Erel Levine

The let-7 –Imp axis regulates ageing of the Drosophila testis stem-cell niche

Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. Here we demonstrate that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals.

Recruitment of RNA polymerase II by the pioneer transcription factor PHA-4

Multifunctional pioneers Proteins surround cellular DNA to silence gene expression. Early in development, proteins such as pioneer transcription factors facilitate the opening of this silenced chromatin structure. Hsu et al. describe an additional role for the PHA-4 pioneer transcription factor in nematode worms. PHA-4 recruited RNA polymerase II to target promoters before transcriptional onset, and this activity preceded its chromatin-opening duty. The multifunctional role identified for PHA-4 may be shared by other pioneer factors. Science, this issue p. 1372 A transcription factor involved in cell-fate changes also facilitates chromatin opening. Pioneer transcription factors initiate cell-fate changes by binding to silent target genes. They are among the first factors to bind key regulatory sites and facilitate chromatin opening. Here, we identify an additional role for pioneer factors. In early Caenorhabditis elegans foregut development, the pioneer factor PHA-4/FoxA binds promoters and recruits RNA polymerase II (Pol II), often in a poised configuration in which Pol II accumulates near transcription start sites. At a later developmental stage, PHA-4 promotes chromatin opening. We found many more genes with poised RNA polymerase than had been observed previously in unstaged embryos, revealing that early embryos accumulate poised Pol II and that poising is dynamic. Our results suggest that Pol II recruitment, in addition to chromatin opening, is an important feature of PHA-4 pioneer factor activity.

Homeostasis in C. elegans sleep is characterized by two behaviorally and genetically distinct mechanisms

Biological homeostasis invokes modulatory responses aimed at stabilizing internal conditions. Using tunable photo- and mechano-stimulation, we identified two distinct categories of homeostatic responses during the sleep-like state of Caenorhabditis elegans (lethargus). In the presence of weak or no stimuli, extended motion caused a subsequent extension of quiescence. The neuropeptide Y receptor homolog, NPR-1, and an inhibitory neuropeptide known to activate it, FLP-18, were required for this process. In the presence of strong stimuli, the correlations between motion and quiescence were disrupted for several minutes but homeostasis manifested as an overall elevation of the time spent in quiescence. This response to strong stimuli required the function of the DAF-16/FOXO transcription factor in neurons, but not that of NPR-1. Conversely, response to weak stimuli did not require the function of DAF-16/FOXO. These findings suggest that routine homeostatic stabilization of sleep may be distinct from homeostatic compensation following a strong disturbance. DOI: http://dx.doi.org/10.7554/eLife.04380.001

Small RNA biology is systems biology.

During the last decade small regulatory RNA (srRNA) emerged as central players in the regulation of gene expression in all kingdoms of life. Multiple pathways for srRNA biogenesis and diverse mechanisms of gene regulation may indicate that srRNA regulation evolved independently multiple times. However, small RNA pathways share numerous properties, including the ability of a single srRNA to regulate multiple targets. Some of the mechanisms of gene regulation by srRNAs have significant effect on the abundance of free srRNAs that are ready to interact with new targets. This results in indirect interactions among seemingly unrelated genes, as well as in a crosstalk between different srRNA pathways. Here we briefly review and compare the major srRNA pathways, and argue that the impact of srRNA is always at the system level. We demonstrate how a simple mathematical model can ease the discussion of governing principles. To demonstrate these points we review a few examples from bacteria and animals.

Large-scale mapping of sequence-function relations in small regulatory RNAs reveals plasticity and modularity

Two decades into the genomics era the question of mapping sequence to function has evolved from identifying functional elements to characterizing their quantitative properties including, in particular, their specificity and efficiency. Here, we use a large-scale approach to establish a quantitative map between the sequence of a bacterial regulatory RNA and its efficiency in modulating the expression of its targets. Our approach generalizes the sort-seq method, introduced recently to analyze promoter sequences, in order to accurately quantify the efficiency of a large library of sequence variants. We focus on two small RNAs (sRNAs) in E. coli, DsrA and RyhB, and their regulation of both repressed and activated targets. In addition to precisely identifying functional elements in the sRNAs, our data establish quantitative relationships between structural and energetic features of the sRNAs and their regulatory activity, and characterize a large set of direct and indirect interactions between nucleotides. A core of these interactions supports a model where specificity can be enhanced by a rigid molecular structure. Both sRNAs exhibit a modular design with limited cross-interactions, dividing the requirements for structural stability and target binding among modules.

Regulating the Many to Benefit the Few: Role of Weak Small RNA Targets

Small regulatory RNAs are central players in the regulation of many cellular processes across all kingdoms of life. Experiments in mouse and human have shown that a typical small RNA may regulate the expression of many different genes, suggesting that small RNAs act as global regulators. It is noted though that most targets respond only weakly to the presence of the small RNA. At the same time, evidence in bacteria and animals suggest that the phenotypes associated with small RNA mutants are only due to a few of their targets. Here we assume that targets regulated by a small RNA to control function is in fact small, and propose that the role of the many other weak targets is to confer robustness to the regulation of these few principal targets. Through mathematical modeling we show that auxiliary targets may significantly buffer both number and kinetic fluctuations of the principal targets, with only minor slowdown in the kinetics of response. Analysis of genomic data suggests that auxiliary targets experience a nonspecific evolutionary pressure, playing a role at the system level. Our work is of importance for studies on small RNA functions, and impacts on the understanding of small RNA evolution.

