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TENT4 enzymes generate 'mixed tails' of diverse nucleotides at 3' ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4-ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for mixed tailing and protection of viral poly(A) tails. We find that the HBV post-transcriptional regulatory element (PRE), specifically the CNGGN-type pentaloop, is critical for TENT4-dependent regulation. HCMV uses a similar pentaloop, an interesting example of convergent evolution. This pentaloop is recognized by the sterile alpha motif domain-containing ZCCHC14 protein, which in turn recruits TENT4. Overall, our study reveals the mechanism of action of PRE, which has been widely used to enhance gene expression, and identifies the TENT4-ZCCHC14 complex as a potential target for antiviral therapeutics.

HBV infection initiates hepatitis B and promotes liver cirrhosis and hepatocellular carcinoma. IFN-α is commonly used in hepatitis B therapy, but how it inhibits HBV is not fully understood. We screened 285 human interferon-stimulated genes (ISGs) for anti-HBV activity using a cell-based assay, which revealed several anti-HBV ISGs. Among these ISGs, SAMD4A was the strongest suppressor of HBV replication. We found the binding site of SAMD4A in HBV RNA, which was a previously unidentified Smaug recognition region (SRE) sequence conserved in HBV variants. SAMD4A binds to the SRE site in viral RNA to trigger its degradation. The SAM domain in SAMD4A is critical for RNA binding and the C-terminal domain of SAMD4A is required for SAMD4A anti-HBV function. Human SAMD4B is a homolog of human SAMD4A but is not an ISG, and the murine genome encodes SAMD4. All these SAMD4 proteins suppressed HBV replication when overexpressed in vitro and in vivo. We also showed that knocking out the Samd4 gene in hepatocytes led to a higher level of HBV replication in mice and AAV-delivered SAMD4A expression reduced the virus titer in HBV-producing transgenic mice. In addition, a database analysis revealed a negative correlation between the levels of SAMD4A/B and HBV in patients. Our data suggest that SAMD4A is an important anti-HBV ISG for use in IFN therapy of hepatitis B and that the levels of SAMD4A/B expression are related to HBV sensitivity in humans.

Copyright © 2022 Santos, Rodrigues, Caneiras and Bárbara. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule fluorescence in situ hybridization (FISH) assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 (also known as SAMD4A and SAMD4B, respectively) affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA-binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial complex I inhibition upon exposure to rotenone, but not strong mitochondrial uncoupling upon exposure to CCCP, rapidly induced the dissolution of Smaug1 MLOs. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMP-activated protein kinase (AMPK). Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK-mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins. This article has an associated First Person interview with the first authors of the paper., This work was supported by Fundação para a Ciência e Tecnologia (FCT) and Nippon Gases Portugal under the Ph.D. studentship in Industry grant PDE/BDE/127785/2016 awarded to CS.

Knowledge about muon tomography has spread in recent years in the geoscientific community and several collaborations between geologists and physicists have been founded. As the data analysis is still mostly done by particle physicists, we address the need of the geoscientific community to participate in the data analysis, while not having to worry too much about the particle physics equations in the background. The result hereof is SMAUG, a toolbox consisting of several modules that cover the various aspects of data analysis in a muon tomographic experiment. In this study we show how a comprehensive geophysical model can be built from basic physics equations. The emerging uncertainties are dealt with by a probabilistic formulation of the inverse problem, which is finally solved by a Monte Carlo Markov Chain algorithm. Finally, we benchmark the SMAUG results against those of a recent study, which however, have been established with an approach that is not easily accessible to the geoscientific community. We show that they reach identical results with the same level of accuracy and precision.

