[Viroids : infectious non coding RNAs]. / Les viroïdes : des ARN infectieux non codants.

Viroids are the smallest non-coding infectious RNAs (between 246 and 401 nucleotides) known to be highly structured and replicate autonomously in the host plants. Although they do not encode any peptides, viroids induce visible symptoms in susceptible host plants. This article provides an overview of their physical and biological properties, the diseases they cause and their significance for the plants. The mechanisms underlying the expression of symptoms in host plants, their detection and various strategies employed for diseases prevention are also developed.

Pathogenic SNPs Affect Both RNA and DNA G-Quadruplexes' Responses to Ligands.

Single nucleotide polymorphisms (SNPs) are common genetic variations that are present in over 1% of the population and can significantly modify the structures of both DNA and RNA. G-quadruplex structures (G4) are formed by the superposition of tetrads of guanines. To date, the impact of SNPs on both G4 ligands' binding efficacies and specificities has not been investigated. Here, using a bioinformatically predicted G4 and SNPs found in the α-synuclein gene as a proof-of-concept, it was demonstrated that SNPs can modulate both DNA and RNA G4s' responses to ligands. Specifically, six widely recognized ligands (Phen-DC3, PDS, 360A, RHPS4, BRACO19, and TMPyP4) were shown to differentially affect both the structure and the polymerase stalling of the different SNPs. This work highlights the importance of choosing the appropriate G4 ligand when dealing with an SNP identified in a G-rich gene.

Development of a highly optimized procedure for the discovery of RNA G-quadruplexes by combining several strategies.

RNA G-quadruplexes (rG4s) are non-canonical secondary structures that are formed by the self-association of guanine quartets and that are stabilized by monovalent cations (e.g. potassium). rG4s are key elements in several post-transcriptional regulation mechanisms, including both messenger RNA (mRNA) and microRNA processing, mRNA transport and translation, to name but a few examples. Over the past few years, multiple high-throughput approaches have been developed in order to identify rG4s, including bioinformatic prediction, in vitro assays and affinity capture experiments coupled to RNA sequencing. Each individual approach had its limits, and thus yielded only a fraction of the potential rG4 that are further confirmed (i.e., there is a significant level of false positive). This report aims to benefit from the strengths of several existing approaches to identify rG4s with a high potential of being folded in cells. Briefly, rG4s were pulled-down from cell lysates using the biotinylated biomimetic G4 ligand BioTASQ and the sequences thus isolated were then identified by RNA sequencing. Then, a novel bioinformatic pipeline that included DESeq2 to identify rG4 enriched transcripts, MACS2 to identify rG4 peaks, rG4-seq to increase rG4 formation probability and G4RNA Screener to detect putative rG4s was performed. This workflow uncovers new rG4 candidates whose rG4-folding was then confirmed in vitro using an array of established biophysical methods. Clearly, this workflow led to the identification of novel rG4s in a highly specific and reliable manner.

Coexistence of Hopf-born rotation and heteroclinic cycling in a time-delayed three-gene auto-regulated and mutually-repressed core genetic regulation network.

In this work, we study the behavior of a time-delayed mutually repressive auto-activating three-gene system. Delays are introduced to account for the location difference between DNA transcription that leads to production of messenger RNA and its translation that result in protein synthesis. We study the dynamics of the system using numerical simulations, computational bifurcation analysis and mathematical analysis. We find Hopf bifurcations leading to stable and unstable rotation in the system, and we study the rotational behavior as a function of cyclic mutual repression parameter asymmetry between each gene pair in the network. We focus on how rotation co-exists with a stable heteroclinic flow linking the three saddles in the system. We find that this coexistence allows for a transition between two markedly different types of rotation leading to strikingly different phenotypes. One type of rotation belongs to Hopf-induced rotation while the other type, belongs to heteroclinic cycling between three saddle nodes in the system. We discuss the evolutionary and biological implications of our findings.

Guanine Nucleotide-Binding Protein-Like 1 (GNL1) binds RNA G-quadruplex structures in genes associated with Parkinson's disease.