Serotonin promotes exploitation in complex environments by accelerating decision-making.

BackgroundFast responses can provide a competitive advantage when resources are inhomogeneously distributed. The nematode Caenorhabditis elegans was shown to modulate locomotion on a lawn of bacterial food in serotonin (5-HT)-dependent manners. However, potential roles for serotonergic signaling in responding to food discovery are poorly understood.ResultsWe found that 5-HT signaling in C. elegans facilitates efficient exploitation in complex environments by mediating a rapid response upon encountering food. Genetic or cellular manipulations leading to deficient serotonergic signaling resulted in gradual responses and defective exploitation of a patchy foraging landscape. Physiological imaging revealed that the NSM serotonergic neurons responded acutely upon encounter with newly discovered food and were key to rapid responses. In contrast, the onset of responses of ADF serotonergic neurons preceded the physical encounter with the food. The serotonin-gated chloride channel MOD-1 and the ortholog of mammalian 5-HT1 metabotropic serotonin receptors SER-4 acted in synergy to accelerate decision-making. The relevance of responding rapidly was demonstrated in patchy environments, where the absence of 5-HT signaling was detrimental to exploitation.ConclusionsOur results implicate 5-HT in a novel form of decision-making, demonstrate its fitness consequences, suggest that NSM and ADF act in concert to modulate locomotion in complex environments, and identify the synergistic action of a channel and a metabotropic receptor in accelerating C. elegans decision-making.

Quantitative effect of target translation on small RNA efficacy reveals a novel mode of interaction

Small regulatory RNAs (sRNAs) in bacteria regulate many important cellular activities under normal conditions and in response to stress. Many sRNAs bind to the mRNA targets at or near the 5′ untranslated region (UTR) resulting in translation inhibition and accelerated degradation. Often the sRNA-binding site is adjacent to or overlapping with the ribosomal binding site (RBS), suggesting a possible interplay between sRNA and ribosome binding. Here we combine quantitative experiments with mathematical modeling to reveal novel features of the interaction between small RNAs and the translation machinery at the 5′UTR of a target mRNA. By measuring the response of a library of reporter targets with varied RBSs, we find that increasing translation rate can lead to increased repression. Quantitative analysis of these data suggests a recruitment model, where bound ribosomes facilitate binding of the sRNA. We experimentally verified predictions of this model for the cell-to-cell variability of target expression. Our findings offer a framework for understanding sRNA silencing in the context of bacterial physiology.

Serotonin-dependent kinetics of feeding bursts underlie a graded response to food availability in C. elegans

Animals integrate physiological and environmental signals to modulate their food uptake. The nematode C. elegans, whose food uptake consists of pumping bacteria from the environment into the gut, provides excellent opportunities for discovering principles of conserved regulatory mechanisms. Here we show that worms implement a graded feeding response to the concentration of environmental bacteria by modulating a commitment to bursts of fast pumping. Using long-term, high-resolution, longitudinal recordings of feeding dynamics under defined conditions, we find that the frequency and duration of pumping bursts increase and the duration of long pauses diminishes in environments richer in bacteria. The bioamine serotonin is required for food-dependent induction of bursts as well as for maintaining their high rate of pumping through two distinct mechanisms. We identify the differential roles of distinct families of serotonin receptors in this process and propose that regulation of bursts is a conserved mechanism of behaviour and motor control.

Sort-seq under the hood: implications of design choices on large-scale characterization of sequence-function relations

BackgroundSort-seq is an effective approach for simultaneous activity measurements in a large-scale library, combining flow cytometry, deep sequencing, and statistical inference. Such assays enable the characterization of functional landscapes at unprecedented scale for a wide-reaching array of biological molecules and functionalities in vivo. Applications of sort-seq range from footprinting to establishing quantitative models of biological systems and rational design of synthetic genetic elements. Nearly as diverse are implementations of this technique, reflecting key design choices with extensive impact on the scope and accuracy the results. Yet how to make these choices remains unclear. Here we investigate the effects of alternative sort-seq designs and inference methods on the information output using mathematical formulation and simulations.ResultsWe identify key intrinsic properties of any system of interest with practical implications for sort-seq assays, depending on the experimental goals. The fluorescence range and cell-to-cell variability specify the number of sorted populations needed for quantitative measurements that are precise and unbiased. These factors also indicate cases where an enrichment-based approach that uses a single sorted population can offer satisfactory results. These predications of our model are corroborated using re-analysis of published data. We explore implications of these results for quantitative modeling and library design.ConclusionsSort-seq assays can be streamlined by reducing the number of sorted populations, saving considerable resources. Simple preliminary experiments can guide optimal experiment design, minimizing cost while maintaining the maximal information output and avoiding latent biases. These insights can facilitate future applications of this highly adaptable technique.