During the maternal-to-zygotic transition (MZT), which encompasses the earliest stages of animal embryogenesis, a subset of maternally supplied gene products is cleared, thus permitting activation of zygotic gene expression. In the Drosophila melanogaster embryo, the RNA-binding protein Smaug (SMG) plays an essential role in progression through the MZT by translationally repressing and destabilizing a large number of maternal mRNAs. The SMG protein itself is rapidly cleared at the end of the MZT by a Skp/Cullin/F-box (SCF) E3-ligase complex. Clearance of SMG requires zygotic transcription and is required for an orderly MZT. Here, we show that an F-box protein, which we name Bard (encoded by CG14317), is required for degradation of SMG. Bard is expressed zygotically and physically interacts with SMG at the end of the MZT, coincident with binding of the maternal SCF proteins, SkpA and Cullin1, and with degradation of SMG. shRNA-mediated knock-down of Bard or deletion of the bard gene in the early embryo results in stabilization of SMG protein, a phenotype that is rescued by transgenes expressing Bard. Bard thus times the clearance of SMG at the end of the MZT., Degradation of the Smaug RNA-binding protein is essential for an orderly maternal-to-zygotic transition (MZT) and requires zygotic transcription. Cao et al. show that the F-box protein Bard is expressed in a narrow time window at the end of the MZT. Upon Bard expression, an SCF E3 ubiquitin ligase complex binds to Smaug, and Smaug is degraded. Loss of Bard results in failure to degrade Smaug, meaning Bard serves as a zygotic timer for Smaug clearance and an orderly MZT.

Smaug is a conserved translational repressor that recognizes specific RNA motifs in a large number of mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs have been shown to form membraneless organelles (MLOs) in several organisms and cell types. Using single-molecule FISH we show here that SDHB and UQCRC1 mRNAs associate with Smaug1 MLOs in the human cell line U2OS. Simultaneous loss of function of Smaug1 and Smaug2 affects both mitochondrial respiration and mitochondrial network morphology. Deletion of specific Smaug1 protein regions resulted in impaired MLO formation that correlates with mitochondrial defects. In addition, rotenone but not the respiratory chain uncoupling agent CCCP rapidly induces Smaug1 MLO dissolution. Finally, metformin elicits a similar effect on Smaug1 MLOs and provokes the release of bounded mRNAs. We propose that mitochondrial activity affects Smaug1 MLO dynamics, thus allowing for regulation of nuclear mRNAs that encode key mitochondrial proteins. Fil: Fernández Alvarez, Ana Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Thomas, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Pascual, Malena Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Habif, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Pimentel, Jerónimo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Corbat, Agustín Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Pessoa, Joao. Universidad de Lisboa; Portugal Fil: la Spina, Pablo Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Boscaglia, Lara. Fundación Instituto Leloir; Argentina Fil: Plessis, Anne. Sorbonne University; Francia. Université Paris Diderot - Paris 7; Francia Fil: Carmo Fonseca, Maria. Universidad de Lisboa; Portugal Fil: Grecco, Hernan Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Casado Pinna, Marta. Instituto de Biomedicina de Valencia; España Fil: Boccaccio, Graciela Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.

Semi-analytic models (SAMs) are a promising means of tracking the physical processes associated with galaxy formation, but many of their approximations have not been rigorously tested. As part of the SMAUG (Simulating Multiscale Astrophysics to Understand Galaxies) project, we compare predictions from the FIRE-2 hydrodynamical "zoom-in" simulations to those from the Santa Cruz SAM run on the same halo merger trees, with an emphasis on the global mass flow cycle. Our study includes 13 halos spanning low-mass dwarfs (M_vir~10^10 M_sun at z=0), intermediate-mass dwarfs (M_vir~10^11 M_sun) and Milky Way-mass galaxies (M_vir~10^12 M_sun). The SAM and FIRE-2 predictions agree relatively well with each other in terms of stellar and interstellar mass, but differ dramatically on circumgalactic mass (the SAM is lower than FIRE-2 by ~3 orders of magnitude for dwarfs). Strikingly, the SAM predicts higher gas accretion rates for dwarfs compared to FIRE-2 by factors of ~10-100, and this is compensated for with higher mass outflow rates in the SAM. We argue that the most severe model discrepancies are caused by the lack of preventative stellar feedback and the assumptions for halo gas cooling and recycling in the SAM. As a first step towards resolving these model tensions, we present a simple yet promising new preventative stellar feedback model in which the energy carried by supernova-driven winds is allowed to heat some fraction of gas outside of halos to at least the virial temperature such that accretion is suppressed., Comment: Accepted to ApJ with minor revisions. More information on the SMAUG project and the other first result papers can be found here: https://www.simonsfoundation.org/flatiron/center-for-computational-astrophysics/galaxy-formation/smaug/papersplash1