RNAs are highly regulated at the post-transcriptional level in neurodegenerative diseases and just a few mutations can significantly affect the fate of neuronal cells. To date, the impact of G-quadruplex (G4) regulation in neurodegenerative diseases like Parkinson's disease (PD) has not been analysed. In this study, in silico potential G4s located in deregulated genes related to the nervous system were initially identified and were found to be significantly enriched. Several G4 sequences found in the 5' untranslated regions (5'UTR) of mRNAs associated with Parkinson's disease were demonstrated to in fact fold in vitro by biochemical assays. Subcloning of the full-length 5'UTRs of these candidates upstream of a luciferase reporter system led to the demonstration that the G4s of both Parkin RBR E3 Ubiquitin Protein Ligase (PRKN) and Vacuolar Protein Sorting-Associated Protein 35 (VPS35) significantly repressed the translation of both genes in SH-SY5Y cells. Subsequently, a strategy of using label-free RNA affinity purification assays with either of these two G4 sequences as bait isolated the Guanine Nucleotide-Binding Protein-Like 1 (GNL1). The latter was shown to have a higher affinity for the G4 sequences than for their mutated version. This study sheds light on new RNA G-quadruplexes located in genes dysregulated in Parkinson disease and a new G4-binding protein, GNL1.

The Small Nuclear Ribonucleoprotein Polypeptide A (SNRPA) binds to the G-quadruplex of the BAG-1 5'UTR.

The protein "BCL-2-associated athanogene-1" (BAG-1), which exists in multiple isoforms, promotes cancer cell survival and is overexpressed in many different cancers. As a result, BAG-1-targeted therapy appears to be a promising strategy with which to treat cancer. It has previously been shown that the 5'UTR of the BAG-1 mRNA contains a guanine rich region that folds into a G-quadruplex structure which can modulate both its cap-dependent and its cap-independent translation. Accumulating data regarding G-quadruplex binding proteins suggest that these proteins can play a central role in gene expression. Consequently, the identification of the proteins that could potentially bind to the G-quadruplex of the BAG-1 mRNA was undertaken. Label-free RNA pulldown assays were performed using protein extracts from colorectal cancer cells and this leads to the detection of RNA G4 binding proteins by LC-MS/MS. The use of G-quadruplex containing RNA, as well as of a mutated version, ensured that the proteins identified were specific for the RNA G-quadruplex structure and not just general RNA binding proteins. Following confirmation of the interaction, the Small Nuclear Ribonucleoprotein Polypeptide A (SNRPA) was shown to bind directly to the BAG-1 mRNA through the G-quadruplex, and knock down experiments in colorectal cancer cells suggested that it can modulate the expression level of BAG-1.

Dynamic Analysis of the Time-Delayed Genetic Regulatory Network Between Two Auto-Regulated and Mutually Inhibitory Genes.

Time delays play important roles in genetic regulatory networks. In this paper, a gene regulatory network model with time delays and mutual inhibition is considered, where time delays are regarded as bifurcation parameters. In the first part of this paper, we analyze the associated characteristic equations and obtain the conditions for the stability of the system and the existence of Hopf bifurcations in five special cases. Explicit formulas are given to determine the direction and stability of the Hopf bifurcation by using the normal form method and the center manifold theorem. Numerical simulations are then performed to illustrate the results we obtained. In the second part of the paper, using time-delayed stochastic numerical simulations, we study the impact of biological fluctuations on the system and observe that, in modest noise regimes, unexpectedly, noise acts to stabilize the otherwise destabilized oscillatory system.

Stability and Hopf bifurcation analysis in a delayed three-node circuit involving interlinked positive and negative feedback loops.

A system involving interlinked positive and negative feedback loops is a flexible motif that can tune itself in gene regulatory networks. It is well known that time delay is inevitable in gene regulatory networks due to transcription and translation not being physically co-located. In this paper, we systematically consider the effect of time delay on the dynamical behavior of the three-node circuit with three time delays. Based on linear stability analysis and bifurcation theory, sufficient conditions for stability of equilibria and oscillatory behaviors via Hopf bifurcation are derived when choosing positive and negative feedback strengths as well as time delays τ1, τ2, τ3 as the bifurcation parameters, respectively. Moreover, stability and direction of the Hopf bifurcation of time delay are studied by using the normal form method and the center manifold theorem. Finally, several examples are performed to illustrate some analytical results we obtained.

