-initiation events in human cells. The single-molecule nature of our data and a total of >2,500-fold coverage of the human genome on 27 million fibers averaging ∼300 kb in length allow us to identify initiation sites and their firing probability with high confidence. We find that the distribution of human replication initiation is consistent with inefficient, stochastic activation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

Telomeric DNA sequences in beetle taxa vary with species richness.

Telomeres are protective structures at the ends of eukaryotic chromosomes, and disruption of their nucleoprotein composition usually results in genome instability and cell death. Telomeric DNA sequences have generally been found to be exceptionally conserved in evolution, and the most common pattern of telomeric sequences across eukaryotes is (TxAyGz)n maintained by telomerase. However, telomerase-added DNA repeats in some insect taxa frequently vary, show unusual features, and can even be absent. It has been speculated about factors that might allow frequent changes in telomere composition in Insecta. Coleoptera (beetles) is the largest of all insect orders and based on previously available data, it seemed that the telomeric sequence of beetles varies to a great extent. We performed an extensive mapping of the (TTAGG)n sequence, the ancestral telomeric sequence in Insects, across the main branches of Coleoptera. Our study indicates that the (TTAGG)n sequence has been repeatedly or completely lost in more than half of the tested beetle superfamilies. Although the exact telomeric motif in most of the (TTAGG)n-negative beetles is unknown, we found that the (TTAGG)n sequence has been replaced by two alternative telomeric motifs, the (TCAGG)n and (TTAGGG)n, in at least three superfamilies of Coleoptera. The diversity of the telomeric motifs was positively related to the species richness of taxa, regardless of the age of the taxa. The presence/absence of the (TTAGG)n sequence highly varied within the Curculionoidea, Chrysomeloidea, and Staphylinoidea, which are the three most diverse superfamilies within Metazoa. Our data supports the hypothesis that telomere dysfunctions can initiate rapid genomic changes that lead to reproductive isolation and speciation.

Characterization and Comparative Analyses of Mitochondrial Genomes in Single-Celled Eukaryotes to Shed Light on the Diversity and Evolution of Linear Molecular Architecture.

Determination and comparisons of complete mitochondrial genomes (mitogenomes) are important to understand the origin and evolution of mitochondria. Mitogenomes of unicellular protists are particularly informative in this regard because they are gene-rich and display high structural diversity. Ciliates are a highly diverse assemblage of protists and their mitogenomes (linear structure with high A+T content in general) were amongst the first from protists to be characterized and have provided important insights into mitogenome evolution. Here, we report novel mitogenome sequences from three representatives (Strombidium sp., Strombidium cf. sulcatum, and Halteria grandinella) in two dominant ciliate lineages. Comparative and phylogenetic analyses of newly sequenced and previously published ciliate mitogenomes were performed and revealed a number of important insights. We found that the mitogenomes of these three species are linear molecules capped with telomeric repeats that differ greatly among known species. The genomes studied here are highly syntenic, but larger in size and more gene-rich than those of other groups. They also all share an AT-rich tandem repeat region which may serve as the replication origin and modulate initiation of bidirectional transcription. More generally we identified a split version of ccmf, a cytochrome c maturation-related gene that might be a derived character uniting taxa in the subclasses Hypotrichia and Euplotia. Finally, our mitogenome comparisons and phylogenetic analyses support to reclassify Halteria grandinella from the subclass Oligotrichia to the subclass Hypotrichia. These results add to the growing literature on the unique features of ciliate mitogenomes, shedding light on the diversity and evolution of their linear molecular architecture.

Evolution of eukaryotes as a story of survival and growth of mitochondrial DNA over two billion years.

Mitochondria's significance in human diseases and in functioning, health and death of eukaryotic cell has been acknowledged widely. Yet our perspective in cell biology and evolution remains nucleocentric. Mitochondrial DNA, by virtue of its omnipresence and species-level conservation, is used as a barcode in animal taxonomy. This article analyses various levels of containment structures that enclose mitochondrial DNA and advocates a fresh perspective wherein evolution of organic structures of the eukarya domain seem to support and facilitate survival and proliferation of mitochondrial DNA by splitting containers as they age and by directing them along two distinct pathways: destruction of containers with more mutant mitochondrial DNA and rejuvenation of containers with less mutant mitochondrial DNA.

Remodelling of membrane tubules by the actin cytoskeleton.

Inside living cells, the remodelling of membrane tubules by actomyosin networks is crucial for processes such as intracellular trafficking or organelle reshaping. In this review, we first present various in vivo situations in which actin affects membrane tubule remodelling, then we recall some results on force production by actin dynamics and on membrane tubules physics. Finally, we show that our knowledge of the underlying mechanisms by which actomyosin dynamics affect tubule morphology has recently been moved forward. This is thanks to in vitro experiments that mimic cellular membranes and actin dynamics and allow deciphering the physics of tubule remodelling in biochemically controlled conditions, and shed new light on tubule shape regulation.

Dynamic interplay between cell membrane tension and clathrin-mediated endocytosis.