SUMMARY In animal embryos, the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. We show that the maternal proteome represents more than half of the protein-coding capacity of Drosophila melanogaster’s genome, and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). Although the ubiquitin-proteasome system is necessary for clearance of these repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL, and ME31B for degradation early in the MZT and the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG abrogates F-box protein interaction and confers immunity to degradation. Persistent SMG downregulates zygotic re-expression of mRNAs whose maternal contribution is degraded by SMG. Thus, clearance of SMG permits an orderly MZT., Graphical Abstract, In Brief Cao et al. show that 2% of the proteome is degraded in early Drosophila embryos, including a repressive ribonucleoprotein complex. Two E3 ubiquitin ligases separately act on distinct components of this complex to phase their clearance. Failure to degrade a key component, the Smaug RNA-binding protein, disrupts an orderly maternal-to-zygotic transition.

To substantiate the claim of a relationship between generation gland morphology and degree of body armour in cordylid lizards, we studied the nine species in the genus Smaug. We predicted that well armoured species in this clade will have multi-layer generation glands, and lightly armoured species two-layer glands. Gland type was determined using standard histological techniques after sectioning a glandular patch of one adult male per species. A total of 133 specimens were examined for data on tail and occipital spine lengths (which were used as indicators of armour). We found that species with multi-layer generation glands (S. giganteus, S. breyeri, and S. vandami) have relatively long tail and occipital spines, while species with two-layer glands (S. mossambicus, S. regius, S. barbertonensis, S. warreni, and an undescribed species) have relatively short spines. Smaug depressus possesses both multi-layer and two-layer glands, and this variation was linked to regional variation in spine length. An ancestral state reconstruction for the Cordylidae showed that the two-layer state always results from the reduction of layers from a multi-layer precursor, and that reduction always culminates in two-layer glands and not in one-layer glands. This finding suggests that the one-layer state in the Ninurta-Chamaesaura-Pseudocordylus clade is most probably plesiomorphic, and therefore the ancestral state at the Cordylidae and Cordylinae nodes. Given the observed relationship between type of generation gland and body armour, this finding would suggest that the most recent common ancestor of the Cordylidae was lightly armoured.

Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of theDrosophila nanosmRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in thenanos3’ UTR. In a comprehensive mass-spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression ofnanoswas confirmedin vivo. Smaug, Cup and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that ‘coating’ of the RNA by a Me31B•Tral complex may be at the core of repression.

The Rufford Foundation (grant number 10843-1), SANBI’s Threatened Species Programme, National Research Foundation, Endangered Wildlife Trust, University of the Witwatersrand, The Alexander Herp Lab and Tshwane University of Technology provided funding, equipment and vehicles to S.P. for this project.

MicroRNAs (miRNAs), as well as the RNA-binding protein Smaug, recruit the CCR4-NOT deadenylase complex for shortening of the poly(A) tail. It has been believed that ATP is required for deadenylation induced by miRNAs or Smaug, based on the fact that the deadenylation reaction is blocked by ATP depletion. However, when isolated, neither of the two deadenylases in the CCR4-NOT complex requires ATP by itself. Thus, it remains unknown why ATP is required for deadenylation by ribonucleoprotein complexes like miRNAs and Smaug. Herein we found that, in the absence of the ATP-regenerating system, ATP is rapidly consumed into AMP, a strong deadenylase inhibitor, in Drosophila cell lysate. Importantly, hydrolysis of AMP was sufficient to reactivate deadenylation by miRNAs or Smaug, suggesting that AMP accumulation, rather than ATP depletion, caused the inhibition of the deadenylation reaction. Our results indicate that ATP is dispensable for deadenylation induced by miRNAs or Smaug and emphasize caution in the use of ATP depletion methods.