The KRESCENT Program (2005-2015): An Evaluation of the State of Kidney Research Training in Canada.

BACKGROUND: The Kidney Research Scientist Core Education and National Training (KRESCENT) Program was launched in 2005 to enhance kidney research capacity in Canada and foster knowledge translation across the 4 themes of health research. OBJECTIVE: To evaluate the impact of KRESCENT on its major objectives and on the careers of trainees after its first 10 years. METHODS: An online survey of trainees (n = 53) who had completed or were enrolled in KRESCENT was conducted in 2015. Information was also obtained from curriculum vitae (CVs). A bibliometric analysis assessed scientific productivity, collaboration, and impact in comparison with unsuccessful applicants to KRESCENT over the same period. The analysis included a comparison of Canadian with international kidney research metrics from 2000 to 2014. RESULTS: Thirty-nine KRESCENT trainees completed the survey (74%), and 44 trainees (83%) submitted CVs. KRESCENT trainees had a high success rate at obtaining grant funding from the Canadian Institutes of Health Research (CIHR; 79%), and 76% of Post-Doctoral Fellows received academic appointments at the Assistant Professor level within 8 months of completing training. The majority of trainees reported that KRESCENT had contributed significantly to their success in securing CIHR funding (90%), and to the creation of knowledge (93%) and development of new methodologies (50%). Bibliometric analysis revealed a small but steady decline in total international kidney research output from 2000 to 2014, as a percentage of all health research, although overall impact of kidney research in Canada increased from 2000-2005 to 2009-2014 compared with other countries. KRESCENT trainees demonstrated increased productivity, multiauthored papers, impact, and international collaborations after their training, compared with nonfunded applicants. CONCLUSIONS: The KRESCENT Program has fostered kidney research career development and contributed to increased capacity, productivity, and collaboration. To further enhance knowledge creation and translation in kidney research in Canada, programs such as KRESCENT should be sustained via long-term funding partnerships.

MISE EN CONTEXTE: Le programme KRESCENT (Kidney Research Scientist Core Education and National Training) a été lancé en 2005 pour augmenter la capacité de la recherche sur les maladies du rein à travers le Canada, et pour encourager la transmission des connaissances au sein des quatre axes de recherche en santé. OBJECTIFS DE L'ÉTUDE: Cette étude avait pour but d'évaluer les répercussions du programme KRESCENT sur ses principaux objectifs ainsi que des retombées sur la carrière des stagiaires participants, dix ans après sa création. MÉTHODOLOGIE: Un sondage en ligne a été mené en 2015 auprès des stagiaires (n = 53) ayant été admis ou ayant complété le programme KRESCENT. Des renseignements ont également été obtenus par la consultation de curriculum vitae (CV). Une analyse bibliométrique a évalué la productivité scientifique et la collaboration des participants ainsi que les répercussions de leur participation à KRESCENT sur leur carrière. Les données de cette analyse ont été comparées à celles des candidats n'ayant pas été retenus au cours de la même période. L'analyse comprenait également une comparaison des données canadiennes avec celles obtenues en recherche sur les maladies du rein ailleurs dans le monde. RÉSULTATS: Trente-neuf stagiaires du KRESCENT ont complété le sondage en ligne, soit 74% des personnes contactées, et quarante-quatre ont soumis leur CV. De manière générale, les stagiaires du KRESCENT ont obtenu plus facilement des subventions des Instituts de recherche en santé du Canada (IRSC) avec un taux de succès de 79%. De plus, 76% des détenteurs d'une bourse au niveau postdoctoral ont obtenu des charges professorales à titre de professeur adjoint dans les 8 mois suivant leur formation. La très grande majorité des stagiaires (90%) a indiqué que le KRESCENT avait grandement contribué au fait qu'ils aient obtenu les fonds des IRSC, de même qu'à la création de nouveaux savoirs (93% des répondants) et au développement de nouvelles méthodes (50% des répondants). L'analyse bibliométrique a révélé un léger, quoique régulier, déclin de la quantité de résultats en recherche sur les maladies du rein dans le monde entre 2000 et 2014, lorsque converti en pourcentage des résultats totaux en recherche sur la santé. Et ce, bien que l'incidence générale de la recherche sur les maladies du rein ait augmenté au Canada de 2000 à 2005 ainsi qu'entre 2009 et 2014 en comparaison des autres pays. De manière générale, à la suite de leur formation, les stagiaires du KRESCENT ont démontré une plus grande productivité, ont plus souvent participé à la rédaction de publications collectives ou à des collaborations internationales que les demandeurs n'ayant pas reçu de financement. CONCLUSION: Le programme KRESCENT a favorisé le perfectionnement professionnel en recherche sur les maladies du rein et a contribué à augmenter la capacité de recherche, la productivité et la collaboration des participants. Ainsi, pour poursuivre la création de nouveaux savoirs en recherche sur les maladies du rein et faciliter leur transmission auprès des chercheurs canadiens, nous sommes d'avis que les programmes de formation tels que le KRESCENT devraient continuer d'être financés sur le long terme par l'entremise de partenariats.