Deformability of the plasma membrane, the outermost surface of metazoan cells, allows cells to be dynamic, mobile and flexible. Factors that affect this deformability, such as tension on the membrane, can regulate a myriad of cellular functions, including membrane resealing, cell motility, polarisation, shape maintenance, membrane area control and endocytic vesicle trafficking. This review focuses on mechanoregulation of clathrin-mediated endocytosis (CME). We first delineate the origins of cell membrane tension and the factors that yield to its spatial and temporal fluctuations within cells. We then review the recent literature demonstrating that tension on the membrane is a fast-acting and reversible regulator of CME. Finally, we discuss tension-based regulation of endocytic clathrin coat formation during physiological processes.

Reframing cognition: getting down to biological basics.

The premise of this two-part theme issue is simple: the cognitive sciences should join the rest of the life sciences in how they approach the quarry within their research domain. Specifically, understanding how organisms on the lower branches of the phylogenetic tree become familiar with, value and exploit elements of an ecological niche while avoiding harm can be expected to aid understanding of how organisms that evolved later (including Homo sapiens) do the same or similar things. We call this approach basal cognition. In this introductory essay, we explain what the approach involves. Because no definition of cognition exists that reflects its biological basis, we advance a working definition that can be operationalized; introduce a behaviour-generating toolkit of capacities that comprise the function (e.g. sensing/perception, memory, valence, learning, decision making, communication), each element of which can be studied relatively independently; and identify a (necessarily incomplete) suite of common biophysical mechanisms found throughout the domains of life involved in implementing the toolkit. The articles in this collection illuminate different aspects of basal cognition across different forms of biological organization, from prokaryotes and single-celled eukaryotes-the focus of Part 1-to plants and finally to animals, without and with nervous systems, the focus of Part 2. By showcasing work in diverse, currently disconnected fields, we hope to sketch the outline of a new multidisciplinary approach for comprehending cognition, arguably the most fascinating and hard-to-fathom evolved function on this planet. Doing so has the potential to shed light on problems in a wide variety of research domains, including microbiology, immunology, zoology, biophysics, botany, developmental biology, neurobiology/science, regenerative medicine, computational biology, artificial life and synthetic bioengineering. This article is part of the theme issue 'Basal cognition: conceptual tools and the view from the single cell'.

Spontaneous electrical low-frequency oscillations: a possible role in Hydra and all living systems.

As one of the first model systems in biology, the basal metazoan Hydra has been revealing fundamental features of living systems since it was first discovered by Antonie van Leeuwenhoek in the early eighteenth century. While it has become well-established within cell and developmental biology, this tiny freshwater polyp is only now being re-introduced to modern neuroscience where it has already produced a curious finding: the presence of low-frequency spontaneous neural oscillations at the same frequency as those found in the default mode network in the human brain. Surprisingly, increasing evidence suggests such spontaneous electrical low-frequency oscillations (SELFOs) are found across the wide diversity of life on Earth, from bacteria to humans. This paper reviews the evidence for SELFOs in diverse phyla, beginning with the importance of their discovery in Hydra, and hypothesizes a potential role as electrical organism organizers, which supports a growing literature on the role of bioelectricity as a 'template' for developmental memory in organism regeneration. This article is part of the theme issue 'Basal cognition: conceptual tools and the view from the single cell'.

Valuing what happens: a biogenic approach to valence and (potentially) affect.

Valence is half of the pair of properties that constitute core affect, the foundation of emotion. But what is valence, and where is it found in the natural world? Currently, this question cannot be answered. The idea that emotion is the body's way of driving the organism to secure its survival, thriving and reproduction runs like a leitmotif from the pathfinding work of Antonio Damasio through four book-length neuroscientific accounts of emotion recently published by the field's leading practitioners. Yet while Damasio concluded 20 years ago that the homeostasis-affect linkage is rooted in unicellular life, no agreement exists about whether even non-human animals with brains experience emotions. Simple neural animals-those less brainy than bees, fruit flies and other charismatic invertebrates-are not even on the radar of contemporary affective research, to say nothing of aneural organisms. This near-sightedness has effectively denied the most productive method available for getting a grip on highly complex biological processes to a scientific domain whose importance for understanding biological decision-making cannot be underestimated. Valence arguably is the fulcrum around which the dance of life revolves. Without the ability to discriminate advantage from harm, life very quickly comes to an end. In this paper, we review the concept of valence, where it came from, the work it does in current leading theories of emotion, and some of the odd features revealed via experiment. We present a biologically grounded framework for investigating valence in any organism and sketch a preliminary pathway to a computational model. This article is part of the theme issue 'Basal cognition: conceptual tools and the view from the single cell'.

Grounding cognition: heterarchical control mechanisms in biology.

We advance an account that grounds cognition, specifically decision-making, in an activity all organisms as autonomous systems must perform to keep themselves viable-controlling their production mechanisms. Production mechanisms, as we characterize them, perform activities such as procuring resources from their environment, putting these resources to use to construct and repair the organism's body and moving through the environment. Given the variable nature of the environment and the continual degradation of the organism, these production mechanisms must be regulated by control mechanisms that select when a production is required and how it should be carried out. To operate on production mechanisms, control mechanisms need to procure information through measurement processes and evaluate possible actions. They are making decisions. In all organisms, these decisions are made by multiple different control mechanisms that are organized not hierarchically but heterarchically. In many cases, they employ internal models of features of the environment with which the organism must deal. Cognition, in the form of decision-making, is thus fundamental to living systems which must control their production mechanisms. This article is part of the theme issue 'Basal cognition: conceptual tools and the view from the single cell'.