Chakravarty et al. (2019) and Itakura et al. (2019) report that the yeast RNA-binding protein Vts1 can convert into the [SMAUG+] prion state and delay meiosis commitment in response to starvation. It enables budding yeast to optimize their sporulation efficiency depending on how quickly nutrient availability fluctuates in their environment.

The description of Liolaemus choique was based on an exhaustive morphological diagnosis. However, a posterior phylogeographic study suggested that it would be a junior synonym of L. smaug. Herein, we present morphological behavioral and distributional data indicating that L. choique would be considered as a valid species. Therefore, we suggest to revalidate L. choique, as a distinct taxon from L. smaug., Asociación Herpetológica Argentina

Single-molecule fluorescence microscopy probes nanoscale, subcellular biology in real time. Existing methods for analyzing single-particle tracking data provide dynamical information, but can suffer from supervisory biases and high uncertainties. Here, we introduce a new approach to analyzing single-molecule trajectories: the Single-Molecule Analysis by Unsupervised Gibbs sampling (SMAUG) algorithm, which uses nonparametric Bayesian statistics to uncover the whole range of information contained within a single-particle trajectory (SPT) dataset. Even in complex systems where multiple biological states lead to a number of observed mobility states, SMAUG provides the number of mobility states, the average diffusion coefficient of single molecules in that state, the fraction of single molecules in that state, the localization noise, and the probability of transitioning between two different states. In this paper, we provide the theoretical background for the SMAUG analysis and then we validate the method using realistic simulations of SPT datasets as well as experiments on a controlled in vitro system. Finally, we demonstrate SMAUG on real experimental systems in both prokaryotes and eukaryotes to measure the motions of the regulatory protein TcpP in Vibrio cholerae and the dynamics of the B-cell receptor antigen response pathway in lymphocytes. Overall, SMAUG provides a mathematically rigorous approach to measuring the real-time dynamics of molecular interactions in living cells.Statement of SignificanceSuper-resolution microscopy allows researchers access to the motions of individual molecules inside living cells. However, due to experimental constraints and unknown interactions between molecules, rigorous conclusions cannot always be made from the resulting datasets when model fitting is used. SMAUG (Single-Molecule Analysis by Unsupervised Gibbs sampling) is an algorithm that uses Bayesian statistical methods to uncover the underlying behavior masked by noisy datasets. This paper outlines the theory behind the SMAUG approach, discusses its implementation, and then uses simulated data and simple experimental systems to show the efficacy of the SMAUG algorithm. Finally, this paper applies the SMAUG method to two model living cellular systems—one bacterial and one mammalian—and reports the dynamics of important membrane proteins to demonstrate the usefulness of SMAUG to a variety of systems.

Theory and experiment suggest that organisms can pre-adapt to future stressors based on reproducible environmental fluctuations experienced by their ancestors. Yet the mechanisms driving such pre-adaptation remain enigmatic. We report that the [SMAUG+] prion allows yeast to anticipate nutrient repletion after periods of starvation, providing a strong selective advantage. By transforming the landscape of post-transcriptional gene expression, [SMAUG+] regulates the decision between two broad growth and survival strategies: mitotic proliferation or meiotic differentiation into a stress-resistant state. [SMAUG+] is common in laboratory yeast strains, where standard propagation practice produces regular cycles of nutrient scarcity followed by repletion. Distinct [SMAUG+] variants are also widespread in wild yeast isolates from multiple niches, establishing that prion polymorphs can be utilized in natural populations. Our data provide a striking example of how protein-based epigenetic switches can be hidden in plain sight and can establish a transgenerational memory that integrates adaptive prediction into developmental decisions.