Subtle interplay of stochasticity and deterministic dynamics pervades an evolutionary plausible genetic circuit for Bacillus subtilis competence.

Here we study the interplay of stochastic and deterministic dynamics in an evolutionary plausible candidate core genetic circuit for Bacillus subtilis competence. We find that high noise would not necessarily be detrimental to the circuit's ability to deliver the phenotype, due to an unexpected built-in robustness that we further investigate. Also, we find that seemingly subtle deterministic dynamical features of the regulation, unstable and stable limit cycles, while in the presence of biochemical noise, would result in a distinctive new observable in the phenotype. We conduct mathematical analyses of the system's stability at the fixed points and derive some general model-independent consequences. We also show how imperfect time-scale separation in the system would result in observables detrimental to the phenotype, that nature could have harnessed for selection.

Point-cycle bistability and stochasticity in a regulatory circuit for Bacillus subtilis competence.

Bacillus subtilis is a very well-studied organism in biology. Recent results show that an evolutionary plausible alternative competence regulation circuit for this bacterium, despite presenting equivalent functionality, exhibits physiologically important differences. Thus, it is not a priori clear why Nature only selects a specific gene regulation circuit other than a plethora of equivalent others. Here, we use simulations to study this question further. Based on the wild-type Bacillus subtilis circuit, we add a positive autoregulation feedback loop to the intermediate gene comS. We use bifurcation theory to study the dynamical features of the hypothetical gene circuit versus the feedback strength of the added loop, and we rely on stochastic simulations to perform in silico experiments. We discover the existence of a bistable system: a stable limit cycle and a stable fixed point separated by an unstable limit cycle with a varying height of underlying stochastic potential. This structure is absent from the wild type. The coexistence of the unstable limit cycle with stochastic noise endows the circuit with an ability to confine, prevent or switch between its two stable attractors.

Architecture-dependent noise discriminates functionally analogous differentiation circuits.

Gene regulatory circuits with different architectures (patterns of regulatory interactions) can generate similar dynamics. This raises the question of why a particular circuit architecture is selected to implement a given cellular process. To investigate this problem, we compared the Bacillus subtilis circuit that regulates differentiation into the competence state to an engineered circuit with an alternative architecture (SynEx) in silico and in vivo. Time-lapse microscopy measurements showed that SynEx cells generated competence dynamics similar to native cells and reconstituted the physiology of differentiation. However, architectural differences between the circuits altered the dynamic distribution of stochastic fluctuations (noise) during circuit operation. This distinction in noise causes functional differences between the circuits by selectively controlling the timing of competence episodes and response of the system to various DNA concentrations. These results reveal a tradeoff between temporal precision and physiological response range that is controlled by distinct noise characteristics of alternative circuit architectures.