Metazoan embryos undergo a maternal-to-zygotic transition (MZT) during which maternal gene products are eliminated and the zygotic genome becomes transcriptionally active. During this process, RNA-binding proteins (RBPs) and the microRNA-induced silencing complex (miRISC) target maternal mRNAs for degradation. In Drosophila, the Smaug (SMG), Brain tumor (BRAT), and Pumilio (PUM) RBPs bind to and direct the degradation of largely distinct subsets of maternal mRNAs. SMG has also been shown to be required for zygotic synthesis of mRNAs and several members of the miR-309 family of microRNAs (miRNAs) during the MZT. Here, we have carried out global analysis of small RNAs both in wild-type and in smg mutants. Our results show that 85% of all miRNA species encoded by the genome are present during the MZT. Whereas loss of SMG has no detectable effect on Piwi-interacting RNAs (piRNAs) or small interfering RNAs (siRNAs), zygotic production of more than 70 species of miRNAs fails or is delayed in smg mutants. SMG is also required for the synthesis and stability of a key miRISC component, Argonaute 1 (AGO1), but plays no role in accumulation of the Argonaute family proteins associated with piRNAs or siRNAs. In smg mutants, maternal mRNAs that are predicted targets of the SMG-dependent zygotic miRNAs fail to be cleared. BRAT and PUM share target mRNAs with these miRNAs but not with SMG itself. We hypothesize that SMG controls the MZT, not only through direct targeting of a subset of maternal mRNAs for degradation but, indirectly, through production and function of miRNAs and miRISC, which act together with BRAT and/or PUM to control clearance of a distinct subset of maternal mRNAs.

Epigenetic modifications allow cells to quickly alter their gene expression and adapt to different stresses. In addition to direct chromatin modifications, prion-like proteins have recently emerged as a system that can sense and adapt the cellular response to stressful conditions. Interestingly, such responses are maintained through prions' self-templating conformations and transmitted to the progeny of the cell that established a prion trait. Alternatively, mnemons are prion-like proteins which conformational switch encodes memories of past events and yet does not propagate to daughter cells. In this review, we explore the biology of the recently described prions found in Saccharomyces cerevisiae including [ESI+], [SMAUG+], [GAR+], [MOT3+], [MOD+], [LSB+] as well as the Whi3 mnemon. The reversibility of the phenotypes they encode allows cells to remove traits which are no longer adaptive under stress relief and chaperones play a fundamental role in all steps of prion-like proteins functions. Thus, the interplay between chaperones and prion-like proteins provides a framework to establish responses to challenging environments.

A diagnostic tool known as SMAUG allows researchers to gather more precise data on the atmospheric conditions of Hot Jupiter-type exoplanets.

A recent multilocus molecular phylogeny of the large dragon lizards of the genus Smaug Stanley et al. (2011) recovered a south-eastern clade of two relatively lightly-armoured, geographically-proximate species (Smaug warreni (Boulenger, 1908) and S. barbertonensis (Van Dam, 1921)). Unexpectedly, S. barbertonensis was found to be paraphyletic, with individuals sampled from northern Eswatini (formerly Swaziland) being more closely related to S. warreni than to S. barbertonensis from the type locality of Barberton in Mpumalanga Province, South Africa. Examination of voucher specimens used for the molecular analysis, as well as most other available museum material of the three lineages, indicated that the ‘Eswatini’ lineage—including populations in a small area on the northern Eswatini–Mpumalanga border, and northern KwaZulu–Natal Province in South Africa—was readily distinguishable from S. barbertonensis sensu stricto (and S. warreni) by its unique dorsal, lateral and ventral colour patterns. In order to further assess the taxonomic status of the three populations, a detailed morphological analysis was conducted. Multivariate analyses of scale counts and body dimensions indicated that the ‘Eswatini’ lineage and S. warreni were most similar. In particular, S. barbertonensis differed from the other two lineages by its generally lower numbers of transverse rows of dorsal scales, and a relatively wider head. High resolution Computed Tomography also revealed differences in cranial osteology between specimens from the three lineages. The ‘Eswatini’ lineage is described here as a new species, Smaug swazicus sp. nov., representing the ninth known species of dragon lizard. The new species appears to be near-endemic to Eswatini, with about 90% of its range located there. Our study indicates that S. barbertonensis sensu stricto is therefore a South African endemic restricted to an altitudinal band of about 300 m in the Barberton–Nelspruit–Khandizwe area of eastern Mpumalanga Province, while S. warreni is endemic to the narrow Lebombo Mountain range of South Africa, Eswatini and Mozambique. We present a detailed distribution map for the three species, and a revised diagnostic key to the genus Smaug.

Summary Spatiotemporal gene regulation is often driven by RNA-binding proteins that harbor long intrinsically disordered regions in addition to folded RNA-binding domains. We report that the disordered region of the evolutionarily ancient developmental regulator Vts1/Smaug drives self-assembly into gel-like condensates. These proteinaceous particles are not composed of amyloid, yet they are infectious, allowing them to act as a protein-based epigenetic element: a prion [SMAUG+]. In contrast to many amyloid prions, condensation of Vts1 enhances its function in mRNA decay, and its self-assembly properties are conserved over large evolutionary distances. Yeast cells harboring [SMAUG+] downregulate a coherent network of mRNAs and exhibit improved growth under nutrient limitation. Vts1 condensates formed from purified protein can transform naive cells to acquire [SMAUG+]. Our data establish that non-amyloid self-assembly of RNA-binding proteins can drive a form of epigenetics beyond the chromosome, instilling adaptive gene expression programs that are heritable over long biological timescales.

Theory and experiments suggest that organisms would benefit from pre-adaptation to future stressors based on reproducible environmental fluctuations experienced by their ancestors, but the mechanisms driving pre-adaptation remain enigmatic. We report that the [SMAUG+] prion allows yeast to anticipate nutrient repletion after periods of starvation, providing a strong selective advantage. By transforming the landscape of post-transcriptional gene expression, [SMAUG+] regulates the decision between two broad growth and survival strategies: mitotic proliferation or meiotic differentiation into a stress-resistant state. [SMAUG+] is common in laboratory yeast strains, where standard propagation practice produces regular cycles of nutrient scarcity followed by repletion. Distinct [SMAUG+] variants are also widespread in wild yeast isolates from multiple niches, establishing that prion polymorphs can be utilized in natural populations. Our data provide a striking example of how protein-based epigenetic switches, hidden in plain sight, can establish a transgenerational memory that integrates adaptive prediction into developmental decisions.

Post-transcriptional regulation of gene expression plays a critical role in a variety of cellular processes during development. RNA binding proteins are fundamental mediators of post-transcriptional regulations that control mRNA expression by recognizing specific cis acting elements within the target transcripts. Smaug is a highly conserved sequence specific RNA-binding protein that is essential during Drosophila early embryogenesis. Smaug binds Smaug Recognition Elements (SRE) in the target mRNA and recruits additional factors, via protein-protein interactions, that regulate the bound mRNA. An emergent concept that signaling pathways can modulate RBP activity by post-translation modifications adds a new layer in the control of gene expression. During my thesis work, I sought to understand how the Hedgehog pathway regulates Smaug by promoting its phosphorylation. My work shows that HH signaling downregulates Smaug protein levels affecting its ability to repress mRNA translation. This negative effect seems to be dependent on the interaction between Smaug and the HH signal transducer Smoothened. Moreover, Smaug is constitutively phosphorylated in its RNA binding domain, which appears to be necessary for cytoplasmic Smaug foci formation., La régulation post-transcriptionnelle de l'expression génique joue un rôle essentiel dans divers processus cellulaires pendant le développement. Les protéines de liaison à l'ARN (RBP) sont des médiateurs fondamentaux des régulations post-transcriptionnelles qui contrôlent l'expression de l'ARNm en reconnaissant des séquences spécifiques dans les transcrits cibles. Smaug est une protéine de liaison à l'ARN conservée de la levure jusqu’à l’homme qui est essentielle pendant l'embryogenèse précoce de la drosophile. Smaug reconnaît et lie des éléments de reconnaissance de Smaug (SRE) dans ses ARNm cibles et recrute des facteurs supplémentaires, via des interactions protéine-protéine, qui régulent l'ARNm lié. Un concept qui émerge est celui des voies de signalisation pouvant moduler l'activité des RBP par des modifications post-traductionnelles, en ajoutant ainsi une couche supplémentaire dans le contrôle de l'expression des gènes.Au cours de mon travail de thèse, j'ai cherché à mettre en évidence que la voie de signalisation Hedgehog régule Smaug en favorisant sa phosphorylation. Mon travail montre que la signalisation HH diminue les niveaux de protéines Smaug affectant sa capacité à réprimer la traduction de l'ARNm. Cet effet négatif semble dépendre de l'interaction entre Smaug et le transducteur de signal HH, Smoothened. De plus, Smaug est constitutivement phosphorylée dans son domaine de liaison à l'ARN, ce qui semble être nécessaire pour la formation des foci cytoplasmiques de Smaug.