A genetic timer through noise-induced stabilization of an unstable state.

Stochastic fluctuations affect the dynamics of biological systems. Typically, such noise causes perturbations that can permit genetic circuits to escape stable states, triggering, for example, phenotypic switching. In contrast, studies have shown that noise can surprisingly also generate new states, which exist solely in the presence of fluctuations. In those instances noise is supplied externally to the dynamical system. Here, we present a mechanism in which noise intrinsic to a simple genetic circuit effectively stabilizes a deterministically unstable state. Furthermore, this noise-induced stabilization represents a unique mechanism for a genetic timer. Specifically, we analyzed the effect of noise intrinsic to a prototypical two-component gene-circuit architecture composed of interacting positive and negative feedback loops. Genetic circuits with this topology are common in biology and typically regulate cell cycles and circadian clocks. These systems can undergo a variety of bifurcations in response to parameter changes. Simulations show that near one such bifurcation, noise induces oscillations around an unstable spiral point and thus effectively stabilizes this unstable fixed point. Because of the periodicity of these oscillations, the lifetime of the noise-dependent stabilization exhibits a polymodal distribution with multiple, well defined, and regularly spaced peaks. Therefore, the noise-induced stabilization presented here constitutes a minimal mechanism for a genetic circuit to function as a timer that could be used in the engineering of synthetic circuits.

Coordinate regulation of G protein signaling via dynamic interactions of receptor and GAP.

Signal output from receptor-G-protein-effector modules is a dynamic function of the nucleotide exchange activity of the receptor, the GTPase-accelerating activity of GTPase-activating proteins (GAPs), and their interactions. GAPs may inhibit steady-state signaling but may also accelerate deactivation upon removal of stimulus without significantly inhibiting output when the receptor is active. Further, some effectors (e.g., phospholipase C-beta) are themselves GAPs, and it is unclear how such effectors can be stimulated by G proteins at the same time as they accelerate G protein deactivation. The multiple combinations of protein-protein associations and interacting regulatory effects that allow such complex behaviors in this system do not permit the usual simplifying assumptions of traditional enzyme kinetics and are uniquely subject to systems-level analysis. We developed a kinetic model for G protein signaling that permits analysis of both interactive and independent G protein binding and regulation by receptor and GAP. We evaluated parameters of the model (all forward and reverse rate constants) by global least-squares fitting to a diverse set of steady-state GTPase measurements in an m1 muscarinic receptor-G(q)-phospholipase C-beta1 module in which GTPase activities were varied by approximately 10(4)-fold. We provide multiple tests to validate the fitted parameter set, which is consistent with results from the few previous pre-steady-state kinetic measurements. Results indicate that (1) GAP potentiates the GDP/GTP exchange activity of the receptor, an activity never before reported; (2) exchange activity of the receptor is biased toward replacement of GDP by GTP; (3) receptor and GAP bind G protein with negative cooperativity when G protein is bound to either GTP or GDP, promoting rapid GAP binding and dissociation; (4) GAP indirectly stabilizes the continuous binding of receptor to G protein during steady-state GTPase hydrolysis, thus further enhancing receptor activity; and (5) receptor accelerates GDP/GTP exchange primarily by opening an otherwise closed nucleotide binding site on the G protein but has minimal effect on affinity (K(assoc) = k(assoc)/k(dissoc)) of G protein for nucleotide. Model-based simulation explains how GAP activity can accelerate deactivation >10-fold upon removal of agonist but still allow high signal output while the receptor is active. Analysis of GTPase flux through distinct reaction pathways and consequent accumulation of specific GTPase cycle intermediates indicate that, in the presence of a GAP, the receptor remains bound to G protein throughout the GTPase cycle and that GAP binds primarily during the GTP-bound phase. The analysis explains these behaviors and relates them to the specific regulatory phenomena described above. The work also demonstrates the applicability of appropriately data-constrained system-level analysis to signaling networks of this scale.