Post-transcriptional regulation of gene expression plays a critical role in a variety of cellular processes during development. RNA binding proteins are fundamental mediators of post-transcriptional regulations that control mRNA expression by recognizing specific cis acting elements within the target transcripts. Smaug is a highly conserved sequence specific RNA-binding protein that is essential during Drosophila early embryogenesis. Smaug binds Smaug Recognition Elements (SRE) in the target mRNA and recruits additional factors, via protein-protein interactions, that regulate the bound mRNA. An emergent concept that signaling pathways can modulate RBP activity by post-translation modifications adds a new layer in the control of gene expression. During my thesis work, I sought to understand how the Hedgehog pathway regulates Smaug by promoting its phosphorylation. My work shows that HH signaling downregulates Smaug protein levels affecting its ability to repress mRNA translation. This negative effect seems to be dependent on the interaction between Smaug and the HH signal transducer Smoothened. Moreover, Smaug is constitutively phosphorylated in its RNA binding domain, which appears to be necessary for cytoplasmic Smaug foci formation.; La régulation post-transcriptionnelle de l'expression génique joue un rôle essentiel dans divers processus cellulaires pendant le développement. Les protéines de liaison à l'ARN (RBP) sont des médiateurs fondamentaux des régulations post-transcriptionnelles qui contrôlent l'expression de l'ARNm en reconnaissant des séquences spécifiques dans les transcrits cibles. Smaug est une protéine de liaison à l'ARN conservée de la levure jusqu’à l’homme qui est essentielle pendant l'embryogenèse précoce de la drosophile. Smaug reconnaît et lie des éléments de reconnaissance de Smaug (SRE) dans ses ARNm cibles et recrute des facteurs supplémentaires, via des interactions protéine-protéine, qui régulent l'ARNm lié. Un concept qui émerge est celui des voies de signalisation pouvant moduler l'activité des RBP par des modifications post-traductionnelles, en ajoutant ainsi une couche supplémentaire dans le contrôle de l'expression des gènes.Au cours de mon travail de thèse, j'ai cherché à mettre en évidence que la voie de signalisation Hedgehog régule Smaug en favorisant sa phosphorylation. Mon travail montre que la signalisation HH diminue les niveaux de protéines Smaug affectant sa capacité à réprimer la traduction de l'ARNm. Cet effet négatif semble dépendre de l'interaction entre Smaug et le transducteur de signal HH, Smoothened. De plus, Smaug est constitutivement phosphorylée dans son domaine de liaison à l'ARN, ce qui semble être nécessaire pour la formation des foci cytoplasmiques de Smaug.

Dissertação de mestrado em Molecular Genetics, The conserved Hedgehog signalling pathway is central to embryogenesis and adult tissue homeostasis in metazoans, e.g. Drosophila melanogaster and vertebrates. Hedgehog (HH) acts as a morphogen regulating different cell fates depending on its concentration. HH transduction cascade is tightly regulated, and deregulation of HH signalling activity accounts for congenital diseases, carcinogenesis, neurological disorders and cardiovascular pathologies in humans. SMO, an oncogene of the GPCR family, is paramount to the transduction of the HH signal. In response to HH reception, SMO undergoes extensive phosphorylation, which controls its trafficking, leading to its accumulation at the plasma membrane in drosophila and in the primary cilium in vertebrates. By proteomic and genetic screens, the team “Development, Signalisation and Traffic” directed by Anne Plessis at the Institut Jacques Monod (IJM) identified a novel partner of SMO: the RNA binding protein SMAUG. SMAUG is a key component of mRNA storage bodies where it controls the degradation and translation of many mRNA during fly embryonic development. Their data indicated SMAUG may both act as a positive regulator of HH signaling and be regulated by HH signaling. My goals were (i) to further characterize the interaction between SMO and Smaug, (ii) to analyze their colocalisation in fly cells (iii) to understand better its potential role in HH signaling during wing morphogenesis. Using a combination of molecular, cellular and genetic approaches, I (i) confirmed the colocalisation of SMO and SMAUG, and showed that it was due to their direct interaction, (ii) analyzed the implication of known phosphosites of SMO in the regulation of its interaction with SMAUG, and initiated the search for the kinases that were involved in the regulation of SMO/SMAUG interaction. Finally, my in vivo studies on the role of SMAUG revealed a synthetic lethal interaction with a mutant affecting HH signaling but the interpretation of their results remains unclear. Altogether my data have brought novel information on this unexpected relationship which shed novel light on the function and regulation of both SMO and SMAUG., A conservada via de sinalização Hedgehog (HH) é central durante embriogéneses e homeostasia do tecido adulto em metazoários, e.g. Drosophila melanogaster e vertebrados. HH atua como um fator morfogénico, regulando diferentes destinos celulares, o que depende da sua concentração. A cascata de tradução de HH é fortemente regulada e, desregulação da atividade de sinalização de HH é responsável por doenças congénitas, carcinogénese, desordens neurológicas e patologias cardiovasculares em humanos. Smoothened (SMO), um oncogene da família dos recetores acoplados a proteínas G, é fundamental para a transdução do sinal da via HH. Em resposta à receção de HH, SMO é extensivamente fosforilado, o que controla o seu tráfego, levando à sua acumulação na membrana plasmática em drosófila, e no cílio primário em vertebrados. Através the análises proteómicas e genéticas, a equipa de investigação “Desenvolvimento, Sinalização e Tráfego” dirigida por Anne Plessis no Instituto Jacques Monod (IJM) identificou um novo parceiro de SMO: uma proteína de ligação ao ácido ribonucleico (ARN), Smaug. Smaug é um componente crucial dos corpos de armazenamento de ARN mensageiro (ARNm) onde controla a degradação e a tradução de muitos ARNm durante o desenvolvimento embrionário na mosca. Os dados obtidos pela equipa indicam que SMAUG possa atuar como regulador positivo da sinalização da via HH, e ser regulado pela mesma via. Os meus objetivos foram (i) caraterizar em maior detalhe a interação entre SMO e SMAUG, (ii) analisar a co localização entre as duas proteínas em células de mosca derivadas do disco imaginário da asa (estrutura que dá origem à asa adulta), (iii) de forma a compreender melhor o potencial papel da relação entre as duas proteínas durante a morfogénese da asa. Através da combinação de técnicas moleculares, celulares e genéticas, eu (i) confirmei a co localização de SMO com SMAUG, e mostrei que esta co localização é devida à interação direta entre as duas proteínas, (ii) analisei a implicação de sítios de fosforilação em SMO, descritos na literatura, na regulação da interação de SMO com SMAUG, e iniciei a procura pelas cinases que estão envolvidas na regulação da interação SMO/SMAUG. Finalmente, os meus estudos in vivo sobre o papel de SMAUG revelaram uma interação sintática letal com uma mutação que afeta a via de sinalização de HH. Contudo, a interpretação destes resultados permanece pouco clara. Em conjunto, os meus dados trouxeram informação adicional sobre esta relação inesperada, contribuindo para a compreensão da função e regulação de ambos SMO and SMAUG.

This chapter describes the major perspectives identified in post-viewing surveys relating to The Desolation of Smaug, conducted between January and July 2014, and The Battle of the Five Armies, conducted between January and May 2015. The chapter notes the emergence of increasingly critical and indeed polarised perspectives on the cinematic value, quality and impact of Jackson’s second and third Hobbit films. These critiques often expressed a more fundamental concern about the detrimental impact of key creative decisions that were seen to reflect underlying commercial imperatives. The chapter thus documents the crystallisation and intensification of audience ambivalence toward the processes and imperatives governing the cinematic adaptation, serialisation and blockbusterisation of Tolkien’s original novel.